Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Differential modulation of T-type voltage gated calcium channels by G-protein coupled receptors. Hildebrand, Michael Earl 2008

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2008_fall_hildebrand_michael.pdf [ 24.35MB ]
Metadata
JSON: 24-1.0066474.json
JSON-LD: 24-1.0066474-ld.json
RDF/XML (Pretty): 24-1.0066474-rdf.xml
RDF/JSON: 24-1.0066474-rdf.json
Turtle: 24-1.0066474-turtle.txt
N-Triples: 24-1.0066474-rdf-ntriples.txt
Original Record: 24-1.0066474-source.json
Full Text
24-1.0066474-fulltext.txt
Citation
24-1.0066474.ris

Full Text

   DIFFERENTIAL MODULATION OF T-TYPE VOLTAGE GATED CALCIUM CHANNELS BY G-PROTEIN COUPLED RECEPTORS  by  Michael Earl Hildebrand  B.Sc., University College of the Fraser Valley, 2001    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies  (Neuroscience)    THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  July 2008    © Michael Earl Hildebrand, 2008    ii ABSTRACT  T-type voltage-gated calcium (Ca2+) channels play critical roles in controlling neuronal excitability, firing patterns, and synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein coupled receptors (GPCRs) remains largely unexplored. Investigations into T-type modulation within native neuronal systems have been complicated by the presence of multiple GPCR subtypes and a lack of pharmacological tools to separate currents generated by the three T-type isoforms; Cav3.1, Cav3.2, and Cav3.3.  We hypothesize that specific Cav3 subtypes play unique roles in neuronal physiology due to their differential functional coupling to specific GPCRs. Co-expression of T-type channel subtypes and GPCRs in a heterologous system allowed us to identify the specific interactions between muscarinic acetylcholine (mAChR) or metabotropic glutamate (mGluR) GPCRs and individual Cav3 isoforms.  Perforated patch recordings demonstrated that activation of Gαq/11-coupled GPCRs had a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes.  Further study of the inhibition of Cav3.3 channels by a specific Gαq/11-coupled mAChR (M1) revealed that this reversible inhibition was associated with a concomitant increase in inactivation kinetics.  Pharmacological and genetic experiments indicated that the M1 receptor-mediated inhibition of Cav3.3 occurs specifically through a Gαq/11 signaling pathway that interacts with two distinct regions of the Cav3.3 channel. As hypothesized, the potentiation of Cav3.1 channels by a Gαq/11-coupled mGluR (mGluR1) initially characterized in the heterologous system was also observed in a native neuronal system: the cerebellar Purkinje cell (PC).  In recordings on PCs within acute cerebellar slices, we demonstrated that the potentiation of Cav3.1 currents by mGluR1 activation is strongest near the threshold of T-type currents, enhancing the excitability of PCs.  Ultrafast two-photon Ca2+ imaging demonstrated that the functional coupling between mGluR1 and T-type transients occurs within dendritic spines, where synaptic integration and plasticity occurs.  A subset of these experiments utilized physiological synaptic activation and specific mGluR1 antagonists in wild-type and Cav3.1 knock-out mice to show that the mGluR1-mediated potentiation of Cav3.1 T-type currents may promote synapse-specific Ca2+ signaling in response to bursts of excitatory inputs.   iii TABLE OF CONTENTS  Abstract................................................................................................................... ii Table of Contents .................................................................................................. iii List of Tables ..........................................................................................................vi List of Figures....................................................................................................... vii List of Abbreviations .......................................................................................... viii Acknowledgements .................................................................................................x Dedication ...............................................................................................................xi Co-Authorship Statement ................................................................................... xii 1 Introduction..........................................................................................................1 1.1 Voltage-gated calcium channel overview......................................................................... 1 1.1.1 Historical context .......................................................................................................... 1 1.1.2 General calcium channel biochemical composition ..................................................... 3 1.1.3 Calcium channel classes ............................................................................................... 5 1.2 T-type voltage-gated calcium channels ............................................................................ 8 1.2.1 T-type channel pharmacology....................................................................................... 8 1.2.2 T-type channel biophysical properties ........................................................................ 10 1.2.3 T-type channel expression .......................................................................................... 14 1.2.4 T-type channel physiological roles ............................................................................. 16 1.2.5 T-type channels in human disease .............................................................................. 19 1.3 Modulation of voltage-gated calcium channels ............................................................. 21 1.3.1 G-protein-coupled receptors ....................................................................................... 21 1.3.2 Modulation of HVA calcium channels ....................................................................... 23 1.3.3 Modulation of T-type calcium channels ..................................................................... 28 1.4 Cerebellar Purkinje cells................................................................................................. 32 1.4.1 Cerebellum overview.................................................................................................. 32 1.4.2 Cerebellar Purkinje cell physiology............................................................................ 36 1.4.3 mGluR1 expression and function ............................................................................... 38 1.4.4 T-type expression and function................................................................................... 40 1.5 Thesis hypotheses and objectives.................................................................................... 43 1.5.1 Hypotheses.................................................................................................................. 43 1.5.2 Objectives ................................................................................................................... 43 1.6 References......................................................................................................................... 45  2 Selective Inhibition of CaV3.3 T-Type Calcium Channels by GαQ/11-Coupled Muscarinic Acetylcholine Receptors.......................................66 2.1 Introduction...................................................................................................................... 66   iv 2.2 Results ............................................................................................................................... 67 2.2.1 Muscarinic M1 receptors selectively inhibit Cav3.3 T-type calcium channels........... 67 2.2.2 Muscarinic M1 receptors dose-dependently modulate Cav3.3 biophysical properties.............................................................................................................................. 70 2.2.3 Inhibition of Cav3.3 channels by M1 receptors requires Gαq/11 ................................. 73 2.2.4 Constitutively active Gαq/11 proteins modulate Cav3.3 T-type calcium channels ...... 73 2.2.5 Gαq/11 inhibits Cav3.3 channels through an unidentified non-classical pathway........ 75 2.2.6 Gαq/11-coupled muscarinic receptors selectively inhibit Cav3.3 channels.................. 78 2.2.7 Two distinct Cav3.3 channel regions are involved in M1-mediated inhibition .......... 78 2.3 Discussion ......................................................................................................................... 83 2.3.1 Differential effects of mAChRs on T-type calcium channel isoforms ....................... 83 2.3.2 Functional effects of M1 receptor activation on Cav3.3 currents ............................... 84 2.3.3 Signal transduction pathway of M1 receptor-mediated Cav3.3 inhibition.................. 85 2.3.4 Gαq/11-mediated inhibition of Cav3.3 involves two discrete channel regions ............ 86 2.4 Experimental procedures ................................................................................................ 87 2.4.1 Molecular biology....................................................................................................... 87 2.4.2 Cell culture and transfection ....................................................................................... 87 2.4.3 Electrophysiological recordings and analysis............................................................. 88 2.4.4 Solutions, drugs, and perfusion................................................................................... 89 2.5 Acknowledgements .......................................................................................................... 90 2.6 References......................................................................................................................... 91 3 Functional Coupling Between mGluR1 and Cav3.1 T-Type Calcium Channels Enhances Cerebellar Purkinje Cell Excitability and Local Signaling.................................................................................................................96 3.1 Introduction...................................................................................................................... 96 3.2 Results ............................................................................................................................... 97 3.2.1 Subtype-specific modulation of recombinant T-type calcium channels by mGluR1a activation ............................................................................................................. 97 3.2.2 Cav3.1-mediated T-type calcium currents are potentiated by mGluR1 activation in cerebellar Purkinje neurons ............................................................................................. 99 3.2.3 mGluR1 potentiates T-type currents through an increase in maximal current and a shift in the voltage-dependence of activation........................................................... 103 3.2.4 mGluR1 activation increases PC excitability via effects on T-type calcium currents............................................................................................................................... 103 3.2.5 mGluR1 potentiates T-type currents through a G-protein-, tyrosine phosphatase-, and calcium- dependent pathway independently of phospholipase C and its downstream effectors.................................................................. 109 3.2.6 mGluR1 potentiates T-type calcium transients at synaptic sites .............................. 109 3.2.7 Parallel fiber inputs trigger T-type calcium transients in spines that are potentiated by mGluR1 activation ..................................................................................... 114   v 3.3 Discussion ....................................................................................................................... 117 3.3.1 mGluR1 potentiation of Cav3.1 T-type calcium channels ........................................ 117 3.3.2 Signal transduction pathway..................................................................................... 117 3.3.3 Physiological implications of alterations in T-type biophysical properties.............. 118 3.3.4 mGluR1 potentiation of Cav3.1-mediated calcium influx in response to synaptic activity ................................................................................................................. 119 3.4 Experimental procedures .............................................................................................. 120 3.4.1 HEK 293 cell culture, transfection, and electrophysiology ...................................... 120 3.4.2 Animals ..................................................................................................................... 120 3.4.3 Slice preparation ....................................................................................................... 120 3.4.4 Electrophysiological recordings................................................................................ 121 3.4.5 Compounds and perfusion ........................................................................................ 122 3.4.6 Two-photon imaging................................................................................................. 123 3.5 Acknowledgements ........................................................................................................ 124 3.6 References....................................................................................................................... 125 4 Discussion..........................................................................................................130 4.1 Overall significance and strengths ............................................................................... 130 4.1.1 T-type calcium channel modulation in a heterologous system................................. 130 4.1.2 T-type calcium channel modulation in cerebellar Purkinje cells.............................. 131 4.2 Specific inhibition of Cav3.3 channels by Gαq/11-coupled receptors.......................... 131 4.2.1 Working hypothesis .................................................................................................. 131 4.2.2 Possible limitations and weaknesses......................................................................... 133 4.3 Potentiation of Cav3.1 currents by mGluR1................................................................ 135 4.3.1 Working hypotheses.................................................................................................. 135 4.3.2 Possible limitations and weaknesses......................................................................... 141 4.4 Conclusions..................................................................................................................... 143 4.4.1 General conclusions .................................................................................................. 143 4.4.2 Possible relevance to human disease ........................................................................ 143 4.4.3 Future directions ....................................................................................................... 145 4.5 References....................................................................................................................... 148 Appendix 1: Contributions of T-Type Calcium Channels to the Pathophysiology of Pain Signaling ....................................................................154 Appendix 2: Activation of Corticotropin-Releasing Factor Receptor 1 Selectively Inhibits CaV3.2 T-Type Calcium Channels ...................................161 Appendix 3: Other PhD Publications ...............................................................175 Appendix 4: UBC Research Certificates of Approval.....................................176   vi LIST OF TABLES  Table 2.1 - Effects of Receptor Activation on T-Type Channel Kinetic and Voltage- Dependent Properties ................................................................................................................ 68 Table 2.2 - Effects of M1 Receptor Activation on Chimeric T-Type Channel Inactivation Kinetics.................................................................................................................. 83 Table 3.2 - Effects of DHPG on Purkinje Cell T-Type Biophysical Properties ................ 102   vii LIST OF FIGURES  Figure 1.1 - Calcium channel classifications and phylogeny.................................................... 3 Figure 1.2 - Structural features of the α1 calcium channel subunit. ....................................... 4 Figure 1.3 - Representative differences between T-type and HVA calcium channels........... 6 Figure 1.4 - Alternative splicing of intracellular exons in human Cav3 channels. .............. 12 Figure 1.5 - Modulation sites of the α1 calcium channel subunit. ......................................... 25 Figure 1.6 - Circuitry within the cerebellar cortex................................................................. 35 Figure 2.1 - T-type calcium channels are differentially modulated by M1 receptors. ........ 69 Figure 2.2 - Mechanistic properties of inhibition of Cav3.3 currents by M1 receptors....... 71 Figure 2.3 - Inhibition of Cav3.3 channels by M1 requires Gαq/11 signaling. ....................... 74 Figure 2.4 - The Gαq/11 subtypes of Gα proteins specifically cause inhibition of Cav3.3 currents........................................................................................................................... 76 Figure 2.5 - Inhibition of Cav3.3 channels by M1 does not require PIP2 signaling. ............ 77 Figure 2.6 - Inhibition of Cav3.3 currents occurs specifically through Gαq/11-coupled mAChRs. .................................................................................................................................... 79 Figure 2.7 - Regions II and IV of human Cav3.3 channels are required and appear sufficient for M1-mediated inhibition. ..................................................................................... 80 Figure 3.1 - Recombinant T-type calcium channels are differentially modulated by mGluR1a receptors.................................................................................................................... 98 Figure 3.2 - T-type calcium channels in cerebellar PCs are reversibly potentiated by mGluR1 activation. .................................................................................................................. 101 Figure 3.3 - T-type calcium currents are potentiated by mGluR1 through an increase in maximal current and a small shift in the voltage-dependence of activation.................. 104 Figure 3.4 - DHPG lowers the threshold for PC T-type-dependent calcium spikes and AP firing. ........................................................................................................................... 107 Figure 3.5 - T-type calcium currents are potentiated by mGluR1 through a signaling pathway that involves G-proteins, intracellular calcium, and tyrosine phosphatases. ..... 110 Figure 3.6 - DHPG mediates an increase in T-type calcium transients in PCs.................. 112 Figure 3.7 - A burst of PF stimulation modulates Cav3.1 calcium channels via mGluR1 receptors.................................................................................................................... 116 Figure 4.1 - Amino acid sequence alignments. ...................................................................... 135 Figure 4.2 - Proposed intracellular signaling microdomains within Purkinje cell spines. ................................................................................................................................. 137   viii LIST OF ABBREVIATIONS  ACSF:   artificial cerebrospinal fluid AMPA:   α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AOD:    acousto-optic deflectors AP:   action potential Ba2+:   barium BBS:   bicarbonate-buffered saline Ca2+:   calcium CAMKII:   calcium/calmodulin kinase II CCh:   carbachol Cd2+:   cadmium CF:   climbing fiber CNS:   central nervous system CPA:   cyclopiazonic acid CRFR:   corticotrophin-releasing factor receptor DAG:   diacylglycerol DCN:   deep cerebellar nuclei DRG:   dorsal root ganglion EPSP:   excitatory postsynaptic potential Gαt:   transducin GAERS:   genetic absence epilepsy rat from Strasbourg GC:   granule cell GDP-β-S:   guanosine-5'-O-(2-thiodiphosphate) GPCR:   G-protein coupled receptor HEK:   human embryonic kidney HVA:   high voltage-activated IGE:   idiopathic generalized epilepsy Ih:   hyperpolarization-activated inward current IL:   leak current IP3:   inositol-1,4,5-trisphosphate IP3R:   IP3 receptor K+:   potassium KO:   knock-out LTD:   long term depression   ix LTP:   long term potentiation LVA:   low voltage-activated mAChR:   muscarinic acetylcholine receptor MAPK:   mitogen-activated protein kinase MF:   mossy fiber mGluR:   metabotropic glutamate receptor Na+:   sodium Ni2+:   nickel NMDA:   N-methyl D-aspartate nRT:   thalamic reticular neuron palpeptide:   N-terminal palmitoylated decapeptide PC:   Purkinje cell PF:   parallel fiber PIP2:   phosphatidylinositol-4,5-bisphosphonate PKA:   protein kinase A PKC:   protein kinase C PLC:   phospholipase C PMA:   phorbol 12-myristate 13-acetate PNS:   peripheral nervous system POIs:   points of interest PTK:   protein tyrosine kinase PTX:   pertussis toxin RGS:   regulator of G-protein signaling RN:   input resistance RS:   series resistance SCG:   superior cervical ganglion sEPSC:   slow excitatory postsynaptic current sER:   smooth endoplasmic reticulum SWD:   spike-and-wave discharge TC:   thalamocortical relay neuron TRPC:   transient receptor potential channel UCN:   urocortin wt:   wild-type   x ACKNOWLEDGEMENTS  I would like to thank my supervisor, Terry Snutch, for the guidance, patience and encouragement that he gave me during my PhD studies.  No matter how busy he was, Terry’s door was always open when I needed to chat with him.  Several other very knowledgeable mentors, including Esperanza Garcia, Philippe Isope, and Tony Stea, taught me many techniques and were a constant source of support.   Thank you.  Many thanks to my supervisory committee, Steve Kehl, Tim Murphy, and Brian MacVicar, for their direction, advice, and feedback on this thesis. A special thanks to all of the past and present members of the Snutch lab.  Besides the great scientific collaborations and discussions I had with many of you, the laughter that could be found with you all helped me survive the inevitable rough patches that are a part of scientific research.  I would also like to thank Dave Parker, Elizabeth Tringham, Francesco Belardetti, Diana Janke, Johnny Lam and the many others at Neuromed Pharmaceuticals who provided me with useful reagents as well as technical advice.  Thank you to Gerald Zamponi and Jawed Hamid for the chimeric channels that added a key piece of the puzzle to the modulation story found in Chapter 2, and to Stephane Dieudonne and the other colleagues of Philippe Isope who provided reagents, experiments, and feedback on the study found in Chapter 3.   All other research experiments were performed in the Michael Smith Laboratories.  I am also very grateful to the Michael Smith Foundation for Health Research and the Natural Sciences and Engineering Research Council of Canada for providing me with trainee funding during my graduate studies. I would like to thank all of the family and friends that helped me maintain sanity and balance during the marathon that is grad school.  A special thanks to my parents and grandparents, who taught me the value of hard work, perseverance, and integrity.  Last but not least, I would like to thank my wife, Sara, for proofreading every one of my papers and for unconditionally supporting me at every stage of this process.   xi DEDICATION   To my saviour, Jesus Christ      my loving wife, Sara     and my precious son, Joshua   xii CO-AUTHORSHIP STATEMENT Along with input and feedback from other researchers, I designed and performed all of the experiments and analyzed the experimental results for all experiments, except for those shown in Figure 3.2E,F; Figure 3.6; and Figure 3.7.  In other words, I performed all experiments except for the electrophysiological recordings on PCs that involved Ca2+ imaging or wild-type, Cav2.3 KO and Cav3.1 KO mice.  As stated in the section 2.4.1 of Chapter 2, I also did not generate the Cav3.1-Cav3.3 chimeric channels used in Figure 2.7 or any other channel-plasmid constructs. In terms of manuscript preparation, I wrote the entire manuscript for Chapter 2, with subsequent editing from Terry Snutch and other authors.  For Chapter 3, I wrote the first draft for all sections except for section 3.3 and parts of sections 3.2 and 3.4 (written by Philippe Isope and Stephane Dieudonne).  I offered editing and feedback on the sections that I did not write for Chapter 3. I designed and wrote the entire review article shown in Appendix 1, with subsequent editing by Terry Snutch.  For the research article in Appendix 2, I helped plan and design several critical experiments, I performed the experiments on the modulation of all three human T-type channels by UCN as well as the recovery from inactivation experiments, and I helped edit the paper and wrote a small section of the discussion. Chapter 1: Introduction  1 1 INTRODUCTION  1.1 Voltage-gated calcium channel overview 1.1.1 Historical context With more than 39,000 publications currently listed in PubMed, the field of voltage-gated calcium (Ca2+) channel research has developed exponentially from its inception over 50 years ago through detailed characterizations in native systems and to the present molecular structure-function analyses of multiple channel classes.  A recurrent theme throughout this rapidly-expanding trajectory has been that the selective modulation of Ca2+ channel activity by various cellular signaling pathways contributes to a wide variety of physiological functions. The first evidence concerning the existence of specific Ca2+ channel-mediated activity emerged in the 1950s.  In the era when Hodgkin and Huxley were characterizing the sodium (Na+)-driven action potentials (APs) in the squid giant axon, Bernard Katz, Paul Fatt, and Bernard Ginsborg discovered a novel form of electrical excitability in the large muscle cells of crab and crayfish that was independent of Na+ gradients and appeared to involve Ca2+ spikes (Fatt and Ginsborg, 1958; Fatt and Katz, 1953).  As with many novel and unexpected scientific discoveries, initial progress and acceptance was slow.  Two key contributors in this “incubation period” of Ca2+ channel discovery were Harald Reuter and Albrecht Fleckenstein.  Reuter performed voltage-clamp recordings on Purkinje fibers of the heart to directly demonstrate a transmembrane Ca2+ current linked to excitation-contraction coupling (Reuter, 1967) while Fleckenstein discovered that certain small organic compounds including verapamil and nifedipine (a dihydropyridine) blocked the Ca2+ signaling relating to excitation-contraction coupling (reviewed in (Fleckenstein, 1983)).  The dihydropyridines were later shown to be antagonists of voltage-gated Ca2+ channels and proved crucial in the purification and characterization of Ca2+ channels. In the late 1970s, voltage-clamp studies in many large invertebrate preparations started to open up the Ca2+ channel field.  Building on the Ca2+ hypothesis of synaptic transmission by Katz and Miledi (Katz and Miledi, 1970), Rodolfo Llinas and colleagues used recordings on the squid stellate ganglion to reveal presynaptic, inward Ca2+ channel currents that were directly linked to postsynaptic potentials, and thus, neurotransmitter release (Llinas et al., 1976).  Llinas went on to apply these findings to mammalian neuronal systems, where he characterized the electrophysiological properties and Ca2+ channel activity in cells such as the cerebellar Purkinje cell (PC) (Llinas and Sugimori, 1980a, b).  Concomitantly, Susuma Hagiwara characterized Ca2+ conductances from various invertebrate tissues ranging from the egg cell membrane of starfish to barnacle giant muscle fibers (Hagiwara et al., 1975).  Together, these studies provided the first evidence for the existence of more than one type of voltage-gated Ca2+ channel. The proliferation of patch clamp recording technology in the 1980s enabled the Ca2+ channel field to explode, as single-cell voltage-clamp recordings were now possible on various types of neurons Chapter 1: Introduction  2 throughout the brain, spinal cord, and peripheral nervous system (PNS).  Building on Hagiwara’s work, Carbone and Lux, along with Llinas and others, divided Ca2+ channel currents into two groupings, low voltage-activated (LVA) and high voltage-activated (HVA) conductances, based on the level of depolarization required to first activate the channels (Carbone and Lux, 1984).  Subsequent extensive work in various native systems by Michael Adams, Clay Armstrong, Kurt Beam, Bruce Bean, Peter Hess, Ed McCleskey, Baldamero Olivera, and Richard Tsien led to the identification of multiple Ca2+ channel classes based on biophysical differences and differential sensitivities to pharmacological tools and venom toxins (reviewed in (Tsien and Barrett, 2005)).  The Ca2+ channel classes included L-type channels that were long lasting and had a large unitary conductance, N-type channels recorded from neurons and that were non L-type, P (and Q)-type channels that were described from cerebellar Purkinje cells, R-type channels that were toxin-resistant, and T-type channels that formed tiny and transient currents.  T-type channels were thought to exclusively contribute to LVA currents, while all other classes contributed to HVA currents (Fig. 1.1). The understanding of the molecular components underlying native Ca2+ channel currents began with the biochemical purification of the skeletal muscle (L-type) Ca2+ channel complex by Bill Catterall, Kevin Campbell, Franz Hoffman and others.  Channel purification revealed that functional L-type Ca2+ channels contained multiple subunits including a pore-forming α1 subunit (based on dihydropyridine pore blockers binding to this subunit), and was followed by the cloning of the pore-forming α1S (now Cav1.1) channel subunit by a team led by Shosaku Numa (reviewed in (Dolphin, 2006)).  Terry Snutch subsequently showed that distinct from skeletal muscle, the nervous system expressed a family of Ca2+ channel α1 subunits and he was able to identify and clone neuronal L-type channels (Cav1.2/α1C; Cav1.3/α1D), the Cav2.1/α1Α channel that underlies P/Q-type currents, the Cav2.2/α1B channel that underlies N-type currents, and the Cav2.3/α1E channel that partially underlies R-type currents (Snutch et al., 1990; Soong et al., 1993).  The final chapter in the Ca2+ channel molecular cloning effort was achieved by in silico alignment searches of the C. elegans, rat and human genomes and led to the identification of three mammalian T-type channels (Cav3.1/α1G; Cav3.2/α1H; Cav3.3/α1I) as well as an additional L-type channel (Cav1.4/α1F) (Cribbs et al., 1998; Lee et al., 1999b; McRory et al., 2004; McRory et al., 2001; Perez-Reyes et al., 1998) (reviewed in (Snutch et al., 2005)).  Figure 1.1 summarizes the primary sequence relationships between all known Ca2+ channel α1 subunits and their corresponding historical native classifications.  Chapter 1: Introduction  3 LVAT-type/ L-Type P/Q-type N-type R-type HVA  Figure 1.1 - Calcium channel classifications and phylogeny. Phylogenetic relationships between primary sequences of cloned Ca2+ channels.  Cav α1 subunits are shown in black, with historical channel names in parentheses.  Only membrane spanning segments and pore-forming loops are compared in this phylogeny.  Calcium channel classes belonging to the HVA historical grouping are shown in magenta while Ca2+ channel classes belong to the LVA historical grouping are shown in blue.  1.1.2 General calcium channel biochemical composition Functional HVA Ca2+ channels appear to be comprised of a multi-subunit complex containing a large (190 to 250 kDa) pore-forming α1 subunit, an intracellular β subunit, a transmembrane, disulfide- linked α2δ subunit, and a γ subunit.  Although the α1 subunit is responsible for most channel biophysical and modulation properties, the auxiliary subunits (4 β subunit genes, four α2δ subunit genes, and eight γ subunit genes cloned to date) increase channel diversity and functional specialization by altering the trafficking, voltage-dependence, and kinetics of specific channel complexes (Stea et al., 1994) (reviewed in (Arikkath and Campbell, 2003)).  The importance of these auxiliary subunits is emphasized by the fact that spontaneous mutations within them can cause epileptic and ataxic phenotypes (reviewed in (Adams and Snutch, 2007)). The determination of the amino acid composition of the Ca2+ channel α1 subunits revealed that they are members of a gene superfamily of transmembrane ion channel proteins that includes voltage- gated potassium (K+) and Na+ channels.  Calcium channels are most closely related to Na+ channel α subunits, with each being composed of four homologous domains each containing six hydrophobic transmembrane segments (termed S1 to S6) and a pore-forming loop between S5 and S6 segments (Fig. 1.2).  The pore-forming loop is responsible for the specificity and permeation properties of the channel, Chapter 1: Introduction  4 while regularly arranged positively charged amino acids in the S4 transmembrane segments contribute to the voltage-sensitive gating of the channel.  Due to their critical functions, the transmembrane segments and pore regions are well conserved across all 10 Ca2+ channel α1 subunit genes (Fig. 1.1).  Overall, amino acid sequences of α1 subunits are over 70% identical within a channel subfamily (e.g. - Cav1), while less than 40% identical between channel subfamilies (e.g. - Cav1 versus Cav2) (reviewed in (Catterall et al., 2005)).  Most divergence occurs within putative cytoplasmic regions of the channel, such as the domain II - domain III linker and the C-terminal tail, where there is also significant variation in size.  Of all Ca2+ channel groupings, T-type channels have the least amount of sequence identity compared to the other classes.  In fact, cloned T-type channels lack entire structural motifs that are conserved in all HVA Ca2+ channel classes, including the auxiliary β subunit binding site in the domain I-II linker and an EF hand Ca2+ binding motif in the C-terminus.  Thus, the typical HVA auxiliary subunits likely do not have an essential role in forming functional native T-type currents and, as discussed below, the modulation of T-type channels by second messenger-dependent pathways also differs from the HVA Ca2+ channels. 1 2 3 4 5 6 + + + + + + + + + + + + + + + + Voltage Sensor Pore-Forming Loop Domain: I II III IV N-terminus C-terminus intracellular extracellular  Figure 1.2 - Structural features of the α1 calcium channel subunit. Common structural features of all four-domain Ca2+ channel α1 subunits include four homologous domains (brown labels) composed of six transmembrane segments (labeled 1 to 6 in domain I) and a pore forming loop (blue region) between segments 5 and 6.  The 4th transmembrane segments (red) have regularly-spaced, positively charged amino acids and form the voltage sensor.  The N-terminus, C- terminus and interdomain linkers are all intracellular (cytoplasmic).  Chapter 1: Introduction  5 1.1.3 Calcium channel classes Original classifications divided Ca2+ channels into HVA and LVA subgroups (Fig. 1.1).  T-type channels were defined as underlying LVA currents and could be distinguished based upon the small depolarizations required for channel opening and their small single channel conductance, slow deactivation kinetics and fast activation and inactivation kinetics.  Conversely, all other Ca2+ channel subtypes formed HVA currents generally characterized by larger depolarizations required for opening, larger conductances, faster deactivation kinetics and variable kinetics and voltage-dependence of activation and inactivation.  The HVA currents also possess a larger amplitude when extracellular Ca2+ is replaced with Ba2+, while permeability of Ca2+ versus Ba2+ through native LVA currents is more variable and often equal.  However, the identification of “low-threshold” Cav1.3 L-type currents (reviewed in (Lipscombe et al., 2004)) and Cav2.3 currents (Bourinet et al., 1996b) has led to some confusion concerning the “low voltage-activated” classification.  In this regard, “T-type” is the preferred term used when specifically describing the Cav3 channel grouping.  Figure 1.3 includes representative characteristics of a T-type channel (Cav3.1) versus a typical HVA Ca2+ channel (Cav1.2) to highlight some main biophysical differences between these two Ca2+ channel groupings. Chapter 1: Introduction  6  Figure 1.3 - Representative differences between T-type and HVA calcium channels. All properties, except for the single channel recording in B, were measured by the author from HEK cells transiently expressing a population of typical T-type channels (Cav3.1, blue) or typical HVA channels (Cav1.2, magenta) using whole-cell voltage clamp recordings at room temperature.  A) T-type Ca2+ channels typically activate at much more hyperpolarized potentials than HVA Ca2+ channels.  B) Cell- attached, single channel recordings of native T-type (top) and L-type (bottom) currents in chick sensory neurons using 110 mM Ba2+. Voltage protocols (in mV) are shown above sweeps of current traces.  T- type channels have a much smaller single channel conductance (8 pS) than L-type currents (~ 25 pS). Data adapted with permission from (Fox et al., 1987b).  C-D) Voltage-clamp waveform protocol shown on top.  C) T-type channels have faster activation and inactivation kinetics compared to HVA channels. D) After variable length test depolarizations to maximally open the channels, T-type channels have a slower rate of channel closing (deactivation) upon repolarization when compared to HVA channels. Chapter 1: Introduction  7 The Cav1 Ca2+ channel subfamily (Cav1.1 through Cav1.4) encodes for the L-type class of Ca2+ channels.  These channels are generally characterized by a large single channel conductance, minimal voltage-dependent inactivation and large Ca2+-dependent inactivation.  L-type channels are predominantly expressed in skeletal, cardiac and smooth muscle where they are involved in excitation- contraction coupling and AP propagation (reviewed in (Flucher and Franzini-Armstrong, 1996)).  In other cell types, the large window currents (membrane potentials where channels remain open at equilibrium) of L-type channels allows for tonic Ca2+ influx, providing a critical role in neurotransmitter release from photoreceptor and cochlear hair cells (McRory et al., 2004) (Appendix 3) and in hormone secretion from endocrine cells.  In neurons, L-type channel expression appears to be mostly somatodendritic and its activity is linked to synaptic plasticity and changes in Ca2+-dependent gene expression (Hell et al., 1993) (reviewed in (Lipscombe et al., 2004)). The Cav2 Ca2+ channel subfamily is composed of the P/Q-type, N-type, and R-type classes.  P/Q- type channels are expressed in the heart, pancreas, and pituitary as well as being widely distributed in neurons throughout the central nervous system (CNS) (Starr et al., 1991).  In mammals, P/Q-type are the primary Ca2+ channel responsible for neurotransmitter release from the presynaptic terminals of both central and peripheral neurons (reviewed in (Catterall et al., 2005)).  Native P-type and Q-type currents are distinguished based upon distinct kinetic, pharmacological, and modulatory properties and Snutch and colleagues demonstrated that alternative splicing of the Cav2.1 gene generates both channel types (Bourinet et al., 1999). Cav2.2 channels make up native N-type currents: the only Ca2+ channel class with expression limited to the nervous system (Dubel et al., 1992).  N-type currents are prominent in the presynaptic terminals, dendrites, and cell bodies of both central and sympathetic neurons, and play critical roles in the sensation and transmission of pain.  New classes of drugs that specifically target N-type Ca2+ channels are currently being developed for clinical use in the treatment of chronic and neuropathic pain (reviewed in (Snutch, 2005)). Cav2.3 channels have several properties, such as ion selectivity and voltage-dependence of activation, that resemble native T-type currents (Bourinet et al., 1996b; Li et al., 2007; Soong et al., 1993).  However, Cav2.3 channels also exhibit some properties consistent with native high-threshold R- type currents and there has been some contention in the literature as to how best classify this channel. Cav2.3 channels are widely expressed in the heart, testes, pituitary, and central neurons and are functionally linked to neurotransmitter release, modulating rhythmic firing patterns, synaptic plasticity and neurosecretion (reviewed in (Catterall et al., 2005)). The Cav3 Ca2+ channel subfamily forms the T-type Ca2+ channel class and is composed of three separate genes: Cav3.1, Cav3.2, and Cav3.3 (McRory et al., 2001).  These subtypes/isoforms have a broad Chapter 1: Introduction  8 expression pattern, including the ovaries, placenta, heart, kidney, smooth muscle, liver, adrenal cortex and neurons throughout the PNS and CNS.  In many of these cell types, more than one T-type isoform is expressed.  Non-neuronal functions of T-type channels include smooth muscle contraction, hormone secretion and cardiac pacemaker activity (reviewed in (Catterall et al., 2005)).  The expression and functional roles of T-type channels are discussed in more detail in the following sections.  1.2 T-type voltage-gated calcium channels 1.2.1 T-type channel pharmacology The study of native T-type currents has been hindered by two major issues:  1) some HVA channels actually activate at relatively negative potentials (e.g. Cav1.3 and Cav2.3), and 2) unlike the HVA Ca2+ channel classes, no high affinity specific channel antagonists have been discovered that clearly distinguish T-type currents from HVA currents or that distinguish between individual T-type subtypes.  These issues were compounded in early investigations of native T-type currents, where biophysical properties and sensitivities to pharmacological antagonists such as nickel (Ni2+) varied depending on the cell type.  The recent cloning and characterization of three separate T-type isoforms has helped explain these divergent properties and has provided some clarification on the limitations and suitable uses of the pharmacological tools presently available.  Although specific, high-affinity T-type blockers are lacking, the recent emergence of T-type channels as a potential novel therapeutic target for the treatment of pain, epilepsy, and hypertension (among other disorders) has led to a strong effort to synthesize specific antagonistic compounds.  As an illustration, over a dozen reviews have been published just in the last two years that directly address the development of T-type-specific antagonists and/or the therapeutic potential of T-type channel blockade. While one of the earliest T-type current antagonists to be identified was Ni2+, the sensitivity to this agent is highly variable between different native systems.  For example, Ni2+ inhibits T-type currents in chick skeletal muscle cells with an IC50 of 21 μM (Satoh et al., 1991), while it is a much less effective T-type inhibitor (IC50 = 110 μM) in cerebellar PCs (Kaneda et al., 1990).  Molecular identification of the three Cav3 channels revealed that Cav3.2 is the only T-type isoform highly sensitive to Ni2+, with an IC50 of 12 μM compared to IC50’s of 250 μM and 216 μM for Cav3.1 and Cav3.3, respectively (Lee et al., 1999c).  The use of Cav3.1- Cav3.2 chimeric channels and point-mutated channels demonstrated that the high affinity inhibition of Cav3.2 channels is due to a histidine residue in the S3-S4 loop of domain I that helps form a Ni2+ binding pocket on the extracellular surface of the channel.  Both Cav3.1 and Cav3.3 channels have a glutamine at this residue (Kang et al., 2006).  Nickel blocks Cav1.2 L-type and Cav2.3 R- type channels with a higher potency than either Cav3.1 or Cav3.3 T-type channels (Lee et al., 1999c; Zamponi et al., 1996).   Therefore, low concentrations (e.g. - 50 μM) of Ni2+ can be used to selectively Chapter 1: Introduction  9 block Cav3.2 T-type currents (with minimal blockade of Cav3.1 and Cav3.3-mediated T-type currents), but Cav2.3-mediated R-type currents will also be significantly attenuated at these concentrations. Another polyvalent cation often used for studying T-type function is cadmium (Cd2+).  Low concentrations of Cd2+ (20 to 100 μM) completely block all HVA Ca2+ current classes, while leaving T- type currents relatively unaffected (Berrow et al., 1997; Fox et al., 1987a; Tai et al., 2006).  Thus, application of Cd2+ can be used to study T-type currents in isolation from all other Ca2+ channels.  The specificity of Cd2+ for high affinity HVA channel block is due to the four amino acid (EEEE) selectivity filter located in the four pore-forming loops of HVA channels.  T-type channels have an EEDD sequence at this locus, and Cd2+ sensitivity is conferred to Cav3.1 channels when mutated to either EEED or EEEE (Talavera et al., 2001). Besides divalent cations, the search for selective T-type antagonists present in nature has thus far only revealed a single peptide toxin.  Kurtoxin is a peptide purified from Parabuthus transvaalicus scorpion toxin and was initially described as a selective, high-affinity Cav3.1 and Cav3.2 blocker (Chuang et al., 1998).  This peptide is also known to block voltage-gated Na+ channels but not to inhibit recombinant HVA Ca2+ channels (Chuang et al., 1998).   However, further studies within native thalamic and sympathetic neurons demonstrated that kurtoxin also inhibits N-type, R-type, and L-type currents with nanomolar affinities, thereby reducing its utility as a selective T-type antagonist (Sidach and Mintz, 2002). There are a number of clinical agents that non-specifically target T-type channels.  Mibefradil is the primary example of a therapeutic compound with efficacy in blocking T-type currents.  This agent was in clinical use for treating hypertension and angina through its apparent block of T-type and L-type currents (Massie, 1997), but was later removed from the market for its potentially fatal inhibition of cytochrome enzymes essential for metabolizing other therapeutic compounds (reviewed in (Welker et al., 1998)).  For research purposes, mibefradil was found to selectively inhibit T-type currents (IC50 ranging from 14 nM to 1μM) over HVA currents in some native systems, with state-dependent block causing greater inhibition of T-type currents at more depolarized potentials (McDonough and Bean, 1998). However, other studies showed that mibefradil can potently block R-type currents in the NG108-15 cell line (Randall and Tsien, 1997) and can also block N-type, L-type and P-type current at a concentration of 1 μM in spinal motor neurons (Viana et al., 1997).  Due to the non-specificity of mibefradil action, this compound is now deemed to be a non-specific T-type antagonist. Some dihydropyridines can also block LVA currents at low micromolar concentrations in native systems, but sensitivity is highly variable and may be partly due to the presence of dihydropyridine- sensitive Cav1.3 currents that are low voltage-activated (Akaike et al., 1989) (reviewed in (Yunker, 2003)).  However, dihydropyridines that are less effective at blocking L-type channels (IC50 ~ 40 μM) Chapter 1: Introduction  10 have recently been shown to be reasonably potent T-type blockers (IC50 ~ 1 μM).  Novel specific T-type antagonists might potentially be generated from derivatives of these compounds (Kumar et al., 2002).  A number of other therapeutic agents that have been shown to act on T-type channels at therapeutically- relevant levels, including diphenylbutylpiperidine neuroleptics (such as pimozide and penfluridol) (Santi et al., 2002), a phenylalkylamine antihypertensive (verapamil) (Freeze et al., 2006), the antidepressant trazadone (Kraus et al., 2007), succinimide antiepileptics (such as ethosuximide) (Gomora et al., 2001), volatile anesthetics (such as enflurane) (Joksovic et al., 2005), and the anesthetic/analgesic, nitrous oxide (Todorovic et al., 2001a).  The blockade of T-type channels is likely only partly contributing to the physiological effects of these drugs, as they also act on a spectrum of molecular targets ranging from HVA Ca2+  channels to voltage-gated Na+ and K+ channels to G-protein coupled receptors (GPCRs) and ionotropic receptors.  Thus, the use of these therapeutic agents as specific T-type antagonists is currently very limited.  However, the efficacy of these agents in blocking T-type channels indicates that T-type channels could be important therapeutic targets for the treatment of conditions including epilepsy, pain, cardiac hypertrophy, ischemia, hypertension, cancer, and diabetes (reviewed in (Yunker, 2003)).  In fact, since the discovery of mibefradil, many groups are attempting to design specific synthetic T-type blockers for therapeutic clinical use (Kim et al., 2007; Rhim et al., 2005).  1.2.2 T-type channel biophysical properties T-type channels have many characteristic biophysical properties that are unique among voltage- gated Ca2+ channels.  These include a small single channel conductance, fast activation and inactivation kinetics, a relatively hyperpolarized voltage-dependence of activation and inactivation, and fast deactivation kinetics.  The activation and inactivation kinetics of T-type channels are also strongly voltage-dependent.  This creates a characteristic “crossing over” pattern in successively more depolarized traces of a conventional square-pulse current-voltage protocol.  For neuronal T-type currents, the τinact can range from greater than 100 ms at activation thresholds (between -70 mV and -50 mV) to less than 20 ms at maximal activating potentials (>-30 mV) (reviewed in (Huguenard, 1996)).  Although T-type currents exhibit strong voltage-dependent inactivation, they do not inactivate in a Ca2+-dependent manner similar to HVA Ca2+ channels. After initial cloning, the recombinant T-type isoforms were biophysically well characterized, and these properties have been thoroughly reviewed (Perez-Reyes, 2003).  In brief, comparison of the three rat Cav3 isoforms revealed that Cav3.1 and Cav3.2 have properties similar to “typical” native T-type currents, while Cav3.3 possesses distinct biophysical properties.  The Cav3.1 and Cav3.2 isoforms have fast activation and inactivation kinetics while Cav3.3 channel activation and inactivation kinetics are much slower.  The rat Cav3.3 channels also have faster deactivation kinetics and a more hyperpolarized voltage dependence of activation and inactivation compared with the other two T-type isoforms (McRory Chapter 1: Introduction  11 et al., 2001).  All three isoforms have a characteristically fast recovery from inactivation, with the Cav3.1 and Cav3.3 channels recovering the fastest (Klockner et al., 1999).  A recent paper comparing T-type biophysical parameters at room temperature and a physiological mammalian temperature (37oC) demonstrated that increasing the recording temperature dramatically alters many of these properties in a non-linear, isoform-specific manner (Iftinca et al., 2006).  In this regard, caution should be used when extrapolating specific T-type parameters measured at room temperature to models of physiological neuronal excitability. Alternative splicing creates additional functional diversity in T-type channel activity and the study of these splice variants has provided insight into the roles of different structural regions in T-type gating properties.  Alternative splicing in the human Cav3.1 channel (Fig. 1.4) leads to several variants including: 1) insertion (exon 14, region e) in the domain II-III linker that shifts the voltage dependence of inactivation in the hyperpolarizing direction and also increases inactivation kinetics, 2) variation in exon 25 (a or b forms) and 3) insertion of exon 26 (region c) in the domain III-IV linker that affects the voltage-dependence of activation and inactivation, and the kinetics and voltage-dependence of inactivation, respectively (Chemin et al., 2001a).  The Cav3.1-a and Cav3.1-ae isoforms predominate in human brain regions such as the cerebellum and thalamus (Monteil et al., 2000).  Additional alternative splicing has recently been identified in the Cav3.1 C-terminus and can affect either current kinetics or the window current magnitude, depending upon the specific splice background (Emerick et al., 2006). The human Cav3.3 channel is alternatively spliced (Fig. 1.4) in the domain I-II linker, involving a deletion of exon 9 (35 amino acids), as well as in the carboxyl-terminus, involving a partial deletion of exon 33 (13 amino acids) (Mittman et al., 1999).  These splicing events affect channel activation and inactivation kinetics interdependently, suggesting a possible direct interaction between the two channel regions (Murbartian et al., 2004).  Overall, the above findings demonstrate that a majority of functional T-type splicing occurs in cytoplasmic channel regions and affects both the kinetics and voltage- dependence of channel gating.  The prevalence of cytoplasmic splicing could also be involved in differential sensitivities to intracellular second messenger modulation pathways, creating a mechanism for further channel specialization. Chapter 1: Introduction  12  1 2 3 4 5 6 Domain: I II III IV N-terminus C-terminus intracellular extracellular 14 25,26 38Cav3.1: Cav3.2: Cav3.3: 9 33 34,35 17 25,26 35  Figure 1.4 - Alternative splicing of intracellular exons in human Cav3 channels. Numbers correspond to exons that are alternatively spliced in Cav3.1 (blue), Cav3.2 (green), and Cav3.3 (red) channels.  Numbers in bold represent splicing that results in complete insertion or deletion of the exon, while numbers in italics represent exons that have partial deletions or insertions due to alternative splicing.  The approximate locations of the alternatively spliced exons in the channel structure are indicated by arrows.  Only alternative splicing events that occur in putative intracellular (cytoplasmic) regions and that result in functional channels are shown.  Alternative splice variants were identified and characterized in (Chemin et al., 2001a; Emerick et al., 2006; Monteil et al., 2000; Murbartian et al., 2004; Zhong et al., 2006).  In addition to alternative splicing analyses, chimeric T-type channels and recombinant T-type channels with targeted deletions or point mutations have provided further insights into the biophysical structure-function relationships of Cav3 channels.  The Cav3 domain I-II linker is highly divergent from that of HVA Ca2+ channels and has lost the ability to bind to β ancillary subunits with high affinity.  As hypothesized, replacement of the Cav3.1 domain I-II linker with a Cav2.2 domain I-II linker conferred some aspects of β-regulation to this T-type channel, but surprisingly causes the channel to activate at even lower voltages (Arias et al., 2005).  The Cav3.1- Cav2.2 chimera also had greater functional expression than wild-type (wt) Cav3.1 channels, leading to the hypothesis that the T-type domain I-II Chapter 1: Introduction  13 linker may contain an ER retention signal.  Experiments involving systematic deletion of regions of the domain I-II linker in Cav3.2 channels further support this hypothesis, as deleting the central region of the domain I-II linker increased T-type current density and surface expression (Vitko et al., 2007).  Other deletions led to the postulation that the first 62 amino acids of the Cav3.2 domain I-II linker are involved in regulating the voltage-dependence of activation and inactivation by acting as a gating particle that stabilizes the channel in the closed state (Vitko et al., 2007).  Structural and electrophysiological experiments reveal that the proximal region of the domain I-II linker forms a putative helix-loop-helix structure that acts as a gating brake (Arias et al., 2008).  Thus, the domain I-II linker appears to have a significant role in regulating channel expression and gating within T-type channels.  It should be noted that the distal C-terminus of Cav3.3 channels also affects channel expression and thus multiple intracellular regions are likely involved (Gomora et al., 2002). Other regions of Cav3 channels also appear to play a role in the voltage-dependence of T-type activation, as replacement of domains I, III, or IV in Cav3.1 channels with wt Cav1.2 sequence leads to HVA-like channel gating (an ~ +40 mV shift in V50act) (Li et al., 2004).  Surprisingly, sequence differences in the S4 putative voltage sensor regions in these domains do not account for the large shifts in activation gating.  However, mutations in the outermost positively charged arginine residues of the S4 transmembrane segment in domain IV of Cav3.2 channels (Lam et al., 2005) and domains I, II, and III of Cav3.1 channels (Kurejova et al., 2007) revealed that S4 segments in T-type channels do, in fact, act as activation voltage-sensing domains.  The selectivity filter EEDD locus in the four pore-forming loops of Cav3 channels has also been shown to affect activation properties (Talavera et al., 2001), leading to the overall conclusion that multiple channel regions including the domain I-II linker, S4 segments, and pore- forming loops all contribute to T-type activation gating. The structural determinants of T-type inactivation have been more difficult to elucidate.  High voltage-activated Ca2+ channels are proposed to inactivate by a mechanism similar to the voltage-gated Na+ channel “hinged-lid” mechanism whereby the HVA domain I-II linker acts as a cytoplasmic inactivation particle that docks at a site formed by the S6 segments of all four domains.  In this model, other channel regions such as the C-terminus can regulate inactivation by altering the mobility of the inactivation particle (reviewed in (Stotz et al., 2004)).  Consistent with this model, mutating domain III S6 residues that are well-conserved inactivation determinants in HVA channels shifted the voltage- dependence of activation and altered the activation and inactivation kinetics of Cav3.1 channels (Marksteiner et al., 2001).  Furthermore, mutations in the S6 segment of domain I in Cav3.1 channels and deletion of the last 15 amino acids of the domain I-II linker in Cav3.2 channels also slowed open channel inactivation (Arias et al., 2005; Vitko et al., 2005).  Chimeric Cav3.1- Cav1.2 channel studies showed that a carboxyl region just after domain IV partly determines the fast inactivation of Cav3.1 channels (Staes et al., 2001), suggesting a role for the C-terminus in T-type channel inactivation and supporting a model Chapter 1: Introduction  14 similar to that of HVA channel inactivation.  However, studies involving the slowly-inactivating Cav3.3 channel appear to conflict with this model.  Chimeric studies between Cav3.1 and Cav3.3 channels revealed that multiple structural elements contribute to the slow inactivation of Cav3.3 currents, with inactivation kinetics becoming more “Cav3.3-like” with increased Cav3.3 sequence in the chimeric channels (Park et al., 2004).  Furthermore, inserting the Cav3.1 domain I-II linker into the Cav3.3 channel had no significant effect on inactivation kinetics (Park et al., 2004).  Another recent Cav3.1- Cav3.3 chimeric study was also unable to determine specific structural regions of the Cav3.3 channel directly responsible for its slower inactivation kinetics (Hamid et al., 2006). One caveat in using chimeric and mutated channels to study the structural determinants of T-type inactivation is that macroscopic inactivation is coupled to activation in these channels.  Increases in the closed-to-open transition rate can also increase the rate of macroscopic channel inactivation (Talavera and Nilius, 2006b).  In fact, Talavera and Nilius show that there is a linear relationship between the activation and inactivation rates in many of the above noted T-type inactivation studies, suggesting that some of the identified structures may in fact be structural determinants of activation and not inactivation (Talavera and Nilius, 2006b).  However, specific mutations in Cav3.1 channels that affect activation but not inactivation kinetics demonstrate that macroscopic inactivation is not strictly linked to activation gating (Lam et al., 2005), and therefore, it is likely that activation and inactivation properties are determined by common T-type structural features.  Overall, the understanding of structure-function relationships in T-type channels is incomplete, and currently “we still know very little about the mechanism underlying the basic functional features of T-type channels” (Talavera and Nilius, 2006a).  1.2.3 T-type channel expression T-type channels are highly expressed in a variety of types of neurons throughout the CNS and PNS.  Combining studies of T-type immunohistochemistry (Craig et al., 1999; McKay et al., 2006) with electrophysiological recordings of T-type currents (Isope and Murphy, 2005; Joksovic et al., 2005; Kavalali et al., 1997) reveals that Cav3 channels and their functional currents are predominantly localized to the soma and dendrites of neurons, with the highest expression often occurring in dendritic regions. The functional significance of this subcellular localization will be further explored in the next section. The first characterization of Cav3 subtype-specific distribution was performed in the rat CNS using in situ hybridization.  It was found that the three major isoforms are differentially expressed in the brain (Talley et al., 1999), likely accounting for the observed heterogeneity in pharmacology, modulation, and biophysics amongst native T-type currents (reviewed in (Huguenard, 1996)).  For example, Cav3.1 is predominantly expressed in thalamocortical relay cells (TCs) at high levels, while Cav3.2 and Cav3.3 are highly expressed in thalamic reticular (nRT) neurons.  Cav3.1 is also highly expressed in cerebellar PCs and inferior olive cells, while Cav3.2 is highly expressed in the dentate gyrus Chapter 1: Introduction  15 and dorsal root sensory ganglia.  Cav3.3 subunits have a more limited distribution but are highly expressed in the olfactory bulb and in the CA1/CA3 layers of the hippocampus (Talley et al., 1999). Functional studies have now confirmed many of these specific Cav3 subtype distribution patterns. Recordings on TCs from Cav3.1 knock-out (KO) mice revealed a complete loss of T-type currents (Kim et al., 2001), while RNAi-mediated knock-down of Cav3.2 expression in dorsal root ganglion (DRG) sensory neurons severely attenuated “Cav3.2-like” functional currents (Bourinet et al., 2005). A further observation from the initial in situ characterization was that only a few regions of the brain, such as the olfactory bulb granule cell layer and the CA1/CA3 layers of the hippocampus, displayed significant expression of all three T-type isoforms (Talley et al., 1999).  Contrastingly, a recent immunohistochemical study of Cav3 protein expression in the rat brain questions this finding, with the conclusions that all three isoforms are expressed in many neurons throughout the brain and that heterogeneity is predominantly restricted to subcellular differences in T-type isoform distribution (McKay et al., 2006).  It is important to note that the latter study reported differences in immunolabeling between slices processed in parallel from the same animal, only examined brain regions known to express high levels of functional T-type currents and could not distinguish between T-type channel protein levels (McKay et al., 2006).  Furthermore, where discrepancies exist between the Talley et al. mRNA study and the McKay et al. protein study, the bulk of literature often supports the Talley et al. findings.  For example, the McKay et al. study showed that Cav3.3 is the main Cav3 subtype expressed in cerebellar PCs, with prominent Cav3.3 expression throughout the soma and dendritic arbour and Cav3.1 expression in only the soma (McKay et al., 2006; Molineux et al., 2006), while the Talley et al. study showed that Cav3.1 is selectively expressed in PCs (Talley et al., 1999).  Three other studies have shown prominent Cav3.1 expression in PCs at the protein and mRNA levels, with Cav3.1 protein present at high levels in the soma and dendrites (Craig et al., 1999; Kase et al., 1999; Yunker et al., 2003), and the biophysical properties of T-type currents in PCs resemble Cav3.1 more than Cav3.3 (Isope and Murphy, 2005).  One other immunohistochemical comparison has been performed using purported subtype- specific Cav3.1 and Cav3.3 antibodies  (Yunker et al., 2003).  However, the antibody generated against Cav3.3 has since been reported to be cross-reactive with the endogenous neural cell adhesion protein, NCAM-180 (Chen et al., 2007), providing a warning of the potential pitfalls when generating anti- peptide antibodies for the staining of low abundance membrane proteins within brain slices.  Overall, when exploring T-type channel expression it is critical to examine a combination of mRNA, protein and biophysical evidence (with KO or antisense studies being essential) prior to forming conclusions on the nature of the Cav3 subtypes that are functionally expressed. While Cav3.3 expression is restricted to the nervous system, both the Cav3.1 and Cav3.2 isoforms are expressed in excitable and non-excitable cells outside of the nervous system.  In fact, compared to all other human tissue, Cav3.2 mRNA is expressed most abundantly within the kidney (Cribbs et al., 1998). Chapter 1: Introduction  16 There is also expression of Cav3.2 within the pituitary and pineal glands, liver, adrenal cortex, heart and sperm (Talley et al., 1999) (reviewed in (Darszon et al., 2006; Perez-Reyes, 2003)).  The Cav3.1 subunit is expressed in the ovaries, placenta and heart (reviewed in (Perez-Reyes, 2003)).  1.2.4 T-type channel physiological roles Differences in biophysical properties, alternative splicing, and expression of individual Cav3 subtypes all allow for considerable functional specialization within the nervous system.  The overlap between the voltage-dependence of T-type activation and inactivation at potentials near neuronal resting membrane potentials (McRory et al., 2001) creates the possibility for “window currents”, where a fraction of channels are tonically open at rest.  In hippocampal CA1 neurons, voltage-gated Ca2+ channel activity has been shown to affect resting cytosolic Ca2+ concentrations, at least partly through the activity of T-type channels (Magee et al., 1996).  The presence and relevance of T-type window currents (and T- type currents in general) is highly dependent on the resting membrane potential, as the channels will become completely inactivated at more depolarized potentials (Carter and Sabatini, 2004).  T-type window currents have the possibility of affecting Ca2+-mediated signaling pathways as well as the electrical firing patterns of neurons (Chevalier et al., 2006; Williams et al., 1997). The predominant expression of T-type currents in neuronal dendrites implicates their potential involvement in signal integration at synaptic inputs.  In both pyramidal cortical and hippocampal CA1 neurons, subthreshold excitatory postsynaptic potentials (EPSPs) can cause the activation of T-type currents and a resultant localized increase in dendritic Ca2+ levels (Magee et al., 1995; Markram and Sakmann, 1994).  This T-type activity could act to boost dendritic depolarizations and therefore increase excitability, or conversely, could activate Ca2+-activated K+ currents to cause membrane hyperpolarizations (Wolfart and Roeper, 2002). Dendritic T-type Ca2+ currents can also affect intracellular Ca2+ signaling pathways, potentially leading to synaptic plasticity.  In addition to the above subthreshold, localized T-type Ca2+ transients, robust activation of T-type channels can produce an active low-threshold Ca2+ spike that spreads to adjacent dendrites (Egger et al., 2005).  T-type Ca2+ spikes that are dendritically generated and localized (propagate poorly to the soma) contribute to postsynaptic depolarization, Ca2+ entry and long-term potentiation (LTP) when hippocampal CA1 synapses are stimulated at high frequency (Golding et al., 2002).  T-type channels are also implicated in the induction of associative long term potentiation within young granule cells of the adult hippocampus during similar theta burst induction protocols (Schmidt- Hieber et al., 2004).  Conversely, low-frequency stimulation of CA1 synapses induces long-term depression (LTD) that is abolished with T-type antagonists (25 μM Ni2+, 10 μM nimodipine) (Christie et al., 1997).  A form of LTP that occurs at synapses in the spinal cord during abnormal pain sensitivity Chapter 1: Introduction  17 (hyperalgesia) is also dependent on T-type activity (Ikeda et al., 2003).  Thus, T-type channels are implicated in modulating synaptic strength through either localized or more global dendritic Ca2+ signals in both the CNS and PNS.  However, the use of imperfect pharmacological tools, such as Ni2+, in many studies makes these postulations still quite preliminary.  More thorough investigations involving high- resolution two-photon Ca2+ imaging, Cav3 KO mice, CaV3 RNAi-mediated knock-down, and/or more specific T-type antagonists are required to help elucidate the exact physiological roles of dendritic T-type currents in plasticity and excitability. In addition to modulating plasticity, T-type Ca2+ spikes can also have profound effects on neuronal excitability.  Low-threshold Ca2+ spikes were first identified from brain slices of the inferior olive, where removal of T-type inactivation with hyperpolarization initiated a spontaneous “rebound- burst” spike (Llinas and Yarom, 1981).  T-type channels have now been shown to underlie regenerative low-threshold spikes and burst firing in neurons throughout the CNS, including in the thalamus, inferior olive, cerebellum, hippocampus, cortex, and neocortex (reviewed in (Huguenard, 1996)).  In some neurons, low threshold spikes and burst firing can alter neuronal oscillations, causing the neuron to switch from high frequency tonic firing to a phasic mode with regular intervals of high frequency bursts of spikes (Diana et al., 2007; Suzuki and Rogawski, 1989).  Within the thalamus, T-type mediated changes in rhythmic oscillations underlie physiological sleep-wake gating and pathophysiological epileptic absence seizure activity (see below). Recent work has begun to unveil the individual functional contributions of the Cav3 isoforms to neuronal excitability.  In one voltage-clamp study on HEK cells expressing individual recombinant T- type isoforms, various neuronal AP firing waveforms were employed to demonstrate that the slower inactivation rates and faster deactivation kinetics of Cav3.3 channels likely contribute to sustained rhythmic electrical activities (Chemin et al., 2002).  Indeed, overexpressing Cav3.3 channels in the NG108-15 neuronal cell line induced spontaneous oscillations and repetitive AP firing with a concomitant increase in intracellular Ca2+ concentration (Chevalier et al., 2006).  Conversely, the faster activation and inactivation kinetics and slower deactivation kinetics of Cav3.2 and especially Cav3.1 allow these T-type isoforms to have a greater role in burst firing activity (Chemin et al., 2002).  A recent study purports that rebound burst firing within deep cerebellar nuclei (DCN) neurons is restricted to a subpopulation that exclusively expresses Cav3.1 channels (Molineux et al., 2006).  Additionally, T-type currents in DRG neurons primarily consisting of Cav3.2 channels are responsible for low threshold spikes that are crowned with a repetitive burst of APs (Bourinet et al., 2005; White et al., 1989). Of all brain regions, the physiological roles of T-type channels have been most thoroughly studied in the thalamus.  Thalamic TC and nRT nuclei form a rebound bursting circuit that creates spindle waves which are crucial during slow-wave sleep.  Reciprocal interactions between T-type currents in TC and nRT cells are critical for the initiation and maintenance of this cycle.  During periods Chapter 1: Introduction  18 of inactivity (sleep initiation), T-type-dependent bursts of APs in nRTs cause hyperpolarization of TCs via GABAergic connections.  The hyperpolarizing currents that flow through dendritic ionotropic GABAA receptors and GABAB-activated channels de-inactivate T-type channels to initiate TC burst firing.  Excitatory glutamatergic synapses from TCs onto the ionotropic glutamate receptors of nRTs re- excite the nRT cells and start the cycle again, causing 6-15 Hz rhythmic oscillations (reviewed in (Destexhe and Sejnowski, 2003; McCormick and Contreras, 2001)). Low-threshold burst firing varies between nRT and TC cells, with bursts in nRT cells displaying an accelerando-decelerando pattern of APs and a slower rising phase than TC bursts, resulting in broader bursts (Huguenard and Prince, 1992).  Recent evidence suggests that these functional differences are due to differential expression of Cav3 isoforms.  T-type currents are predominantly located near the soma of TC cells (Coulter et al., 1989) and entirely consist of Cav3.1 channels (Kim et al., 2001).  Knocking out Cav3.1 completely abolishes burst firing in TC cells (Kim et al., 2001).  In contrast, T-type currents are largest in the dendrites of nRT cells (Destexhe et al., 1996) and electrophysiological evidence indicates that they consist of Cav3.3 channels in the dendrites, with a lower density of Cav3.2 channels in the soma (Joksovic et al., 2005).  T-type currents in nRT cells of Cav3.2 KO mice are reduced, but not abolished, indicating that both Cav3.2 and Cav3.3 isoforms are functionally expressed (Joksovic et al., 2006).  The slow onset and broadness of bursts in nRT neurons is proposed to be due to the slower activation and inactivation kinetics of their Cav3.3-mediated T-type currents (Huguenard and Prince, 1992).  Blocking T-type currents with Ni2+ or volatile anesthetics abolishes low threshold bursts in nRT cells (Joksovic et al., 2005).  The essential role of thalamic T-type currents in generating rhythmic sleep oscillations is illustrated in the observation that conditional KO of Cav3.1 in the thalamus, but not the cortex, disrupts AP firing in vitro and destabilizes sleep in vivo (Anderson et al., 2005). The expression of Cav3.1 and Cav3.2 isoforms in circulatory, endocrine, and reproductive tissues suggests that T-type channels also play physiological roles outside of the nervous system.  The Cav3.2 isoform is robustly expressed in the atrial and ventricular myocytes of developing heart tissue, but is generally restricted to expression within conduction Purkinje fiber cells and pacemaker cells in the adult heart of humans and other higher-order mammals (Rosati et al., 2007) (reviewed in (Vassort et al., 2006)).  Although the biophysical properties of T-type currents appear to be suited for generating pacemaker potentials in the atrioventricular and sinoatrial nodes of the heart, experiments involving T- type antagonists and Cav3.2 KO mice have demonstrated that T-type channels can modify heart rate firing frequency but are not the primary pacemaker currents in the heart (L-type Ca2+ channels are the primary pacemaker currents) (Chen et al., 2003a) (reviewed in (Vassort et al., 2006)).  However, Cav3.2 channels play an essential role in the function of non-excitable coronary smooth muscle.  The constitutively constricted coronary arterioles and focal myocardial fibrosis observed in Cav3.2 KO mice Chapter 1: Introduction  19 demonstrate that CaV3.2 currents are essential for coronary arteriole relaxation, possibly through coupling to inhibitory Ca2+-dependent K+ channels (BK)  (Chen et al., 2003a). In addition to their putative roles in coronary smooth muscle, the slow increases in basal intracellular Ca2+ concentrations that are mediated by T-type window currents are also implicated in the function of non-excitable endocrine, kidney and sperm cells.   Within the adrenal cortex, both the angiotensin II-induced secretion of aldosterone from adrenal glomerulosa cells and the adrenocorticotropin hormone-induced secretion of cortisol from zona fasciculata cells are thought to require Cav3.2-mediated window Ca2+ currents (Yao et al., 2006) (reviewed in (Perez-Reyes, 2003)).  T- type window currents are also implicated in regulating the renal efferent arteriole smooth muscle tone and resultant microcirculation levels within the kidney (reviewed in (Hayashi et al., 2007)).  Finally, Cav3.2-mediated Ca2+ window currents play a major role in the sperm acrosome reaction of egg fertilization and may also function in sperm motility and capacitation (reviewed in (Darszon et al., 2006)).  1.2.5 T-type channels in human disease Of the three T-type isoforms, only Cav3.2 has been directly linked to human disease.  Recent studies have shown that missense mutations in Cav3.2 directly contribute to certain forms of epilepsy and autism spectrum disorders. Epilepsy is a general disorder of the nervous system that is primarily characterized by hyperexcitability and hypersynchronization of thalamic and cortical neuronal circuits.  Idiopathic generalized epilepsy (IGE) is a major form of epilepsy that has no clear etiology, is partly caused by complex non-Mendelian genetics, and includes disorders such as juvenile myoclonic epilepsy, juvenile absence epilepsy, and childhood absence epilepsy.  Absence epilepsy is characterized by brief seizures that cause impairments of consciousness through 3 to 6 Hz spike-and-wave discharges (SWDs) mediated by oscillations within the thalamocortical circuit (reviewed in (McCormick and Contreras, 2001)).  As previously discussed in section 1.2.4, T-type channels play critical roles in rhythmogenesis within these circuits. Correlative evidence linking nRT T-type activity with epileptic activity can be found in a rat model of absence epilepsy (GAERS).  In acutely dissociated nRT (but not TC cells) from GAERS rats, T-type currents have significantly higher current amplitude than currents from the nRT of wt rats.  The increase in nRT T-type current is both age-dependent and absent at birth, as are the occurrence of seizures and SWD activity in this model of absence epilepsy (Tsakiridou et al., 1995).  Furthermore, succinimide antiepileptic drugs reduce both T-type currents and associated burst generation in TC and nRT cells (Huguenard and Prince, 1994), providing further evidence for the link between T-type currents Chapter 1: Introduction  20 within thalamic neurons and pathophysiological SWDs.  More significantly concerning human epilepsy, missense mutations have been identified in the Cav3.2 channel gene of human patients with childhood absence epilepsy (Chen et al., 2003b).  Extensive electrophysiological analysis of these mutations as well as other subsequently identified Cav3.2 IGE mutations have demonstrated that most of the mutations increase T-type channel activity by altering biophysical properties or by increasing Cav3.2 surface expression (Heron et al., 2007; Khosravani et al., 2004; Khosravani et al., 2005; Peloquin et al., 2006; Vitko et al., 2007; Vitko et al., 2005).  Interestingly, a majority of the mutations occur in the Cav3.2 channel domain I-II linker (reviewed in (Adams and Snutch, 2007)), which is implicated in channel gating and expression (see section 1.2.2).  As Cav3.2 channels are expressed within the nRT (Talley et al., 1999), the gain-of-function mutations associated with IGE could cause nRT hyperexcitability and an increase in low-threshold bursting that contributes to SWD generation (reviewed in (Khosravani and Zamponi, 2006)).  However, IGEs such as childhood absence epilepsy have a complex polygenic etiology and some Cav3.2 mutations have no detectable effects on channel properties (Peloquin et al., 2006; Vitko et al., 2005).  Extensive alternative splicing of Cav3.2 may cause some mutations to only be functionally relevant in specific splice variants (Fig. 1.4; (Zhong et al., 2006)).  The recent identification of single nucleotide polymorphisms in Cav3.2 that are associated with an increased risk of childhood absence epilepsy indicates that mutations in Cav3.2 can increase susceptibility to IGE in certain ethnicities (Liang et al., 2007).  Although mutations in Cav3.1 have not been associated with human epilepsy to date, Cav3.1 channel activity is linked to epileptic activity.  In Cav3.1 KO mice, both low-threshold Ca2+ spikes and burst firing in response to hyperpolarizations are abolished within TCs, and SWDs that can be induced by baclofen (a GABAB agonist) in wt mice are absent in the Cav3.1 KO mice (Kim et al., 2001). Epileptic Cav2.1 P/Q-type KO or missense mutant mice that have characteristic SWDs and behavioral arrests also have increased T-type currents within TCs (Song et al., 2004; Zhang et al., 2002).  In one example, the SWDs were abolished in Cav2.1/ Cav3.1 double KO animals (Song et al., 2004). Compared to the association between Cav3.2 mutations and IGE, the link between Cav3.2 and autism is relatively unexplored.  Neuroanatomical studies have revealed histological abnormalities in the limbic system and other CNS regions of autism patients, including the hippocampus, amygdala, cerebellum, and cortex; all regions of high T-type expression and function (reviewed in (Adams and Snutch, 2007)).  A recent study by Splawski et al. identified missense mutations within the Cav3.2 channel in 6 out of 461 Caucasian autism patients.  These mutations occur in conserved S4, P loop, and C-terminus channel regions and cause alterations in channel biophysical and current density properties that are predicted to reduce overall neuronal firing (Splawski et al., 2006). Although not related to specific channel mutations, T-type currents are also associated with the pathophysiology of pain signaling.  Evidence from RNAi-mediated knock-down, Cav3.2 KO mice and Chapter 1: Introduction  21 pharmacological experiments reveal that T-type Cav3.2 currents contribute to the excitability of peripheral nociceptors and spinal cord dorsal horn neurons, where they are implicated in pronociceptive mechanisms of somatic pain (reviewed in (Hildebrand and Snutch, 2006)) (Appendix 1).   Blockade of peripheral T-type currents with multiple clinical agents and pharmacological antagonists has been shown to reduce various forms of acute and chronic pain (reviewed in (Snutch and David, 2006)).  Conversely, experiments on Cav3.1 KO mice indicate that this T-type channel isoform plays an antinociceptive role in visceral pain responses via its role in sensory gating within thalamic relay nuclei (Appendix 1).  1.3 Modulation of voltage-gated calcium channels 1.3.1 G-protein-coupled receptors One mechanism of altering ion channel activity in the CNS is through the activation of GPCRs by various neurotransmitter and neuropeptide ligands.  Unlike ionotropic receptors that directly conduct transmembrane currents, GPCRs act on effectors through G-protein-mediated intracellular signaling pathways.  Despite enormous diversity in sequence and functions that range from olfaction to phototransduction to synaptic integration, all GPCRs have a well-conserved tertiary structure consisting of an extracellular N-terminus, seven transmembrane α helices connected by alternating intracellular and extracellular loops, and a cytoplasmic C-terminus.  The selective coupling between specific ligands and the N-terminus, and specific G-proteins and cytoplasmic regions results in a wide array of specific receptor subtypes connected to a multitude of downstream cellular effectors.  Two of the major subfamilies of GPCRs in the brain are the metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs). The mGluR subfamily was originally cloned in the early 1990’s as a novel subfamily of GPCR that lacked amino acid sequence similarity with other conventional GPCRs, such as mAChRs.  It took functional expression of the rat cerebellar cDNA in Xenopus oocytes followed by serial dilutions of the response-evoking cDNA mixtures to painstakingly clone the first member, mGluR1a (Houamed et al., 1991; Masu et al., 1991).  The cloning of additional mGluR members revealed highly conserved regions among the mGluR subfamily that include the N-terminus, transmembrane domains, and intracellular and extracellular loops (reviewed in (Schoepp and Conn, 1993)).  Eight members of the mGluR subfamily have been cloned to date and are classified into 3 groups based on sequence similarity; Group I includes mGluR1 and mGluR5, Group II includes mGluR2 and mGluR3 and Group III includes mGluR4 and mGluR5-8.  Functional characterization within heterologous and native systems has revealed that Group I mGluRs couple to Gαq/11 proteins and phospholipase C (PLC) activation, leading to the hydrolysis of phosphatidylinositol into diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3) (reviewed in (Conn and Pin, 1997)).  Both of these components function as intracellular signals, as DAG is responsible for Chapter 1: Introduction  22 the activation of protein kinase C (PKC) and IP3 increases the intracellular Ca2+ concentration through activation of Ca2+ permeable receptors (IP3Rs) in the membrane of the endoplasmic reticulum.  mGluR1 also couples to other G-proteins to increase cAMP formation and arachidonic acid release when expressed in non-neuronal cell-lines (Nakanishi, 1992).  As observed between T-type isoforms, mGluR1 and mGluR5 have similar sequences and functional properties and are both expressed in various regions of the CNS, yet their expression patterns are largely complementary (Abe et al., 1992).  Both Group II and Group III mGluRs reduce cytosolic cAMP levels by inhibiting adenylate cyclase and can decrease the intracellular Ca2+ concentration, but only Group III receptors also decrease cGMP levels (Hayashi et al., 1992) (reviewed in (Coutinho and Knopfel, 2002)). Activation of mGluRs within the CNS causes a range of electrophysiological effects, including the alteration of both ligand-gated and voltage-gated channel activity.  For example, mGluR activation can cause either the inhibition or activation of voltage-gated K+ and Ca2+ channels, the activation of non- specific cationic currents, the potentiation of glutamatergic ionotropic receptors and the opening of G- protein activated K+ channels (reviewed in (Anwyl, 1999)).  Through these mechanisms, mGluR activity is implicated in modulating neuronal excitability, neurotransmitter release, synaptic integration and synaptic plasticity (reviewed in (Coutinho and Knopfel, 2002)).  However, the precise intracellular pathways that link mGluR activation to specific effectors within native systems remain to be identified. Similar to mGluRs, mAChRs are divided into groups based on their sequence similarity and downstream effector coupling.  Of the five mAChRs cloned to date, M1, M3, and M5 are classified as Group I mAChRs that couple to Gαq/11 to activate PLC and its downstream pathways.  Besides the IP3- and DAG-mediated signals, the breakdown of membrane phosphatidylinositol-4,5-bisphosphonate (PIP2) by PLC can directly alter channel activity, as PIP2 can act to stabilize or inhibit voltage-gated channels (reviewed in (Suh and Hille, 2005)).  Similar to mGluR1, Group I mAChRs have been shown to increase arachidonic acid levels by activating phospholipase A2 (reviewed in (Lanzafame et al., 2003)).  The other two mAChRs, M2 and M4, are classified as Group II and couple to PTX-sensitive Gαi/o proteins that inhibit adenylate cyclase to decrease cAMP.  Group II mAChRs can also activate G-protein-coupled K+ channels through a direct Gβγ-mediated pathway, leading to membrane hyperpolarization (reviewed in (Ishii and Kurachi, 2006)).  mAChRs are implicated in similar types of ion channel modulation and resultant neuronal processes as mGluRs.  Further, both mGluR and mAChR receptors can desensitize through uncoupling between the receptor and relevant G-proteins (timescale of ~ 2 to 3 minutes) or through phosphorylation-induced internalization of the receptor (timescale of ~ 10 to 30 minutes) (reviewed in (Ishii and Kurachi, 2006)). Besides the variability in subtype and functional coupling of GPCRs, signaling heterogeneity is also generated in the nervous system through differential expression of specific G-protein isoforms. Chapter 1: Introduction  23 There are four main families of the intracellular signaling Gα proteins (Gαi/o, Gαs, Gαq, and Gα13), yet 16 specific Gα genes have been identified within these families (reviewed in (Tedford and Zamponi, 2006)).  GPCR activation results in the release of cytoplasmic GTP-bound Gα proteins from membrane- bound Gβγ complexes that can act directly on nearby effectors, such as ion channels (reviewed in (Dascal, 2001)).  To date, there are five cloned subtypes of Gβ and 12 different Gγ subunits (reviewed in (Tedford and Zamponi, 2006)).  1.3.2 Modulation of HVA calcium channels The functional coupling between GPCR activation and Ca2+ channel activity was first studied over 25 years ago, when Kathleen Dunlap and Gerald Fischback demonstrated that various neurotransmitters decreased AP duration in cultured chick DRG neurons by inhibiting voltage-gated Ca2+ conductances (Dunlap and Fischbach, 1978, 1981).  Subsequent characterization of various native GPCR-Ca2+ channel interactions revealed that GPCR activation typically inhibits neuronal HVA Ca2+ channel activity and that this inhibition can occur through both membrane-delimited, voltage-dependent and cytoplasmic, voltage-independent pathways. The Ca2+ currents responsible for neurotransmitter release within presynaptic terminals primarily consist of Cav2.1 P/Q-type channels in the CNS and Cav2.2 N-type channels in the PNS.  Studies that followed Dunlap and Fishbach’s initial discovery demonstrated that both the P/Q-type and N-type currents could be inhibited by numerous neurotransmitters, including acetylcholine, dopamine, glutamate, norepinephrine and serotonin all acting through metabotropic receptors.  Thus, it was shown that neurotransmitters themselves can inhibit further neurotransmitter release through the inhibition of presynaptic Ca2+ channels.  This GPCR-mediated inhibition of Ca2+ channels is voltage-dependent, with stronger inhibition occurring at more hyperpolarized potentials.  Either strong voltage-clamped depolarizations (> +100 mV) or rapid physiological trains of APs can temporarily reverse this voltage- dependent inhibition to produce “facilitation” (reviewed in (Elmslie, 2003)).  Single channel recordings in small, detached membrane patches demonstrated that voltage-dependent Ca2+ channel inhibition is membrane-delimited and independent of soluble cytoplasmic signals.  The inhibition also involves a reduction in the open probability at the single channel level that produces the characteristic slowing of activation kinetics observed at the whole-cell level (Lipscombe et al., 1989).  Furthermore, the voltage- dependent inhibition of native Cav2 currents is accompanied by a concomitant positive shift in the voltage-dependence of channel activation and is usually PTX-sensitive, indicating an involvement of Gαi/o proteins.  However, the demonstration of voltage-dependent inhibition through PTX-insensitive pathways revealed that the type of Gα subunit is not the critical intracellular signal (reviewed in (Elmslie, 2003)). Chapter 1: Introduction  24 Terry Snutch and colleagues were the first to propose that voltage-dependent inhibition is due to the direct binding of Gβγ subunits to Cav2 channels independent of Gα activity (Bourinet et al., 1996a). Shortly thereafter, two groups demonstrated that injection or overexpression of Gβγ subunits can reconstitute voltage-dependent inhibition of P/Q-type and N-type channels in both neurons and heterologous systems (Herlitze et al., 1996; Ikeda, 1996).  Subsequent studies identified both the N- terminus and domain I-II linker of Cav2 channels as being essential for direct Gβγ binding and voltage- dependent inhibition (reviewed in (Dolphin, 2003; Tedford and Zamponi, 2006)) (Fig. 1.5).  The binding of Gβγ to the domain I-II linker was shown to be antagonized by the binding of the accessory Ca2+ channel β subunit (Bourinet et al., 1996a) as well as by the phosphorylation of specific residues within the domain I-II linker by PKC (Hamid et al., 1999; Zamponi et al., 1997).  Further characterization at the single channel level demonstrated that Gβγ binding stabilizes the closed state of Cav2 channels to create a “reluctant” state with a prolonged latency to first opening.  The extent of voltage-dependent Ca2+ channel inhibition is highly variable within neuronal systems, likely due to differences in primary sequence, cellular electrical properties, and colocalization between specific GPCRs, Gβγ and Cav2 channel subtypes (reviewed in (Dolphin, 2003; Tedford and Zamponi, 2006)). Chapter 1: Introduction  25  1 2 3 4 5 6 Domain: I II III IV β auxilliary subunit binding (HVA only) Gβγ voltage-dependent inhibition (Cav2 only) Gβ2γ inhibition and CAMKII potentiation (at Ser1198) (Cav3.2 only) N-terminus C-terminus intracellular extracellular High affinity inhibition by Ni2+ and Zn2+ at His191 (Cav3.2 only) Potentiation by reducing agents through relief of Zn2+ blockade (Cav3.2 only)  Figure 1.5 - Modulation sites of the α1 calcium channel subunit. Labeled Ca2+ channel modulation sites (not comprehensive) include a binding site for the Ca2+ channel β accessory subunit in the domain I-II linker of all HVA Ca2+ channels (region indicated by green line). Gβγ binding to the N-terminus, domain I-II linker, and possibly the C-terminus of Cav2 channels (region indicated by red line) causes voltage-dependent inhibition, which is antagonized by PKC binding to overlapping regions (not shown).  Cav3.2 T-type channels are inhibited by Gβ2γ proteins binding to the domain II-III linker, while binding of CAMKII to the domain II-III linker causes the potentiation of Cav3.2 channels (region indicated by dark blue line).  High affinity binding of Ni2+ or Zn2+ to His191 of Cav3.2 channels mediates a specific inhibition, while endogenous reducing agents (L-cysteine) selectively potentiate Cav3.2 channels through relief of tonic Zn2+ blockade (light blue line).  Unlike membrane-associated voltage-dependent inhibition, voltage-independent inhibition of HVA Ca2+ channels can occur through multiple intracellular pathways and is functionally defined as a GPCR-mediated inhibition that does not exhibit facilitation or a characteristic slowing of channel activation kinetics.  Voltage-independent inhibition is often mediated by PTX-insensitive Gαq/11 proteins and can occur through both fast (seconds) and slow (minutes) signaling pathways (Beech et al., 1992). This type of inhibition has been well characterized in peripheral neurons as it relates to the voltage- independent inhibition of N-type Ca2+ currents by mAChRs (see below).  Besides mAChR-mediated effects, activation of ORL1 receptors within nociceptive DRG neurons causes the slow (30 minutes) internalization of N-type channels (along with the receptor) into cytoplasmic vesicles through a PKC- dependent pathway (Altier et al., 2006).  Alternatively, voltage-independent channel internalization can Chapter 1: Introduction  26 occur within seconds, as observed for the GABAB-mediated internalization of N-type channels through a tyrosine kinase-dependent pathway in chick DRGs (Tombler et al., 2006).  Voltage-independent inhibition effects can include a component that is dependent upon the nature of the N-type channel splice-variant.  For example, the Cav2.2 channel isoform most highly expressed in nociceptive DRGs includes exon 37a in its C-terminus.  A specific residue within this region appears to be phosphorylated via GPCR-mediated tyrosine kinase activity to allow for voltage-independent inhibition and a mechanism whereby GABA, opiates and other neurotransmitters inhibit nociceptive Ca2+ currents independent of firing intensity (Raingo et al., 2007).  Besides the activity of classical cytoplasmic kinases, it has also recently been shown that the breakdown of membrane PIP2 by PLC can underlie voltage-independent inhibition.  Wu and coworkers first demonstrated that P/Q-type Ca2+ channels are stabilized in the plasma membrane by PIP2 (Wu et al., 2002).   Following this key discovery, the activation of PLC by Gα/11 and subsequent breakdown of PIP2 was shown to be directly involved in the voltage-independent muscarinic inhibition of both N-type and L-type channels (Gamper et al., 2004; Liu and Rittenhouse, 2003; Liu et al., 2006).  The possible involvement of downstream phospholipase A2 activation and arachidonic acid production in this inhibition pathway is currently in debate (reviewed in (Gamper and Shapiro, 2007)). Muscarinic inhibition of HVA Ca2+ channels generally occurs through Gαi/o-coupled M2/M4 receptors for the voltage-dependent pathway and through Gαq/11-coupled M1/M3/M5 receptors for voltage-independent pathways.  In both PNS superior cervical ganglion (SCG) neurons and CNS striatal neurons, voltage-independent inhibition occurs through M1 receptors, PTX-insensitive Gα proteins, and a Ca2+-sensitive intracellular pathway, while voltage-dependent inhibition occurs through M4 receptors and PTX-sensitive Gα proteins (Beech et al., 1991; Bernheim et al., 1992; Howe and Surmeier, 1995). However, promiscuity also exists between mAChRs and the inhibition pathways.  In rat hippocampal neurons, voltage-dependent inhibition of non-N-type HVA Ca2+ currents surprisingly occurs largely through M3 receptors coupled to PTX-sensitive G-proteins while voltage-independent inhibition of N- type currents in the same cells occurs through M2 receptors (Toselli and Taglietti, 1995).  Unlike the selective inhibition of Cav2 channels through the voltage-dependent pathway, voltage-independent inhibition can also be observed for L-type channels.  Expression of recombinant mAChRs in NIH 3T3 cells revealed that L-type currents are inhibited by M2/M4 receptors through a cAMP- (but not PKA) mediated pathway, while M1/M3/M5 receptors inhibit L-type channels through a PKC-dependent pathway (Pemberton and Jones, 1997).  Recombinant Cav1.2 currents are also inhibited by M1, M3, or M5 activation through a slow voltage-independent pathway that requires Gαq/11 activity but is insensitive to antagonists of phospholipases, protein kinases, and protein phosphatases (Bannister et al., 2002). Chapter 1: Introduction  27 Analysis of GPCR-Ca2+ channel interactions in recombinant expression systems can lead to insights that are often difficult to reach in many native systems where multiple receptors, channels, and signaling pathways co-exist (see above).  These insights can often be relevant to native signal integration.  For example, co-expression of Cav2.2 channels and M1 receptors in HEK cells demonstrated a fast voltage-dependent inhibition through Gβγ proteins and a slower voltage-independent inhibition mediated by Gαq/11 proteins (Melliti et al., 2001).  This both confirmed and clarified the inhibition observed for native N-type currents by M1 receptor activation in SCG neurons (Kammermeier et al., 2000).  Studying the effects of recombinant M1, M3 and M5 receptor activation on Cav2.3 currents in HEK cells revealed that all three receptors stimulate this R-type current through activation of Gαq, DAG, and Ca2+-independent PKCs, while only M3 and M5 receptors cause robust Gβγ-mediated, voltage-dependent inhibition of Cav2.3 currents prior to the stimulation (Bannister et al., 2004; Melliti et al., 2000).  These results led MacVicar and colleagues to identify a similar stimulation of native R-type currents by M1/M3 receptor activation in hippocampal CA1 neurons that also occurs through a Ca2+- independent PKC pathway and ultimately alters neuronal firing patterns (Tai et al., 2006). The modulation of Ca2+ channels by mGluRs has been studied much less thoroughly than for mAChRs.  Like mAChRs, Gαi/o-coupled Group II mGluRs inhibit HVA Ca2+ channels through a membrane-delimited, voltage-dependent pathway (McCool et al., 1996), while activation of Gαq/11- coupled Group I mGluRs cause voltage-independent inhibition of Cav2 currents (McCool et al., 1998). Inhibition of HVA Ca2+ channels by mGluRs has been observed in various regions of the CNS, including hippocampal CA3 pyramidal cells (Swartz and Bean, 1992), dentate gyrus granule cells (Schumacher et al., 2000) and neostriatal neurons (Colwell and Levine, 1999).  Activation of heterologously-expressed mGluR1a receptors in SCG neurons also induces similar effects on N-type currents to those observed for native M1 receptor activation, including Gβγ-mediated, voltage-dependent inhibition and Gαq/11- mediated, voltage-independent inhibition (Kammermeier and Ikeda, 1999).  Also, as observed for Group I mAChRs (Bannister et al., 2004), activation of mGluR1a stimulates Cav2.3 R-type channels through a PKC-mediated pathway (Stea et al., 1995). The stimulation of Cav2.3 currents by GPCR-mediated activation of PKC illustrates another form of Ca2+ channel modulation.  Although voltage-dependent and -independent inhibition of HVA Ca2+ currents are more predominant, Ca2+ channels can also be potentiated by various intracellular kinases and signaling pathways.  For example, both Cav1.2 and Cav2.1 currents can be facilitated through the association of Ca2+/calmodulin complexes (Lee et al., 1999a) (reviewed in (Lee and Catterall, 2005)). Furthermore, besides Cav2.3, PKC can also stimulate Cav2.2 currents but has no effect on Cav2.1 or Cav1.2 currents expressed in oocytes (Stea et al., 1995).  This PKC-mediated stimulation can antagonize Gβγ-mediated inhibition in signaling cross-talk (Zamponi et al., 1997).  Other kinases, such as protein Chapter 1: Introduction  28 kinase A (PKA), Ca2+/calmodulin kinase II (CAMKII), mitogen-activated protein kinase (MAPK), tyrosine kinases, and lipid kinases can also stimulate HVA Ca2+ currents, but the potentiation effects are much more localized and specific than the general inhibition pathways that have been discussed (reviewed in (Bannister et al., 2005)).  1.3.3 Modulation of T-type calcium channels T-type Ca2+ channels were originally thought to be resistant to modulation by intracellular signaling pathways, partly because of the lack of cell dialysis-induced T-type current rundown during whole-cell recordings.  Subsequent studies on T-type modulation in native systems revealed a number of discrepancies, with individual neurotransmitter types being reported to inhibit, stimulate, or have no effect on T-type currents depending upon the tissue and cell type being examined (reviewed in (Yunker, 2003)).  After a lull following their initial cloning and characterization, the study of recombinant T-type channel modulation is currently emerging as an essential tool aimed at shedding further light on modulation within native systems.  Of the three T-type isoforms, Cav3.2 appears to be the most widely modulated, with the domain II-III linker acting as a signaling hotspot.  Contrastingly, very little is presently known concerning the modulation of Cav3.1 and Cav3.3 channels.  Because of the vast diversity of neurotransmitter-activated GPCRs and the functional heterogeneity within each GPCR family, the modulation of T-type channels by all possible GPCRs will not be discussed in detail here (see (Chemin et al., 2006; Yunker, 2003) for extensive reviews).  This section will focus on the modulation of T-type currents by downstream cellular signaling pathways, including G-proteins, kinases, and phosphatases.  The effects of both endogenous reducing agents and bioactive lipids on T-type currents will also be discussed.  This section will end with a discussion on our current known status concerning the modulation of T-type channels by mAChR- and mGluR-mediated pathways. The Gβγ-mediated inhibition of Ca2+ channels was originally thought to be restricted to Cav2 channels because of a lack of observed voltage-dependent inhibition for the L-type and T-type Ca2+ channel classes.  However, the inhibition of LVA Ca2+ currents by dopamine D1 receptors in adrenal glomerulosa cells was shown to involve Gβγ subunits (Drolet et al., 1997).  In a pioneering paper within the field of T-type channel modulation, Wolfe et al. co-expressed specific Gβγ proteins with Cav3 isoforms to demonstrate that Cav3.2, but not Cav3.1, is specifically inhibited by Gβγ proteins that contain Gβ2  (Wolfe et al., 2003).  Experiments using Cav3.1- Cav3.2 chimeric channels and fusion proteins demonstrated that Gβ2γ proteins directly bind to the domain II-III linker of Cav3.2 channels and that this binding is both necessary and sufficient for inhibition (Fig. 1.5).  Unlike the Gβγ-mediated inhibition of Cav2 channels, the inhibition of Cav3.2 is voltage-independent and not associated with a concomitant change in channel kinetics or voltage dependence (Wolfe et al., 2003).  A follow-up study using purified Chapter 1: Introduction  29 recombinant Gβγ subunits revealed that the Gβ2γ-mediated inhibition of Cav3.2 channels involves a reduction in the open probability of the channels, with no effects on membrane expression or active- channel gating properties, and also identified specific residues within the Gβ2 subunit that confer this specific inhibition (DePuy et al., 2006).  We have recently linked this specific Gβγ-mediated inhibition of Cav3.2 to activation of a native GPCR by showing that activation of the endogenous urocortin receptor, CRFR1, causes the selective inhibition of both rat and human Cav3.2 currents through the Gβγ- mediated pathway (Tao et al., 2008) (Appendix 2). The study of the modulation of T-type Ca2+ channels by CAMKII is a clear example of how experiments using recombinant subtypes can further our understanding of modulation within native systems.  In the early 1990s, peak T-type currents levels were shown to be sensitive to intracellular Ca2+ concentration in cardiac cells (Tseng and Boyden, 1991).  Shortly thereafter, Barrett and colleagues demonstrated that in bovine adrenal glomerulosa cells, T-type currents are potentiated by Ca2+-dependent CAMKII activity through a hyperpolarizing shift in their voltage-dependence of activation (Lu et al., 1994).  Cell-attached patch recordings confirmed that CAMKII activation did indeed underlie the Ca2+- mediated augmentation of T-type currents by increasing the single channel open probability (Barrett et al., 2000).  Upon the molecular cloning of the three Cav3 subtypes, the potentiation of T-type currents by CAMKII activation was recapitulated in HEK cells by co-expression of Cav3.2, but not Cav3.1, with CAMKIIγc (Wolfe et al., 2002).  Similar to their study on Gβγ-mediated modulation, the Barrett lab used chimeric channels, point mutations, and fusion proteins to reveal that CAMKII specifically modulates Cav3.2 channels by directly interacting with the domain II-III linker and phosphorylating a specific serine residue (Ser1198, Fig. 1.5) (Welsby et al., 2003).  They then used the results from the HEK system to design immunohistochemical experiments in adrenal glomerulosa cells that demonstrated that activation of the Ang II receptor in vivo results in an increased phosphorylation of the Ser1198 residues of native Cav3.2 channels (Yao et al., 2006).  Thus, the potentiation of Cav3.2 channels by CAMKII is functionally implicated in the AngII receptor-mediated regulation of aldosterone synthesis and secretion within adrenal cells. In contrast to the specific effects of CAMKII on Cav3.2 channels, the effects of PKA and PKC on T-type currents are much less clear.  Activation of GPCRs specific for growth hormones (Chen et al., 2000), serotonin (Kim et al., 2006), and acetylcholine (Pemberton and Jones, 1997) all cause an increase in T-type currents mediated by cAMP and downstream PKA activity.  However, many of these modulation studies are either incomplete or have significant experimental limitations, such as the inappropriate use of small whole-cell currents (~20 pA; low signal-noise ratio) for pharmacological studies (Chen et al., 2000).  Recombinant Cav3.2 channels are stimulated by PKA activity when expressed in Xenopus oocytes, but this effect has an extremely slow time course (> 1 hour) and could not be repeated in experiments using HEK cells (Kim et al., 2006).  Over an even longer time course, Chapter 1: Introduction  30 incubation of cultured rat chromaffin cells with cAMP in the media for three to five days induces high density expression of Ni2+-sensitive T-type currents that requires protein synthesis but is independent of PKA activity (Novara et al., 2004). Activation of PKC has been reported to stimulate, inhibit or have no effect on native T-type currents, depending on the system examined (reviewed in (Chemin et al., 2006)).  In rat DRG neurons and bovine adrenal glomerulosa cells, PKC activation causes an inhibition of native T-type currents (Rossier et al., 1995; Schroeder et al., 1990).  Interestingly, the phorbol 12-myristate 13-acetate (PMA)- induced inhibition observed in DRG neurons is only observed at temperatures close to the mammalian physiological range (Schroeder et al., 1990).  Within recordings on Xenopus oocytes at room temperature, activation of PKC by PMA causes a slow potentiation of all 3 Cav3 isoforms with no effect on other properties including no changes in membrane expression (Park et al., 2006).  A recent paper explored the variable PKA- and PKC-dependent effects on Cav3 isoforms in oocytes and HEK cells and determined that potentiation by chemical-induction of these kinases occurs at physiological relevant temperatures (37oC) within HEK cells, but not at room temperature, because of a proposed deficit in kinase trafficking to the plasma membrane at lower temperatures (Chemin et al., 2007a).  However, the chemical activation of kinases by compounds such as PMA may be more temperature sensitive than endogenous GPCR pathways, as PMA failed to induce potentiation of Cav2.3 currents at room temperature (Chemin et al., 2007a), but activation of M1 receptors triggered robust PKC-mediated stimulation of Cav2.3 currents at room temperature in the same expression system (Bannister et al., 2004). The activation of intracellular phosphatases can also modulate T-type channel activity.  T-type currents in mouse spermatogenic cells are facilitated in a voltage-dependent manner through a tyrosine phosphatase-dependent pathway (Arnoult et al., 1997).  Spermatogenic T-type currents are highly Ni2+- sensitive, suggesting that they likely consist of Cav3.2 channels (Arnoult et al., 1998), and are proposed to be activated to a higher conductance state following tyrosine phosphatase activity (Arnoult et al., 1997).  Conversely, blocking tyrosine kinase activity with genistein (>10 μM) caused inhibition of T- type currents in NG180-15 cells (Morikawa et al., 1998).  However, it has recently been shown that genistein directly inhibits Cav3.1 T-type channels independently of its action on tyrosine kinases in this concentration range (IC50 = 25 μM) (Kurejova and Lacinova, 2006). Redox modulation provides another well characterized mechanism for altering T-type activity and associated neuronal excitability within native systems.  Todorovic and colleagues have shown that both recombinant and native DRG T-type currents consisting of Cav3.2 channels are selectively and potently stimulated by the endogenous reducing agent, L-cysteine (Todorovic et al., 2001b).  This enhancement of T-type currents by L-cysteine at physiologically relevant concentrations is associated Chapter 1: Introduction  31 with an increase in burst firing and DRG excitability in vitro (Nelson et al., 2005) and hyperalgesia in vivo (Todorovic et al., 2001b).  L-cysteine also increases the overall excitability and burst firing of thalamic nRT neurons through the enhancement of native Cav3.2-mediated T-type currents (Joksovic et al., 2006).  The selective potentiation of Cav3.2 channels by reducing agents appears to be due to chelation of endogenous Zn2+ ions that selectively bind to an extracellular histidine residue on Cav3.2 channels, but not Cav3.1 or Cav3.3 channels (Fig. 1.5) (Nelson et al., 2007b).  Thus, the redox-mediated enhancement of DRG T-type channels is in fact a removal of specific inhibition of Cav3.2 channels by endogenous Zn2+ ions (Traboulsie et al., 2007).  Related to this phenomenon, the endogenous redox agent, ascorbate (vitamin C), has recently been shown to selectively and potently inhibit Cav3.2 channels in HEK cells and in native DRG and nRT neurons, revealing a novel mechanism of action of vitamin C in the PNS and CNS (Nelson et al., 2007a). T-type channels have recently been shown to be inhibited by several classes of natural lipids. The endogenous cannabinoid, anandamide, inhibits all three Cav3 isoforms at submicromolar concentrations independently from the activation of its endogenous GPCRs, CB1 and CB2, and their downstream second messenger pathways.  Inside-out patch recordings with anandamide applied to the intracellular surface revealed that the inhibition is due to direct action on T-type currents and includes an acceleration of inactivation kinetics and a hyperpolarizing shift in the voltage dependence of inactivation (Chemin et al., 2001b).  Arachidonic acid is an ω-6 polyunsaturated fatty acid that is a product of anandamide hydrolysis and is produced by many GPCR pathways to act as an intracellular signaling agent.  Like anandamide, arachidonic acid has been shown to inhibit both Cav3.1 and Cav3.2 channels through a membrane-delimited pathway that includes both a reduction in channel availability as well as a negative shift in the voltage-dependence of inactivation (Talavera et al., 2004; Zhang et al., 2000).  In another example, the common dietary ω-3 cis-polyunsaturated fatty acids, DHA and LNA, have also been shown to inhibit native T-type currents through a similar mechanism (Danthi et al., 2005).  A recent paper has tied these findings on “bioactive lipids” together through a systematic study that identified the hydroxyl group and alkyl chain of anandamide and the degree of unsaturation, but not alkyl chain length, of fatty acids as key determinants for Cav3 channel inhibition (Chemin et al., 2007b). Relevant to the present proposal, the activation of mAChRs has been variously shown to inhibit, stimulate, or have no effect on native T-type currents.  Transfection of individual recombinant mAChRs into NIH 3T3 fibroblast cells revealed that both M3 and M5 receptors increase T-type current amplitudes, while activation of M2 and M4 receptors has no effect and M1 receptors cause an increase in T-type currents albeit only after blocking PKC activity (Pemberton et al., 2000).  Thus, heterogeneity in T-type modulation appears partially dependent on the subtype of mAChR co-expressed.  Activation of mAChRs with a general muscarinic agonist (carbachol) augments Ni2+-sensitive T-type currents via increasing their open probability in hippocampal CA3 pyramidal neurons through a PTX-insensitive Chapter 1: Introduction  32 pathway (Fisher and Johnston, 1990; Toselli and Lux, 1989).  Muscarinic agonists also stimulate Ni2+- insensitive T-type currents with kinetics similar to Cav3.1 channels in hippocampal interneurons (Fraser and MacVicar, 1991).  However, interpretation of muscarinic effects in the hippocampus is complicated by an incomplete pharmacology that often does not eliminate contaminating R-type currents, which are potently potentiated by M1/M3 receptor activation (Tai et al., 2006).  Recordings on Cav3.2-like currents in hen granulosa cells revealed a robust carbachol-mediated inhibition of T-type currents (Wan et al., 1996).  Similarly, T-type currents in rat DRGs (primarily consisting of Cav3.2) are inhibited by acetylcholine (Formenti and Sansone, 1991).  In contrast, highly Ni2+-sensitive T-type currents in magnocellular cholinergic basal forebrain neurons are not affected by muscarinic agonists (Allen and Brown, 1993).  These discrepancies illustrate the need to study the interactions between specific mAChRs and Cav3 subtypes in more well-defined systems. The modulation of T-type channels by mGluRs is even less well characterized.  Thus far, one voltage-clamp study has shown that T-type currents within retinal ganglion cells can be enhanced by activation of mGluR2 through a G-protein-dependent, but CTX and PTX-insensitive pathway (Robbins et al., 2003).  However, the biophysical properties of the recorded currents in this paper do not appear to be completely characteristic of T-type channels and the possibility of the currents being mediated by R- type Cav2.3 Ca2+ channels has not been ruled out.  In isolated hippocampal dendritic segments, activation of mGluRs had no significant effect on the recorded T-type currents (Kavalali et al., 1997).  The dependence of N-methyl D-aspartate (NMDA) receptor-independent LTP on both Ni2+-sensitive Ca2+ currents and mGluR1 activity provides indirect evidence of a possible interaction between mGluR1 and T-type currents (Wang et al., 1997), but more extensive experiments are needed on this putative receptor-channel interaction.  1.4 Cerebellar Purkinje cells 1.4.1 Cerebellum overview One area of the CNS wherein both mGluR1 receptors and T-type channels are functionally relevant is the cerebellum.  Cerebellar PCs express a robust T-type current that is predominantly localized to the dendritic tree (Isope and Murphy, 2005).  mGluR1 receptors are also highly expressed at synaptic sites within the PC dendritic tree, and serve critical functions in modulating synaptic plasticity, retrograde signaling, excitability and synaptic pruning within PCs (reviewed in (Knopfel and Grandes, 2002)).  The roles of both of these proteins in PC physiology will be further discussed in the following sections. The cerebellum is a highly folded brain region lying between the brain stem and cortex that is involved in balance; motor planning, initiation, and execution; motor learning; and some forms of Chapter 1: Introduction  33 cognition (reviewed in (Barlow, 2002; Boyden et al., 2004; Ghez and Thach, 2000; Ito, 2001)).  The highly-conserved cerebellum allows vertebrates to perform complicated movements and to calibrate these movements in time and space as the body and surrounding environment continually change. Despite such sophisticated functions, the cerebellar cortex has a highly repeated, uniform laminar structure that is oriented in parasagittal planes and composed of three layers of well-defined cell types (Fig. 1.6A).  The cerebellar PCs are responsible for the sole output of the cerebellar cortex.   In the mature cerebellar cortex, the PCs align in a single layer (the Purkinje cell layer) with an extensive apical dendritic arbourization (forming the molecular layer) in the parasagittal plane.  This elaborate dendritic tree is the largest and most highly branched of all neurons in the CNS, yet is generally confined to a single plane perpendicular to the long axis of the cerebellar folia.  The innermost layer beneath the Purkinje cell layer is called the granule cell layer and is composed of excitatory granule cells (GCs) and inhibitory golgi cells.  Because of their small size and high density in the cerebellar cortex, GCs compose approximately half the neurons in the entire brain (reviewed in (Boyden et al., 2004)).  The excitatory axons of GCs pass through the PC layer and bifurcate in the molecular layer, where they run perpendicular to the PC dendritic tree and parallel to the long axis of the cerebellar folia, giving them their name: parallel fibers (PFs).  The PFs can pass through up to ~ 1,000 PC dendritic trees and the distal dendrites of individual PCs have a plethora of PF synaptic connections, ranging from approximately 175,000 in rats to 15 million PF synapses in humans (reviewed in (Altman and Bayer, 1997; Harvey and Napper, 1991)).  The molecular layer also consists of inhibitory interneurons called stellate cells and basket cells that form GABAergic connections with the PC dendritic shafts or axon hillock, respectively. The CNS and PNS are connected to the cerebellum through two well-defined excitatory inputs. The first is formed by moderately thin, myelinated axons called climbing fibers (CFs) that pass directly from the inferior olive of the medulla, through the white matter and granule cell layer, to the PC’s proximal dendrites (Fig. 1.6B).  Individual CFs branch and innervate 10 to 15 PCs (there are over 10 times less cells in the inferior olive compared to PCs), but a single PC is only innervated by a single CF at maturity, due to pruning of multiple CF innervations during the first few weeks of postnatal development.  The CF wraps around the primary and secondary proximal dendrites of the PC in the inner two-thirds of the molecular layer and forms ~ 1,500 synaptic contact sites with a high release probability (reviewed in (Schmolesky et al., 2002)).  The other main excitatory input carries both sensory and motor signals from various regions of the CNS and PNS to the cerebellum through brainstem relays in the pontine and reticular nuclei (Fig. 1.6B).  The axons from these nuclei are called mossy fibers (MFs) and are thick and heavily myelinated.  The MFs branch extensively and directly synapse with GC dendrites. The cluster of nerve endings at the end of the MFs form giant presynaptic terminal rosettes called glomeruli that each form excitatory glutamatergic synaptic connections with the dendrites of many GCs, Chapter 1: Introduction  34 resulting in an individual MF being electrically connected to hundreds of GCs.  The GCs then relay the MF signal to multiple PCs via their PFs (Fig. 1.6).  The output of the cerebellum consists of PC axons projecting through the granule cell layer and white matter to make inhibitory GABAergic connections with several neurons of the DCN.  Collaterals from the excitatory CF and MF cerebellar inputs also directly synapse with DCN neurons to generate output signals that are subsequently modulated by inhibitory PC activity (reviewed in (Ito, 2001)).  Signals from the DCN neurons are relayed to the motor cortex via the thalamus, and are thought to be the start of the motor command pathway (reviewed in (Barlow, 2002)). The unique repeating functional units within the cytoarchitecture of the cerebellar cortex create an ideal model system for studying physiological and molecular mechanisms of learning within the CNS. As each of these units contain the same cell types that are synaptically connected in the same manner (see above), it is presumed that the precise inputs and outputs of these units will vary depending on location within the cerebellum but the mechanisms of signal processing will remain constant.  Therefore, studying a simple model of motor learning can reveal important insights into the underlying mechanisms of more complex motor learning behaviors (reviewed in (Boyden et al., 2004; Ito, 2001)).  Many simple motor-learning paradigms, such as eyeball movement reflexes (e.g. - the vestibulo-ocular reflex), have now been well characterized at the underlying cerebellar cellular circuitry level.  A distinct type of cellular plasticity called long-term depression (LTD) has also been thoroughly studied at the PF-PC synapse.  As discussed in further sections, these cerebellar features allow for the linkage between molecular signaling mechanisms, synaptic plasticity and in vivo learning (reviewed in (Evans, 2007)). Chapter 1: Introduction  35   Figure 1.6 - Circuitry within the cerebellar cortex. A) Three-dimensional configuration of the cerebellar cortex.  The soma of PCs (brown) form the Purkinje cell layer, while the extensive dendritic arbourizations of the PCs are oriented in the parasagittal plane and form the molecular layer.  Granule cells (GCs; light green) form the innermost granule cell layer.   The axons of GCs (light green) project through the Purkinje cell layer to the molecular layer, where the bifurcate and run parallel to the long axis of the cerebellum.  Inhibitory GABAergic interneurons, including stellate cells (blue), basket cells (red), and golgi cells (purple), are also found in the cerebellar cortex and form synaptic connections with various cellular elements.  B) The glutamatergic excitatory (green plus sign) inputs onto PCs (brown) consist of GC parallel fibers (PFs; light green) that synapse onto the distal dendrites of many PCs per individual PF and climbing fibers (CFs; dark green) that form thousands of synaptic connections with the proximal dendrites of the PC. Climbing fibers project directly from the inferior olive of the brainstem, while mossy fibers (MFs; yellow) from various brain regions project onto many GCs, which relay the excitatory signals to many PCs via their PFs.  The axon of PCs projects to deep cerebellar nuclei neurons (DCN; purple) via inhibitory (black minus sign) GABAergic synaptic connections and form the sole output of the cerebellum.  DCN neurons relay signals to the motor cortex via the thalamus. Adapted with permission from Purves et al., 2001, Neuroscience, 2nd edition, 416-417. Chapter 1: Introduction  36 1.4.2 Cerebellar Purkinje cell physiology The electrical and chemical activity of the cerebellar PC is shaped by strong but low frequency excitatory input from an individual CF and weak but high frequency excitatory inputs from multiple PFs. For simplicity, the effects of GABAergic interneurons at the PC axon hillock (basket cells) and dendritic tree (stellate cells) will not be discussed here (for more information, see (Evans, 2007)).  Because the CF entwines the PC proximal dendrite and forms thousands of synaptic contacts, CF excitation results in the largest recorded EPSP (~ 25 mV) yet observed in the CNS.  Due to the amplitude of this EPSP, CF firing always results in a series of fast and slow postsynaptic spikes, termed the complex spike (reviewed in (Schmolesky et al., 2002)).  This complex spike is evoked by glutamate binding to ligand-gated α- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) ionotropic receptors to generate the large EPSP that causes sufficient depolarization to open voltage-gated Ca2+ channels and generate a putative dendritic Ca2+ spike (Knopfel et al., 1991).  The putative Ca2+ spike in the PC dendrites excites voltage- gated Na+ channels that are predominantly localized in the soma, initiating the generation of a burst of somatic Na+-driven APs (Llinas and Sugimori, 1980a, b).  The large Ca2+ transients that are generated by the CF-induced complex spike synchronously spread over the entire PC dendritic tree through the activation of dendritic Ca2+ channels (Miyakawa et al., 1992), proposed to consist of mainly P-type channels (Mintz et al., 1992; Usowicz et al., 1992; Watanabe et al., 1998).  As the CF discharges at a low, irregular rate around 1 Hz that is much lower than the overall PC firing rate, the CF’s contribution to the total spike output of PCs is small (reviewed in (Ito, 2002)).  As we will discuss shortly, the CF- induced complex spike and dendritic Ca2+ transients serve additional purposes within the PC. Although individual MFs excite many GCs which in turn excite (via their PFs) many PCs, the electrical excitation at an individual PF-PC synapse is quite small.  Furthermore, a majority of the PF-PC synapses appear to be electrically silent (Isope and Barbour, 2002) in order to create well-defined PF receptive fields that are specifically coupled to the local CF input (Ekerot and Jorntell, 2001).  The EPSPs that are produced at PF-PC synapses dissipate over the long distances of passive propagation between the distal dendrites, where they are generated, and the soma, where they are summated (Roth and Hausser, 2001).  Temporal and/or spatial summation of distal PF-induced EPSPs is therefore required to induce PC firing (Brunel et al., 2004), and the conjunctive action of many MFs is needed to shape cerebellar PC output. Mature PCs also exhibit spontaneous activity in the absence of PF and CF synaptic inputs. Much of this spontaneous activity is generated by the functional interplay between various voltage-gated ion channels in the PC dendrites (Womack and Khodakhah, 2002) (Swensen and Bean, 2003) that is independent of the back-propogation of somatic Na+-driven APs (Womack and Khodakhah, 2004).  Like PC firing in vivo, this spontaneous in vitro firing can involve different modes and can reach rates of over 100 Hz.  The spontaneous activity in PCs includes periods of rhythmic firing as well as periods of burst Chapter 1: Introduction  37 firing, separated by silent periods (Womack and Khodakhah, 2002).  This “trimodal” spontaneous firing pattern could be related to the membrane bistability that is observed under certain in vivo conditions (Loewenstein et al., 2005), with CF inputs controlling the transitions between firing states (McKay et al., 2007; Shin et al., 2007).  Thus, the spontaneous activity of PCs will combine with the synaptic inputs to determine the overall firing output of PCs. The electrical output of cerebellar PCs is also shaped by chemically-mediated changes in synaptic connections.  The most well studied of these mechanisms is the LTD that occurs postsynaptically at the PF-PC synapse, termed PF-LTD.  PF-LTD is produced by the activation of a bundle of PFs in conjunction with CF stimulation, leading to a sustained decrease in PF-PC EPSP amplitude (Ekerot and Kano, 1985; Ito et al., 1982).  The reduction in EPSP amplitude can also be observed when an individual PF and PC are simultaneously depolarized during paired recordings (Casado et al., 2002).  Electrophysiological and Ca2+ imaging experiments have shown that CF activity causes a widespread dendritic depolarization and resultant Ca2+ influx through voltage-gated Ca2+ channels necessary for PF-LTD (reviewed in (Eilers et al., 1996; Ito, 2002)).  Thus, one model of motor learning suggests that the CF input to the PC provides a “teacher” signal that weakens synaptic inputs when error signals occur (reviewed in (Ito, 2001; Schmolesky et al., 2002)).  The activation of PF-PC synapses also produces local Ca2+ signals, and the concurrent activation of PFs and CFs causes a supralinear increase in cytoplasmic Ca2+ that reaches up to 10 μM, which is much greater than the sum of the two independent Ca2+ inputs (Wang et al., 2000).  Thus, some mechanism of “coincidence detection” creates a supralinear Ca2+ increase at the PF-PC synapse.  The large increase in synaptic intracellular Ca2+ drives a kinase activation/ phosphatase suppression system that ultimately results in the reduction of functional AMPA receptors (GluR2/GluR3 subunits) in the postsynaptic density via direct phosphorylation by PKC and subsequent internalization (Linden, 2001; Linden and Connor, 1991; Xia et al., 2000).  As AMPA receptors exclusively mediate glutamate-driven fast excitatory transmission at the PF-PC synapses (reviewed in (Eilers et al., 1996)), their internalization leads to the sustained reduction in EPSP amplitude that underlies PF-LTD. Relevant to the importance of intracellular Ca2+ in PF-PC synaptic plasticity, Ca2+ levels are tightly regulated within PCs (reviewed in (Inoue, 2003)).  High concentrations of endogenous Ca2+ buffers, such as calbindin and parvalbumin, are expressed in PC dendrites (Schmidt et al., 2003), which enables a rapid return to baseline Ca2+ levels after large dendritic Ca2+ transients (Sugimori and Llinas, 1990).  One group has proposed that saturation of the large concentration of mobile, high-affinity Ca2+ buffer forms the coincidence detector behind the supralinear Ca2+  increase (Maeda et al., 1999). However, Ca2+ channels are also strongly implicated in this coincidence detector mechanism (Miyakawa et al., 1992; Wang et al., 2000). Chapter 1: Introduction  38 The mechanism that links PF activity to PF-LTD is currently being investigated.  Just as depolarization of the PC dendrite can replace the CF stimulation requirement for PF-LTD induction, so can glutamate application replace the PF stimulation requirement (reviewed in (Ito, 2002)).  Glutamate activates both AMPA receptors and mGluRs at the PF-PC synapse, and the activation of mGluR1 has been shown to be an essential component of PF-LTD.  1.4.3 mGluR1 expression and function The mGluR1 receptor was originally cloned from the cerebellum (Houamed et al., 1991; Masu et al., 1991) and subsequent in situ hybridization and immunohistochemical studies have confirmed a high level of mGluR1 expression in cerebellar PCs compared to other regions of the brain (Baude et al., 1993; Lin et al., 1997).  The expression of mGluR1 increases in PCs during the first few weeks of development and remains at a high expression level in adult rodents while the expression of the other Group I mGluR, mGluR5, is very low or absent in the adult rat cerebellum (Casabona et al., 1997; Catania et al., 1994; Shigemoto et al., 1992).  Viewing anti-mGluR1a stained cerebellar slices with a combination of light and electron microscopy has revealed that mGluR1a is expressed in the PC soma and dendrites of P10 to P12 rats, while mGluR1a expression increases and becomes localized to PC synapses in the molecular layer during later development (Lopez-Bendito et al., 2001).  In the adult rat, mGluR1a is preferentially localized at the periphery of the PF-PC postsynaptic density (Baude et al., 1993; Lopez-Bendito et al., 2001).  There is also evidence for expression of the mGluR1b isoform in a certain population of PCs (Mateos et al., 2000). The study of intracellular signaling at the PF-PC synapse has progressed with the development of high resolution Ca2+ imaging techniques.  Confocal laser scanning microscopy revealed that subthreshold excitation of PFs can induce Ca2+ transients in a PC dendritic branchlet and its associated spines, allowing for non-electrical synaptic integration (Eilers et al., 1995).  Two-photon laser scanning microscopy experiments in the same year revealed that the smallest unit of integration within the PC is the dendritic spine, with PF-mediated activation of voltage-gated Ca2+ channels being localized to specific spines (Denk et al., 1995).  A breakthrough in the understanding of PF-LTD was made when it was discovered that repetitive stimulation of PF synapses causes a localized Ca2+ transient in PC dendritic microdomains that is dependent on the activation of mGluR1 and the downstream IP3-mediated Ca2+ release from internal stores (Finch and Augustine, 1998; Takechi et al., 1998).  This Ca2+ transient is specialized for local integration, as it is much more restricted than electrical signaling and ranges from confinement in individual spines to spinodendritic compartments depending on the level of stimulation (Takechi et al., 1998).  The mGluR1-mediated Ca2+ transients at PF-PC synapses were shown to be a critical component of PF-LTD (Finch and Augustine, 1998), with some studies showing an absolute requirement of IP3-mediated Ca2+ release in this process (Miyata et al., 2000), while other studies Chapter 1: Introduction  39 showing that PF-LTD can be elicited in the absence of IP3 signaling (Narasimhan et al., 1998).  One study showed that (along with conjunctive activation of CFs) activation of a sparse number of PFs caused a supralinear Ca2+ increase restricted to single spines and mediated by an mGluR1 and IP3- dependent pathway.  Meanwhile, activation of a dense PF bundle induced widespread LTD throughout dendritic branchlets that is mediated by voltage-gated Ca2+ channel activation and not mGluR1 or IP3 activity (Wang et al., 2000).  Thus, both IP3-mediated Ca2+ release and Ca2+ channels are implicated in the Ca2+ signals that underlie PF-LTD. In addition to IP3-mediated signaling, mGluR1 also contributes to local synaptic integration at PF-PC synapses through its activation of a slow excitatory postsynaptic current (sEPSC) (reviewed in (Knopfel and Grandes, 2002)).  High frequency stimulation of PFs produces an mGluR1-mediated sEPSC and associated elevation in intracellular Ca2+ that is independent of IP3 receptor activation (Tempia et al., 1998) and is abolished in mGluR1 KO mice (Conquet et al., 1994).  Subsequent analysis directly demonstrated that the sEPSC is mediated by cation-permeable ion channels that allow influx of both Na+ and Ca2+ into PC dendrites (Tempia et al., 2001).  Interestingly, the activation of the sEPSC by mGluR1 occurs independently from activation of PLC or PKC, but does require the activation of G- proteins and tyrosine phosphatases (Canepari and Ogden, 2003; Canepari et al., 2001; Hirono et al., 1998).  Blockade of TRPC1 (transient receptor potential channel) activity with non-functional mutants or specific antibodies in the recording pipette attenuates the sEPSC in PCs, indicating that they are mediated by the TRPC1 cation channel (Kim et al., 2003).  However, discrepancies in the single channel conductance and pharmacology between the native recorded sEPSC and TRPC1 currents indicate that other unidentified cationic channels may contribute to the sEPSC (Canepari et al., 2004).  The potential direct contribution of this mGluR1-mediated sEPSC to physiological PF-LTD remains to be tested. Along with in vitro experiments, mGluR1 activity has also been directly linked to PF-LTD in vivo.  Cerebellar LTD recorded in vivo was verified to require the concomitant stimulation of CF and PF inputs (Ito et al., 1982) and was blocked with an mGluR1 antagonist (Gao et al., 2003).  A direct linkage between in vivo PF-LTD and cerebellar motor learning was demonstrated in experiments on transgenic mice that specifically express a PKC antagonist in their cerebellar PCs.  Both PF-LTD and adaptation of the vestibulo-ocular reflex are disrupted in these PKC-deficient transgenic animals (De Zeeuw et al., 1998).  Studies on mGluR1 KO mice have also yielded exciting links between PF-LTD and in vivo motor learning.  The mGluR1 KO mice exhibit severe deficits in motor coordination and spatial learning as well as impaired PF-LTD, yet they have no gross anatomical or basic electrophysiological impairments (Aiba et al., 1994; Conquet et al., 1994).  Furthermore, injection of mGluR1-specific antibodies into the cerebellum of wt rats blocks the glutamate-induced sEPSC and PF-LTD (Shigemoto et al., 1994) and human Hodgkin’s disease patients with autoantibodies against mGluR1 develop cerebellar ataxia due to a loss of PCs, a disruption of PF-LTD, and a resultant impairment of motor learning (Coesmans et al., Chapter 1: Introduction  40 2003; Sillevis Smitt et al., 2000).  The most convincing evidence for a role of mGluR1 in the above processes is that rescuing the mGluR1 KO mice with expression of a mGluR1a transgene driven by a PC-specific promoter restores normal cerebellar LTD, interlimb coordination, and associative conditioning motor learning, while not affecting the deficits in non-spatial hippocampus-dependent learning (Ichise et al., 2000; Kishimoto et al., 2002). It has also been demonstrated that mGluR1 is activated in response to CF stimulation and generates the same sEPSC and IP3-mediated Ca2+ increase as that observed at PF-PC synapses (Dzubay and Otis, 2002).  The mGluR1-mediated sEPSC at CF synapses is first detectable during the second week of postnatal development, a period of CF pruning from multiple innervations to singular innervation (reviewed in (Altman and Bayer, 1997)).  Aberrant multiple CF innervation has been observed in adult mice that have had either mGluR1, Gαq, PLCβ4 or PKCγ genetically knocked out, indicating that the mGluR1-mediated signaling pathway is involved in the physiological process of CF pruning (Kano et al., 1997) (reviewed in (Hashimoto and Kano, 2005)).  Abnormalities in CF synapse elimination first appear during the third postnatal week in the mGluR1 KO mice, indicating a role of the mGluR1 pathway in the late phases of CF pruning.  This role was verified in recovery experiments where reintroducing mGluR1a expression specifically within the PCs of mGluR1 KO mice restores proper CF monoinnervation (Ichise et al., 2000).  Besides their putative role in CF pruning, mGluR1 receptors also participate in the newly identified LTD that occurs at CF-PC synapses (Weber et al., 2003).  1.4.4 T-type expression and function Compared to the well studied roles of mGluR1 in PC physiology, relatively little is known concerning the functional contributions of T-type Ca2+ channels.  As discussed in section 1.2.3, the consensus from in situ hybridization and immunohistochemical experiments is that T-type channels are robustly expressed in the soma and dendrites of PCs.  The primary subtype appears to be Cav3.1 with the potential expression of Cav3.3 in a subset of neurons (Craig et al., 1999; Kase et al., 1999; McKay et al., 2006; Talley et al., 1999; Yunker et al., 2003).  The biophysical properties of T-type currents recorded from PCs include low sensitivity to Ni2+, fast inactivation kinetics, and slow deactivation kinetics that are more characteristic of Cav3.1 currents than either Cav3.2 or Cav3.3 currents (Isope and Murphy, 2005; Kaneda et al., 1990). T-type Ca2+ channel activity was first identified in PCs using sharp electrode intracellular recordings in adult rat cerebellar slices.  These recordings on mature PCs revealed a prominent inward- rectifying hyperpolarization-activated inward current (Ih) as well as a putative low threshold Ca2+ conductance that is de-inactivated by hyperpolarization (Crepel and Penit-Soria, 1986).  The presence of T-type currents in mammalian PCs was initially controversial, as one single channel recording study Chapter 1: Introduction  41 failed to identify LVA Ca2+ currents in acute cerebellar slices from adult guinea pigs (Usowicz et al., 1992).  T-type currents were however subsequently identified in PCs of both juvenile and adult rats and mice through recordings on acutely dissociated and primary cultured PCs, PCs from slice cultures and PCs from acute brain slices (Bossu et al., 1989; Gruol et al., 1992; Hirano and Hagiwara, 1989; Isope and Murphy, 2005; Kaneda et al., 1990; Mouginot et al., 1997; Raman and Bean, 1999; Watanabe et al., 1998).  These native PC currents possess all of the hallmark T-type Ca2+ channel biophysical properties, including low threshold activation ranging between -60 mV and -40 mV, small single channel conductance between 7 and 9 pS, relatively hyperpolarizing voltage dependence of inactivation, fast activation and inactivation kinetics, and relatively slow deactivation kinetics (Bossu et al., 1989; Hirano and Hagiwara, 1989; Isope and Murphy, 2005; Kaneda et al., 1990; Mouginot et al., 1997).  Cell- attached recordings on PCs from newborn rat slice cultures demonstrated that T-type currents are distributed more densely on the dendritic membranes compared to the somatic membrane (Mouginot et al., 1997).  This finding is consistent with the observation that T-type currents are only present in PCs that have developed a dendritic structure in a cell culture model of PC development (Gruol et al., 1992). A recent study by Isope and Murphy furthered these investigations by using a combination of two photon Ca2+ imaging and voltage-clamp recordings on PCs from juvenile rat acute brain slices to show that T- type Ca2+ currents are present in both the spines and dendrites of PCs (Isope and Murphy, 2005).  These T-type currents have a large peak amplitude (> - 2 nA) that increases with developmental age, demonstrating that T-type currents are functionally expressed in adult rodent PCs (Isope and Murphy, 2005).  Several studies have now reached the consensus that P-type and T-type currents comprise the vast majority (up to 95%) of Ca2+ channel currents in cerebellar PCs (Isope and Murphy, 2005; Swensen and Bean, 2003; Usowicz et al., 1992; Watanabe et al., 1998). Recent studies have implicated T-type Ca2+ channels as having a significant physiological role in dendritic Ca2+ spikes and burst firing within the soma and proximal dendrites of PCs (reviewed in (Cavelier and Bossu, 2003)).  For over 25 years it has been generally accepted that Na+-driven APs are produced at the PC soma while Ca2+-driven APs are produced in the PC dendrites and that both are generally restricted to their respective compartments (Llinas and Sugimori, 1980a, b).  In accordance with this notion, Na+-driven APs that are initiated in the axosomatic region of PCs (of a rat cerebellar slice culture) propagate very poorly into the dendritic tree, exponentially decreasing in amplitude with distance from the soma (Pouille et al., 2000).  However, Ca2+-driven dendritic low threshold spikes only decrease in a linear manner as they approach the soma and can propagate directly to the soma in ~ 20% of PCs examined (Pouille et al., 2000).  The dendritic Ca2+ spikes originally studied in guinea pig PCs include both Ca2+-dependent plateau potentials as well as Ca2+ spikes (Llinas and Sugimori, 1980a, b), and P-type channels are proposed to generate these Ca2+ spikes (Usowicz et al., 1992; Watanabe et al., 1998).  Examining PCs from rat organotypic cerebellar slice cultures revealed that T-type Ca2+ channels Chapter 1: Introduction  42 underlie dendritic Ca2+ spikes while P-type Ca2+ channels underlie a plateau potential that is unmasked when K+ channels are blocked (Pouille et al., 2000).  In fact, pharmacological blockade of the P-type current promoted propagation of the low threshold Ca2+ spikes to the PC soma (increased from 20% to 80% of PCs) (Cavelier et al., 2002b).  The robust P-type dendritic currents in PCs have been shown to activate Ca2+-dependent BK and SK K+ channels, which induces after-hyperpolarizing potentials and alters the frequency of PC firing (Edgerton and Reinhart, 2003; Womack et al., 2004).  It is proposed that dendrosomatic propagation of T-type-dependent Ca2+ spikes is inhibited by this activation of Ca2+- dependent K+ channels, and that the low threshold spikes may underlie the CF-induced complex spike (Cavelier et al., 2003; Cavelier et al., 2002b).  In separate experiments on acutely dissociated PCs and PCs from acute cerebellar slices, it has been shown that P-type currents are required to sustain the spontaneous firing of PCs, while T-type currents make a substantial contribution to Ca2+ currents generated during interspike intervals of spontaneous bursting (Swensen and Bean, 2003; Womack and Khodakhah, 2004). Although the above studies implicate T-type Ca2+ channels in generating Ca2+-dependent bursting in PC dendrites, with potentially significant physiological implications, several limitations of these studies should be noted.  Firstly, the overall structure and native composition of PCs is not well maintained in most of the in vitro systems currently used.  Acutely dissociated PCs lack the dendritic tree where T-type Ca2+ channels predominate (Swensen and Bean, 2003), while cerebellar slice cultures are taken from newborn rats and subsequent dendritic growth and channel expression is determined by in vitro culture conditions (Cavelier et al., 2002a; Cavelier et al., 2003; Cavelier et al., 2002b; Pouille et al., 2000).  Furthermore, most electrophysiological recordings have been performed in current clamp mode, where a lack of high affinity, specific T-type antagonists limits the conclusions that can be made.  A thorough investigation into the role of T-type Ca2+ channels in generating dendritic burst firing and altering excitability within intact PCs of acute brain slices has been lacking, likely due to the space- clamp complications that arise in adult PCs. To date, no published study has examined the functional roles of T-type Ca2+ channels in distal dendritic PF-PC synapses.  The supralinear Ca2+ increase required for PF-LTD implicates the possible involvement of Ca2+ channels due to their highly nonlinear dependence of activation on membrane potential.  It has been shown that activation of AMPA receptors at PF synapses can cause sufficient depolarization to open voltage-gated Ca2+ channels (Denk et al., 1995; Ito, 2002), and coincident depolarization due to the CF-mediated complex spike would likely increase open probability.  It is also known that mGluR1 can directly bind to P/Q-type Ca2+ channels and TRPC1 channels and is colocalized with both of these in PC dendrites (Kim et al., 2003; Kitano et al., 2003).  However, the majority of research on dendritic signaling and plasticity in PCs has involved synaptic stimulation, current clamp recordings, and Ca2+ imaging and the direct contributions of low voltage-activated Ca2+ channels in these Chapter 1: Introduction  43 processes is poorly understood.  The potential interaction within PC distal dendrites between mGluR1 receptors and nearby T-type Ca2+ channels has significant implications concerning cerebellar integration, excitability, and plasticity.  1.5 Thesis hypotheses and objectives 1.5.1 Hypotheses T-type Ca2+ channel isoforms have very distinct expression patterns in the mammalian CNS at both the cellular and subcellular levels which are likely correlated to their colocalization with different neurotransmitter receptor subtypes and downstream intracellular signaling pathways.  This selective combination of coupling between T-type Ca2+ channel isoforms and signaling pathways is hypothesized to form the basis for T-type Ca2+ channels serving unique physiological functions in the CNS.  In this thesis I specifically hypothesized: 1) that the three major Cav3 T-type isoforms are differentially modulated by the activation of specific GPCRs, 2) that functional T-type currents in cerebellar PCs mainly consist of Cav3.1 channels, and 3) that in PCs the Cav3.1-mediated T-type currents are modulated by mGluR1 and that this pathway serves unique roles in neuronal excitability and synaptic integration.  1.5.2 Objectives In order to address the above hypotheses, the following scientific objectives were generated: 1) To study the functional effects of muscarinic and glutamatergic Gαq/11-protein coupled receptor activation on specific T-type isoforms in a heterologous system in order to identify possible modulatory interactions.  Any observed modulation of recombinant T-type channels will be characterized in terms of its effects on the channels’ biophysical properties, the regions of the T-type channel that are involved in the modulatory interaction, and the intracellular signaling pathway mediating the effect.  2) To perform whole-cell voltage-clamp recordings on PCs from acute cerebellar slices of rats and Ca2+ channel KO mice to determine what specific Cav3 isoform underlies functional T-type currents within PCs.  The biophysical, pharmacological, and modulatory profile of the PC T-type currents will also be analyzed to complement the KO experiments in forming a firm conclusion. 3) To apply the information gained from Objective 1 to a native neuronal system where a specific T-type isoform and GPCR are functionally expressed: cerebellar PCs.  Specific pharmacological antagonists and agonists will be used in the same cerebellar recordings as in Objective 2 to investigate the modulation of T-type currents by mGluR1 and the second messenger pathway that mediates this modulation.  The Chapter 1: Introduction  44 biophysical characteristics of the T-type modulation will be investigated with various voltage-clamp protocols and two-photon Ca2+ imaging will be used to determine the localization of the modulation. Combining current clamp recordings and synaptic PF stimulation with the above techniques will enable the investigation into how the observed T-type modulation affects excitability and local signaling within PCs. Chapter 1: Introduction  45  1.6 References Abe, T., Sugihara, H., Nawa, H., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1992). Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. J Biol Chem 267, 13361-13368. Adams, P.J., and Snutch, T.P. (2007). Calcium channelopathies: voltage-gated calcium channels. In Calcium signalling and disease - molecular pathology of calcium, E. Carafoli, and M. Brini, eds. (New York: Springer), pp. 215-251. Aiba, A., Kano, M., Chen, C., Stanton, M.E., Fox, G.D., Herrup, K., Zwingman, T.A., and Tonegawa, S. (1994). Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79, 377-388. Akaike, N., Kostyuk, P.G., and Osipchuk, Y.V. (1989). Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J Physiol 412, 181-195. Allen, T.G., and Brown, D.A. (1993). M2 muscarinic receptor-mediated inhibition of the Ca2+ current in rat magnocellular cholinergic basal forebrain neurones. J Physiol 466, 173-189. Altier, C., Khosravani, H., Evans, R.M., Hameed, S., Peloquin, J.B., Vartian, B.A., Chen, L., Beedle, A.M., Ferguson, S.S., Mezghrani, A., et al. (2006). ORL1 receptor-mediated internalization of N-type calcium channels. Nat Neurosci 9, 31-40. Altman, J., and Bayer, S.A. (1997). Development of the cerebellar system : in relation to its evolution, structure, and functions (Boca Raton: CRC Press). Anderson, M.P., Mochizuki, T., Xie, J., Fischler, W., Manger, J.P., Talley, E.M., Scammell, T.E., and Tonegawa, S. (2005). Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc Natl Acad Sci U S A 102, 1743-1748. Anwyl, R. (1999). Metabotropic glutamate receptors: electrophysiological properties and role in plasticity. Brain Res Brain Res Rev 29, 83-120. Arias, J.M., Murbartian, J., Vitko, I., Lee, J.H., and Perez-Reyes, E. (2005). Transfer of beta subunit regulation from high to low voltage-gated Ca2+ channels. FEBS Lett 579, 3907-3912. Arias, O., II, Vitko, I., Fortuna, M., Baumgart, J.P., Sokolova, S., Shumilin, I.A., Van Deusen, A., Soriano-Garcia, M., Gomora, J.C., and Perez-Reyes, E. (2008). Characterization of the Gating Brake in the I-II Loop of Cav3.2 T-type Ca2+ Channels. J Biol Chem 283, 8136-8144. Arikkath, J., and Campbell, K.P. (2003). Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr Opin Neurobiol 13, 298-307. Arnoult, C., Lemos, J.R., and Florman, H.M. (1997). Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation. Embo J 16, 1593-1599. Arnoult, C., Villaz, M., and Florman, H.M. (1998). Pharmacological properties of the T-type Ca2+ current of mouse spermatogenic cells. Mol Pharmacol 53, 1104-1111. Bannister, R.A., Melliti, K., and Adams, B.A. (2002). Reconstituted slow muscarinic inhibition of neuronal (Cav1.2c) L-type Ca2+ channels. Biophys J 83, 3256-3267. Chapter 1: Introduction  46 Bannister, R.A., Melliti, K., and Adams, B.A. (2004). Differential modulation of CaV2.3 Ca2+ channels by Galphaq/11-coupled muscarinic receptors. Mol Pharmacol 65, 381-388. Bannister, R.A., Meza, U., and Adams, B.A. (2005). Phosphorylation-dependent regulation of voltage- gated Ca2+ channels. In Voltage-gated calcium channels, G.W. Zamponi, ed. (New York: Landes Bioscience), pp. 168-182. Barlow, J.S. (2002). The cerebellum and adaptive control (Cambridge, U.K. ; New York, NY: Cambridge University Press). Barrett, P.Q., Lu, H.K., Colbran, R., Czernik, A., and Pancrazio, J.J. (2000). Stimulation of unitary T- type Ca(2+) channel currents by calmodulin-dependent protein kinase II. Am J Physiol Cell Physiol 279, C1694-1703. Baude, A., Nusser, Z., Roberts, J.D., Mulvihill, E., McIlhinney, R.A., and Somogyi, P. (1993). The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11, 771-787. Beech, D.J., Bernheim, L., and Hille, B. (1992). Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons. Neuron 8, 97-106. Beech, D.J., Bernheim, L., Mathie, A., and Hille, B. (1991). Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci U S A 88, 652- 656. Bernheim, L., Mathie, A., and Hille, B. (1992). Characterization of muscarinic receptor subtypes inhibiting Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci U S A 89, 9544- 9548. Berrow, N.S., Brice, N.L., Tedder, I., Page, K.M., and Dolphin, A.C. (1997). Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line. Eur J Neurosci 9, 739-748. Bossu, J.L., Fagni, L., and Feltz, A. (1989). Voltage-activated calcium channels in rat Purkinje cells maintained in culture. Pflugers Arch 414, 92-94. Bourinet, E., Alloui, A., Monteil, A., Barrere, C., Couette, B., Poirot, O., Pages, A., McRory, J., Snutch, T.P., Eschalier, A., and Nargeot, J. (2005). Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. Embo J 24, 315-324. Bourinet, E., Soong, T.W., Stea, A., and Snutch, T.P. (1996a). Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc Natl Acad Sci U S A 93, 1486-1491. Bourinet, E., Soong, T.W., Sutton, K., Slaymaker, S., Mathews, E., Monteil, A., Zamponi, G.W., Nargeot, J., and Snutch, T.P. (1999). Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. Nat Neurosci 2, 407-415. Bourinet, E., Zamponi, G.W., Stea, A., Soong, T.W., Lewis, B.A., Jones, L.P., Yue, D.T., and Snutch, T.P. (1996b). The alpha 1E calcium channel exhibits permeation properties similar to low-voltage- activated calcium channels. J Neurosci 16, 4983-4993. Boyden, E.S., Katoh, A., and Raymond, J.L. (2004). Cerebellum-dependent learning: the role of multiple plasticity mechanisms. Annu Rev Neurosci 27, 581-609. Chapter 1: Introduction  47 Brunel, N., Hakim, V., Isope, P., Nadal, J.P., and Barbour, B. (2004). Optimal information storage and the distribution of synaptic weights: perceptron versus Purkinje cell. Neuron 43, 745-757. Canepari, M., Auger, C., and Ogden, D. (2004). Ca2+ ion permeability and single-channel properties of the metabotropic slow EPSC of rat Purkinje neurons. J Neurosci 24, 3563-3573. Canepari, M., and Ogden, D. (2003). Evidence for protein tyrosine phosphatase, tyrosine kinase, and G- protein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons. J Neurosci 23, 4066-4071. Canepari, M., Papageorgiou, G., Corrie, J.E., Watkins, C., and Ogden, D. (2001). The conductance underlying the parallel fibre slow EPSP in rat cerebellar Purkinje neurones studied with photolytic release of L-glutamate. J Physiol 533, 765-772. Carbone, E., and Lux, H.D. (1984). A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones. Nature 310, 501-502. Carter, A.G., and Sabatini, B.L. (2004). State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons. Neuron 44, 483-493. Casabona, G., Knopfel, T., Kuhn, R., Gasparini, F., Baumann, P., Sortino, M.A., Copani, A., and Nicoletti, F. (1997). Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain. Eur J Neurosci 9, 12-17. Casado, M., Isope, P., and Ascher, P. (2002). Involvement of presynaptic N-methyl-D-aspartate receptors in cerebellar long-term depression. Neuron 33, 123-130. Catania, M.V., Landwehrmeyer, G.B., Testa, C.M., Standaert, D.G., Penney, J.B., Jr., and Young, A.B. (1994). Metabotropic glutamate receptors are differentially regulated during development. Neuroscience 61, 481-495. Catterall, W.A., Perez-Reyes, E., Snutch, T.P., and Striessnig, J. (2005). International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev 57, 411-425. Cavelier, P., Beekenkamp, H., Shin, H.S., Jun, K., and Bossu, J.L. (2002a). Cerebellar slice cultures from mice lacking the P/Q calcium channel: electroresponsiveness of Purkinje cells. Neurosci Lett 333, 64-68. Cavelier, P., and Bossu, J.L. (2003). Dendritic low-threshold Ca2+ channels in rat cerebellar Purkinje cells: possible physiological implications. Cerebellum 2, 196-205. Cavelier, P., Desplantez, T., Beekenkamp, H., and Bossu, J.L. (2003). K+ channel activation and low- threshold Ca2+ spike of rat cerebellar Purkinje cells in vitro. Neuroreport 14, 167-171. Cavelier, P., Pouille, F., Desplantez, T., Beekenkamp, H., and Bossu, J.L. (2002b). Control of the propagation of dendritic low-threshold Ca2+ spikes in Purkinje cells from rat cerebellar slice cultures. J Physiol 540, 57-72. Chemin, J., Mezghrani, A., Bidaud, I., Dupasquier, S., Marger, F., Barrere, C., Nargeot, J., and Lory, P. (2007a). Temperature-dependent modulation of CaV3 T-type calcium channels by protein kinases C and A in mammalian cells. J Biol Chem 282, 32710-32718. Chapter 1: Introduction  48 Chemin, J., Monteil, A., Bourinet, E., Nargeot, J., and Lory, P. (2001a). Alternatively spliced alpha(1G) (Ca(V)3.1) intracellular loops promote specific T-type Ca(2+) channel gating properties. Biophys J 80, 1238-1250. Chemin, J., Monteil, A., Perez-Reyes, E., Bourinet, E., Nargeot, J., and Lory, P. (2002). Specific contribution of human T-type calcium channel isotypes (alpha1G, alpha1H and alpha1I) to neuronal excitability. J Physiol 540, 3-14. Chemin, J., Monteil, A., Perez-Reyes, E., Nargeot, J., and Lory, P. (2001b). Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide. Embo J 20, 7033-7040. Chemin, J., Nargeot, J., and Lory, P. (2007b). Chemical determinants involved in anandamide-induced inhibition of T-type calcium channels. J Biol Chem 282, 2314-2323. Chemin, J., Traboulsie, A., and Lory, P. (2006). Molecular pathways underlying the modulation of T- type calcium channels by neurotransmitters and hormones. Cell Calcium 40, 121-134. Chen, C., Xu, R., Clarke, I.J., Ruan, M., Loneragan, K., and Roh, S.G. (2000). Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes. Immunol Cell Biol 78, 356-368. Chen, C.C., Lamping, K.G., Nuno, D.W., Barresi, R., Prouty, S.J., Lavoie, J.L., Cribbs, L.L., England, S.K., Sigmund, C.D., Weiss, R.M., et al. (2003a). Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels. Science 302, 1416-1418. Chen, Y., Lu, J., Pan, H., Zhang, Y., Wu, H., Xu, K., Liu, X., Jiang, Y., Bao, X., Yao, Z., et al. (2003b). Association between genetic variation of CACNA1H and childhood absence epilepsy. Ann Neurol 54, 239-243. Chen, Y., Sharp, A.H., Hata, K., Yunker, A.M., Polo-Parada, L., Landmesser, L.T., and McEnery, M.W. (2007). Site-directed antibodies to low-voltage-activated calcium channel CaV3.3 (alpha1I) subunit also target neural cell adhesion molecule-180. Neuroscience 145, 981-996. Chevalier, M., Lory, P., Mironneau, C., Macrez, N., and Quignard, J.F. (2006). T-type CaV3.3 calcium channels produce spontaneous low-threshold action potentials and intracellular calcium oscillations. Eur J Neurosci 23, 2321-2329. Christie, B.R., Schexnayder, L.K., and Johnston, D. (1997). Contribution of voltage-gated Ca2+ channels to homosynaptic long-term depression in the CA1 region in vitro. J Neurophysiol 77, 1651-1655. Chuang, R.S., Jaffe, H., Cribbs, L., Perez-Reyes, E., and Swartz, K.J. (1998). Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin. Nat Neurosci 1, 668-674. Coesmans, M., Smitt, P.A., Linden, D.J., Shigemoto, R., Hirano, T., Yamakawa, Y., van Alphen, A.M., Luo, C., van der Geest, J.N., Kros, J.M., et al. (2003). Mechanisms underlying cerebellar motor deficits due to mGluR1-autoantibodies. Ann Neurol 53, 325-336. Colwell, C.S., and Levine, M.S. (1999). Metabotropic glutamate receptor modulation of excitotoxicity in the neostriatum: role of calcium channels. Brain Res 833, 234-241. Conn, P.J., and Pin, J.P. (1997). Pharmacology and functions of metabotropic glutamate receptors. Annu Rev Pharmacol Toxicol 37, 205-237. Chapter 1: Introduction  49 Conquet, F., Bashir, Z.I., Davies, C.H., Daniel, H., Ferraguti, F., Bordi, F., Franz-Bacon, K., Reggiani, A., Matarese, V., Conde, F., and et al. (1994). Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1. Nature 372, 237-243. Coulter, D.A., Huguenard, J.R., and Prince, D.A. (1989). Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current. J Physiol 414, 587-604. Coutinho, V., and Knopfel, T. (2002). Metabotropic glutamate receptors: electrical and chemical signaling properties. Neuroscientist 8, 551-561. Craig, P.J., Beattie, R.E., Folly, E.A., Banerjee, M.D., Reeves, M.B., Priestley, J.V., Carney, S.L., Sher, E., Perez-Reyes, E., and Volsen, S.G. (1999). Distribution of the voltage-dependent calcium channel alpha1G subunit mRNA and protein throughout the mature rat brain. Eur J Neurosci 11, 2949-2964. Crepel, F., and Penit-Soria, J. (1986). Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells. An in vitro study. J Physiol 372, 1-23. Cribbs, L.L., Lee, J.H., Yang, J., Satin, J., Zhang, Y., Daud, A., Barclay, J., Williamson, M.P., Fox, M., Rees, M., and Perez-Reyes, E. (1998). Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res 83, 103-109. Danthi, S.J., Enyeart, J.A., and Enyeart, J.J. (2005). Modulation of native T-type calcium channels by omega-3 fatty acids. Biochem Biophys Res Commun 327, 485-493. Darszon, A., Lopez-Martinez, P., Acevedo, J.J., Hernandez-Cruz, A., and Trevino, C.L. (2006). T-type Ca2+ channels in sperm function. Cell Calcium 40, 241-252. Dascal, N. (2001). Ion-channel regulation by G proteins. Trends Endocrinol Metab 12, 391-398. De Zeeuw, C.I., Hansel, C., Bian, F., Koekkoek, S.K., van Alphen, A.M., Linden, D.J., and Oberdick, J. (1998). Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the vestibulo-ocular reflex. Neuron 20, 495-508. Denk, W., Sugimori, M., and Llinas, R. (1995). Two types of calcium response limited to single spines in cerebellar Purkinje cells. Proc Natl Acad Sci U S A 92, 8279-8282. DePuy, S.D., Yao, J., Hu, C., McIntire, W., Bidaud, I., Lory, P., Rastinejad, F., Gonzalez, C., Garrison, J.C., and Barrett, P.Q. (2006). The molecular basis for T-type Ca2+ channel inhibition by G protein beta2gamma2 subunits. Proc Natl Acad Sci U S A 103, 14590-14595. Destexhe, A., Contreras, D., Steriade, M., Sejnowski, T.J., and Huguenard, J.R. (1996). In vivo, in vitro, and computational analysis of dendritic calcium currents in thalamic reticular neurons. J Neurosci 16, 169-185. Destexhe, A., and Sejnowski, T.J. (2003). Interactions between membrane conductances underlying thalamocortical slow-wave oscillations. Physiol Rev 83, 1401-1453. Diana, M.A., Otsu, Y., Maton, G., Collin, T., Chat, M., and Dieudonne, S. (2007). T-type and L-type Ca2+ conductances define and encode the bimodal firing pattern of vestibulocerebellar unipolar brush cells. J Neurosci 27, 3823-3838. Dolphin, A.C. (2003). G protein modulation of voltage-gated calcium channels. Pharmacol Rev 55, 607- 627. Chapter 1: Introduction  50 Dolphin, A.C. (2006). A short history of voltage-gated calcium channels. Br J Pharmacol 147 Suppl 1, S56-62. Drolet, P., Bilodeau, L., Chorvatova, A., Laflamme, L., Gallo-Payet, N., and Payet, M.D. (1997). Inhibition of the T-type Ca2+ current by the dopamine D1 receptor in rat adrenal glomerulosa cells: requirement of the combined action of the G betagamma protein subunit and cyclic adenosine 3',5'- monophosphate. Mol Endocrinol 11, 503-514. Dubel, S.J., Starr, T.V., Hell, J., Ahlijanian, M.K., Enyeart, J.J., Catterall, W.A., and Snutch, T.P. (1992). Molecular cloning of the alpha1 subunit of an omega-conotoxin-sensitive calcium channel. Proc Natl Acad Sci U S A 89, 5058-5062. Dunlap, K., and Fischbach, G.D. (1978). Neurotransmitters decrease the calcium component of sensory neurone action potentials. Nature 276, 837-839. Dunlap, K., and Fischbach, G.D. (1981). Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones. J Physiol 317, 519-535. Dzubay, J.A., and Otis, T.S. (2002). Climbing fiber activation of metabotropic glutamate receptors on cerebellar Purkinje neurons. Neuron 36, 1159-1167. Edgerton, J.R., and Reinhart, P.H. (2003). Distinct contributions of small and large conductance Ca2+- activated K+ channels to rat Purkinje neuron function. J Physiol 548, 53-69. Egger, V., Svoboda, K., and Mainen, Z.F. (2005). Dendrodendritic synaptic signals in olfactory bulb granule cells: local spine boost and global low-threshold spike. J Neurosci 25, 3521-3530. Eilers, J., Augustine, G.J., and Konnerth, A. (1995). Subthreshold synaptic Ca2+ signalling in fine dendrites and spines of cerebellar Purkinje neurons. Nature 373, 155-158. Eilers, J., Plant, T., and Konnerth, A. (1996). Localized calcium signalling and neuronal integration in cerebellar Purkinje neurones. Cell Calcium 20, 215-226. Ekerot, C.F., and Jorntell, H. (2001). Parallel fibre receptive fields of Purkinje cells and interneurons are climbing fibre-specific. Eur J Neurosci 13, 1303-1310. Ekerot, C.F., and Kano, M. (1985). Long-term depression of parallel fibre synapses following stimulation of climbing fibres. Brain Res 342, 357-360. Elmslie, K.S. (2003). Neurotransmitter modulation of neuronal calcium channels. J Bioenerg Biomembr 35, 477-489. Emerick, M.C., Stein, R., Kunze, R., McNulty, M.M., Regan, M.R., Hanck, D.A., and Agnew, W.S. (2006). Profiling the array of Cav3.1 variants from the human T-type calcium channel gene CACNA1G: alternative structures, developmental expression, and biophysical variations. Proteins 64, 320-342. Evans, G.J. (2007). Synaptic signalling in cerebellar plasticity. Biol Cell 99, 363-378. Fatt, P., and Ginsborg, B.L. (1958). The ionic requirements for the production of action potentials in crustacean muscle fibres. J Physiol 142, 516-543. Fatt, P., and Katz, B. (1953). The electrical properties of crustacean muscle fibres. J Physiol 120, 171- 204. Chapter 1: Introduction  51 Finch, E.A., and Augustine, G.J. (1998). Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites. Nature 396, 753-756. Fisher, R., and Johnston, D. (1990). Differential modulation of single voltage-gated calcium channels by cholinergic and adrenergic agonists in adult hippocampal neurons. J Neurophysiol 64, 1291-1302. Fleckenstein, A. (1983). History of calcium antagonists. Circ Res 52, I3-16. Flucher, B.E., and Franzini-Armstrong, C. (1996). Formation of junctions involved in excitation- contraction coupling in skeletal and cardiac muscle. Proc Natl Acad Sci U S A 93, 8101-8106. Formenti, A., and Sansone, V. (1991). Inhibitory action of acetylcholine, baclofen and GTP-gamma-S on calcium channels in adult rat sensory neurons. Neurosci Lett 131, 267-272. Fox, A.P., Nowycky, M.C., and Tsien, R.W. (1987a). Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones. J Physiol 394, 149-172. Fox, A.P., Nowycky, M.C., and Tsien, R.W. (1987b). Single-channel recordings of three types of calcium channels in chick sensory neurones. J Physiol 394, 173-200. Fraser, D.D., and MacVicar, B.A. (1991). Low-threshold transient calcium current in rat hippocampal lacunosum-moleculare interneurons: kinetics and modulation by neurotransmitters. J Neurosci 11, 2812- 2820. Freeze, B.S., McNulty, M.M., and Hanck, D.A. (2006). State-dependent verapamil block of the cloned human Ca(v)3.1 T-type Ca(2+) channel. Mol Pharmacol 70, 718-726. Gamper, N., Reznikov, V., Yamada, Y., Yang, J., and Shapiro, M.S. (2004). Phosphatidylinositol-4,5- bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels. J Neurosci 24, 10980-10992. Gamper, N., and Shapiro, M.S. (2007). Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci 8, 921-934. Gao, W., Dunbar, R.L., Chen, G., Reinert, K.C., Oberdick, J., and Ebner, T.J. (2003). Optical imaging of long-term depression in the mouse cerebellar cortex in vivo. J Neurosci 23, 1859-1866. Ghez, C., and Thach, W.T. (2000). The cerebellum. In Principles of Neural Science, E.R. Kandel, J.H. Schwartz, and T.M. Jessell, eds. (New York, NY: McGraw-Hill Companies), pp. 832-852. Golding, N.L., Staff, N.P., and Spruston, N. (2002). Dendritic spikes as a mechanism for cooperative long-term potentiation. Nature 418, 326-331. Gomora, J.C., Daud, A.N., Weiergraber, M., and Perez-Reyes, E. (2001). Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol Pharmacol 60, 1121-1132. Gomora, J.C., Murbartian, J., Arias, J.M., Lee, J.H., and Perez-Reyes, E. (2002). Cloning and expression of the human T-type channel Cav3.3: insights into prepulse facilitation. Biophys J 83, 229-241. Gruol, D.L., Deal, C.R., and Yool, A.J. (1992). Developmental changes in calcium conductances contribute to the physiological maturation of cerebellar Purkinje neurons in culture. J Neurosci 12, 2838- 2848. Chapter 1: Introduction  52 Hagiwara, S., Ozawa, S., and Sand, O. (1975). Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. J Gen Physiol 65, 617-644. Hamid, J., Nelson, D., Spaetgens, R., Dubel, S.J., Snutch, T.P., and Zamponi, G.W. (1999). Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels. J Biol Chem 274, 6195-6202. Hamid, J., Peloquin, J.B., Monteil, A., and Zamponi, G.W. (2006). Determinants of the differential gating properties of Cav3.1 and Cav3.3 T-type channels: a role of domain IV? Neuroscience 143, 717- 728. Harvey, R.J., and Napper, R.M. (1991). Quantitative studies on the mammalian cerebellum. Prog Neurobiol 36, 437-463. Hashimoto, K., and Kano, M. (2005). Postnatal development and synapse elimination of climbing fiber to Purkinje cell projection in the cerebellum. Neurosci Res 53, 221-228. Hayashi, K., Wakino, S., Sugano, N., Ozawa, Y., Homma, K., and Saruta, T. (2007). Ca2+ channel subtypes and pharmacology in the kidney. Circ Res 100, 342-353. Hayashi, Y., Tanabe, Y., Aramori, I., Masu, M., Shimamoto, K., Ohfune, Y., and Nakanishi, S. (1992). Agonist analysis of 2-(carboxycyclopropyl)glycine isomers for cloned metabotropic glutamate receptor subtypes expressed in Chinese hamster ovary cells. Br J Pharmacol 107, 539-543. Hell, J.W., Westenbroek, R.E., Warner, C., Ahlijanian, M.K., Prystay, W., Gilbert, M.M., Snutch, T.P., and Catterall, W.A. (1993). Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol 123, 949-962. Herlitze, S., Garcia, D.E., Mackie, K., Hille, B., Scheuer, T., and Catterall, W.A. (1996). Modulation of Ca2+ channels by G-protein beta gamma subunits. Nature 380, 258-262. Heron, S.E., Khosravani, H., Varela, D., Bladen, C., Williams, T.C., Newman, M.R., Scheffer, I.E., Berkovic, S.F., Mulley, J.C., and Zamponi, G.W. (2007). Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants. Ann Neurol 62, 560-568. Hildebrand, M.E., and Snutch, T.P. (2006). Contributions of T-type calcium channels to the pathophysiology of pain signaling. Drug Discovery Today: Disease Mechanisms 3, 335-341. Hirano, T., and Hagiwara, S. (1989). Kinetics and distribution of voltage-gated Ca, Na and K channels on the somata of rat cerebellar Purkinje cells. Pflugers Arch 413, 463-469. Hirono, M., Konishi, S., and Yoshioka, T. (1998). Phospholipase C-independent group I metabotropic glutamate receptor-mediated inward current in mouse purkinje cells. Biochem Biophys Res Commun 251, 753-758. Houamed, K.M., Kuijper, J.L., Gilbert, T.L., Haldeman, B.A., O'Hara, P.J., Mulvihill, E.R., Almers, W., and Hagen, F.S. (1991). Cloning, expression, and gene structure of a G protein-coupled glutamate receptor from rat brain. Science 252, 1318-1321. Howe, A.R., and Surmeier, D.J. (1995). Muscarinic receptors modulate N-, P-, and L-type Ca2+ currents in rat striatal neurons through parallel pathways. J Neurosci 15, 458-469. Huguenard, J.R. (1996). Low-threshold calcium currents in central nervous system neurons. Annu Rev Physiol 58, 329-348. Chapter 1: Introduction  53 Huguenard, J.R., and Prince, D.A. (1992). A novel T-type current underlies prolonged Ca(2+)-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12, 3804-3817. Huguenard, J.R., and Prince, D.A. (1994). Intrathalamic rhythmicity studied in vitro: nominal T-current modulation causes robust antioscillatory effects. J Neurosci 14, 5485-5502. Ichise, T., Kano, M., Hashimoto, K., Yanagihara, D., Nakao, K., Shigemoto, R., Katsuki, M., and Aiba, A. (2000). mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination. Science 288, 1832-1835. Iftinca, M., McKay, B.E., Snutch, T.P., McRory, J.E., Turner, R.W., and Zamponi, G.W. (2006). Temperature dependence of T-type calcium channel gating. Neuroscience 142, 1031-1042. Ikeda, H., Heinke, B., Ruscheweyh, R., and Sandkuhler, J. (2003). Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 299, 1237-1240. Ikeda, S.R. (1996). Voltage-dependent modulation of N-type calcium channels by G-protein beta gamma subunits. Nature 380, 255-258. Inoue, T. (2003). Dynamics of calcium and its roles in the dendrite of the cerebellar Purkinje cell. Keio J Med 52, 244-249. Ishii, M., and Kurachi, Y. (2006). Muscarinic acetylcholine receptors. Curr Pharm Des 12, 3573-3581. Isope, P., and Barbour, B. (2002). Properties of unitary granule cell-->Purkinje cell synapses in adult rat cerebellar slices. J Neurosci 22, 9668-9678. Isope, P., and Murphy, T.H. (2005). Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T-type calcium channels. J Physiol 562, 257-269. Ito, M. (2001). Cerebellar long-term depression: characterization, signal transduction, and functional roles. Physiol Rev 81, 1143-1195. Ito, M. (2002). The molecular organization of cerebellar long-term depression. Nat Rev Neurosci 3, 896- 902. Ito, M., Sakurai, M., and Tongroach, P. (1982). Climbing fibre induced depression of both mossy fibre responsiveness and glutamate sensitivity of cerebellar Purkinje cells. J Physiol 324, 113-134. Joksovic, P.M., Bayliss, D.A., and Todorovic, S.M. (2005). Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane. J Physiol 566, 125- 142. Joksovic, P.M., Nelson, M.T., Jevtovic-Todorovic, V., Patel, M.K., Perez-Reyes, E., Campbell, K.P., Chen, C.C., and Todorovic, S.M. (2006). CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus. J Physiol 574, 415-430. Kammermeier, P.J., and Ikeda, S.R. (1999). Expression of RGS2 alters the coupling of metabotropic glutamate receptor 1a to M-type K+ and N-type Ca2+ channels. Neuron 22, 819-829. Kammermeier, P.J., Ruiz-Velasco, V., and Ikeda, S.R. (2000). A voltage-independent calcium current inhibitory pathway activated by muscarinic agonists in rat sympathetic neurons requires both Galpha q/11 and Gbeta gamma. J Neurosci 20, 5623-5629. Chapter 1: Introduction  54 Kaneda, M., Wakamori, M., Ito, C., and Akaike, N. (1990). Low-threshold calcium current in isolated Purkinje cell bodies of rat cerebellum. J Neurophysiol 63, 1046-1051. Kang, H.W., Park, J.Y., Jeong, S.W., Kim, J.A., Moon, H.J., Perez-Reyes, E., and Lee, J.H. (2006). A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels. J Biol Chem 281, 4823- 4830. Kano, M., Hashimoto, K., Kurihara, H., Watanabe, M., Inoue, Y., Aiba, A., and Tonegawa, S. (1997). Persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking mGluR1. Neuron 18, 71-79. Kase, M., Kakimoto, S., Sakuma, S., Houtani, T., Ohishi, H., Ueyama, T., and Sugimoto, T. (1999). Distribution of neurons expressing alpha 1G subunit mRNA of T-type voltage-dependent calcium channel in adult rat central nervous system. Neurosci Lett 268, 77-80. Katz, B., and Miledi, R. (1970). Further study of the role of calcium in synaptic transmission. J Physiol 207, 789-801. Kavalali, E.T., Zhuo, M., Bito, H., and Tsien, R.W. (1997). Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments. Neuron 18, 651-663. Khosravani, H., Altier, C., Simms, B., Hamming, K.S., Snutch, T.P., Mezeyova, J., McRory, J.E., and Zamponi, G.W. (2004). Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J Biol Chem 279, 9681-9684. Khosravani, H., Bladen, C., Parker, D.B., Snutch, T.P., McRory, J.E., and Zamponi, G.W. (2005). Effects of Ca(v)3.2 channel mutations linked to idiopathic generalized epilepsy. Ann Neurol 57, 745- 749. Khosravani, H., and Zamponi, G.W. (2006). Voltage-gated calcium channels and idiopathic generalized epilepsies. Physiol Rev 86, 941-966. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W., and Shin, H.S. (2001). Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31, 35-45. Kim, H.S., Kim, Y., Doddareddy, M.R., Seo, S.H., Rhim, H., Tae, J., Pae, A.N., Choo, H., and Cho, Y.S. (2007). Design, synthesis, and biological evaluation of 1,3-dioxoisoindoline-5-carboxamide derivatives as T-type calcium channel blockers. Bioorg Med Chem Lett 17, 476-481. Kim, J.A., Park, J.Y., Kang, H.W., Huh, S.U., Jeong, S.W., and Lee, J.H. (2006). Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A. J Pharmacol Exp Ther 318, 230-237. Kim, S.J., Kim, Y.S., Yuan, J.P., Petralia, R.S., Worley, P.F., and Linden, D.J. (2003). Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426, 285-291. Kishimoto, Y., Fujimichi, R., Araishi, K., Kawahara, S., Kano, M., Aiba, A., and Kirino, Y. (2002). mGluR1 in cerebellar Purkinje cells is required for normal association of temporally contiguous stimuli in classical conditioning. Eur J Neurosci 16, 2416-2424. Kitano, J., Nishida, M., Itsukaichi, Y., Minami, I., Ogawa, M., Hirano, T., Mori, Y., and Nakanishi, S. (2003). Direct interaction and functional coupling between metabotropic glutamate receptor subtype 1 and voltage-sensitive Cav2.1 Ca2+ channel. J Biol Chem 278, 25101-25108. Chapter 1: Introduction  55 Klockner, U., Lee, J.H., Cribbs, L.L., Daud, A., Hescheler, J., Pereverzev, A., Perez-Reyes, E., and Schneider, T. (1999). Comparison of the Ca2 + currents induced by expression of three cloned alpha1 subunits, alpha1G, alpha1H and alpha1I, of low-voltage-activated T-type Ca2 + channels. Eur J Neurosci 11, 4171-4178. Knopfel, T., and Grandes, P. (2002). Metabotropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells. Cerebellum 1, 19-26. Knopfel, T., Vranesic, I., Staub, C., and Gahwiler, B.H. (1991). Climbing Fibre Responses in Olivo- cerebellar Slice Cultures. II. Dynamics of Cytosolic Calcium in Purkinje Cells. Eur J Neurosci 3, 343- 348. Kraus, R.L., Li, Y., Jovanovska, A., and Renger, J.J. (2007). Trazodone inhibits T-type calcium channels. Neuropharmacology 53, 308-317. Kumar, P.P., Stotz, S.C., Paramashivappa, R., Beedle, A.M., Zamponi, G.W., and Rao, A.S. (2002). Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity. Mol Pharmacol 61, 649-658. Kurejova, M., and Lacinova, L. (2006). Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel. Arch Biochem Biophys 446, 20-27. Kurejova, M., Lacinova, L., Pavlovicova, M., Eschbach, M., and Klugbauer, N. (2007). The effect of the outermost basic residues in the S4 segments of the Ca(V)3.1 T-type calcium channel on channel gating. Pflugers Arch 455, 527-539. Lam, A.D., Chikina, M.D., McNulty, M.M., Glaaser, I.W., and Hanck, D.A. (2005). Role of Domain IV/S4 outermost arginines in gating of T-type calcium channels. Pflugers Arch 451, 349-361. Lanzafame, A.A., Christopoulos, A., and Mitchelson, F. (2003). Cellular signaling mechanisms for muscarinic acetylcholine receptors. Receptors Channels 9, 241-260. Lee, A., and Catterall, W.A. (2005). Ca2+-dependent modulation of voltage-gated Ca2+ channels. In Voltage-gated calcium channels, G.W. Zamponi, ed. (New York: Landes Bioscience), pp. 183-193. Lee, A., Wong, S.T., Gallagher, D., Li, B., Storm, D.R., Scheuer, T., and Catterall, W.A. (1999a). Ca2+/calmodulin binds to and modulates P/Q-type calcium channels. Nature 399, 155-159. Lee, J.H., Daud, A.N., Cribbs, L.L., Lacerda, A.E., Pereverzev, A., Klockner, U., Schneider, T., and Perez-Reyes, E. (1999b). Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19, 1912-1921. Lee, J.H., Gomora, J.C., Cribbs, L.L., and Perez-Reyes, E. (1999c). Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J 77, 3034-3042. Li, J., Stevens, L., Klugbauer, N., and Wray, D. (2004). Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels. J Biol Chem 279, 26858-26867. Li, L., Bischofberger, J., and Jonas, P. (2007). Differential gating and recruitment of P/Q-, N-, and R- type Ca2+ channels in hippocampal mossy fiber boutons. J Neurosci 27, 13420-13429. Chapter 1: Introduction  56 Liang, J., Zhang, Y., Chen, Y., Wang, J., Pan, H., Wu, H., Xu, K., Liu, X., Jiang, Y., Shen, Y., and Wu, X. (2007). Common polymorphisms in the CACNA1H gene associated with childhood absence epilepsy in Chinese Han population. Ann Hum Genet 71, 325-335. Lin, F.F., Varney, M., Sacaan, A.I., Jachec, C., Daggett, L.P., Rao, S., Flor, P., Kuhn, R., Kerner, J.A., Standaert, D., et al. (1997). Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype. Neuropharmacology 36, 917-931. Linden, D.J. (2001). The expression of cerebellar LTD in culture is not associated with changes in AMPA-receptor kinetics, agonist affinity, or unitary conductance. Proc Natl Acad Sci U S A 98, 14066- 14071. Linden, D.J., and Connor, J.A. (1991). Participation of postsynaptic PKC in cerebellar long-term depression in culture. Science 254, 1656-1659. Lipscombe, D., Helton, T.D., and Xu, W. (2004). L-type calcium channels: the low down. J Neurophysiol 92, 2633-2641. Lipscombe, D., Kongsamut, S., and Tsien, R.W. (1989). Alpha-adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calcium-channel gating. Nature 340, 639- 642. Liu, L., and Rittenhouse, A.R. (2003). Arachidonic acid mediates muscarinic inhibition and enhancement of N-type Ca2+ current in sympathetic neurons. Proc Natl Acad Sci U S A 100, 295-300. Liu, L., Zhao, R., Bai, Y., Stanish, L.F., Evans, J.E., Sanderson, M.J., Bonventre, J.V., and Rittenhouse, A.R. (2006). M1 muscarinic receptors inhibit L-type Ca2+ current and M-current by divergent signal transduction cascades. J Neurosci 26, 11588-11598. Llinas, R., Steinberg, I.Z., and Walton, K. (1976). Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate. Proc Natl Acad Sci U S A 73, 2918-2922. Llinas, R., and Sugimori, M. (1980a). Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. J Physiol 305, 197-213. Llinas, R., and Sugimori, M. (1980b). Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. J Physiol 305, 171-195. Llinas, R., and Yarom, Y. (1981). Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances. J Physiol 315, 549-567. Loewenstein, Y., Mahon, S., Chadderton, P., Kitamura, K., Sompolinsky, H., Yarom, Y., and Hausser, M. (2005). Bistability of cerebellar Purkinje cells modulated by sensory stimulation. Nat Neurosci 8, 202-211. Lopez-Bendito, G., Shigemoto, R., Lujan, R., and Juiz, J.M. (2001). Developmental changes in the localisation of the mGluR1alpha subtype of metabotropic glutamate receptors in Purkinje cells. Neuroscience 105, 413-429. Lu, H.K., Fern, R.J., Nee, J.J., and Barrett, P.Q. (1994). Ca(2+)-dependent activation of T-type Ca2+ channels by calmodulin-dependent protein kinase II. Am J Physiol 267, F183-189. Chapter 1: Introduction  57 Maeda, H., Ellis-Davies, G.C., Ito, K., Miyashita, Y., and Kasai, H. (1999). Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons. Neuron 24, 989-1002. Magee, J.C., Avery, R.B., Christie, B.R., and Johnston, D. (1996). Dihydropyridine-sensitive, voltage- gated Ca2+ channels contribute to the resting intracellular Ca2+ concentration of hippocampal CA1 pyramidal neurons. J Neurophysiol 76, 3460-3470. Magee, J.C., Christofi, G., Miyakawa, H., Christie, B., Lasser-Ross, N., and Johnston, D. (1995). Subthreshold synaptic activation of voltage-gated Ca2+ channels mediates a localized Ca2+ influx into the dendrites of hippocampal pyramidal neurons. J Neurophysiol 74, 1335-1342. Markram, H., and Sakmann, B. (1994). Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels. Proc Natl Acad Sci U S A 91, 5207-5211. Marksteiner, R., Schurr, P., Berjukow, S., Margreiter, E., Perez-Reyes, E., and Hering, S. (2001). Inactivation determinants in segment IIIS6 of Ca(v)3.1. J Physiol 537, 27-34. Massie, B.M. (1997). Mibefradil: a selective T-type calcium antagonist. Am J Cardiol 80, 23I-32I. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., and Nakanishi, S. (1991). Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. Mateos, J.M., Benitez, R., Elezgarai, I., Azkue, J.J., Lazaro, E., Osorio, A., Bilbao, A., Donate, F., Sarria, R., Conquet, F., et al. (2000). Immunolocalization of the mGluR1b splice variant of the metabotropic glutamate receptor 1 at parallel fiber-Purkinje cell synapses in the rat cerebellar cortex. J Neurochem 74, 1301-1309. McCool, B.A., Pin, J.P., Brust, P.F., Harpold, M.M., and Lovinger, D.M. (1996). Functional coupling of rat group II metabotropic glutamate receptors to an omega-conotoxin GVIA-sensitive calcium channel in human embryonic kidney 293 cells. Mol Pharmacol 50, 912-922. McCool, B.A., Pin, J.P., Harpold, M.M., Brust, P.F., Stauderman, K.A., and Lovinger, D.M. (1998). Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells. J Neurophysiol 79, 379-391. McCormick, D.A., and Contreras, D. (2001). On the cellular and network bases of epileptic seizures. Annu Rev Physiol 63, 815-846. McDonough, S.I., and Bean, B.P. (1998). Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons. Mol Pharmacol 54, 1080-1087. McKay, B.E., Engbers, J.D., Mehaffey, W.H., Gordon, G.R., Molineux, M.L., Bains, J.S., and Turner, R.W. (2007). Climbing fiber discharge regulates cerebellar functions by controlling the intrinsic characteristics of purkinje cell output. J Neurophysiol 97, 2590-2604. McKay, B.E., McRory, J.E., Molineux, M.L., Hamid, J., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons. Eur J Neurosci 24, 2581-2594. McRory, J.E., Hamid, J., Doering, C.J., Garcia, E., Parker, R., Hamming, K., Chen, L., Hildebrand, M., Beedle, A.M., Feldcamp, L., et al. (2004). The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution. J Neurosci 24, 1707-1718. Chapter 1: Introduction  58 McRory, J.E., Santi, C.M., Hamming, K.S., Mezeyova, J., Sutton, K.G., Baillie, D.L., Stea, A., and Snutch, T.P. (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem 276, 3999-4011. Melliti, K., Meza, U., and Adams, B. (2000). Muscarinic stimulation of alpha1E Ca channels is selectively blocked by the effector antagonist function of RGS2 and phospholipase C-beta1. J Neurosci 20, 7167-7173. Melliti, K., Meza, U., and Adams, B.A. (2001). RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line. J Physiol 532, 337-347. Mintz, I.M., Adams, M.E., and Bean, B.P. (1992). P-type calcium channels in rat central and peripheral neurons. Neuron 9, 85-95. Mittman, S., Guo, J., Emerick, M.C., and Agnew, W.S. (1999). Structure and alternative splicing of the gene encoding alpha1I, a human brain T calcium channel alpha1 subunit. Neurosci Lett 269, 121-124. Miyakawa, H., Lev-Ram, V., Lasser-Ross, N., and Ross, W.N. (1992). Calcium transients evoked by climbing fiber and parallel fiber synaptic inputs in guinea pig cerebellar Purkinje neurons. J Neurophysiol 68, 1178-1189. Miyata, M., Finch, E.A., Khiroug, L., Hashimoto, K., Hayasaka, S., Oda, S.I., Inouye, M., Takagishi, Y., Augustine, G.J., and Kano, M. (2000). Local calcium release in dendritic spines required for long-term synaptic depression. Neuron 28, 233-244. Molineux, M.L., McRory, J.E., McKay, B.E., Hamid, J., Mehaffey, W.H., Rehak, R., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons. Proc Natl Acad Sci U S A 103, 5555-5560. Monteil, A., Chemin, J., Bourinet, E., Mennessier, G., Lory, P., and Nargeot, J. (2000). Molecular and functional properties of the human alpha(1G) subunit that forms T-type calcium channels. J Biol Chem 275, 6090-6100. Morikawa, H., Fukuda, K., Mima, H., Shoda, T., Kato, S., and Mori, K. (1998). Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108-15 cells. Pflugers Arch 436, 127-132. Mouginot, D., Bossu, J.L., and Gahwiler, B.H. (1997). Low-threshold Ca2+ currents in dendritic recordings from Purkinje cells in rat cerebellar slice cultures. J Neurosci 17, 160-170. Murbartian, J., Arias, J.M., and Perez-Reyes, E. (2004). Functional impact of alternative splicing of human T-type Cav3.3 calcium channels. J Neurophysiol 92, 3399-3407. Nakanishi, S. (1992). Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597-603. Narasimhan, K., Pessah, I.N., and Linden, D.J. (1998). Inositol-1,4,5-trisphosphate receptor-mediated Ca mobilization is not required for cerebellar long-term depression in reduced preparations. J Neurophysiol 80, 2963-2974. Nelson, M.T., Joksovic, P.M., Perez-Reyes, E., and Todorovic, S.M. (2005). The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors. J Neurosci 25, 8766-8775. Chapter 1: Introduction  59 Nelson, M.T., Joksovic, P.M., Su, P., Kang, H.W., Van Deusen, A., Baumgart, J.P., David, L.S., Snutch, T.P., Barrett, P.Q., Lee, J.H., et al. (2007a). Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate. J Neurosci 27, 12577-12583. Nelson, M.T., Woo, J., Kang, H.W., Vitko, I., Barrett, P.Q., Perez-Reyes, E., Lee, J.H., Shin, H.S., and Todorovic, S.M. (2007b). Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels. J Neurosci 27, 8250-8260. Novara, M., Baldelli, P., Cavallari, D., Carabelli, V., Giancippoli, A., and Carbone, E. (2004). Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins. J Physiol 558, 433-449. Park, J.Y., Kang, H.W., Jeong, S.W., and Lee, J.H. (2004). Multiple structural elements contribute to the slow kinetics of the Cav3.3 T-type channel. J Biol Chem 279, 21707-21713. Park, J.Y., Kang, H.W., Moon, H.J., Huh, S.U., Jeong, S.W., Soldatov, N.M., and Lee, J.H. (2006). Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density. J Physiol 577, 513-523. Peloquin, J.B., Khosravani, H., Barr, W., Bladen, C., Evans, R., Mezeyova, J., Parker, D., Snutch, T.P., McRory, J.E., and Zamponi, G.W. (2006). Functional analysis of Ca3.2 T-type calcium channel mutations linked to childhood absence epilepsy. Epilepsia 47, 655-658. Pemberton, K.E., Hill-Eubanks, L.J., and Jones, S.V. (2000). Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells. Pflugers Arch 440, 452- 461. Pemberton, K.E., and Jones, S.V. (1997). Inhibition of the L-type calcium channel by the five muscarinic receptors (m1-m5) expressed in NIH 3T3 cells. Pflugers Arch 433, 505-514. Perez-Reyes, E. (2003). Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83, 117-161. Perez-Reyes, E., Cribbs, L.L., Daud, A., Lacerda, A.E., Barclay, J., Williamson, M.P., Fox, M., Rees, M., and Lee, J.H. (1998). Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391, 896-900. Pouille, F., Cavelier, P., Desplantez, T., Beekenkamp, H., Craig, P.J., Beattie, R.E., Volsen, S.G., and Bossu, J.L. (2000). Dendro-somatic distribution of calcium-mediated electrogenesis in purkinje cells from rat cerebellar slice cultures. J Physiol 527 Pt 2, 265-282. Raingo, J., Castiglioni, A.J., and Lipscombe, D. (2007). Alternative splicing controls G protein- dependent inhibition of N-type calcium channels in nociceptors. Nat Neurosci 10, 285-292. Raman, I.M., and Bean, B.P. (1999). Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons. J Neurosci 19, 1663-1674. Randall, A.D., and Tsien, R.W. (1997). Contrasting biophysical and pharmacological properties of T- type and R-type calcium channels. Neuropharmacology 36, 879-893. Reuter, H. (1967). The dependence of slow inward current in Purkinje fibres on the extracellular calcium-concentration. J Physiol 192, 479-492. Chapter 1: Introduction  60 Rhim, H., Lee, Y.S., Park, S.J., Chung, B.Y., and Lee, J.Y. (2005). Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers. Bioorg Med Chem Lett 15, 283- 286. Robbins, J., Reynolds, A.M., Treseder, S., and Davies, R. (2003). Enhancement of low-voltage-activated calcium currents by group II metabotropic glutamate receptors in rat retinal ganglion cells. Mol Cell Neurosci 23, 341-350. Rosati, B., Dun, W., Hirose, M., Boyden, P.A., and McKinnon, D. (2007). Molecular basis of the T- and L-type Ca2+ currents in canine Purkinje fibres. J Physiol 579, 465-471. Rossier, M.F., Aptel, H.B., Python, C.P., Burnay, M.M., Vallotton, M.B., and Capponi, A.M. (1995). Inhibition of low threshold calcium channels by angiotensin II in adrenal glomerulosa cells through activation of protein kinase C. J Biol Chem 270, 15137-15142. Roth, A., and Hausser, M. (2001). Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings. J Physiol 535, 445-472. Santi, C.M., Cayabyab, F.S., Sutton, K.G., McRory, J.E., Mezeyova, J., Hamming, K.S., Parker, D., Stea, A., and Snutch, T.P. (2002). Differential inhibition of T-type calcium channels by neuroleptics. J Neurosci 22, 396-403. Satoh, R., Nakabayashi, Y., and Kano, M. (1991). Pharmacological properties of two types of calcium channel in embryonic chick skeletal muscle cells in culture. Neurosci Lett 122, 233-236. Schmidt-Hieber, C., Jonas, P., and Bischofberger, J. (2004). Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus. Nature 429, 184-187. Schmidt, H., Brown, E.B., Schwaller, B., and Eilers, J. (2003). Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching. Biophys J 84, 2599-2608. Schmolesky, M.T., Weber, J.T., De Zeeuw, C.I., and Hansel, C. (2002). The making of a complex spike: ionic composition and plasticity. Ann N Y Acad Sci 978, 359-390. Schoepp, D.D., and Conn, P.J. (1993). Metabotropic glutamate receptors in brain function and pathology. Trends Pharmacol Sci 14, 13-20. Schroeder, J.E., Fischbach, P.S., and McCleskey, E.W. (1990). T-type calcium channels: heterogeneous expression in rat sensory neurons and selective modulation by phorbol esters. J Neurosci 10, 947-951. Schumacher, T.B., Beck, H., Steffens, R., Blumcke, I., Schramm, J., Elger, C.E., and Steinhauser, C. (2000). Modulation of calcium channels by group I and group II metabotropic glutamate receptors in dentate gyrus neurons from patients with temporal lobe epilepsy. Epilepsia 41, 1249-1258. Shigemoto, R., Abe, T., Nomura, S., Nakanishi, S., and Hirano, T. (1994). Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells. Neuron 12, 1245-1255. Shigemoto, R., Nakanishi, S., and Mizuno, N. (1992). Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J Comp Neurol 322, 121-135. Chapter 1: Introduction  61 Shin, S.L., Hoebeek, F.E., Schonewille, M., De Zeeuw, C.I., Aertsen, A., and De Schutter, E. (2007). Regular patterns in cerebellar Purkinje cell simple spike trains. PLoS ONE 2, e485. Sidach, S.S., and Mintz, I.M. (2002). Kurtoxin, a gating modifier of neuronal high- and low-threshold ca channels. J Neurosci 22, 2023-2034. Sillevis Smitt, P., Kinoshita, A., De Leeuw, B., Moll, W., Coesmans, M., Jaarsma, D., Henzen-Logmans, S., Vecht, C., De Zeeuw, C., Sekiyama, N., et al. (2000). Paraneoplastic cerebellar ataxia due to autoantibodies against a glutamate receptor. N Engl J Med 342, 21-27. Snutch, T.P. (2005). Targeting chronic and neuropathic pain: the N-type calcium channel comes of age. NeuroRx 2, 662-670. Snutch, T.P., and David, L.S. (2006). T-type calcium channels: an emerging therapeutic target for the treatment of pain. Drug Development Research 67, 404-415. Snutch, T.P., Leonard, J.P., Gilbert, M.M., Lester, H.A., and Davidson, N. (1990). Rat brain expresses a heterogeneous family of calcium channels. Proc Natl Acad Sci U S A 87, 3391-3395. Snutch, T.P., Peloquin, J., Mathews, E., and McRory, J.E. (2005). Molecular properties of voltage-gated calcium channels. In Voltage-gated calcium channels, G.W. Zamponi, ed. (New York: Landes Bioscience), pp. 61-94. Song, I., Kim, D., Choi, S., Sun, M., Kim, Y., and Shin, H.S. (2004). Role of the alpha1G T-type calcium channel in spontaneous absence seizures in mutant mice. J Neurosci 24, 5249-5257. Soong, T.W., Stea, A., Hodson, C.D., Dubel, S.J., Vincent, S.R., and Snutch, T.P. (1993). Structure and functional expression of a member of the low voltage-activated calcium channel family. Science 260, 1133-1136. Splawski, I., Yoo, D.S., Stotz, S.C., Cherry, A., Clapham, D.E., and Keating, M.T. (2006). CACNA1H mutations in autism spectrum disorders. J Biol Chem 281, 22085-22091. Staes, M., Talavera, K., Klugbauer, N., Prenen, J., Lacinova, L., Droogmans, G., Hofmann, F., and Nilius, B. (2001). The amino side of the C-terminus determines fast inactivation of the T-type calcium channel alpha1G. J Physiol 530, 35-45. Starr, T.V., Prystay, W., and Snutch, T.P. (1991). Primary structure of a calcium channel that is highly expressed in the rat cerebellum. Proc Natl Acad Sci U S A 88, 5621-5625. Stea, A., Soong, T.W., and Snutch, T.P. (1995). Determinants of PKC-dependent modulation of a family of neuronal calcium channels. Neuron 15, 929-940. Stea, A., Tomlinson, W.J., Soong, T.W., Bourinet, E., Dubel, S.J., Vincent, S.R., and Snutch, T.P. (1994). Localization and functional properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels. Proc Natl Acad Sci U S A 91, 10576-10580. Stotz, S.C., Jarvis, S.E., and Zamponi, G.W. (2004). Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels. J Physiol 554, 263-273. Sugimori, M., and Llinas, R.R. (1990). Real-time imaging of calcium influx in mammalian cerebellar Purkinje cells in vitro. Proc Natl Acad Sci U S A 87, 5084-5088. Chapter 1: Introduction  62 Suh, B.C., and Hille, B. (2005). Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15, 370-378. Suzuki, S., and Rogawski, M.A. (1989). T-type calcium channels mediate the transition between tonic and phasic firing in thalamic neurons. Proc Natl Acad Sci U S A 86, 7228-7232. Swartz, K.J., and Bean, B.P. (1992). Inhibition of calcium channels in rat CA3 pyramidal neurons by a metabotropic glutamate receptor. J Neurosci 12, 4358-4371. Swensen, A.M., and Bean, B.P. (2003). Ionic mechanisms of burst firing in dissociated Purkinje neurons. J Neurosci 23, 9650-9663. Tai, C., Kuzmiski, J.B., and MacVicar, B.A. (2006). Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons. J Neurosci 26, 6249-6258. Takechi, H., Eilers, J., and Konnerth, A. (1998). A new class of synaptic response involving calcium release in dendritic spines. Nature 396, 757-760. Talavera, K., and Nilius, B. (2006a). Biophysics and structure-function relationship of T-type Ca2+ channels. Cell Calcium 40, 97-114. Talavera, K., and Nilius, B. (2006b). Evidence for common structural determinants of activation and inactivation in T-type Ca2+ channels. Pflugers Arch 453, 189-201. Talavera, K., Staes, M., Janssens, A., Droogmans, G., and Nilius, B. (2004). Mechanism of arachidonic acid modulation of the T-type Ca2+ channel alpha1G. J Gen Physiol 124, 225-238. Talavera, K., Staes, M., Janssens, A., Klugbauer, N., Droogmans, G., Hofmann, F., and Nilius, B. (2001). Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G). J Biol Chem 276, 45628-45635. Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E., and Bayliss, D.A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19, 1895-1911. Tao, J., Hildebrand, M.E., Liao, P., Liang, M.C., Tan, G., Li, S., Snutch, T.P., and Soong, T.W. (2008). Activation of corticotropin-releasing factor receptor 1 selectively inhibits CaV3.2 T-type calcium channels. Mol Pharmacol. Tedford, H.W., and Zamponi, G.W. (2006). Direct G protein modulation of Cav2 calcium channels. Pharmacol Rev 58, 837-862. Tempia, F., Alojado, M.E., Strata, P., and Knopfel, T. (2001). Characterization of the mGluR(1)- mediated electrical and calcium signaling in Purkinje cells of mouse cerebellar slices. J Neurophysiol 86, 1389-1397. Tempia, F., Miniaci, M.C., Anchisi, D., and Strata, P. (1998). Postsynaptic current mediated by metabotropic glutamate receptors in cerebellar Purkinje cells. J Neurophysiol 80, 520-528. Todorovic, S.M., Jevtovic-Todorovic, V., Mennerick, S., Perez-Reyes, E., and Zorumski, C.F. (2001a). Ca(v)3.2 channel is a molecular substrate for inhibition of T-type calcium currents in rat sensory neurons by nitrous oxide. Mol Pharmacol 60, 603-610. Chapter 1: Introduction  63 Todorovic, S.M., Jevtovic-Todorovic, V., Meyenburg, A., Mennerick, S., Perez-Reyes, E., Romano, C., Olney, J.W., and Zorumski, C.F. (2001b). Redox modulation of T-type calcium channels in rat peripheral nociceptors. Neuron 31, 75-85. Tombler, E., Cabanilla, N.J., Carman, P., Permaul, N., Hall, J.J., Richman, R.W., Lee, J., Rodriguez, J., Felsenfeld, D.P., Hennigan, R.F., and Diverse-Pierluissi, M.A. (2006). G protein-induced trafficking of voltage-dependent calcium channels. J Biol Chem 281, 1827-1839. Toselli, M., and Lux, H.D. (1989). Opposing effects of acetylcholine on the two classes of voltage- dependent calcium channels in hippocampal neurons. Exs 57, 97-103. Toselli, M., and Taglietti, V. (1995). Muscarine inhibits high-threshold calcium currents with two distinct modes in rat embryonic hippocampal neurons. J Physiol 483 ( Pt 2), 347-365. Traboulsie, A., Chemin, J., Chevalier, M., Quignard, J.F., Nargeot, J., and Lory, P. (2007). Subunit- specific modulation of T-type calcium channels by zinc. J Physiol 578, 159-171. Tsakiridou, E., Bertollini, L., de Curtis, M., Avanzini, G., and Pape, H.C. (1995). Selective increase in T- type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. J Neurosci 15, 3110-3117. Tseng, G.N., and Boyden, P.A. (1991). Different effects of intracellular Ca and protein kinase C on cardiac T and L Ca currents. Am J Physiol 261, H364-379. Tsien, R.W., and Barrett, C.F. (2005). A brief history of calcium channel discovery. In Voltage-gated calcium channels, G.W. Zamponi, ed. (New York: Landes Bioscience), pp. 27-47. Usowicz, M.M., Sugimori, M., Cherksey, B., and Llinas, R. (1992). P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells. Neuron 9, 1185-1199. Vassort, G., Talavera, K., and Alvarez, J.L. (2006). Role of T-type Ca2+ channels in the heart. Cell Calcium 40, 205-220. Viana, F., Van den Bosch, L., Missiaen, L., Vandenberghe, W., Droogmans, G., Nilius, B., and Robberecht, W. (1997). Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones. Cell Calcium 22, 299-311. Vitko, I., Bidaud, I., Arias, J.M., Mezghrani, A., Lory, P., and Perez-Reyes, E. (2007). The I-II loop controls plasma membrane expression and gating of Ca(v)3.2 T-type Ca2+ channels: a paradigm for childhood absence epilepsy mutations. J Neurosci 27, 322-330. Vitko, I., Chen, Y., Arias, J.M., Shen, Y., Wu, X.R., and Perez-Reyes, E. (2005). Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel. J Neurosci 25, 4844-4855. Wan, X., Desilets, M., Soboloff, J., Morris, C., and Tsang, B.K. (1996). Muscarinic activation inhibits T- type Ca2+ current in hen granulosa cells. Endocrinology 137, 2514-2521. Wang, S.S., Denk, W., and Hausser, M. (2000). Coincidence detection in single dendritic spines mediated by calcium release. Nat Neurosci 3, 1266-1273. Wang, Y., Rowan, M.J., and Anwyl, R. (1997). LTP induction dependent on activation of Ni2+-sensitive voltage-gated calcium channels, but not NMDA receptors, in the rat dentate gyrus in vitro. J Neurophysiol 78, 2574-2581. Chapter 1: Introduction  64 Watanabe, S., Takagi, H., Miyasho, T., Inoue, M., Kirino, Y., Kudo, Y., and Miyakawa, H. (1998). Differential roles of two types of voltage-gated Ca2+ channels in the dendrites of rat cerebellar Purkinje neurons. Brain Res 791, 43-55. Weber, J.T., De Zeeuw, C.I., Linden, D.J., and Hansel, C. (2003). Long-term depression of climbing fiber-evoked calcium transients in Purkinje cell dendrites. Proc Natl Acad Sci U S A 100, 2878-2883. Welker, H.A., Wiltshire, H., and Bullingham, R. (1998). Clinical pharmacokinetics of mibefradil. Clin Pharmacokinet 35, 405-423. Welsby, P.J., Wang, H., Wolfe, J.T., Colbran, R.J., Johnson, M.L., and Barrett, P.Q. (2003). A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II. J Neurosci 23, 10116-10121. White, G., Lovinger, D.M., and Weight, F.F. (1989). Transient low-threshold Ca2+ current triggers burst firing through an afterdepolarizing potential in an adult mammalian neuron. Proc Natl Acad Sci U S A 86, 6802-6806. Williams, S.R., Toth, T.I., Turner, J.P., Hughes, S.W., and Crunelli, V. (1997). The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones. J Physiol 505 ( Pt 3), 689-705. Wolfart, J., and Roeper, J. (2002). Selective coupling of T-type calcium channels to SK potassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. J Neurosci 22, 3404-3413. Wolfe, J.T., Wang, H., Howard, J., Garrison, J.C., and Barrett, P.Q. (2003). T-type calcium channel regulation by specific G-protein betagamma subunits. Nature 424, 209-213. Wolfe, J.T., Wang, H., Perez-Reyes, E., and Barrett, P.Q. (2002). Stimulation of recombinant Ca(v)3.2, T-type, Ca(2+) channel currents by CaMKIIgamma(C). J Physiol 538, 343-355. Womack, M., and Khodakhah, K. (2002). Active contribution of dendrites to the tonic and trimodal patterns of activity in cerebellar Purkinje neurons. J Neurosci 22, 10603-10612. Womack, M.D., Chevez, C., and Khodakhah, K. (2004). Calcium-activated potassium channels are selectively coupled to P/Q-type calcium channels in cerebellar Purkinje neurons. J Neurosci 24, 8818- 8822. Womack, M.D., and Khodakhah, K. (2004). Dendritic control of spontaneous bursting in cerebellar Purkinje cells. J Neurosci 24, 3511-3521. Wu, L., Bauer, C.S., Zhen, X.G., Xie, C., and Yang, J. (2002). Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419, 947-952. Xia, J., Chung, H.J., Wihler, C., Huganir, R.L., and Linden, D.J. (2000). Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins. Neuron 28, 499-510. Yao, J., Davies, L.A., Howard, J.D., Adney, S.K., Welsby, P.J., Howell, N., Carey, R.M., Colbran, R.J., and Barrett, P.Q. (2006). Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+/calmodulin-dependent protein kinase II. J Clin Invest 116, 2403-2412. Yunker, A.M. (2003). Modulation and pharmacology of low voltage-activated ("T-Type") calcium channels. J Bioenerg Biomembr 35, 577-598. Chapter 1: Introduction  65 Yunker, A.M., Sharp, A.H., Sundarraj, S., Ranganathan, V., Copeland, T.D., and McEnery, M.W. (2003). Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117, 321-335. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T.P. (1997). Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit. Nature 385, 442-446. Zamponi, G.W., Bourinet, E., and Snutch, T.P. (1996). Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites. J Membr Biol 151, 77-90. Zhang, Y., Cribbs, L.L., and Satin, J. (2000). Arachidonic acid modulation of alpha1H, a cloned human T-type calcium channel. Am J Physiol Heart Circ Physiol 278, H184-193. Zhang, Y., Mori, M., Burgess, D.L., and Noebels, J.L. (2002). Mutations in high-voltage-activated calcium channel genes stimulate low-voltage-activated currents in mouse thalamic relay neurons. J Neurosci 22, 6362-6371. Zhong, X., Liu, J.R., Kyle, J.W., Hanck, D.A., and Agnew, W.S. (2006). A profile of alternative RNA splicing and transcript variation of CACNA1H, a human T-channel gene candidate for idiopathic generalized epilepsies. Hum Mol Genet 15, 1497-1512.   Chapter 2: Inhibition of Cav3.3 by mAChRs   66 2 SELECTIVE INHIBITION OF CAV3.3 T-TYPE CALCIUM CHANNELS BY GαQ/11-COUPLED MUSCARINIC ACETYLCHOLINE RECEPTORS*  2.1 Introduction T-type Ca2+ channels play critical roles in shaping the electrical, chemical and plastic properties of neurons throughout the CNS and PNS.  In thalamic nRT and TC neurons, T-type channels are involved in rhythmic rebound burst firing and spindle waves associated with slow-wave sleep (Anderson et al., 2005; Destexhe and Sejnowski, 2003; Huguenard and Prince, 1992; Kim et al., 2001; Tsakiridou et al., 1995).  Studies on KO mice and a rat model of absence epilepsy indicate that altering T-type activity within thalamic cells can contribute to pathological conditions such as sleep disorders and epilepsy (Anderson et al., 2005; Destexhe and Sejnowski, 2003; Huguenard and Prince, 1992; Kim et al., 2001; Tsakiridou et al., 1995).   Certain human epilepsies appear to be associated with T-type Ca2+ channel point mutations conferring channel gain-of-function phenotypes (Khosravani et al., 2004; Khosravani et al., 2005; Peloquin et al., 2006; Vitko et al., 2005).  T-type channels also play crucial roles in dendritic integration and Ca2+ spiking in hippocampal pyramidal cells (Christie et al., 1995; Thompson and Wong, 1991).  Within the olfactory bulb, T-type channels are implicated in modulating Ca2+ transients and synaptic release at dendrodendritic synapses (Egger et al., 2003, 2005).  In the periphery, antisense oligonucleotides and pharmacological approaches have implicated T-type channels in contributing to both acute and chronic nociceptive behaviors (Bourinet et al., 2005; Hildebrand and Snutch, 2006). Previous studies have identified three main subtypes of T-type Ca2+ channel α1 subunits (Cav3.1, Cav3.2 and Cav3.3) and characterized their voltage-dependent, kinetic, and pharmacological properties (Cribbs et al., 1998; Lee et al., 1999; McRory et al., 2001; Monteil et al., 2000; Perez-Reyes et al., 1998; Santi et al., 2002).   Cav3.1 and Cav3.2 channels display “typical” T-type properties, including relatively small conductance, fast activation and inactivation kinetics and slow deactivation kinetics, while Cav3.3 channels uniquely display a larger conductance, much slower activation and inactivation kinetics as well as faster deactivation kinetics (Lee et al., 1999; McRory et al., 2001).   Some of the distinct biophysical properties associated with Cav3.3 T-type currents have been observed in certain populations of native T- type currents (Huguenard and Prince, 1992; Joksovic et al., 2005; Lee et al., 1999; McRory et al., 2001). The biophysical differences between the T-type channels likely enables them to differentially shape and modulate firing patterns, with the more slowly inactivating Cav3.3 currents able to produce longer bursts of spikes and tonic firing patterns (Chevalier et al., 2006; McRory et al., 2001; Murbartian et al., 2004).  * A version of this chapter has been published.  Hildebrand, M.E., David, L.S., Hamid, J., Mulatz, K., Garcia, E., Zamponi, G.W., and Snutch, T.P. (2007). Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11- coupled muscarinic acetylcholine receptors. J Biol Chem 282, 21043-21055.  Chapter 2: Inhibition of Cav3.3 by mAChRs   67 Although the basic properties of both cloned and native T-type channels have now been largely characterized, there remains relatively little information concerning their modulation by GPCR-linked pathways.  Neurotransmitters such as acetylcholine have been shown to either attenuate or stimulate low threshold Ca2+ currents depending on the type of native cells examined, and sometimes multiple forms of modulation can be observed within the same cell type (Castillo et al., 1999; Chemin et al., 2006; Fraser and MacVicar, 1991; Pemberton et al., 2000; Wan et al., 1996).  Multiple T-type Ca2+ channel subtypes are expressed in most native cells (Talley et al., 1999; Yunker et al., 2003) although pharmacological tools with the specificity needed to separate these currents have not been generated.  In this regard, the description of the modulation of specific T-type Ca2+ channels in heterologous systems will provide insights crucial towards further investigations within native systems.  This approach is also well-suited for GPCR studies as most neurotransmitters activate multiple receptor subtypes in neurons. Within thalamic nRT, hippocampal pyramidal and olfactory granule cells, there is evidence for the expression of both T-type Ca2+ channels and Gαq/11-coupled mAChRs (Castillo et al., 1999; Levey et al., 1994; Levey et al., 1995; McKay et al., 2006; Plummer et al., 1999; Talley et al., 1999; Wei et al., 1994; Yunker et al., 2003).  As both T-type Ca2+ currents and mAChRs have been independently shown to play important physiological roles within these cell types, their functional coupling could be relevant to a number of neuronal processes.  In the present paper we studied the modulatory effects of mAChRs on the three main subtypes of low threshold T-type Ca2+ channels expressed in the mammalian nervous system.  We find the selective modulation of Cav3.3 Ca2+ channels by Gαq/11-coupled mAChRs and combined pharmacological, genetic and chimeric channel approaches to examine the G-protein-mediated pathway and structural regions responsible for the distinct Cav3.3 signaling characteristics.  2.2 Results 2.2.1 Muscarinic M1 receptors selectively inhibit Cav3.3 T-type calcium channels To investigate the potential for T-type Ca2+ channel modulation by mAChRs, we transiently transfected HEK cell lines stably expressing individual subtypes of recombinant rat brain T-type channels with the human muscarinic M1 receptor.  Perforated patch recordings with β-Escin demonstrated that activation of M1 with 1 mM carbachol (CCh) caused a rapid (< 30 sec) and robust inhibition of exogenously expressed rat brain Cav3.3 T-type channel peak currents (-45% +/- 2%, n=34) (Fig. 2.1C,F).  Only a small subpopulation of stable Cav3.3 cells (<10%) were not affected by CCh application (likely representing cells untransfected with the M1 receptor).  Activation of M1 with 1 mM CCh had no significant effect (p>0.05) on the voltage-dependence of Cav3.3 currents, but significantly increased both the rates of activation and inactivation (p<0.001; Table 2.1).  In contrast to the clear inhibition of Cav3.3 T-type currents, activation of M1 receptors with 1 mM CCh largely had no effect on Chapter 2: Inhibition of Cav3.3 by mAChRs   68 the peak current amplitude of either rat brain Cav3.1 (-2.1% +/- 2.0%, n=18) or Cav3.2 channels (-0.1% +/- 2.3%, n=17) (Fig. 2.1A,B,D,E). In a small subset of both Cav3.1 and Cav3.2 currents we noted a stimulation induced by M1 activation (Cav3.1 = 35% +/- 12%, n=4; Cav3.2 = 36% +/- 12%, n=5), with a slower time course to equilibrium of greater than one minute (n=3 and n=4, respectively).  For the prevalent null effect on Cav3.1 and Cav3.2 currents, 1 mM CCh application had no significant effect on channel activation and inactivation kinetics or the voltage-dependence of activation (p>0.05; Table 2.1). Different Cav3.3 T-type channel isoforms with distinct carboxyl termini have been identified from both the rat and human brain (Lee et al., 1999; McRory et al., 2001; Monteil et al., 2000; Murbartian et al., 2002).  To test whether inhibition of the Cav3.3 channel by M1 receptors was restricted to the rat brain short carboxyl terminus isoform (McRory et al., 2001), we also examined the longer human Cav3.3 isoform (Monteil et al., 2000) transiently co-transfected into HEK cells with the M1 receptor.  Similar to that for the shorter rat brain isoform, application of 1 mM CCh resulted in significant inhibition of the human Cav3.3 peak current amplitude (-28% +/- 2%, n=15) and also significantly increased activation and inactivation kinetics (p<0.001; Fig. 2.5A; Table 2.1).  Additionally, similar to that for the rat Cav3.1 T-type channel, application of 1 mM CCh to HEK cells co-transfected with the human Cav3.1 channel and M1 receptor had no significant effect on peak current amplitude (- 0.3% +/- 2.0%, n=9) or channel kinetics (for 100% cells tested; p>0.05; Fig. 2.7B; Table 2.1).  Overall, the differential modulation of T-type Ca2+ channel subtypes mediated by M1 receptors was consistent across both rat and human recombinant T-type channels. Table 2.1 - Effects of Receptor Activation on T-Type Channel Kinetic and Voltage- Dependent Properties * p < 0.02, ** p < 0.001 τact  (ms) τinact  (ms) V50act  (mV) τact  (ms) τinact  (ms) V50act  (mV) Cav3.3 + M1  (Inhibition) 6.0 +/- 0.4, n=27 86 +/- 6, n=27 -51 +/- 2, n=13 4.1 +/- 0.3, n=27** 31 +/- 2, n=27** -49 +/- 1, n=13 human Cav3.3 + M1 (Whole-Cell; Inhibition) 9.7 +/- 0.8, n=10 117 +/- 6, n=10 -44 +/- 1, n=4 5.4 +/- 0.6, n=10** 41 +/- 5, n=10** -47 +/- 1, n=4 Cav3.3 + M2  (No Effect) 6.1 +/- 0.7, n=10 81 +/- 9, n=10 -53 +/- 3, n=4 5.5 +/- 0.7, n=10 76 +/- 9, n=10 -52 +/- 3, n=4 Cav3.3 + M3  (Inhibition) 9.5 +/- 0.9, n=9 104 +/- 13, n=9 -53 +/- 4, n=4 6.2 +/- 0.6, n=9* 56 +/- 6, n=8* -50 +/- 4, n=4 Cav3.3 + M4  (No Effect) 6.7 +/- 0.9, n=6 110 +/- 18, n=6 -50 +/- 1, n=5 6.0 +/- 0.9, n=6 101 +/- 16, n=6 -51 +/- 2, n=5 Cav3.3 + M5  (Inhibition) 8.6 +/- 0.9, n=6 127 +/- 23, n=7 -48 +/- 1, n=5 5.6 +/- 0.6, n=6 49 +/- 6, n=7* -45 +/- 1, n=5 Cav3.3 + Control Plasmid 6.0 +/- 0.4, n=5 78 +/- 13, n=5 5.2 +/- 0.2, n=5 71 +/- 12, n=5 Cav3.1 + M1 (No Effect) 2.3 +/- 0.2, n=17 19 +/- 2, n=17 -36 +/- 2, n=17 2.1 +/- 0.2, n=17 17 +/- 1, n=17 -39 +/- 3, n=11 human Cav3.1 + M1 (Whole-Cell; No Effect) 2.0 +/- 0.4, n=9 14 +/- 1, n=9 -42 +/- 2, n=6 1.2 +/- 0.1, n=9 13 +/- 1, n=9 -51 +/- 3, n=4 Cav3.2 + M1 (No Effect) 4.7 +/- 0.2, n=16 36 +/- 2, n=15 -42 +/- 1, n=7 4.3 +/- 0.2, n=16 34 +/- 2, n=15 -40 +/- 2, n=7 2mM Ca2+ Control 1 mM Carbachol Chapter 2: Inhibition of Cav3.3 by mAChRs   69 0 50 100 150 200 0.5 0.6 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 150 200 0.5 0.6 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt Time (s) 1 mM CCh Control CCh 50 ms 50 0 pA CCh Control 20 ms 10 0 pA CCh Control 20 ms5 0 pA A D B C F 0 50 100 150 200 0.5 0.6 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt Time (s) 1mM CCh Cav3.1 + M1 ECav3.2 + M1 Cav3.3 + M1  Figure 2.1 - T-type calcium channels are differentially modulated by M1 receptors. A,B) Representative perforated patch current traces during depolarizing pulses from -110 mV to -30 mV demonstrating no effect on Cav3.1 currents (A) and Cav3.2 currents (B) when M1 is activated with 1 mM CCh.  D,E) Normalized peak current levels during perfusion of control recording solution (2 mM Ca2+) followed by 1 mM CCh for Cav3.1 (+M1) currents (D) and Cav3.2 (+M1) currents (E).  Perfusion of CCh usually had no effect on Cav3.1 peak current amplitudes (-2.1% +/- 2.0%, n=18) and Cav3.2 peak current amplitudes (-0.1% +/- 2.3%, n=17).   C) Representative perforated patch current traces during depolarizing pulses from -110 mV to -40 mV showing inhibition of Cav3.3 currents by M1. F) Normalized peak current levels during perfusion of control recording solution (2 mM Ca2+) followed by 1 mM CCh for Cav3.3 (+M1) currents.  Perfusion of CCh caused a 45% (+/- 2%, n=34) decrease in Cav3.3 currents.  All data points correspond to mean +/- S.E. Chapter 2: Inhibition of Cav3.3 by mAChRs   70 2.2.2 Muscarinic M1 receptors dose-dependently modulate Cav3.3 biophysical properties Perforated patch recordings on stable rat Cav3.3 cells transiently transfected with M1 receptors revealed that the CCh-induced inhibition of peak current levels was reversible over a time course of about 2 minutes (n=13; Fig. 2.2A,B).  As previously mentioned, activation of M1 receptors with 1 mM CCh caused a significant (p<0.001) increase in inactivation kinetics (Control, τinact=86 +/- 6, n=27; 1 mM CCh, τinact=31 +/- 2, n=27).  Along with peak current inhibition, the CCh-induced increase in Cav3.3 inactivation kinetics was reversible (Fig. 2.2A,C).  Both the M1 receptor-induced inhibition of Cav3.3 peak currents and the increased inactivation rate would be predicted to reduce the total amount of Ca2+ flowing through Cav3.3 T-type channels during a cellular depolarization.  The effect of M1 receptor activation on total Ca2+ influx was determined by integrating the area over Cav3.3 current traces during 200 ms depolarizing pulses to peak potential before and after 1 mM CCh application.  Normalizing these Ca2+ influx values to control levels showed a 77% +/- 2% (n=20) reduction in Ca2+ influx mediated by M1 receptor activation (Fig. 2.2D). As activation of M1 receptors increases the kinetics of Cav3.3 activation and inactivation, it is possible that this signaling pathway modulates Cav3.3 function via acting upon the open and/or inactivated states.  A protocol that involved perfusion of 1 mM CCh for 50 seconds (time to reach normal inhibition equilibrium) in the absence of test pulses, followed by regular 0.2 Hz test pulses to peak potential examined whether the M1 receptor-mediated inhibitory effect is use-dependent.  Figure 2.2E shows that this “no depolarization” protocol displayed the same level of inhibition of Cav3.3 currents observed in the regular 0.2 Hz experiments, indicating that M1 effects on Cav3.3 are use- independent.  Increasing the test pulse frequency to 0.5 Hz also caused no significant (p>0.05) change in the level of Cav3.3 inhibition, indicating that the M1 effects on Cav3.3 are also frequency-independent (Fig. 2.2E).  Another possibility is that M1 receptor activation inhibits Cav3.3 currents by shifting steady- state inactivation to more hyperpolarized potentials, reducing the proportion of channels available (in the closed state) to open at the holding potential of -110 mV.  A protocol with a 1 second prepulse to -140 mV to remove accumulated channel inactivation demonstrated no significant (p>0.05) difference in inhibition compared to the control protocol, suggesting that the inhibitory effect is not due to changes in the steady-state inactivation of Cav3.3 channels (Fig. 2.2E). Control experiments with mock transfections of an empty control vector or with a pre-incubated mAChR antagonist (atropine) demonstrated that the CCh-induced inhibition of Cav3.3 currents is mediated specifically via the transfected M1 receptor (Fig. 2.2E).  Testing the effects of varying concentrations of CCh on stable Cav3.3 cells with transfected M1 receptors revealed that the inhibitory effect is dose-dependent (Fig. 2.2F).  The IC50 for inhibition of Cav3.3 currents by CCh = 27 μM, consistent with that reported for phophatidyl inositol hydrolysis triggered by M1 receptor activation in both HEK 293 and CHO cells (Hogger et al., 1995; Schwarz et al., 1993). Chapter 2: Inhibition of Cav3.3 by mAChRs   71  50 ms2 00  p A 1 2 3 0 100 200 300 400 30 40 50 60 70 80 90 100 τ in ac t ( m s) Time (s) 1mM CCh 1 2 3 0 50 100 150 200 0.2 0.4 0.6 0.8 1.0 1.2 N or m al iz ed  C a In flu x Time (s) 1mM CCh 0.1 1 10 100 1000 0 10 20 30 40 50 %  In hi bi tio n [Carbachol] (μM) 0 100 200 300 400 -400 -500 -600 -700 -800 -900 1 Pe ak  C ur re nt  (p A ) Time (s) 1mM CCh 2 3 E A C F B D -50 -40 -30 -20 -10 0 10 Co nt ro l ( 0. 2 Hz ) No  D ep ol ar iza tio n 0. 5 Hz  P ul se s -1 40  m V Pr ep ul se Ve ct or  C on tro l  50  u M  A tro pi ne  %  M od ul at io n of  C av 3. 3 by  M 1  * * Chapter 2: Inhibition of Cav3.3 by mAChRs   72        Figure 2.2 - Mechanistic properties of inhibition of Cav3.3 currents by M1 receptors. A) The inhibition of Cav3.3 channels by M1 is reversible.  Representative Cav3.3 perforated patch current traces during depolarizing pulses from –110 mV to –40 mV before (1), during (2), and after (3) perfusion of 1 mM CCh.  Note the increase in inactivation kinetics when CCh is applied (Table 2.1). B) A plot of peak current amplitude (for same cell as in A) showing the rate of inhibition by 1 mM CCh perfusion and the rate of washout, with the selected traces from A (1,2,3) labeled.  C) Application of 1 mM CCh increases Cav3.3 inactivation kinetics in a reversible manner.  The inactivating component of every trace from B was fit with an exponential equation to give τinact.  D) Application of 1 mM CCh dramatically reduces the amount of Ca2+ influx through Cav3.3 channels.  The effects of M1 activation on normalized Ca2+ influx is shown for all Cav3.3 cells.  Perfusion of 1 mM CCh caused a 77% (+/- 2%, n=20) decrease in Ca2+ influx.  E) Inhibition of Cav3.3 currents by 1 mM CCh occurs through M1 receptors.  Control experiments show elimination of the inhibition due to 1 mM CCh when a muscarinic antagonist (50 μM atropine) is co-applied, or when a control vector (pBluescript) is transfected instead of M1.  A lack of depolarizing test pulses during initial CCh perfusion, increase in test pulse frequency to 0.5 Hz, or a hyperpolarizing prepulse to -140 mV for 1 second had no significant (p>0.02) effect on the magnitude of Cav3.3 inhibition by M1.  F) CCh inhibited Cav3.3 currents in a dose-dependent manner. CCh concentration vs. percentage block data was fit with a Hill equation and the IC50 for CCh inhibition of Cav3.3 currents was 27 μM.  All data points correspond to mean +/- S.E. Chapter 2: Inhibition of Cav3.3 by mAChRs   73 2.2.3 Inhibition of Cav3.3 channels by M1 receptors requires Gαq/11 The inhibitory effect of M1 receptor activation on Cav3.3 currents could occur either through Gβγ- or Gα-mediated processes.  To test for the involvement of Gβγ, a membrane-targeted version of the C-terminus of βARK, MAS-GRK3ct (Kammermeier et al., 2000), was co-transfected with M1 receptors into the stable Cav3.3 HEK cell line.  Control experiments showed that the MAS-GRK3ct construct was able to completely abolish the well described Gβγ-dependent inhibition of N-type Ca2+ channels (data not shown).  MAS-GRK3ct only partially reduced M1-mediated Cav3.3 current inhibition (-24.8% +/- 3.4%, n=10) in most cells, suggesting that inhibition is distinct from the previously reported pure Gβγ- mediated inhibition of N- and P/Q-type and Cav3.2 Ca2+ channels (Dolphin, 2003; Wolfe et al., 2003) (Fig. 2.3A,D).  A smaller subset of MAS-GRK3ct co-transfected cells displayed no exponential inhibitory effect (-9.3% +/- 3.0%, n=7).   Co-expression of transducin (Gαt), which also buffers Gβγ signaling (Kammermeier et al., 2000), caused the same reduction in M1-mediated inhibition of Cav3.3 currents (-25.1% +/- 2.5%, n=10), with a small number of cells not being inhibited at all (7.0% +/- 10.1%, n=3) (Fig. 2.3B,D). In contrast to the partial effect of Gβγ signaling antagonists, co-expression of the Regulator of G-Protein Signaling 2, RGS2 – an effector antagonist for Gαq/11 (Heximer et al., 1997), completely prevented the M1 receptor-induced inhibition of Cav3.3 currents for all cells examined.   In perforated patch recordings of Cav3.3 cells co-transfected with M1 and RGS2, application of 1 mM CCh either had no effect (Fig. 2.3C,D; 1% +/- 5%, n=7), or caused a stimulation of Cav3.3 currents (30% +/- 9%, n=5).  2.2.4 Constitutively active Gαq/11 proteins modulate Cav3.3 T-type calcium channels To further test whether active Gαq/11 G-proteins are sufficient to produce inhibition of Cav3.3 currents, stable Cav3.3 cells were transiently transfected with various constitutively active Gα subunit constructs.  These constructs contain missense mutations that confer constitutive activity by reducing GTPase activity.  If Gαq/11 is the downstream signal of M1 receptor activation mediating the effects on Cav3.3 currents then it is hypothesized that activation of the co-expressed Gαq or Gα11 mutants by dialysis of GTP would cause a reduction in current amplitude and an increase in inactivation kinetics. Similar to a study that analyzed inhibition of KCNQ2/KCNQ3 channels by Gαq/11 (Suh et al., 2004), we used constitutively active Gαq (Gαq-Q209L) and Gα11 (Gα11-Q209L) mutants to test for the hypothesized effect and a constitutively active Gα protein (Gα13-Q226L) that does not couple to the same downstream effectors (PLC) as a negative control.  We also performed controls wherein empty vectors were transfected. Chapter 2: Inhibition of Cav3.3 by mAChRs   74 0 50 100 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1mM CCh N or m al iz ed  C ur re nt Time (s) Cav3.3 + M1 + MAS-GRK3ct Cav3.3 + M1 +  Gαt Cav3.3 + M1 +  RGS2 A C B D -50 -40 -30 -20 -10 0 10 Co nt ro l   R GS 2 M AS -G RK 3c t PT X St au ro sp or in e H9 Ch el er yth rin e G o 69 76 G en ist ei n O ka da ic Ac id W or tm an ni n Rp -c AM Ps BA PT A- AM *  * %  M od ul at io n of  C av 3. 3 b y M 1  * G α t  Figure 2.3 - Inhibition of Cav3.3 channels by M1 requires Gαq/11 signaling. A) In perforated patch recordings of Cav3.3 stable cells co-transfected with M1 and a membrane targeted form of the C-terminus of βARK that sequesters active Gβγ subunits (MAS-GRK3ct), application of 1 mM CCh caused inhibition of Cav3.3 currents (-24.8% +/- 3.4%, n=10).  B) Similarly, inhibition by perfusion of 1 mM CCh was observed for Cav3.3 cells co-transfected with the Gβγ buffer Gαtransducin (Gαt) and M1 (-25.1% +/- 2.5%, n=10).  C) In perforated patch recordings of Cav3.3 cells co-transfected with M1 and RGS2 (antagonist of active Gαq/11 subunits), application of 1 mM CCh predominantly had no effect (1% +/- 5%, n=7) on Cav3.3 currents.  D) Bar graph comparing various genetic and pharmacological manipulations to control conditions where stable Cav3.3 cells are transfected with M1 and inhibited by 1 mM CCh.  Inhibitors of serine/threonine kinases (500 nM staurosporine, n=7; 50 μM H9, n=6), PKC (10 μM Chelerythrine, n=5; 500 nM Go 6976, n=6), tyrosine kinases (10 μM genistein, n=7), phosphatases (100 nM okadaic acid, n= 6), phosphoinositide-3-kinases (200 nM wortmannin, n=7), PTX-sensitive Gα proteins (0.5 μg/mL PTX, n=6), cAMP (10 μM Rp-cAMPs, n=5) and internal Ca2+ (10 μM BAPTA-AM, n=5) had no significant (p>0.02) effect on the inhibition of Cav3.3 currents by M1. RGS2, MAS-GRK3ct, and Gαt (p<0.001) caused a significant elimination or reduction in the inhibition of Cav3.3 currents by M1.  All data points correspond to mean +/- S.E. Chapter 2: Inhibition of Cav3.3 by mAChRs   75 By comparing traces 30 seconds after forming the whole-cell configuration with traces 2 minutes after whole-cell in Figure 2.4A-D, we found that dialysis of the cell with the GTP-containing pipette internal solution caused both a significant reduction in peak current levels and an increase in inactivation kinetics only for the Gαq-Q209L and Gα11-Q209L transfections.  The ratio of peak current levels at 2 minutes divided by the peak current levels at 30 seconds was significantly reduced (p<0.001) for Gαq-Q209L (n=16) and Gα11-Q209L (n=15) compared to the control transfection (n=18), while the Gα13-Q226L (n=17) transfection current ratio was not significantly altered ((p>0.05), Fig. 2.4E)).  The rates of inactivation (τinact) were determined during depolarizing steps from -110 mV to -30 mV for all transfection types. The τιnact was significantly faster (p<0.001) for Gαq-Q209L and Gα11-Q209L compared to control transfections, while the τιnact was not significantly different for Gα13-Q226L (p>0.02; Fig. 2.4F).  2.2.5 Gαq/11 inhibits Cav3.3 channels through an unidentified non-classical pathway The active GTP-bound form of Gαq/11 causes the activation of PLC, which then produces IP3- and DAG/PKC-mediated signals.  Various pharmacological antagonists were used to investigate the role of potential cellular signals downstream of Gαq/11.  Specific inhibitors of PKC including 10 μM chelerythrine (n=5) and 500 nM Go 6976 (n=6) had no significant (p>0.05) effect on the M1-mediated inhibition of Cav3.3 currents (Fig. 2.3D).  To ensure pharmacological activity of these antagonists, the PKC-mediated stimulation of Cav3.2 channels by 300 nM PMA (65% +/- 17%, n=7; see (Park et al., 2006)) was shown to be significantly (p<0.02) abolished by both 10 μM chelerythrine (-15% +/- 6%, n=5) and 500 nM Go 6976 (3% +/- 5%, n=5) (data not shown).  Inhibitors of serine/threonine kinases (500 nM staurosporine, n=7; 50 μM H9, n=6), tyrosine kinases (10 μM genistein, n=7), phosphatases (100 nM okadaic acid, n= 6), phosphoinositide-3-kinases (200 nM wortmannin, n=7), PTX-sensitive Gα proteins (0.5 μg/mL PTX, n=6), cAMP (10 μM Rp-cAMPs, n=5) and internal Ca2+ (10 μM BAPTA-AM, n=5) also had no significant (p>0.02) effect on the inhibition of Cav3.3 currents by M1 receptors (Fig. 2.3D).  In this regard, classical Gαq/11 downstream effectors such as PKC, PKA, and increased cytosolic Ca2+ concentration appear not to be directly involved in the M1 receptor-mediated inhibition of Cav3.3 T- type Ca2+ currents.  Phospholipase C activity has recently been shown to directly inhibit voltage-gated ion channels through the depletion of membrane PIP2 levels, which are thought to stabilize active channels in the membrane (Suh and Hille, 2005).  Dialyzing cells with a PIP2 antibody (50 μg/mL) to reduce available PIP2 levels had no significant effect (p>0.05) on the M1-receptor mediated inhibition of Cav3.3 currents (Fig. 2.5B,D).  Similarly, dialyzing cells with synthetic PIP2 (200 μM di-C8 PIP2) to saturate membrane PIP2 levels also had no significant effect (p>0.05) on M1 receptor-mediated inhibition of Cav3.3 currents (Fig. 2.5C,D). Chapter 2: Inhibition of Cav3.3 by mAChRs   76 E A C F B D Tra ns fec ted  Co ntr ol Ga lph a1 3-Q 22 6L Ga lph aq -Q 20 9L Ga lph a1 1-Q 20 9L 0 20 40 60 80 100 120 **τ in ac t ( m s) 50 ms 25 0 pA Cav3.3: Transfected Control 20 0 pA 50 ms Cav3.3 + Gα11-Q209L 50 ms 25 0 pA Cav3.3 + Gα13-Q226L 50 ms 25 0 pA Cav3.3 + Gαq-Q209L Tra ns fec ted  Co ntr ol Ga lph a1 3-Q 22 6L Ga lph aq -Q 20 9L Ga lph a1 1-Q 20 9L 0.0 0.2 0.4 0.6 0.8 1.0 ** C ur re nt  R at io  Figure 2.4 - The Gαq/11 subtypes of Gα proteins specifically cause inhibition of Cav3.3 currents. A-D) Representative whole-cell current traces of stable Cav3.3 cells transfected with various control or Gα plasmids during depolarizing steps from -110 mV to -30 mV.  Traces were obtained 30 seconds (black) and 2 minutes (grey) after the whole-cell conformation was formed, using an internal solution that contained 4 mM ATP and 0.3 mM GTP.  The stable Cav3.3 cells were mock-transfected with empty plasmid (A) or transfected with the constitutively active forms (lack of GTPase activity) of Gα-proteins: Gα13-Q226L (B), Gαq-Q209L (C), Gα11-Q209L (D).  E) Gαq-Q209L and Gα11-Q209L cause a time-dependent reduction in Cav3.3 current magnitude.  The peak current levels at 2 minutes were divided by the peak current levels at 30 seconds to determine the level of inhibition due to internal solution dialysis for the various types of transfected Cav3.3 cells, as described above.  The Cav3.3 currents co-transfected with Gαq-Q209L and Gα11-Q209L had a significant (p<0.001) reduction in current ratio compared to the control transfection, while the Gα13-Q226L transfection caused no significant (p>0.05) change.  F) The rate of inactivation (τinact) was determined during depolarizing steps from -110 mV to -30 mV for all transfection types.  The τιnact was significantly (p<0.001) faster for Gαq-Q209L and Gα11-Q209L compared to control transfections, while the τιnact was not significantly (p>0.02) different for Gα13-Q226L. All data points correspond to mean +/- S.E. Chapter 2: Inhibition of Cav3.3 by mAChRs   77  0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1mM CCh N or m al iz ed  C ur re nt Time (s) 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1mM CCh N or m al iz ed  C ur re nt Time (s) 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt Time (s) 1mM CCh A C B D -40 -30 -20 -10 0 10 Co ntr ol di- c8  PI (4, 5)P 2 PIP 2 A b %  M od ul at io n of  C av 3. 3 by  M 1  Cav3.3 + M1 Cav3.3 + M1 + PIP2 Abs Cav3.3 + M1 + di-C8 PIP2  Figure 2.5 - Inhibition of Cav3.3 channels by M1 does not require PIP2 signaling. A) In whole-cell recordings of HEK 293 cells co-transfected with human Cav3.3 and M1, application of 1 mM CCh caused inhibition of Cav3.3 currents (-27.7% +/- 1.5%, n=15).  B) Dialyzing cells with 50 μg/mL PIP2 antibody for 10+ minutes had no effect on the inhibition of human Cav3.3 currents by M1 receptor activation (-30.8% +/- 3.9%, n=7).  C) Dialyzing cells with 200 μM di-C8 PIP2 for 5+ minutes had no effect on the inhibition of human Cav3.3 currents by M1 receptor activation (-29.0% +/- 1.8%, n=6).  D) Bar graph showing that attenuating PIP2 signaling with either PIP2 antibodies or di-C8 PIP2 had no significant effect (p>0.05) on M1-mediated inhibition compared to whole-cell recordings from control human Cav3.3 + M1 cells. Chapter 2: Inhibition of Cav3.3 by mAChRs   78 As a positive control for di-C8 PIP2 activity and as previously shown (Bian et al., 2001), dialysis of 200 μM di-C8 PIP2 into HEK 293 cells stably expressing HERG K+ channels caused a significant (p<0.02) stimulation of K+ channel currents (n=8) compared to control recordings (n=5; data not shown). Taken together, these results indicate that inhibition of Cav3.3 by M1 receptors occurs either directly through Gαq/11, or a downstream pathway that is independent of PIP2 metabolism and other classical effectors.  2.2.6 Gαq/11-coupled muscarinic receptors selectively inhibit Cav3.3 channels If inhibition of Cav3.3 T-type Ca2+ channels by M1 receptors is primarily dependent on Gαq/11 signaling, then all Gαq/Gα11-coupled mAChRs should similarly inhibit Cav3.3 currents, while Gαi- coupled mAChRs should have no effect.  Indeed, activation of co-expressed Gαi-coupled M2 and M4 receptors with 1 mM CCh had no effect on Cav3.3 current amplitude (M2 = -4% +/- 2%, n=11; M4 = - 4% +/- 3%, n=8) or kinetics (Fig. 2.6A,C,E,G; Table 2.1).  In contrast, upon transfection of either the Gαq/Gα11-coupled M3 or M5 receptor subtypes into stable Cav3.3 cells perforated patch recordings revealed a significant CCh-mediated inhibition (M3 = -25% +/- 3%, n=10; M5 = -31% +/- 3%, n=10) as well as a concomitant increase in both activation and inactivation kinetics (Fig. 2.6B,D,F,H; Table 2.1). Overall, experiments with genetically encoded antagonists of Gαq/11 (RGS2) and Gβγ (MAS- GRK3ct) and genetically-encoded Gα subtypes, as well as inhibition experiments with various mAChRs all support the assertion that inhibition of Cav3.3 channels by mAChRs specifically occurs through Gαq/11.  2.2.7 Two distinct Cav3.3 channel regions are involved in M1-mediated inhibition Most modulation of Ca2+ channels by intracellular signaling pathways involves physical interactions between various effectors and cytoplasmic channel domains (Wolfe et al., 2003; Zamponi and Snutch, 2002).  Chimeric T-type Ca2+ channels between human Cav3.1 and human Cav3.3 were generated to determine the molecular regions of the Cav3.3 channel involved in the M1 receptor- mediated inhibition (Fig. 2.7).  The Cav3.1 and Cav3.3 full-length channels were initially divided into 4 approximately equal portions and chimeric channels were constructed using restriction enzyme digestion and re-ligation (see (Hamid et al., 2006)).  The four channel portions were named as followed: Region 1 = amino-terminus + domain I, Region 2 = domain I-II linker, domain II + the first 39-63 amino acids of the domain II-III linker,  Region 3 = remainder of the domain II-III linker + domain III, and Region 4 = the domain III-IV linker, domain IV + the carboxyl-terminus.  Chimeric channel names were assigned based on whether the chimera contained Cav3.1 (G) or Cav3.3 (I) sequence in each of the four regions described (e.g., the chimeric Cav3.3 channel that contained Region 2 from Cav3.1 is called IGII). Chapter 2: Inhibition of Cav3.3 by mAChRs   79 CCh Control 50 ms 40 0 pA 50 ms 40 0 pA C A B D G E F H 50 ms 40 0 pA Cav3.3 + M2 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh 0 50 100 150 0.6 0.7 0.8 0.9 1.0 1.1 1.2 N or m al iz ed  C ur re nt Time (s) 1mM CCh Cav3.3 + M4 Cav3.3 + M5 50 ms 20 0 pA Control CCh Cav3.3 + M3  Figure 2.6 - Inhibition of Cav3.3 currents occurs specifically through Gαq/11-coupled mAChRs. A,C) Representative perforated patch current traces during depolarizing pulses from -110 mV to -40 mV showing no effect on Cav3.3 currents by the Gαi-coupled M2 and M4 receptors, respectively.  Traces during control perfusion and perfusion of 1 mM CCh are indistinguishable and CCh application had no significant effect on channel kinetics (Table 2.1).  E,G) Averaged time course of normalized peak current levels during perfusion of control recording solution (2 mM Ca2+) followed by 1 mM CCh for Cav3.3 (+M2/M4) currents.  Perfusion of CCh had no effect on Cav3.3 currents for M2 (E; -4% +/- 2%, n=11) and M4 (G; -4% +/- 3%, n=8) receptors.  B,D) Representative perforated patch current traces during depolarizing pulses from –110 mV to –40 mV showing inhibition of Cav3.3 currents by the Gαq/11-coupled M3 and M5 receptors, respectively.  Activating the receptors with 1 mM CCh also significantly (p<0.02) increased channel kinetics (Table 2.1).  F,H) Averaged time course of normalized peak current levels during perfusion of control recording solution (2 mM Ca2+) followed by 1 mM CCh for Cav3.3 plus either M3 or M5 receptors.  Perfusion of CCh caused a 25% +/- 3% decrease (n=10) of Cav3.3 currents with co-transfected M3 receptors (F), and a 31% +/- 3% decrease (n=10) of Cav3.3 currents with co-transfected M5 receptors.  Only a small number of cells (n=2 for both M3 and M5) were not inhibited by CCh.  All data points correspond to mean +/- S.E. Chapter 2: Inhibition of Cav3.3 by mAChRs   80 0 50 100 150 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt T im e (s) 1m M  CCh 0 50 100 150 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt T im e (s) 1m M  CCh C A B D I I I I 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 0 50 100 150 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt T im e (s) 1m M  CCh I G I G 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 0 50 100 150 0.7 0.8 0.9 1.0 1.1 N or m al iz ed  C ur re nt T im e (s) 1m M  CCh G G G 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 G G I G 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 I E -30 -20 -10 0 10 III I GI II IG II IIG I III G IG IG GG GG IG GG GI GG GG IG GG GI GI GI ** ** ** * * %  M od ul at io n by  M 1  Chapter 2: Inhibition of Cav3.3 by mAChRs   81      Figure 2.7 - Regions II and IV of human Cav3.3 channels are required and appear sufficient for M1-mediated inhibition. A-D) (Left) Schematic diagrams of the various chimeric channels that were co-transfected into HEK cells with M1.  The blue transmembrane domains and intra/extra-cellular regions correspond to Cav3.3 (labeled I) sequences, while the red transmembrane domains and intra/extra-cellular regions correspond to Cav3.1 (labeled G) sequences.  (Middle) The effect of activating M1 receptors with 1 mM CCh on the normalized peak current levels of chimeric channel types shown to the left.  Inclusion of Cav3.1 sequence at Regions II and IV (C) eliminated M1-mediated inhibition and attenuated the effect on inactivation kinetics, while inclusion of Cav3.3 sequence at Regions II and IV (D) restored M1-mediated inhibition to a level that was not significantly (p>0.05) different from IIII inhibition levels (see Table 2.1).  (Right) Insets include chimeric whole-cell current traces during depolarizing pulses from –110 mV to peak potential before (line arrow) and after (block arrow) application of 1 mM CCh.  Traces are representative of the various chimeras in terms of activation and inactivation kinetics as well as magnitude of inhibition.  For inset scale bars, x = 50 ms, y = 100 pA.  E) Histogram where GIII, IGII, IIGI, IIIG, and IGIG inhibition values were statistically compared to the IIII control while IGGG, GIGG, GGIG, and GGGI values were compared to the GGGG control and the GIGI value was compared to both the IIII and GGGG controls. * indicates significant difference (<0.02) compared to IIII inhibition, while ** indicates a significant difference (p<0.02) compared to GGGG modulation levels.  All data points correspond to mean +/- S.E.  Chapter 2: Inhibition of Cav3.3 by mAChRs   82 Co-expression of M1 receptors with chimeric GIII and IIGI T-type channels both showed a similar degree of M1 receptor-mediated peak current inhibition compared to that of the inhibition of the wt Cav3.3 channel (IIII) (Fig. 2.7A,E).  In contrast, when the IGII chimera was co-transfected with M1 receptors, application of 1 mM CCh resulted in a significantly attenuated degree of inhibition (-5.6% +/- 2.1%, n=11, (p<0.001)) compared to the wt IIII channel (-26.9% +/- 2.3%, n=9; Fig. 2.7E). Interestingly, while the chimeric IGII channels exhibited lowered M1 receptor-mediated inhibition they still possessed significantly increased inactivation kinetics (p<0.001; Table 2.2).  Finally, while the IIIG chimeric channels showed similar degree of M1 receptor-mediated peak current inhibition compared to the wt IIII, the rate of inhibition was notably slower (not shown).  The changes in the rate of inhibition for IIIG and the significant decrease in the amount of inhibition for IGII suggested that both Regions 2 and 4 might be involved in the M1-induced inhibition of Cav3.3 channels.  To explore this, a double chimera (IGIG) was co-transfected into HEK cells with M1 receptors.  Figures 2.7C and 2.7E show that the inhibiting effect of 1 mM CCh application on peak current amplitude was completely abolished for the IGIG chimera (0.9% +/- 2.5%, n=8).  Activation of M1 with 1 mM CCh still caused a significant increase (p<0.001) in the inactivation kinetics of IGIG, but the τinact decreased by less than 25% for IGIG while it decreased by 40-65% for all the single chimeric and wt Cav3.3 channels (Table 2.2). The chimeric channel loss-of-function experiments indicate that both Regions 2 and 4 are involved in the M1-mediated inhibition of current amplitude and increase in inactivation kinetics of Cav3.3 currents.  In gain-of-function experiments, substitution of either Region 2 (GIGG) or Region 4 (GGGI) into the Cav3.1 channel resulted in 1 mM CCh-induced inhibition (GIGG = -14.3% +/- 0.8%, n=7; GGGI = -9.1% +/- 2.6%, n=9) that was significantly different (p<0.001 and p<0.02, respectively) when compared to GGGG (-0.3% +/- 2.2%, n=9; Fig. 2.7B,E; Table 2.2).  In contrast, inclusion of either Region 1 or Region 3 of Cav3.3 into Cav3.1 resulted in no significant change (p>0.05) in M1-mediated inhibition when compared to GGGG (Fig. 2.7E).  Although both GIGG and GGGI were inhibited by M1, the level of inhibition was significantly lower (p<0.001) than the inhibition of IIII by M1 (Fig. 2.7E). When the effect of 1 mM CCh application on GIGI current amplitude was tested, M1 activation was found to produce a significant level of GIGI inhibition (-25.1% +/- 2.4%, n=11; p<0.001) compared to GGGG that was not significantly (p>0.05) different from the inhibition of IIII (Fig. 2.7A,B,D,E). Application of 1 mM CCh also significantly increased (p<0.001) the rate of inactivation for the GIGI Cav3.1 chimera, but not for GIGG, GGGI, or the other Cav3.1 single chimeras (Table 2.2).  Overall, the combined substitution of Regions 2 and 4 from the Cav3.3 channel into the Cav3.1 channel completely restores M1-induced inhibition together with the associated increase in channel inactivation kinetics. Chapter 2: Inhibition of Cav3.3 by mAChRs   83 Table 2.2 - Effects of M1 Receptor Activation on Chimeric T-Type Channel Inactivation Kinetics  * p < 0.02, ** p < 0.001  2.3 Discussion In the present paper we systematically explored the effects of activated mAChRs on the three main T-type Ca2+ channel isoforms expressed in the mammalian nervous system and report for the first time the differential modulation between a G-protein signaling pathway and Cav3.3 T-type Ca2+ channels.  Most studies on T-type Ca2+ channel modulation have involved the Cav3.2 (α1H) subtype, revealing specific modulatory responses to Gβ2γ, CAMKII, and redox modulation that are not observed for the Cav3.1 and Cav3.3 T-type Ca2+ channel isoforms (Joksovic et al., 2006; Welsby et al., 2003; Wolfe et al., 2003).  The exclusive inhibition of Cav3.3 channels by Gαq/11-coupled mAChRs is the first example of specific, GPCR-mediated modulation of a T-type Ca2+ channel subtype other than for Cav3.2.  2.3.1 Differential effects of mAChRs on T-type calcium channel isoforms Examination of the literature shows that activation of mAChRs can result in multiple effects on native T-type Ca2+ currents including causing stimulation (Fisher and Johnston, 1990; Fraser and MacVicar, 1991; Pemberton et al., 2000), inhibition (Wan et al., 1996), or having no effect (Allen and Brown, 1993).  Given the heterogeneous nature of native low threshold Ca2+ currents, without investigating interactions between specific mAChR gene products and specific T-type Ca2+ channel isoforms, the published differences in modulation are nearly impossible to interpret.  Our results using exogenous expression of cloned T-type Ca2+ channels indicates that M1 receptor activation has a robust inhibitory effect on Cav3.3 currents and has either no effect or a small stimulation on both Cav3.1 and Cav3.2 currents.  Similarly, experiments examining native Cav3.2 Ca2+ channels in NIH3T3 cells transiently transfected with mAChRs demonstrated that M1 receptor activation had either no effect or a stimulatory effect if a PKC inhibitor was applied (Pemberton et al., 2000).  Active Gβ2γ subunits have been shown to specifically inhibit Cav3.2 currents (DePuy et al., 2006; Wolfe et al., 2003) and the lack of inhibition of Cav3.2 channels by M1 receptors in our study is likely due to the absence of any functional coupling between M1 receptors and Gβ2 proteins (Dippel et al., 1996).  We also found that all Gαq/11- coupled mAChR subtypes (M1, M3, and M5) cause attenuation of Cav3.3 currents while Gαi-coupled IIII IG II IIIG IG IG GG GG GI GG GG GI GI GI Control − τinact  (ms) 117 +/- 6, n=10 86 +/- 7, n=11 103 +/- 11, n=10 62 +/- 2, n=8 14 +/- 1, n=9 20 +/- 1, n=7 38 +/- 3, n=8 58 +/- 2, n=10 1mM CCh - τinact  (ms) 41 +/- 5, n=10** 43 +/- 3, n=11** 58 +/- 4, n=10* 47 +/- 3, n=8* 13 +/- 1, n=9 17 +/- 2, n=7 32 +/- 2, n=8 38 +/- 1, n=10** Chapter 2: Inhibition of Cav3.3 by mAChRs   84 M2 and M4 receptors had no effect on Cav3.3 currents.  Thus it is likely that any stimulation of T-type Ca2+ currents by mAChRs in native systems does not involve Cav3.3 channels.  Experiments testing the effects of recombinant M2-M5 receptors on the Cav3.2 and Cav3.1 Ca2+ channel isoforms in a heterologous system are required to further facilitate the possibility of interactions between these T-type channels and mAChRs.  2.3.2 Functional effects of M1 receptor activation on Cav3.3 currents Activation of M1 receptors dramatically altered Cav3.3 currents by both reversibly attenuating peak current levels and increasing the rate of inactivation, resulting in a significant reduction in the influx of Ca2+.  The relationship between these effects was explored using both structural channel chimeras and classical gating property studies.  In chimeric studies (see section 2.3.4), the activation of M1 receptors primarily caused an increase in inactivation kinetics of the IGII chimera and conversely, primarily a decrease in peak current levels for the GIGG chimera.  Both this isolation of the two specific M1 receptor-mediated effects and the gating results discussed below suggest that the effects of M1 on current amplitude and inactivation kinetics are complementary but distinct phenomena.  For gating studies, reduction of Cav3.3 current magnitude by M1 receptor activation was equally robust when the Cav3.3 channels were held in various states: (1) during a prolonged hyperpolarization with no test depolarizations (channels mostly in closed state), (2) after a strong hyperpolarizing prepulse to -140 mV, and (3) during 200 ms test depolarizations to peak potential at 0.2 Hz and 0.5 Hz.  Combining this lack of use-dependence with the observed reduction in peak current amplitude and the increase in activation and inactivation kinetics indicates that all states of the Cav3.3 channel are subject to modulation by M1 receptor activation.  The acceleration of Cav3.3 channel kinetics by M1 receptor activation also supports the hypothesis that modulation affects channel biophysical properties and not channel density via internalization, which has recently been shown to occur for the voltage-independent, GPCR-mediated inhibition of N-type Ca2+ channels on a relatively fast timescale (Tombler et al., 2006).  Physiologically, the combined decrease in Cav3.3 peak currents and the increased activation and inactivation kinetics would be predicted to alter neuronal firing patterns and perhaps eliminate rhythmic oscillations (Chemin et al., 2002; Chevalier et al., 2006).  In support of this notion, the concomitant reduction in peak current and increase in inactivation kinetics of Cav3.3 currents triggered by anandamide has been shown to completely eliminate the sustained, rhythmic Cav3.3 current during an AP voltage clamp experiment with an oscillating thalamic waveform (Chemin et al., 2001).  Chapter 2: Inhibition of Cav3.3 by mAChRs   85 2.3.3 Signal transduction pathway of M1 receptor-mediated Cav3.3 inhibition Use of genetically-encoded antagonists of Gβγ (MAS-GRK3ct and Gαt) and Gαq/11 (RGS2) demonstrated that Gβγ may partially contribute to the M1-mediated inhibition of Cav3.3 currents, while Gαq/11 is absolutely required for complete inhibition.  The potential involvement of both Gαq/11 and Gβγ in a non-classical, voltage-independent mechanism of Ca2+ channel inhibition by mAChR has been previously described for HVA Ca2+ channels.  In rat SCG sympathetic neurons application of a muscarinic agonist causes the voltage-independent inhibition of endogenous N-type Ca2+ channels that is abolished by co-expression of RGS2, Gαt, or MAS-GRK3ct and exhibits a time course similar to the Cav3.3 inhibition reported here (Kammermeier et al., 2000).  As Gβγ is a cofactor for PLCβ activity, a possible explanation is that sequestering Gβγ reduces PLCβ activity (Rhee and Bae, 1997).   Although Gβγ may potentiate the inhibitory effect of M1 receptor activation, transfection of constitutively active Gαq/11 mutants into stable Cav3.3 cells demonstrated that active Gαq/11 alone is sufficient to induce the inhibition of Cav3.3 currents.  In support of this notion, only Gαq/11-coupled mAChRs (M1, M3, and M5) inhibited Cav3.3 currents, while Gαi-coupled M2 and M4 receptors that activate Gβγ signaling have no effect on Cav3.3 currents.  Unlike that reported for the attenuation of Cav3.2 channels by Gβ2γ, this novel form of T-type Ca2+ channel inhibition involves the Gαq/11 subunit and also affects channel kinetics. This inhibitory mechanism for the Cav3.3 T-type isoform may be applicable to all Gαq/11-coupled receptors as we have also found a similar inhibition of Cav3.3 channels by mGluR1a receptors (Hildebrand et al., 2005) (Fig. 3.1). Pharmacological antagonists eliminated the potential involvement of various intracellular signals downstream of Gαq/11 activation that may be involved in the inhibition of Cav3.3 by M1 receptor activation.  Abolishing the activity of PKC, serine/threonine kinases (including PKA), tyrosine kinases, phosphatases, phosphoinositide-3-kinases and intracellular Ca2+ signaling all had no effect on inhibition. This profile of M1/Gαq/11-mediated Ca2+ channel inhibition resistant to common antagonists of cytoplasmic signaling is not unique and has been reported for the inhibition of L-type channels by Gαq- coupled M1/3/5 receptors in HEK cells (Bannister et al., 2002).  Like the inhibition of Cav3.3 via M1 receptors, this inhibition is voltage-independent, relatively slow kinetically (τon=13 seconds), and insensitive to antagonists of protein kinases and protein phosphatases (Bannister et al., 2002). A more recent explanation for the Gαq/11-mediated inhibition of ion channels including voltage- gated K+ channels and HVA Ca2+ channels has emerged wherein channel activity is suppressed through the depletion of membrane PIP2 levels via PLC activity (Gamper et al., 2004; Suh et al., 2004; Wu et al., 2002).  In these studies, Gαq/11-mediated inhibition was shown to be inhibited via dialysis of synthetic PIP2 or a PIP2-specific antibody into the cytoplasm.  In our experiments, adding di-C8 PIP2 or the PIP2 antibody into the internal pipette solution and dialyzing cells for up to 25 minutes had no significant Chapter 2: Inhibition of Cav3.3 by mAChRs   86 effect on M1-mediated inhibition of Cav3.3 channels, suggestive of another to-be-defined mechanism whereby Gαq/11 signaling causes the inhibition of voltage-gated ion channels.  Further biophysical and biochemical experiments are required to clarify the nature of the intracellular messengers and/or scaffolding proteins that can modulate Cav3.3 T-type Ca2+ channels and also whether Gαq/11 can interact directly with the channel through a novel mechanism.  2.3.4 Gαq/11-mediated inhibition of Cav3.3 involves two discrete channel regions Replacing both Regions 2 and 4 in the Cav3.3 channel with the corresponding Cav3.1 T-type Ca2+ channel sequences abrogated both the M1 receptor-mediated peak current inhibition and concomitant increase in inactivation kinetics.  Conversely, substituting Regions 2 and 4 from Cav3.3 into Cav3.1 conferred M1 receptor-mediated inhibition and increased inactivation kinetics.  These data suggest that Regions 2 and 4 of the Cav3.3 channel are both necessary and sufficient to recapitulate M1 receptor- mediated channel modulation.  Region 2 of the Cav3.1 and Cav3.3 sequence contains the highly divergent domain I-II linker, the highly conserved domain II and 39-63 amino acids of the domain II-III linker while Region 4 contains most of the III-IV linker, the highly conserved domain IV and the highly divergent C-terminus.  Based on their putatively intracellular regions and their high divergence between the two T-type isoforms, the I-II linker, proximal region of the II-III linker, the III-IV linker, and the C- terminus are all candidates for modulation sites within Regions 2 and 4.  Interestingly, the only identified sites of alternative splicing within the rat and human Cav3.3 channel occur both in the I-II linker and the C-terminal regions (Mittman et al., 1999; Murbartian et al., 2002) (Fig. 1.4).  The effects of these splicing variations on the biophysical properties (activation kinetics) of the human Cav3.3 channel are interdependent rather than additive, suggesting a possible direct interaction between the I-II linker and C- terminus that affects channel kinetics (Murbartian et al., 2004).  Both the human and rat Cav3.3 channels inhibited by M1 receptor activation in our study lack exon 9 located in the I-II linker, while both the rat and human Cav3.1 channels have a 10 amino acid insertion in this region in a manner similar to that for the + exon 9 Cav3.3 splice variant.  Thus, several observations suggest that the I-II linker may be a target region in the inhibition of Cav3.3 by M1 and some evidence points to a possible role for the C-terminus. However, as multiple structural determinants contribute to the slow inactivation kinetics of Cav3.3 compared to Cav3.1 in a nearly additive manner (Hamid et al., 2006), and M1 activation dramatically speeds up Cav3.3 inactivation kinetics, it is also possible that multiple intracellular loci within Regions 2 and 4 of the Cav3.3 channel may be involved in the M1-mediated effect. In summary, we find that activation of known Gαq/11-coupled mAChRs results in the selective inhibition of Cav3.3 T-type Ca2+ currents with a concomitant increase in inactivation kinetics.  The Gαq/11-mediated signaling pathway appears to be mediated via two disparate regions of the Cav3.3 channel.  Functional interactions between mAChRs and Cav3.3 Ca2+ channels could potentially impact Chapter 2: Inhibition of Cav3.3 by mAChRs   87 firing patterns of various cell types including thalamic nRT cells.  Both biophysical and pharmacological evidence suggests that primarily Cav3.3 channels compose dendritic T-type currents in nRT cells (Joksovic et al., 2005) while immunostaining suggests the presence of M3 receptors in these cells (Levey et al., 1994).  This raises the possibility that the inhibition of Cav3.3 T-type Ca2+ channels by M3 receptors in the dendrites of nRT cells could be involved in cholinergic modulation of thalamic firing patterns.  2.4 Experimental procedures 2.4.1 Molecular biology Human Cav3.1- Cav3.3 T-type Ca2+ channel α1 subunit chimeras were constructed as described in detail by Hamid et al (Hamid et al., 2006).  2.4.2 Cell culture and transfection HEK 293H (Invitrogen, ON, Canada) cells were grown in standard DMEM (10% fetal bovine serum and 50 U/ml penicillin-streptomycin) to ~80% confluence and maintained at 37oC in a humidified incubator with 95% atmosphere and 5% CO2.  The generation of stable T-type cell lines (in HEK 293, tsa-201) expressing rat brain Cav3.1, Cav3.2, or Cav3.3 α1 subunits has been previously described (Santi et al., 2002).  Stable cell lines were transiently transfected with human muscarinic M1, M2, M3, M4, or M5 cDNAs (all in pcDNA3.1) using Lipofectamine (Invitrogen).  As a reporter for transfection, all transient transfections included co-transfection with either CD8 or pEGFP marker plasmids at a 1:4 molar ratio compared to receptor and/or channel plasmid DNA, unless otherwise indicated. Lipofectamine-mediated transfections used 1 to 1.25 μg of DNA/35 mm dish and 5 μL of Lipofectamine/dish.  In G-protein experiments, stable Cav3.3 cells were co-transfected with M1 receptors and equal amounts of either MAS-GRK3ct (in pcDNA3.1), Gαt (in pcDNA3.1) or RGS2 (in pEGFP) using Lipofectamine.  Only MAS-GRK3ct and Gαt transfections required co-transfection with marker plasmids as RGS2 expression could be directly detected with fluorescence. In Gα transfection experiments, stable Cav3.3 cells were transfected with constitutively active mutants of Gαq, Gα11 or Gα13 (Gαq-Q209L, Gα11-Q209L, and Gα13-Q226L, respectively, all in pcDNA3.1) using Lipofectamine.  Twelve to 18 hours after transfection the media was changed from Opti-Mem I to regular DMEM and cells were transferred to a 28oC incubator.  The M1 to M5, Gαq-Q209L, Gα11-Q209L, and Gα13-Q226L cDNAs were all obtained from the UMR cDNA Resource Center (Rolla, MO) while the RGS2 and MAS-GRK3ct constructs were a generous gift from Dr. Brett Adams. In separate experiments, HEK 293H cells were co-transfected with M1 and wt or chimeric human Cav3.1 or Cav3.3 channels using standard Ca2+ phosphate transfection with 2 μg of total Chapter 2: Inhibition of Cav3.3 by mAChRs  88 cDNA/dish, 0.15 to 0.4 μg channel cDNA/dish, and 0.2 μg M1 cDNA/dish.  In a subset of these experiments involving co-transfection of wt Cav3.3 and M1, either 200 μM di-C8 PI(4,5)P2 (Echelon Biosciences Inc., Salt Lake City, UT) or 50 μg/mL PI(4,5)P2 IgG2b antibody (~1:30 dilution) (Assay Designs, Ann Arbor, MI) was included in the internal solution to explore the role of PIP2 signaling.  As the PIP2 antibody was supplied in a PBS buffer solution containing 10% calf serum and 0.05% sodium azide, the control Cav3.3 + M1 cells were recorded in an internal solution containing a 1:30 dilution of PBS with 10% fetal bovine serum and 0.05% sodium azide.  Electrophysiological recordings for all experiments were performed 24-48 hours after transfection.  Transiently transfected cells were selected for CD8 or pEGFP expression using either adherence of Dynabeads (Dynal, Great Neck, NY) or fluorescence of EGFP under UV light.  2.4.3 Electrophysiological recordings and analysis Macroscopic currents were recorded using the perforated patch-clamp technique to reduce current rundown and to preserve cytoplasmic signaling pathways.  The external recording solution contained (in mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 40 TEACl, 92 CsCl, 10 glucose, pH=7.4, while the internal pipette solution contained (in mM): 120 CsMethanesulfonate, 11 EGTA, 10 HEPES, 2 MgCl2, 75-100 μM β-Escin pH=7.2.  For these perforated patch recordings, experimental recording did not begin until the RS was below 20 MΩ and constant, as measured by amplifier compensation. Whole-cell recordings were used for the transiently transfected wt or chimeric human Cav3.1 and Cav3.3 channel experiments as well as the Gα transfection experiments.  The internal solution for these recordings contained (in mM): 120 CsMethanesulfonate, 11 EGTA, 10 HEPES, 2 MgCl2, 4 Mg-ATP, 0.3 Na-GTP. Macroscopic currents were recorded using Axopatch 200A and 200B amplifiers (Axon Instruments, Foster City, CA), controlled and monitored with Pentium 4 personal computers running pClamp software version 9 (Axon Instruments).  Patch pipettes (borosilicate glass BF150-86-10; Sutter Instruments, Novato, CA), were pulled using a Sutter P-87 puller and polished with a Narishige (Tokyo, Japan) microforge, with typical resistances of 3-6 MΩ when filled with internal solution. The bath was connected to the ground via a 3 M KCl agar bridge. Data were low-pass filtered at 2 kHz using the built-in Bessel filter of the amplifier, with sampling at 10 kHz.   The amplifier was also used for capacitance and series resistance compensation between 70% and 85% on every cell.  Leak subtraction of capacitance and leakage current was performed on-line using a P/5 protocol, or else performed with Clampfit (Axon Instruments) during offline analysis.  Figures and fittings utilized the software program Microcal Origin (Version 7.5, Northampton, MA).  All recordings were performed at room temperature (20-22oC). Chapter 2: Inhibition of Cav3.3 by mAChRs  89 The voltage-dependence of activation for Cav3.1, Cav3.2, and Cav3.3 currents were measured by a series of 100 ms to 220 ms depolarizing pulses applied from a holding potential of -110 mV to membrane potentials from -80 mV to +10 mV, increasing at 5 mV increments, with 2 seconds between pulses.  The potential that elicited peak currents (“peak potential”, ranging from -45 to -25 mV) was obtained from this protocol and used in subsequent protocols.  Series resistance was also monitored with a 5 ms depolarizing pulse to -105 mV immediately before the test pulse to ensure that this variable was relatively constant and any changes in peak current levels were not due to significant changes in Rs. Effects of saturating concentrations of mAChR agonist (1 mM CCh) on stable T-type currents were then investigated using steps to peak potential every 5 seconds (0.2 Hz) from a holding potential of -110 mV. These depolarizing steps were 80 ms in duration for Cav3.1 and Cav3.2, and 200 ms in duration for Cav3.3.  The -140 mV prepulse protocol for Cav3.3 included a 1 second prepulse to -140 mV to remove any accumulated channel inactivation.  To quantify the percent of channel inhibition, stimulation, or washout during CCh or control solution perfusion, the peak current magnitude at equilibrium was averaged (2-5 values).  When distinct effects were observed (i.e. - stimulation versus no effect of M1 on Cav3.1 currents) all cells displaying a >10% modulating effect with a clear exponential time course were grouped into one group, while the rest of the cells were grouped into the “no effect” group. Current-voltage relationships were fitted with the modified Boltzmann equation: I = [Gmax*(Vm- Erev)]/[1+exp({Vm-V0.5a}/ka)], where Vm is the test potential, V0.5a is the half-activation potential, Erev is the extrapolated reversal potential, Gmax is the maximum slope conductance, and ka reflects the slope of the activation curve.  Data from CCh concentration-response studies were fitted with the equation y = [(A1- A2)/{1+(x/xo)P} + A2] where A1 is initial amplitude (= 0) and A2 is final block value, xo is IC50 (concentration causing 50% inhibition of currents), and P gives a measure of the steepness of the curve. The activation and inactivation rates during steps to peak potential were well described by single exponential curves to give τact and τinact values, respectively. Statistical significance was tested with Student’s T-tests with significance being determined at a confidence interval of p<0.02.  2.4.4 Solutions, drugs, and perfusion A 25 mM stock of β-Escin (in dH2O) was prepared fresh, with dilution to working stocks in intracellular solution.  Carbachol was added directly to the extracellular recording solution. Wortmannin, okadaic acid, genistein, and H9 were all obtained from Tocris Cookson (Ellisville, MO). Rp-cAMPs was obtained from BioMol International (Plymouth Meeting, PA).  BAPTA-AM was obtained from Molecular Probes (Oregon, USA).  Unless otherwise stated, all other drugs were obtained from Sigma-Aldrich (St. Louis, MO).  Drugs were dissolved in either dH2O or DMSO, according to Chapter 2: Inhibition of Cav3.3 by mAChRs  90 manufacturer’s solubility data.  The highest concentration of DMSO in the recording solution did not exceed 0.1%, a concentration that did not detectably affect T-type properties (data not shown).  Gravity- driven perfusion occurred at a rate of approximately 400 μl/minute, and the outputs of the manifold were placed within close proximity of the cell, resulting in the cell being bathed in new solutions with minimal delay (within 1 sec).  2.5 Acknowledgements We thank Dr. Brett Adams for the kind gifts of MAS-GRK3ct and EGFP-RGS2, and Drs. Aaron Beedle, Arnaud Monteil and Philippe Lory for the human Cav3.1 and Cav3.3 channels used in the chimeric channel experiments.  Thanks also to Dr. Colin Thacker, Tracy Evans, Paul Adams, and Dr. John Tyson for molecular biology support, to Diana Janke and Dr. David Parker for providing stable cell lines, and to Dr. Philippe Isope for comments on the manuscript.  The work is supported by operating grants from the Canadian Institutes of Health Research and Canada Research Tier 1 Chairs to T.P.S and G.W.Z., and trainee fellowships from the Natural Sciences and Engineering Research Council of Canada and Michael Smith Foundation for Health Research to M.E.H.  L.S.D. is supported by a fellowship from the Heart and Stroke Foundation of Canada.  Chapter 2: Inhibition of Cav3.3 by mAChRs  91 2.6 References Allen, T.G., and Brown, D.A. (1993). M2 muscarinic receptor-mediated inhibition of the Ca2+ current in rat magnocellular cholinergic basal forebrain neurones. J Physiol 466, 173-189. Anderson, M.P., Mochizuki, T., Xie, J., Fischler, W., Manger, J.P., Talley, E.M., Scammell, T.E., and Tonegawa, S. (2005). Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc Natl Acad Sci U S A 102, 1743-1748. Bannister, R.A., Melliti, K., and Adams, B.A. (2002). Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels. Biophys J 83, 3256-3267. Bian, J., Cui, J., and McDonald, T.V. (2001). HERG K(+) channel activity is regulated by changes in phosphatidyl inositol 4,5-bisphosphate. Circ Res 89, 1168-1176. Bourinet, E., Alloui, A., Monteil, A., Barrere, C., Couette, B., Poirot, O., Pages, A., McRory, J., Snutch, T.P., Eschalier, A., and Nargeot, J. (2005). Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. Embo J 24, 315-324. Castillo, P.E., Carleton, A., Vincent, J.D., and Lledo, P.M. (1999). Multiple and opposing roles of cholinergic transmission in the main olfactory bulb. J Neurosci 19, 9180-9191. Chemin, J., Monteil, A., Perez-Reyes, E., Bourinet, E., Nargeot, J., and Lory, P. (2002). Specific contribution of human T-type calcium channel isotypes (alpha(1G), alpha(1H) and alpha(1I)) to neuronal excitability. J Physiol 540, 3-14. Chemin, J., Monteil, A., Perez-Reyes, E., Nargeot, J., and Lory, P. (2001). Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide. Embo J 20, 7033-7040. Chemin, J., Traboulsie, A., and Lory, P. (2006). Molecular pathways underlying the modulation of T- type calcium channels by neurotransmitters and hormones. Cell Calcium 40, 121-134. Chevalier, M., Lory, P., Mironneau, C., Macrez, N., and Quignard, J.F. (2006). T-type CaV3.3 calcium channels produce spontaneous low-threshold action potentials and intracellular calcium oscillations. Eur J Neurosci 23, 2321-2329. Christie, B.R., Eliot, L.S., Ito, K., Miyakawa, H., and Johnston, D. (1995). Different Ca2+ channels in soma and dendrites of hippocampal pyramidal neurons mediate spike-induced Ca2+ influx. J Neurophysiol 73, 2553-2557. Cribbs, L.L., Lee, J.H., Yang, J., Satin, J., Zhang, Y., Daud, A., Barclay, J., Williamson, M.P., Fox, M., Rees, M., and Perez-Reyes, E. (1998). Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res 83, 103-109. DePuy, S.D., Yao, J., Hu, C., McIntire, W., Bidaud, I., Lory, P., Rastinejad, F., Gonzalez, C., Garrison, J.C., and Barrett, P.Q. (2006). The molecular basis for T-type Ca2+ channel inhibition by G protein beta2gamma2 subunits. Proc Natl Acad Sci U S A 103, 14590-14595. Destexhe, A., and Sejnowski, T.J. (2003). Interactions between membrane conductances underlying thalamocortical slow-wave oscillations. Physiol Rev 83, 1401-1453. Dippel, E., Kalkbrenner, F., Wittig, B., and Schultz, G. (1996). A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta. Proc Natl Acad Sci U S A 93, 1391-1396. Chapter 2: Inhibition of Cav3.3 by mAChRs  92 Dolphin, A.C. (2003). G protein modulation of voltage-gated calcium channels. Pharmacol Rev 55, 607- 627. Egger, V., Svoboda, K., and Mainen, Z.F. (2003). Mechanisms of lateral inhibition in the olfactory bulb: efficiency and modulation of spike-evoked calcium influx into granule cells. J Neurosci 23, 7551-7558. Egger, V., Svoboda, K., and Mainen, Z.F. (2005). Dendrodendritic synaptic signals in olfactory bulb granule cells: local spine boost and global low-threshold spike. J Neurosci 25, 3521-3530. Fisher, R., and Johnston, D. (1990). Differential modulation of single voltage-gated calcium channels by cholinergic and adrenergic agonists in adult hippocampal neurons. J Neurophysiol 64, 1291-1302. Fraser, D.D., and MacVicar, B.A. (1991). Low-threshold transient calcium current in rat hippocampal lacunosum-moleculare interneurons: kinetics and modulation by neurotransmitters. J Neurosci 11, 2812- 2820. Gamper, N., Reznikov, V., Yamada, Y., Yang, J., and Shapiro, M.S. (2004). Phosphatidylinositol [correction] 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels. J Neurosci 24, 10980-10992. Hamid, J., Peloquin, J.B., Monteil, A., and Zamponi, G.W. (2006). Determinants of the differential gating properties of Cav3.1 and Cav3.3 T-type channels: a role of domain IV? Neuroscience 143, 717- 728. Heximer, S.P., Watson, N., Linder, M.E., Blumer, K.J., and Hepler, J.R. (1997). RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc Natl Acad Sci U S A 94, 14389-14393. Hildebrand, M.E., David, L.S., and Snutch, T.P. (2005). Differential modulation of T-type calcium channels by metabotropic glutamate receptors. In Biophysical Society-49th Annual Meeting (Long Beach, California). Hildebrand, M.E., and Snutch, T.P. (2006). Contributions of T-type calcium channels to the pathophysiology of pain signaling. Drug Discovery Today: Disease Mechanisms 3, 335-341. Hogger, P., Shockley, M.S., Lameh, J., and Sadee, W. (1995). Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors. J Biol Chem 270, 7405- 7410. Huguenard, J.R., and Prince, D.A. (1992). A novel T-type current underlies prolonged Ca(2+)-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12, 3804-3817. Joksovic, P.M., Bayliss, D.A., and Todorovic, S.M. (2005). Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane. J Physiol 566, 125- 142. Joksovic, P.M., Nelson, M.T., Jevtovic-Todorovic, V., Patel, M.K., Perez-Reyes, E., Campbell, K.P., Chen, C.C., and Todorovic, S.M. (2006). CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus. J Physiol 574, 415-430. Kammermeier, P.J., Ruiz-Velasco, V., and Ikeda, S.R. (2000). A voltage-independent calcium current inhibitory pathway activated by muscarinic agonists in rat sympathetic neurons requires both Galpha q/11 and Gbeta gamma. J Neurosci 20, 5623-5629. Chapter 2: Inhibition of Cav3.3 by mAChRs  93 Khosravani, H., Altier, C., Simms, B., Hamming, K.S., Snutch, T.P., Mezeyova, J., McRory, J.E., and Zamponi, G.W. (2004). Gating effects of mutations in the Cav3.2 T-type calcium channel associated with childhood absence epilepsy. J Biol Chem 279, 9681-9684. Khosravani, H., Bladen, C., Parker, D.B., Snutch, T.P., McRory, J.E., and Zamponi, G.W. (2005). Effects of Cav3.2 channel mutations linked to idiopathic generalized epilepsy. Ann Neurol 57, 745-749. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W., and Shin, H.S. (2001). Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31, 35-45. Lee, J.H., Daud, A.N., Cribbs, L.L., Lacerda, A.E., Pereverzev, A., Klockner, U., Schneider, T., and Perez-Reyes, E. (1999). Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19, 1912-1921. Levey, A.I., Edmunds, S.M., Heilman, C.J., Desmond, T.J., and Frey, K.A. (1994). Localization of muscarinic m3 receptor protein and M3 receptor binding in rat brain. Neuroscience 63, 207-221. Levey, A.I., Edmunds, S.M., Koliatsos, V., Wiley, R.G., and Heilman, C.J. (1995). Expression of m1-m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation. J Neurosci 15, 4077-4092. McKay, B.E., McRory, J.E., Molineux, M.L., Hamid, J., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Cav3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons. Eur J Neurosci 24, 2581-2594. McRory, J.E., Santi, C.M., Hamming, K.S., Mezeyova, J., Sutton, K.G., Baillie, D.L., Stea, A., and Snutch, T.P. (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem 276, 3999-4011. Mittman, S., Guo, J., Emerick, M.C., and Agnew, W.S. (1999). Structure and alternative splicing of the gene encoding alpha1I, a human brain T calcium channel alpha1 subunit. Neurosci Lett 269, 121-124. Monteil, A., Chemin, J., Leuranguer, V., Altier, C., Mennessier, G., Bourinet, E., Lory, P., and Nargeot, J. (2000). Specific properties of T-type calcium channels generated by the human alpha 1I subunit. J Biol Chem 275, 16530-16535. Murbartian, J., Arias, J.M., Lee, J.H., Gomora, J.C., and Perez-Reyes, E. (2002). Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1). FEBS Lett 528, 272-278. Murbartian, J., Arias, J.M., and Perez-Reyes, E. (2004). Functional impact of alternative splicing of human T-type Cav3.3 calcium channels. J Neurophysiol 92, 3399-3407. Park, J.Y., Kang, H.W., Moon, H.J., Huh, S.U., Jeong, S.W., Soldatov, N.M., and Lee, J.H. (2006). Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density. J Physiol 577, 513-523. Peloquin, J.B., Khosravani, H., Barr, W., Bladen, C., Evans, R., Mezeyova, J., Parker, D., Snutch, T.P., McRory, J.E., and Zamponi, G.W. (2006). Functional analysis of Ca3.2 T-type calcium channel mutations linked to childhood absence epilepsy. Epilepsia 47, 655-658. Chapter 2: Inhibition of Cav3.3 by mAChRs  94 Pemberton, K.E., Hill-Eubanks, L.J., and Jones, S.V. (2000). Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells. Pflugers Arch 440, 452- 461. Perez-Reyes, E., Cribbs, L.L., Daud, A., Lacerda, A.E., Barclay, J., Williamson, M.P., Fox, M., Rees, M., and Lee, J.H. (1998). Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 391, 896-900. Plummer, K.L., Manning, K.A., Levey, A.I., Rees, H.D., and Uhlrich, D.J. (1999). Muscarinic receptor subtypes in the lateral geniculate nucleus: a light and electron microscopic analysis. J Comp Neurol 404, 408-425. Rhee, S.G., and Bae, Y.S. (1997). Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem 272, 15045-15048. Santi, C.M., Cayabyab, F.S., Sutton, K.G., McRory, J.E., Mezeyova, J., Hamming, K.S., Parker, D., Stea, A., and Snutch, T.P. (2002). Differential inhibition of T-type calcium channels by neuroleptics. J Neurosci 22, 396-403. Schwarz, R.D., Davis, R.E., Jaen, J.C., Spencer, C.J., Tecle, H., and Thomas, A.J. (1993). Characterization of muscarinic agonists in recombinant cell lines. Life Sci 52, 465-472. Suh, B.C., and Hille, B. (2005). Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15, 370-378. Suh, B.C., Horowitz, L.F., Hirdes, W., Mackie, K., and Hille, B. (2004). Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq. J Gen Physiol 123, 663- 683. Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E., and Bayliss, D.A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19, 1895-1911. Thompson, S.M., and Wong, R.K. (1991). Development of calcium current subtypes in isolated rat hippocampal pyramidal cells. J Physiol 439, 671-689. Tombler, E., Cabanilla, N.J., Carman, P., Permaul, N., Hall, J.J., Richman, R.W., Lee, J., Rodriguez, J., Felsenfeld, D.P., Hennigan, R.F., and Diverse-Pierluissi, M.A. (2006). G protein-induced trafficking of voltage-dependent calcium channels. J Biol Chem 281, 1827-1839. Tsakiridou, E., Bertollini, L., de Curtis, M., Avanzini, G., and Pape, H.C. (1995). Selective increase in T- type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. J Neurosci 15, 3110-3117. Vitko, I., Chen, Y., Arias, J.M., Shen, Y., Wu, X.R., and Perez-Reyes, E. (2005). Functional characterization and neuronal modeling of the effects of childhood absence epilepsy variants of CACNA1H, a T-type calcium channel. J Neurosci 25, 4844-4855. Wan, X., Desilets, M., Soboloff, J., Morris, C., and Tsang, B.K. (1996). Muscarinic activation inhibits T- type Ca2+ current in hen granulosa cells. Endocrinology 137, 2514-2521. Wei, J., Walton, E.A., Milici, A., and Buccafusco, J.J. (1994). m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC. J Neurochem 63, 815-821. Chapter 2: Inhibition of Cav3.3 by mAChRs  95 Welsby, P.J., Wang, H., Wolfe, J.T., Colbran, R.J., Johnson, M.L., and Barrett, P.Q. (2003). A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II. J Neurosci 23, 10116-10121. Wolfe, J.T., Wang, H., Howard, J., Garrison, J.C., and Barrett, P.Q. (2003). T-type calcium channel regulation by specific G-protein betagamma subunits. Nature 424, 209-213. Wu, L., Bauer, C.S., Zhen, X.G., Xie, C., and Yang, J. (2002). Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419, 947-952. Yunker, A.M., Sharp, A.H., Sundarraj, S., Ranganathan, V., Copeland, T.D., and McEnery, M.W. (2003). Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development. Neuroscience 117, 321-335. Zamponi, G.W., and Snutch, T.P. (2002). Modulating modulation: crosstalk between regulatory pathways of presynaptic calcium channels. Mol Interv 2, 476-478.   Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  96 3 FUNCTIONAL COUPLING BETWEEN MGLUR1 AND CAV3.1 T-TYPE CALCIUM CHANNELS ENHANCES CEREBELLAR PURKINJE CELL EXCITABILITY AND LOCAL SIGNALING*  3.1 Introduction T-type Ca2+ channels are expressed in a wide range of tissues, including in the nervous, cardiovascular and endocrine systems (Perez-Reyes, 2003).  At the molecular level the T-type Ca2+ channel family is composed of three main subtypes (Cav3.1, Cav3.2, and Cav3.3) characterized by similar low thresholds of activation, but with distinct biophysical and modulatory properties and unique cellular and subcellular expression patterns (Talley et al., 1999).  Recent knock-down studies suggest that individual T-type channel subtypes might play specific physiological roles.  For example, Cav3.2 KO mice show developmental defects in coronary arteries (Chen et al., 2003) while siRNA-mediated Cav3.2 silencing in sensory neurons has antinociceptive effects (Bourinet et al., 2005).  Furthermore, in Cav3.1 KO mice, the typical burst firing mode of thalamic relay cells is absent and defects in sleep behavior are observed (Anderson et al., 2005). T-type currents play a critical role in the soma and dendrites of neurons in initiating dendritic boosting and Ca2+ spikes (Egger et al., 2005), in altering neuronal excitability and firing patterns (Crunelli et al., 2006; Kim et al., 2001) and in modulating synaptic integration and plasticity (Christie et al., 1997; Ikeda et al., 2003).  Each T-type Ca2+ channel isoform likely plays different roles in these effects depending upon the combination of its cellular and subcellular localization (McKay et al., 2006) and unique biophysical properties (Chemin et al., 2002; Kozlov et al., 1999; McRory et al., 2001).  The differential modulation of T-type Ca2+ channel isoforms may constitute an additional substrate of functional specialization and heterogeneity.  While the Cav3.2 isoform is specifically modulated by Gβ2γ proteins, CAMKII, and redox changes (Joksovic et al., 2006; Welsby et al., 2003; Wolfe et al., 2003), Cav3.2 and Cav3.1/Cav3.3 are modulated in opposite directions by lysophosphatidic acid (Iftinca et al., 2007).  Gαq/11-coupled mAChRs inhibit Cav3.3 channels but not Cav3.1 or Cav3.2 channels (Hildebrand et al., 2007).  Overall, these results suggest that subtype-specific modulation of T-type Ca2+ channels may participate in system-specific specializations, although explorations into these contributions in native neuronal systems is thus far lacking. Cerebellar PCs express the Cav3.1 and Cav3.3 T-type Ca2+ channel isoforms (Hartmann et al., 2004; Isope and Murphy, 2005; McKay et al., 2006).  We recently combined whole-cell recordings with  * A version of this chapter has been submitted for publication.  Hildebrand, M.E., Isope, P., Garcia, E., Feltz, A., Schneider, T., Hescheler, J., Kano, M., Sakimura, K., Dieudonne, S., Snutch, T.P. (2008) Functional coupling between mGluR1 and Cav3.1 T-type calcium channels enhances cerebellar Purkinje cell excitability and local signaling.  Neuron.  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  97 Ca2+ imaging to demonstrate the presence of Cav3.1-like T-type currents/transients within both spines and dendrites of PCs (Isope and Murphy, 2005).  Although their exact functions in PCs remains to be elucidated, T-type currents have been linked to the generation of dendritic low-threshold spikes and in altering interspike and interburst intervals during spontaneous dendritic burst firing (Pouille et al., 2000; Swensen and Bean, 2003; Womack and Khodakhah, 2004).  PCs also show the highest relative expression of the G-protein Gαq/11 in the CNS (Hartmann et al., 2004; Tanaka et al., 2000).  In PCs, Gαq/11 is linked to the mGluR1 subtype of metabotropic glutamate receptors.  mGluR1 receptors play a central role in the induction of short-term and long-term plasticity at the PF synapse and also control cerebellar motor learning (Aiba et al., 1994; Conquet et al., 1994; Finch and Augustine, 1998; Ichise et al., 2000; Kishimoto et al., 2002; Takechi et al., 1998).  Activation of mGluR1 causes PLC translocation and IP3-mediated local Ca2+ release in spines and dendrites (Finch and Augustine, 1998; Takechi et al., 1998).  mGluR1 has also been shown to affect intracellular Ca2+ levels within signaling domains of PC dendrites by activating a sEPSC (potentially TRPC1) (Kim et al., 2003; Kitano et al., 2003; Tempia et al., 2001). Here, we show that mGluR1a receptor activation differentially modulates recombinant T-type isoforms, potentiating Cav3.1 and Cav3.2 while inhibiting Cav3.3.  In PCs of acute rodent slices we characterize an mGluR1-mediated potentiation of Cav3.1 T-type currents that we find occurs through a G-protein and protein tyrosine phosphatase-dependent pathway, is localized to dendritic compartments, and that alters PC excitability.  Finally, we show that this potentiation of Cav3.1 by mGluR1 can be triggered by trains of PF stimulation, suggesting a new potential role for the Cav3.1 T-type Ca2+ channel isoform in cerebellar synaptic integration.  3.2 Results 3.2.1 Subtype-specific modulation of recombinant T-type calcium channels by mGluR1a activation We initially tested whether activation of mGluR1a, a GPCR coupled to Gαq/11, could modulate T-type Ca2+ channels exogenously expressed in HEK cells.  Transfection of mGluR1a into HEK 293 cell lines that stably expressed each of the three recombinant rat brain T-type isoforms showed a distinct pattern of modulation; activation of mGluR1a with 100 μM glutamate caused a potentiation of Cav3.1 and Cav3.2 currents and an inhibition of Cav3.3 currents (Fig. 3.1).  The time course of the potentiation of Cav3.1 and Cav3.2 currents was slow (Cav3.1, τonset = 95 +/- 15 s, n=6; Cav3.2, τonset = 225 +/- 22 s, n=5) and included a “lag” period compared to the rapid onset inhibition of the Cav3.3 currents (τonset = 16 +/- 2 s, n=14), suggesting that the intracellular pathway mediating the two effects could be different (Fig. 3.1). Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  98  0 50 100 150 200 250 300 350 400 0.9 1.0 1.1 1.2 1.3 1.4 100 μM Glutamate N or m al iz ed  C ur re nt Time (s) 0 50 100 150 200 250 300 350 400 0.9 1.0 1.1 1.2 1.3 1.4 N or m al iz ed  C ur re nt Time (s) 100 μM Glutamate 0 50 100 150 200 0.7 0.8 0.9 1.0 1.1 100 μM Glutamate N or m al iz ed  C ur re nt Time (s) C A B Cav3.1- mGluR1a 50  p A 20 ms Cav3.2- mGluR1a 25 ms 10 0 pA 25 0 pA 25 ms Cav3.3- mGluR1a  Figure 3.1 - Recombinant T-type calcium channels are differentially modulated by mGluR1a receptors. A-C) Left Panels: Representative voltage-clamped current traces during depolarizing pulses from a holding potential of –110 mV to –30 mV showing potentiation of Cav3.1 (A) and Cav3.2 (B) currents and inhibition of Cav3.3 (C) currents by activation of mGluR1a receptors with 100 μM glutamate.  Open shapes indicate traces during control perfusion while closed shapes indicate traces during 100 μM glutamate perfusion.  Right Panels: Normalized peak current levels during perfusion of control recording solution (2 mM Ca2+) followed by 100 μM glutamate for Cav3.1 (A), Cav3.2 (B), and Cav3.3 (C) currents.  The potentiation shown for Cav3.1 (A) and Cav3.2 (B) channels was observed in ~30% of cells tested, while the mGluR1a -induced inhibition of Cav3.3 currents was observed in ~70% of cells. mGluR1a activation caused an average potentiation of Cav3.1 currents by 29.6% (+/-5.8%; n=8), potentiation of Cav3.2 currents by 41.8% (+/-8.8%; n=6), and inhibition of Cav3.3 currents by 28.7% (+/- 1.9%, n=33).  For Cav3.1 and Cav3.2 currents, glutamate had no effect on all excluded cells, while for Cav3.3 100 μM glutamate caused either no effect or inhibition followed by a recovery in the cells not shown (data not shown, see section 3.4 for further details). Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  99  Cav3.1 (Fig. 3.1A) and Cav3.2 (Fig. 3.1B) current kinetics were not significantly (p>0.05) affected during the mGluR1a-induced potentiation, while the inhibition of Cav3.3 by mGluR1a was accompanied by a robust increase in activation and inactivation kinetics (Fig. 3.1C; for Cav3.3: Control, τact = 6.4 +/- 0.4 ms, n=25, τinact = 76 +/- 8 ms, n=13; 100 μM Glutamate τact = 4.4 +/- 0.3 ms, n=25, τinact = 39 +/- 5 ms, n=13, p<0.02).  We note that a subset of cells for all three T-type isoforms showed no modulation by mGluR1a, which could be explained by a lack of functional expression of mGluR1a in some transfected cells and/or the heterogeneous efficacy of the downstream signaling cascade.  3.2.2 Cav3.1-mediated T-type calcium currents are potentiated by mGluR1 activation in cerebellar Purkinje neurons We next examined the modulation of T-type currents by mGluR1 in a native system, cerebellar PCs.  In PCs, mGluR1 is highly expressed both perisynaptically at PF and at CF contacts (Baude et al., 1993; Lopez-Bendito et al., 2001).  Additionally, we have recently shown that T-type channels are expressed in PC dendrites and spines, where they evoke Ca2+ entry upon low threshold depolarization (Isope and Murphy, 2005).  In the present study, T-type currents were recorded in whole-cell voltage clamp in PCs prepared from cerebellar slices of male Wistar rats aged P8 to P12.  At this age, mGluR1 and T-type currents are both expressed (Isope and Murphy, 2005; Shigemoto et al., 1992) and the dendritic arbour is small enough to alleviate space clamp problems (Roth and Hausser, 2001; Sacco and Tempia, 2002).  A combination of pharmacological antagonists were used to isolate T-type currents from potential contamination by Na+, K+, and HVA Ca2+ currents (as previously described (Isope and Murphy, 2005) and discussed in section 3.4).  To ensure the quality of the space-clamp, modulatory effects were studied on T-type currents of moderate amplitude (213 +/- 35 pA, n=19) elicited every 10 seconds from a holding potential of -75 mV (with a 500 ms prepulse to -90 mV to remove inactivation) and depolarizing test pulses ranging between -50 mV and -35 mV (Fig. 3.2A-C).  Activation of mGluR1 receptors by bath-application of DHPG (20 μM) caused a robust and reversible increase in T-type peak current amplitude (51 +/- 7 %, n=19; reversed by 84 +/- 11 %, n=5; Fig. 3.2A,B) in all PCs tested.  The DHPG- induced potentiation of T-type currents did not involve a significant change in the activation (τact) or inactivation (τinact) kinetics of the current (Fig. 3.2A, Table 3.1).  The increase in T-type currents was not due to changes in passive electrical properties, as Rs and leak currents (IL; at Vh = -75 mV) remained relatively constant between the measured conditions (Control, Rs = 10.4 +/- 1.5 MΩ, IL = -204 +/- 46 pA; DHPG for 2 minutes, Rs = 10.4 +/- 1.8 MΩ, IL = -233 +/- 49 pA (n=9)). Bath application of DHPG tonically activates mGluR1 receptors to produce robust and reproducible maximal effects, but this form of receptor activation may lead to receptor desensitization Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  100 and does not accurately mimic a physiological activation.  To overcome this limitation, we used brief puffs of DHPG (see section 3.4) previously shown to reproduce the sEPSC responses when mGluR1 was synaptically activated by brief tetanic stimulation of PFs (Tempia et al., 2001).  In our experiments, puffing on 100 μM DHPG caused a fast potentiation of T-type currents (45 +/- 12 %, n=6) that reached a maximal level within 10 seconds (Fig. 3.2D).  An increase in leak current (likely due to the activation of sEPSCs) was observed for the first two pulses after the DHPG puff (IL = -279 +/- 132 pA, n=6) but leak values then returned to control levels (IL = -171 +/- 94 pA, n=6) and the T-type potentiation remained. Similar to bath application of DHPG (not shown), the DHPG puff-induced increase in T-type currents was reversed (by 94 +/- 9 %, n=6) upon prolonged perfusion of control solution. Since DHPG activates both mGluR1 and mGluR5 receptors, we assessed the specificity of the effect using co-application of DHPG with a group 1 antagonist (500 μM MCPG) or LY367385 (100 μM), a specific mGluR1 antagonist.  In both conditions, the T-type potentiation was completely blocked, indicating that the DHPG-induced potentiation of T-type currents occurs specifically through mGluR1 receptors (Fig. 3.5B,F).  Our exogenous expression experiments in transfected HEK cells indicated that both Cav3.1 and Cav3.2 T-type channel isoforms were capable of being upregulated by mGluR1 activation (Fig. 3.1).  As PCs appear to express the Cav3.1 and Cav3.3 T-type Ca2+ isoforms but not Cav3.2 (Hartmann et al., 2004; Isope and Murphy, 2005; McKay et al., 2006), the observed T-type current upregulation in PCs was predicted to be due to native Cav3.1 channel modulation.  To test this notion we examined PCs in Cav3.1 KO mice (P8 to P12).  Figure 3.2 (E,F) shows that under our measurement conditions PCs from Cav3.1 KO mice completely lacked T-type whole cell currents during depolarizing test pulses to -40 mV that normally induce robust currents in the wt mice (Cav3.1 KO = 12 +/- 16 pA, n=4; wt = 1280 +/- 85 pA at - 40 mV, n=3).  Thus, T-type currents of immature rodent PCs are principally mediated by Cav3.1 channels. We note that R-type Ca2+ currents show some voltage-dependent and kinetic properties similar to those for Cav3.1 and that R-type channels may also be expressed in PCs (Meacham et al., 2003).  In order to rule out the possibility that in wt mice the potentiating effect of mGluR1 activation might in part be carried by R-type Ca2+ currents (Cav2.3) we examined a strain of Cav2.3 KO mice.  Figure 3.2F shows that the DHPG-induced Ca2+ current potentiation in PCs was similar in Cav2.3 KO and wt mice (Fig. 3.2F).  Taken together, our findings suggest that mGluR1 receptor activation in PCs specifically and selectively potentiates T-type currents mediated by Cav3.1 channels.  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  101  Figure 3.2 - T-type calcium channels in cerebellar PCs are reversibly potentiated by mGluR1 activation. A) Representative voltage-clamped current traces for rat PC T-type currents during depolarizing pulses from -90 mV to -43 mV before (1), during (2), and after (3) activation of endogenous mGluR1 receptors with 20 μM DHPG.  B) Normalized peak current time course for the cell shown in A demonstrating the reversibility of DHPG-induced potentiation.  C) Average normalized peak T-type current time course during control perfusion followed by bath perfusion of 20 μM DHPG. T-type currents increased by 51 % +/- 7 % (n=19) after 100 to 120 seconds of DHPG application.  D) Micropressure (50 ms, 10 PSI) pulses of DHPG (100 μM) through a puff pipette triggered a rapid potentiation of T-type current amplitude by 45 % +/- 12 % (n=6).  E) Representative current traces during depolarizations from -80 mV to -40 mV in both wt (black trace) and Cav3.1 -/- (grey trace) mice.  F) Left: Quantification of peak PC T-type currents in Cav3.1 -/- mice (n=4) compared to wt mice (n=4) during depolarizing steps to -40 mV. Right: DHPG causes robust and equal T-type potentiation in wt mice (n=3) and Cav2.3 -/- mice (n=4). Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  102 Table 3.2 - Effects of DHPG on Purkinje Cell T-Type Biophysical Properties  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  103 3.2.3 mGluR1 potentiates T-type currents through an increase in maximal current and a shift in the voltage-dependence of activation The effect of mGluR1 activation (via DHPG bath perfusion) on the voltage-dependence of T- type activation was studied using a series of test depolarizations ranging from -70 mV to - 5 mV.  In order to minimize the effects of contaminating HVA Ca2+ currents (especially P-type) and K+ currents in this broader voltage range, a stronger pharmacological cocktail was used that included 300 μM Cd2+, 10- 20 mM TEA+, and 5-10 mM 4-AP.  Figure 3.3A shows a representative cell where mGluR1 activation with DHPG (30 μM) caused an increase in the remaining rat T-type currents at all potentials.  The DHPG-mediated increase in peak current was significant (p<0.05) at potentials between -45 mV and -20 mV (Fig. 3.3B).  DHPG application also caused a significant (-2.4 mV, n=7, p<0.02) shift in the half- activation potential (V50act) of the T-type currents (Fig. 3.3C, Table 3.1) but no significant (p>0.05) effect on the voltage-dependence of T-type steady-state inactivation (Fig. 3.3D) or the voltage dependence of deactivation kinetics (Fig. 3.3E).  These results indicate that the DHPG potentiation effect is mediated both by an increase in maximal current and a shift in the voltage-dependence of activation, suggesting a change in open probability and/or conductance of the channels.  The strong voltage-dependence of these deactivation kinetics (Randall and Tsien, 1997) combined with the fact that 50 μM Ni2+ was included to block R-type currents provide further confirmation that the DHPG-potentiated low threshold currents are due to T-type, and not R-type, channels.  3.2.4 mGluR1 activation increases PC excitability via effects on T-type calcium currents The current-voltage relationships shown in Figure 3.3B indicates that the maximum mGluR1- mediated potentiation occurs at potentials between -45 mV and -40 mV (110 % increase, n=7 at -40 mV compared to 22 % increase, n=7 at -20 mV).  These potentials are very close to the somatic threshold for the generation of sodium spikes and we hypothesized that the subthreshold potentiation of T-type currents by mGluR1 could shorten the first spike latency during a depolarizing event.  To test this hypothesis, current clamp experiments were performed using the same pharmacological cocktail described in voltage-clamp experiments in order to isolate changes in the rat PC membrane potential that are due to T-type activity.  Hyperpolarized initial resting potential values (-82.6 +/- 1.1 mV, n=5) were obtained via constant current injection, averaging -191 +/- 17 pA (n=5) to ensure the availability of T- type currents (Fig. 3.4).  In the absence of voltage-gated Na+, K+, and HVA Ca2+ currents (validated by voltage-clamp IV curves; data not shown), injection of depolarizing currents (relative to holding currents; averaging 154 pA +/- 9 pA, n=5) resulted in T-type-mediated Ca2+ spikes and a sustained plateau potential with minimal repolarization (due to the blockade of K+ currents; (Swensen and Bean, 2003) (Fig. 3.4A)). Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  104     Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  105      Figure 3.3 - T-type calcium currents are potentiated by mGluR1 through an increase in maximal current and a small shift in the voltage-dependence of activation. A) Representative voltage-clamped current traces during depolarizations from -90 mV to potentials ranging from -70 mV to -5 mV before (left) and after (right) mGluR1 was activated with 30 μM DHPG, in the presence of a cocktail containing 300 μM Cd2+, 10-20 mM TEA+, and 5-10 mM 4-AP.  Recordings were from PCs of P8 to P12 Wistar rats.  B-E) Control recordings are represented by black squares while recordings in DHPG are represented by grey circles.  B) Normalized IV curve showing that DHPG increases maximal current, resulting in a significant potentiation of T-type currents at potentials between -45 mV and -20 mV (n=7). * indicates significance at p<0.05 compared to control current values. C) Normalized conductance curve for PC T-type currents generated by fitting the IV curves in B with a modified Boltzmann equation (see Methods).  DHPG application caused a small, but significant (p<0.02) shift of ~ 2 mV in the V50act for these currents (Table 1).  D) Activation of mGluR1 with 20 μM DHPG had no significant (p>0.05) effect on the steady-state inactivation (V50inact) of T-type currents within PCs (Table 1).  E) Perfusion of 20 μM DHPG had no significant (p>0.05) effects on the kinetics of T-type channel deactivation (Table 1).  Inset: Waveform protocol showed on top and representative current traces shown below for a PC under control conditions. For scale bar, X axis = 20 ms, Y axis = 200 pA. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  106 Bath application of 20 μM DHPG significantly (p<0.02) decreased the temporal latency (by 82 ms, n=7) and the voltage threshold (by 6.9 mV, n=7) of these T-type-dependent Ca2+ spikes and significantly (p<0.02) increased their maximal slope (dV/dtmax) in a reversible manner (Fig. 3.4A,B,D). The ability of DHPG to increase T-type spike activity by decreasing spike threshold and latency was not due to changes in passive properties, as RN and Rs were not significantly (p>0.05) altered during experiments (data not shown; Fig. 3.4D). Purkinje cells are known to undergo transitions from a hyperpolarized inactive state to a depolarized state, where they fire spontaneously both in vitro (Williams et al., 2002) and in vivo (Loewenstein et al., 2005).  Ih has been shown to prevent PC bistability (Williams et al., 2002) but endogenous conductances promoting the bistable behavior have not been analyzed in detail.  T-type currents are ideally suited to boost the transition from hyperpolarized to depolarized potentials (Williams et al., 1997).  We conducted experiments, using physiological internal and external solutions, to test the participation of T-type currents in rat PC state transitions. A relatively hyperpolarized resting potential (- 76.1 mV, Fig. 3.4C,D) was maintained with constant injection of current (-228 +/- 28 pA, n=8) to ensure availability of T-type currents, and an average depolarizing injection of 165 +/- 25 pA, n=8 induced AP firing during control recordings.  Application of 20 μM DHPG caused a significant (p<0.05) decrease in the voltage threshold for AP firing by 4.0 mV (n= 8) and decreased the first spike latency by 41 ms (n=8) (Fig. 3.4C,D).  These DHPG-mediated increases in PC membrane excitability were completely reversed with the co-perfusion of the T-type antagonist mibefradil (10 μM; Fig. 3.4C,D).  While relatively high concentrations of antagonists such as mibefradil are required to block T-type currents in slice recordings (as confirmed by voltage-clamp recordings, data not shown), mibefradil’s > 100-fold higher affinity for T-type currents over P-type currents in PCs suggests that T-type Ca2+ currents will be selectively blocked at this concentration (McDonough and Bean, 1998).  To support these results, 200 μM Ni2+ was also used as a T-type antagonist predicted to have no effect on either P/Q-type Ca2+ channels or voltage-gated Na+ channels (Kuo et al., 2004; Zamponi et al., 1996).  When co-applied with DHPG, 200 μM Ni2+ either reversed the DHPG effect (n=3) as was observed for mibefradil or completely eliminated AP firing in the current injection range used (n=3) (data not shown).  As observed for the effects of DHPG on T- type spike thresholds, the effects of DHPG on overall PC excitability were not due to changes in RN or RS (data not shown; Fig. 3.4D).  These findings suggest that activation of mGluR1 boosts subthreshold membrane depolarization via an increase in T-type currents, leading to a decrease in the threshold and the latency of the first spike at the soma of the PC. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  107  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  108       Figure 3.4 - DHPG lowers the threshold for PC T-type-dependent calcium spikes and AP firing. A) Top Panels: Current clamp traces from a representative rat PC during hyperpolarizing and depolarizing current injection pulses before (left), during (middle), and after (right) DHPG application. Similar to voltage-clamp experiments, T-type conductances were isolated with 300 nM TTX, 50 μM Ni2+, 20 mM TEA, 5 mM 4-AP, and 50 μM Cd2+ in the external solution and a Cs+-based internal solution.  Bottom Panels: Current injection protocol for above traces showing steps to -100, -50, 0, 50, 100, 110, 120, and 130 pA.  B) Current injection steps from A (to 130 pA, grey traces) that induced T- type-dependent Ca2+ spikes in all conditions.  Bath application of 20 μM DHPG significantly (p<0.02) decreased both the spike latency and the voltage threshold for T-type-dependent Ca2+ spike activation (see panel D).  Washout of DHPG reversed the changes in the latency and voltage threshold of T-type- dependent Ca2+ spikes (see panel D).  C) Current clamp traces from a representative PC in ACSF solution with no voltage-gated ion channel antagonists during a depolarizing current injection step that elicited AP firing during control recordings (thin line), perfusion of 20 μM DHPG (thick line), and finally co-application of 20 μM DHPG with 10 μM mibefradil (dashed line).  Application of DHPG decreased the latency and voltage threshold for firing of the first AP, which was reversed upon application of the T-type antagonist, mibefradil.  D) Top: Table of passive and active properties before (Control), during (20 mM DHPG), and after (Wash) perfusion of 20 μM DHPG.  I Threshold = first current injection step where T-type spikes are observed; V Threshold = voltage of inflection point where T-type spikes are initiated; Spike Latency = time point, from start of depolarizing current step, at which T-type spike inflection point occurs; dV/dtmax= maximum slope of T-type spike as determined by differentiation (see section 3.4); Vrest = membrane potential before depolarizing current step.  V threshold, Spike Latency, dV/dtmax and Vrest were measured from a constant depolarizing current injection step that elicited T-type spikes under all conditions for a given cell.  Bottom: Table of the same parameters derived for first AP firing in physiological saline, except that the spike latency and V threshold are now calculated from the inflection point of the first AP.  * represents significance at p<0.02 compared to control values. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  109 3.2.5 mGluR1 potentiates T-type currents through a G-protein-, tyrosine phosphatase-, and calcium- dependent pathway independently of phospholipase C and its downstream effectors  Multiple intracellular pathways are known to be associated with mGluR1 receptors in PCs (Knopfel and Grandes, 2002) and we set out to study the identity of the signaling pathway leading to T- type current potentiation.  Inclusion of 2 mM GDP-β-S in the pipette solution to block G-protein activity (Gα and Gβγ) completely abolished the potentiation of rat T-type currents by DHPG (Fig. 3.5C,F). However, blocking PLC with either 1 μM U73122 or 10 μM edelfosine, PKC with 1 to 2.5 μM staurosporine, or IP3Rs with 1 μM xestospongin C all had no significant (p>0.05) effect on the DHPG- mediated increase in T-type currents (Fig. 3.5F).  Using a similar approach, Canepari and Ogden showed that activation of sEPSCs by mGluR1 was dependent on G-protein activation but independent of PLC activation (Canepari and Ogden, 2003).  In these experiments, the authors found that sEPSC activation depended upon tyrosine phosphatase activity.  In our experiments, blocking tyrosine phosphatase activity with either 1 mM Na3VO4 or its more potent analog, 100 μM bpV(phen), caused a significant (p<0.02) attenuation of T-type current potentiation by DHPG (Fig. 3.5E,F).  Interestingly, the potentiation of T- type currents by mGluR1 also depended on intracellular Ca2+ concentration [Ca2+]i, because buffering [Ca2+]i with 20 mM intracellular BAPTA in the recording pipette significantly (p<0.02) reduced the effect (Fig. 3.5D,F).  To test whether the Ca2+-permeable sEPSC was directly involved in the potentiation of T-type currents by mGluR1, antagonists of this current (250 μM IEM 1460 or 100 μM NA-spermine; (Canepari et al., 2004)) were pre- and co-applied with DHPG and found to have no significant effect on the potentiation magnitude (Fig. 3.5F).  Thus, it appears that mGluR1 activates a G-protein and tyrosine- phosphatase-dependent pathway upstream of PLC activation that positively modulates both sEPSC and T-type currents independently of each other.  3.2.6 mGluR1 potentiates T-type calcium transients at synaptic sites mGluR1 receptors and the intracellular pathway leading to the sEPSC are principally localized at the PF-PC synapse in dendritic spines.  We used two photon microscopy Ca2+ imaging to determine the localization of the DHPG potentiation effect.  Experiments were performed on P9 to P12 rats with heparin (4 mg/ml) included in the patch pipette in order to block the contaminating effect of [Ca2+]i increase through the mGluR1-activated IP3R pathway.  Pharmacological antagonists were also used to specifically isolate T-type Ca2+ transients during the whole-cell voltage clamp recordings (see section 3.4.6).  In the example shown in Figure 3.6, 40 points-of-interest (POIs) were imaged at a frame rate close to 1 kHz (Fig. 3.6A).  As previously shown (Isope and Murphy, 2005), T-type channel mediated Ca2+ transients were observed throughout the PC dendritic tree and the soma (Fig. 3.6). Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  110 0 1 2 3 4 5 0.8 1.0 1.2 1.4 1.6 N or m al iz ed  C ur re nt Time (min) 20 μM DHPG CONTROL 0 1 2 3 4 5 0.8 1.0 1.2 1.4 1.6 20μM DHPG N or m al iz ed  C ur re nt Time (min) 2 mM GDP-β-S 0 1 2 3 4 5 0.8 1.0 1.2 1.4 1.6 20μM DHPG N or m al iz ed  C ur re nt Time (min) 100 μM LY367385 0 1 2 3 4 5 0.8 1.0 1.2 1.4 1.6 20μM DHPG N or m al iz ed  C ur re nt Time (min) 20 mM BAPTA 0 1 2 3 4 5 0.8 1.0 1.2 1.4 1.6 20μM DHPG N or m al iz ed  C ur re nt Time (min) 100 μM bpV(phen) A B C D E F -- Na3VO4 bpV(phen) NA-spermine IEM 1460 Xesto C BAPTA Staurosporine Edelfosine U73122 GDP-  -S LY367385 MCPG Control (DHPG) 0 20 40 60 80 100 120 β % Stimulation by 20μM DHPG * * * * * *   Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  111      Figure 3.5 - T-type calcium currents are potentiated by mGluR1 through a signaling pathway that involves G-proteins, intracellular calcium, and tyrosine phosphatases. A) Control normalized time course showing the effects of DHPG application on voltage-clamped rat PC T-type currents in the absence of any other antagonists (as shown in Figure 3.2C).  B) DHPG stimulates T-type currents specifically through mGluR1 receptors.  Blocking mGluR1a with perfusion of 100 μM LY 367385 (n=8) for 20 minutes before and during DHPG application abolished potentiation. C) Potentiation of T-type currents via mGluR1 requires G-protein activation.  Substitution of 2 mM GDP-β-S (n=5) for GTP in the intracellular pipette solution for 10 minutes in the whole-cell conformation eliminated DHPG-induced potentiation.  D) The T-type potentiation pathway involves Ca2+-dependent processes.  Buffering intracellular Ca2+ through the inclusion of 20 mM BAPTA (n=6) in the internal solution (whole-cell for 5+ minutes) attenuated the DHPG-induced potentiation. E) Potentiation of T-type currents by DHPG requires tyrosine phosphatase activity.  Blocking tyrosine phosphatases via perfusion of 100 μM bpV(phen) (n=6) for 10 minutes before and during DHPG application attenuated the potentiation effect.  F) Histogram showing potentiation values compared to the control (DHPG) potentiation value for the above results as well as for other antagonists.  Blocking group I mGluR receptors with 500 μM MCPG (n=6) and blocking tyrosine phosphatases with 1 mM Na3VO4 (n=5) also significantly (p<0.02) reduced the DHPG-induced increase.  Blocking PLC with 1 μM U73122 (n=6) or 10 μM edelfosine (n=7), serine/threonine kinases (such as PKC) with 1 to 2.5 μM staurosporine (n=8), IP3Rs with 1 μM xestospongin C (n=6), and sEPSC currents with 250 μM IEM 1460 (n=6) or 100 μM NA-spermine (n=5) all caused no significant (p>0.05) change in the level of DHPG-mediated increase in T-type currents.  All potentiation values were calculated 100 to 120 seconds after initiation of DHPG application, except for NA-spermine and edelfosine, where the effect was calculated 60 to 80 seconds into potentiation (equivalent time from start of potentiation to other groups), because the effect was delayed.  * indicates significance at p<0.02. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  112   Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  113  Figure 3.6 - DHPG mediates an increase in T-type calcium transients in PCs. A) Two-photon image of a patch-clamped rat cerebellar PC.  The dendritic regions outlined by circles are sites of imaging.  Scale bar = 10 μM.  B) DHPG causes an increase in voltage-clamped low-threshold Ca2+ transients in spines and proximal dendrites when IP3Rs are blocked.  The Ca2+ transients (ΔF/Red, see Methods) at individual POIs during depolarizing steps to -45 mV at the soma are shown before (black) and after (red) application of 20 μM DHPG.  Numbers refer to labels from A.  Traces were smoothed.  The dotted vertical line indicates onset of the depolarizing pulse.  Recordings were performed in the presence of 4 mg/mL heparin to block IP3Rs.  C) The increase in low threshold Ca2+ transients by mGluR1 activation coincides with the potentiation of T-type somatic currents.  Three successive depolarizing pulses at the onset of the DHPG effect are represented, starting 2 min. after the beginning of the application, with 1 min. between pulses. Average Ca2+ transients in all the POIs in the PC are shown in red.  The red dotted line represents the standardized baseline Ca2+ level before the pulse; the red solid line identifies the peak of fluorescence before the DHPG effect.  In black, current recorded at the soma. The black dotted line represents current baseline while the black solid line identifies the peak current before the DHPG effect.  D) Upper panel, the average Ca2+ transient in all imaged spines during the control period is shown in black (10 min.) while the average Ca2+ transient after onset of the DHPG effect is shown in red (5 min).  Middle panel, Mean current in control period (black) and during DHPG application (red). Lower panel, holding potentials.  All data shown above in this figure are from the same cell.  E) Left: DHPG causes a potentiation of voltage-clamped T-type Ca2+ transients in the spines, dendrites, and proximal dendrites of PCs when IP3Rs are not blocked.  Proximal dendrites, n=20/5 cells, dendrites, n=40/5 cells, spines, n=55/5 cells, soma, n=6/5cells. ** p<0.01, ****p<0.001, (Wilcoxon rank test).  Right: DHPG causes a potentiation of T-type Ca2+ transients in the spines and proximal dendrites of PCs when IP3Rs are blocked with heparin inclusion (4 mg/ml) in the patch pipette.  Variation of the peak Ca2+ transient (ΔF/red) normalized to the density of current at the soma (under control conditions; see section 3.4.6) in different compartments of the cell during the control period (black bars) and DHPG application (white bars).  Proximal dendrites, n=33/5 cells, dendrites, n=52/5 cells, spines, n=84/5 cells, soma, n=9/5 cells. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  114 Strikingly, during DHPG (20 µM) application, the increase in T-type current recorded at the soma was correlated with: 1) an increase in fluorescence transients in spines but not systematically in their parent dendrites 2) an increase in fluorescence transients in proximal dendrites, and 3) no observed increase in Ca2+ entry in the soma (Fig. 3.6B,C,E).  Figure 3.6D shows the average Ca2+ transient from all spines identified in Figure 3.6A both in the control period and during DHPG application.  These findings suggest that the effect is localized in the vicinity of mGluR1 receptors, in spines and proximal dendrites.  Since the size of the voxel of the 2-photon imaging is greater than the volume of the spine, spine measurements are contaminated by signal coming from the parent dendrite.  Because this signal is not potentiated by DHPG, the increase in fluorescence transients observed in spines is underestimated. This most likely explains the larger effect of DHPG on the T-type current than on the fluorescence transient.  In order to quantitatively assess the localization of the DHPG effect, the PC was divided into four compartments: soma, proximal dendrites, dendrites and spines.  All the POIs for all the cells recorded were then pooled by compartment, normalized by the density of the current in each cell, and averaged together.  Histograms in Figure 3.6E show that the Ca2+ transients are larger in spines and that the DHPG effect is significant only in spines (p<0.001, wilcoxon rank test) and proximal dendrites (p<0.01, wilcoxon rank test).  We also tested the influence of IP3-mediated release of Ca2+ from internal stores by performing the imaging experiments with no heparin in the pipette.  When the IP3R component was not blocked, DHPG also increased the Ca2+ transients in the shaft of the spiny branchlets (Fig. 3.6E), suggesting that Ca2+ released from internal stores can relay the effect of spine mGluR1 receptor activation to the dendritic shafts.  3.2.7 Parallel fiber inputs trigger T-type calcium transients in spines that are potentiated by mGluR1 activation We have shown that T-type Ca2+ transients and currents evoked by step depolarizations at synaptic sites in PC dendrites are modulated by bath and “puff” applications of DHPG.  We then assessed the physiological relevance of this modulation during trains of PF stimulation.  High frequency trains, a physiological mode of GC discharge (Chadderton et al., 2004; Jorntell and Ekerot, 2006), are known to be necessary to activate mGluR1 (Takechi et al., 1998).  The corresponding EPSPs should be large enough to activate T-type Ca2+ channels (Roth and Hausser, 2001).  Experiments were carried out in older wt or Cav3.1 mice (P16 to P30 mice) using a K+-based internal solution and a near physiological temperature (32°C).  We combined trains of PF stimulation at 100 Hz every 30 sec with PC current clamp recordings (at a holding potential of -75 mV) and two-photon Ca2+ imaging.  Heparin (4 mg/ml) was included in the patch pipette and cyclopiazonic acid (CPA, 6 μM) in the bath to ensure a complete block of internal Ca2+ stores, as mGluR1 also increases intracellular Ca2+ levels through a well- documented activation of IP3Rs (Finch and Augustine, 1998; Takechi et al., 1998) .  AM 251, a Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  115 cannabinoid antagonist, was also perfused to prevent presynaptic depression of the PF input (Crepel and Daniel, 2007; Maejima et al., 2005).  In wt animals, all cells tested displayed a small and graded local Ca2+ transient in PC spines and spiny branchlets (Fig. 3.7A,B).  This signal became detectable after the third or the fourth stimulation in the 100 Hz train (Fig. 3.7C; n=6) and increased after each of the following stimulations.  In all of the recordings, the size of the first EPSP in the train was used to monitor the strength of the stimulation (mean unitary EPSP size: 2.2 mV, n=6; Fig. 3.7).  It should be noted that regenerative Ca2+ spikes were never induced in the dendrites when stimulating the PFs only. Using the Cav3.1 KO mice, we show that a major component of the local Ca2+ transients evoked by a train of PF stimulation in target spines is mediated by Cav3.1 T-type Ca2+ channels, since the mean ΔF/R was 4.67 ± 1.2 % (n=6) in wt mice and 1 ± 0.3 % in KO mice (n=6; Fig. 3.7D).  In wt mice, perfusion of JNJ16259685 (1.5 μM), a very potent and specific antagonist of the mGluR1 receptor (Knopfel, 2007), strongly reduced local Ca2+ transients (mean ΔF/R=2.45 ± 0.9 %; Fig. 3.7C,D), suggesting that the train of PF stimulation underlies an mGluR1 receptor-mediated potentiation of the local Ca2+ transient.  This effect occurred with a fast time course (Fig. 3.7C) that is consistent with the penetration kinetics of the antagonist in the slice.  Moreover, in Cav3.1 KO mice, no effect was observed after application of JNJ16259685 (1.5 μM) (mean ΔF/R=1.2 ± 0.28%; Fig. 3.7D).  Thus, bursts of PF EPSPs can activate Cav3.1-mediated Ca2+ transients that are strongly potentiated by mGluR1 activation, and therefore, activation of the mGluR1 receptor by trains of PFs can locally and selectively modulate Ca2+ signaling in PC dendrites.  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  116  Figure 3.7 - A burst of PF stimulation modulates Cav3.1 calcium channels via mGluR1 receptors. A) Schematic drawing of experimental arrangement.  The stimulation pipette is in blue while the recording pipette is in black, with an inset representative EPSP recorded at the soma following PF stimulation.  B) Fluorescence transients (ΔF/R) in spiny branchlets of the mouse PC positioned on a contrast enhanced 2-photon section.  In red, average fluorescence of 3-7 POIs in the regions of interest outlined with white dashed-lines.  Note the local and graded Ca2+ transients. In blue, position of the stimulations delivered in PFs (11 stimulations/100 Hz).  C) Left panel, reduction (red dashed line) of the PF-induced Ca2+ transient following perfusion of JNJ16259685 (1.5 µM).  Each trace is an average of 5 consecutives trials.  Fluorescence traces are an averaging of all responsive spiny branchlets.  Right panel, time course of the size the first EPSP in the train (black) and change of Ca2+ fluorescence (red) following perfusion of JNJ16259685 (1.5 µM) in all of the cells tested (n=6).  D) Effect of JNJ16259685 application on the change of Ca2+ fluorescence in wt (n=6) and Cav3.1 -/- mice (n=6).  Left panel, individual cells, Right panel, average histograms. *p<0.05 Wilcoxon Matched-Pairs Signed-Ranks Test. **p<0.01 Wilcoxon test. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  117 3.3 Discussion 3.3.1 mGluR1 potentiation of Cav3.1 T-type calcium channels We report here that recombinant and native Cav3.1 T-type Ca2+ currents are potentiated by mGluR1 activation.  In recombinant expression systems mGluR1 modulation is shown to be subtype- specific, with a potentiation of Cav3.1 and Cav3.2 isoforms and an inhibition of Cav3.3 channels.  The latter is consistent with the known inhibition of Cav3.3 channels by Gαq/11-coupled mAChRs (Hildebrand et al., 2007). In PCs from young rodents (P7-P15) T-type currents can be isolated and are mainly mediated by the Cav3.1 channels.  As previously reported (Isope and Murphy, 2005), young PC T-type currents have a low sensitivity to Ni2+, indicating little functional expression of Cav3.2, and their biophysical properties match closer with Cav3.1 rather than Cav3.3 currents.  We show here that T-type Ca2+ currents are undetectable in PCs from young Cav3.1 KO mice.  These findings are in agreement with in situ hybridization on 250-350 g Sprague Dawley rats that revealed very high expression of Cav3.1 in PCs, with Cav3.2 and Cav3.3 signals below detection levels (Talley et al., 1999).  However, an immunohistochemistry study showed high expression of Cav3.3 in PCs of P14 to P21 Sprague Dawley rats (Molineux et al., 2006) suggesting that Cav3.3 expression may increase during the third week of cerebellar development.  Indeed, in our imaging experiments in 3-4 week old Cav3.1 KO mice, a small component of the PF-induced subthreshold Ca2+ transient remained that may be mediated by Cav3.3 (Fig. 3.7D). When DHPG, an agonist of mGluR1 receptors, was applied onto acute cerebellar slices from young rats or mice, PC T-type Ca2+ currents were robustly and reversibly potentiated as expected if Cav3.1 subunits, but not Cav3.3 subunits, predominate.  As observed in the heterologous system expressing Cav3.1, mGluR1-mediated potentiation did not alter the activation or inactivation kinetics of the T-type current.  Using fast optical mapping of Ca2+ influx we show that the T-type mediated Ca2+ influx is widespread in the PC soma and dendrites. However, the mGluR1-mediated potentiation is restricted to the spines and proximal dendrites, where these metabotropic receptors are found in association with PF and CF synapses, respectively (Lopez-Bendito et al., 2001).  3.3.2 Signal transduction pathway As shown in Figure 3.1, activation of mGluR1a with glutamate in the HEK expression system causes a potentiation of Cav3.1 and Cav3.2 currents with a slow and delayed onset.  In contrast, mGluR1a activation causes a rapid and saturating inhibition of the Cav3.3 currents similar to that shown for the muscarinic modulation of Cav3.3 T-type currents in the same system (Hildebrand et al., 2007).  Since the same Gαq/11 protein is coupled to the metabotropic receptors that cause these modulations of Cav3.1, Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  118 Cav3.2, and Cav3.3 currents, we postulate that different downstream transduction pathways in combination with specific channel isoforms determines the sign of the T-type modulation (see also (Iftinca et al., 2007)).  Indeed, we have previously shown that the intracellular pathway leading to the inhibition of the Cav3.3 currents is independent from classical PLC/IP3R pathways, Ca2+-independent, but partially Gβγ-dependant.  In contrast to this “inhibition” pathway, the mGluR1-mediated “potentiation” of Cav3.1 currents within PCs is also PLC/IP3R/Ca2+ store-independent, but depends on tyrosine phosphatase activity and intracellular Ca2+ signals, as blocking tyrosine phosphatases or buffering Ca2+ using high concentration of BAPTA attenuates the T-type current stimulation.  Although non-classical, this PLC-independent, tyrosine phosphatase-dependent pathway was already described at the PF-PC synapse by Canepari and Ogden (2003) between the mGluR1 receptor and the sEPSC, identified as the non-specific cationic channel TRPC1 (Kim et al., 2003), and also between mGluR1 and TRP channels in hippocampal inhibitory interneurons (Topolnik et al., 2006).  Canepari and Ogden showed that photolysis of glutamate in cerebellar slices activates the sEPSC via a G protein and protein tyrosine kinase/phosphatase pathway.  Protein tyrosine kinase (PTK) inhibitor enhanced the sEPSC while protein tyrosine phosphatase inhibitor blocked this current.  We similarly observed a non- significant potentiation of the T-type current using 2.5 µM staurosporine, a broad antagonist of protein kinases like PKC and PTK. Since sEPSC antagonists (IEM 1460 or NA-spermine) have no effect on the T-type current modulation, the sEPSC channels and T-type channels appear to be independently regulated by mGluR1 activity.  Blocking IP3Rs also does not alter the mGluR1-mediated potentiation of T-type currents, leading to the hypothesis that the intracellular Ca2+ signal required for this T-type potentiation could be due to Ca2+ influx through the T-type channel itself, with possible contributions from either the sEPSC channels or IP3Rs not being ruled out.  In support of this notion, it was observed that the mGluR1- mediated potentiation of T-type Ca2+ influx spread further along the dendritic shaft from the putative mGluR1 synaptic PF and CF microdomains when IP3Rs were not blocked with heparin, consistent with past literature on mGluR1-induced, IP3R-dependent Ca2+ waves (Nakamura et al., 1999).  3.3.3 Physiological implications of alterations in T-type biophysical properties Our data indicate that the mGluR1-dependent modulation of Cav3.1 channels involves a modification of channel gating properties rather than a change in channel expression; the effect can be induced within seconds by agonist application and the potentiation involves a shift in the activation curve (Fig. 3.3).  In support of a direct T-type channel interaction, tyrosine phosphatase activity has been shown to facilitate Cav3 currents through a proposed transition to a high conductance state in spermatogenic cells (Arnoult et al., 1997).  The observed alteration of T-type biophysical properties by mGluR1a activation has potential physiological implications. Interestingly, the mGluR1a-induced shift in Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  119 the activation curve of the Cav3.1 channels leads to a more efficient potentiation at potentials between -45 mV and -40 mV.  At -45 mV, the T-type current increased two-fold upon mGluR1 activation while only 20 % at -20 mV.  The stringent recording conditions used for the characterization of these biophysical parameters (see sections 3.2 and 3.4) make it unlikely that poor voltage-clamp may account for the activation shift.  We show here that this activation shift can significantly influence dendritic excitability and synaptic integration.  mGluR1 activation decreases the threshold for T-type Ca2+ spike generation (Fig. 3.4) when other conductances are blocked and reduces the latency of the first spike appearance upon depolarization in physiological conditions.  Parallel fiber and/or CF activation of mGluR1 receptors and subsequent potentiation of T-type Ca2+ conductances may thus promote PC transitions from the hyperpolarized state to the depolarized state, in which synaptic integration and spike coding can proceed (Loewenstein et al., 2005).  3.3.4 mGluR1 potentiation of Cav3.1-mediated calcium influx in response to synaptic activity We imaged Ca2+ influx in response to trains of PF stimulations and found that the response was local and significantly smaller in Cav3.1 KO mice compared to control mice.  Interestingly, the first EPSP in the train did not induce any Ca2+ influx, suggesting that temporal summation is required to reach the threshold for T-type channel activation.  Based on pairs of GC and PC recordings (Isope and Barbour, 2002), we calculate that about 30 PF inputs (mean unitary connection: 8.4 pA; conversion factor: 8.3 µV/pA) are required to generate an EPSP of 2 mV at the soma (mean unitary value for the results in Fig. 3.7).  Roth and Hausser (2001) showed that an EPSP recorded at the soma is attenuated by 6-fold compared to synaptic locations, indicating that a 2 mV EPSP at the soma corresponds to a 12 mV depolarization at synaptic sites.  Because hyperpolarized potentials are necessary to remove the inactivation of T-type currents, 12 mV is indeed too small to span the gap to the foot of the T-type activation curve.  High frequency bursts around 100 Hz may produce sufficient temporal summation to lead to T-type channel opening.  In addition, recent results suggest the existence of a membrane potential overshoot in the spine head when compared to the parent shaft due to the neck resistance (Araya et al., 2006).  This electrical amplification may be regulated, as assessed by measuring the spine-dendrite diffusion coefficient (Bloodgood and Sabatini, 2005; Svoboda et al., 1996) and could permit local T-type channel activation.  By shifting the activation curve and potentiating T-type conductance at spines receiving a burst of EPSPs, mGluR1 activation could efficiently enhance the Ca2+ influx at the active spine relative to nearby inactive spines (see Figs. 3.6 and 3.7).  As suggested recently for sodium channels (Araya et al., 2007), Ca2+ currents could in turn boost spine depolarization, potentially affecting the transmission of the EPSPs to the soma.  Overall, our results unravel a novel mechanism generating Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  120 spine-specific, frequency-dependent Ca2+ signaling that might play an important role in dendritic integration and plasticity.  3.4 Experimental procedures 3.4.1 HEK 293 cell culture, transfection, and electrophysiology All equipment, procedures, and reagents were used as described in detail for the study of modulation of recombinant T-type channels by mAChRs (Hildebrand et al., 2007).  Briefly, HEK 293 cells that stably expressed either rat Cav3.1, Cav3.2 or Cav3.3 subunits were transiently transfected with mGluR1a using Lipofectamine (Invitrogen) and perforated patch recordings (with β-Escin) were performed 48 hours later.  The external recording solution contained (in mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 40 TEACl, 92 CsCl, 10 glucose, pH=7.4, while the internal pipette solution contained (in mM): 120 CsMethanesulfonate, 11 EGTA, 10 HEPES, 2 MgCl2, and 75-100 μM β-Escin (for perforated patch) pH=7.2.  During pharmacology experiments, cells were considered “potentiated” or “inhibited” when they displayed a >10% modulating effect with a clear exponential time course.  All remaining cells were grouped into a “no effect” group.  To quantify the effect of 100 μM glutamate, peak current levels were allowed to reach equilibrium and then 2 to 5 values were averaged.  3.4.2 Animals All experimental procedures involving animals and their care were performed in accordance with recommendations of the Canadian Council on Animal Care and the regulations and policies of the University of British Columbia Animal Care Facility and the University Animal Care Committee.  Mice lacking the cacna1e gene (encoding Cav2.3) and the cacna1g were respectively produced as previously described (Pereverzev et al., 2002) (Petrenko et al., 2007).  All animals were bred under an identical C57/Bl6 background, and litter mate controls were used where possible.  3.4.3 Slice preparation Male Wistar rats (8-12 days old, but mostly 9-10 days old) or male CBL57/Bl6 mice or mutant mice (8-12 days old) were anaesthetized with halothane and decapitated.  The head was immediately chilled over ice and the cerebellar vermis was removed with a scalpel and placed in ice cold bicarbonate- buffered saline (BBS) solution containing (in mM): 120 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 20 glucose, 1 kynurenate, 0.1 picrotoxin.  During cutting, incubating, and recording, slices were constantly bubbled with 95% O2/5% CO2 (carbogen).  The vermis was glued in the sagittal orientation to the stage of a Vibratome 1500 Sectioning System (MO, USA) and 200 to 250 μm sagittal Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  121 slices were cut from the cerebellar vermis, transferred to BBS at 32°C, and allowed to cool down passively to room temperature. For imaging and KO mice experiments, slices were cut in a protecting solution containing (in mM): 130 KGluconate, 14.6 KCl, 2 EGTA, 20 HEPES, 25 Glucose, D-APV 0.05 and 0.00005 minocycline.  Before the transfer into normal BBS solution, slices were soaked in sucrose-based solution containing (in mM): 230 sucrose, 2.5 KCl, 26 NaHCO3, 1.25 NaHPO4, 25 Glucose, 0.8 CaCl2, 8 MgCl2, D-APV 0.05 and 0.00005 minocycline.  For PF stimulation experiments older animals (16-30 days old) were used.  All experiments were performed on Wistar rats, except for the experiments shown in Figure 3.2E,F and Figure 3.7, where wt, Cav2.3, and Cav3.1 KO mice were used as indicated.  3.4.4 Electrophysiological recordings Slices were transferred to a Warner RC-26G recording chamber (total working volume = ~ 250 μL; CT, USA) and perfused with bubbling modified BBS external solution, containing (in mM): 120 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 20 glucose, 1 kynurenate, 0.1 picrotoxin, 0.3 tetrodotoxin, 5 TEACl, 1 4-aminopyridine, 0.05 NiCl2, and 0.02 CdCl2.  The 20 μM Cd2+ was used as an effective antagonist of all HVA Ca2+ currents that left T-type currents unaffected (Tai et al., 2006), while 50 μM Ni2+ blocked R-type but not Cav3.1 or Cav3.3 currents (Lee et al., 1999; Zamponi et al., 1996).  Cerebellar PCs were visually identified using a Zeiss Axioskop 2 microscope with an Achroplan 60 X water immersion lens.  Whole-cell patch clamp recordings from PCs were performed using a Multiclamp 700B amplifier and Digidata 1322A (MDS Analytical Technologies), controlled and monitored with a computer running pClamp9 software (MDS Analytical Technologies).  Patch pipettes (borosilicate glass BF150-86-10; Sutter Instruments; CA, USA), were pulled using a Sutter P-87 puller and had typical resistances of 3-5 MΩ when filled with internal solution. For voltage-clamp experiments, the internal solution contained (in mM): 140 CsMethanesulfonate, 5 TEACl, 0.5 MgCl2, 10 HEPES, 4 MgATP, 0.5 Na3GTP, and 0.3 EGTA, adjusted to pH=7.3, ~290 mOsm.  In specific pharmacology experiments, 0.5 mM Na3GTP was replaced with 2 mM GDP-β-S.  Cells were held at a holding potential of Vh= -75 mV.  Cells with leak current above 600 pA at Vh= -75 mV were discarded.  Data were low-pass filtered at 2 kHz using the built-in Bessel filter of the amplifier, with sampling at 20 kHz.  Series resistance was compensated between 70 and 80 % on every cell.  Leak subtraction of capacitance current and IL was performed on-line using a P/6 protocol with reverse polarity pulses for channel kinetics experiments and with Clampfit9 (MDS Analytical Technologies) during offline analysis for all other experiments.  Both the leak-subtracted and non leak- subtracted traces were acquired through separate channels for all protocols.  Current-voltage Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  122 relationships and channel activation, inactivation, and deactivation rates were analyzed as previously described (Hildebrand et al., 2007). For current clamp experiments, the external solution (termed ACSF) contained (in mM): 125 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 glucose, 1 kynurenate and 0.1 picrotoxin and the internal solution contained (in mM): 150 KGluconate, 4 NaCl, 10 HEPES, 10 MgATP and 0.5 Na3GTP, adjusted to pH=7.3 and 295 mOsm.  For T-type spike experiments, the voltage-clamp internal solution was used.  Recordings were low-pass filtered at 10 kHz, with sampling at 50 kHz.  Input resistance was calculated and averaged from the voltages measured at equilibrium during small depolarizing and hyperpolarizing currents (between -50 pA and +50 pA) that elicited no dramatic voltage-gated changes (see Fig. 3.4A for example).  To determine the inflection point where T-type spikes or APs are initiated, the slopes of the relevant traces were extrapolated and plotted in Microcal Origin (Version 7.5, Northampton, MA) and the inflection point where the slope stops decreasing and starts increasing was determined.  The time value at this point represents the spike latency and the corresponding membrane voltage value from the original trace represents the V threshold.  The maximum slope (dV/dtmax) was also determined from these differentiation plots. All recordings were performed at room temperature (20-24oC) except for PF stimulation where slices were maintained at 32°C.  Figures and fittings utilized the software program Microcal Origin. Statistical significance was determined by Student’s T-Tests or non parametric Wilcoxon rank test, and significant values were set as indicated in the text and figure legends.  3.4.5 Compounds and perfusion (S)-3,5-DHPG, LY367385, (S)-MCPG, U73122, IEM 1460, and SKF 96365 were all obtained from Tocris Cookson (MO, USA).  Edelfosine and bpV(phen) was obtained from Calbiochem (CA, USA).  All other drugs were ordered from Sigma-Aldrich (MO, USA).  Drugs were dissolved in dH2O, equimolar NaOH, or DMSO, according to manufacturers solubility data.  The highest concentration of DMSO in the recording solution did not exceed 0.1%, a concentration that did not detectably affect Ca2+ channel properties.  Cells were gravity perfused with solutions at flow rates > 1ml/minute through high chemical-resistant PTFE tubing (Cole-Parmer Instrument Company; Il, USA).  A closed perfusion system (5-20 ml) was also used to maintain high concentrations of antagonists and agonists during experiments, with separate lines containing control or DHPG solutions.  During washout experiments, the perfusion system was open with solution flowing into a waste container.  For a more physiological activation of mGluR1, DHPG was puffed directly onto the recorded PC using a 50 ms, 10 PSI pressure pulse delivered by a Parker Hannifin Picospritzer III to a patch pipette loaded with 100 μM DHPG and placed within close proximity of the PC.   Control recordings showed that puffing on antagonist was able Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  123 to block T-type currents (puffing 100 μM SKF 96365 inhibited T-type current by 28% +/- 4%, n=4, while puffing control BBS had no effect on T-type currents (2% +/- 1%, n=2)).  3.4.6 Two-photon imaging Calcium transients in PCs were imaged with a custom-built multi-photon laser scanning microscope in which both X and Y scanning are operated by acousto-optic deflectors (AOD; A-A Opto- Electronics; based on the AA.DTS.XY-250 model).  Two-photon excitation was produced by an infrared Ti-Sa pulsed laser (Tsunami pumped by a Millenia VI, Spectra-Physics) set to 825 nm and tuned to 700 fs to mitigate AOD-induced dispersion.  In this non-mechanical scanning microscope, deflection of the laser beam to a specific position is obtained by setting the appropriate acoustic wavelength in the AOD crystal.  Switching the illumination between any two points takes 4 μs permitting random access imaging at high frame rates.  National Instrument boards programmed under Labview were used to implement digital scanning strategies and to synchronize AOD scanning and photon-counting detection with a cooled AsGaP photomultiplier (H7421-40, Hamamatsu). Whole cell recordings were performed with an Axoclamp amplifier (Axon Instruments).  PCs were visualized using a combination of gradient contrast and on-line video contrast enhancement (CoolSnapCf and Metamorph, Roper Scientific) at wavelengths of 670-740 nm.  Pipettes were filled with intracellular solution that was supplemented with a morphological dye (Alexa 594, 10-20 μM , Molecular Probes) and a Ca2+-sensitive dye (Fluo5F 400 μM).  In order to block P/Q-type Ca2+ channels, slices were pre-incubated for at least 30 min in a chamber containing 1 ml of BBS supplemented with 1µM Agatoxin IVA/0.1%BSA (except for the PF stimulation experiment).  Besides the addition of Agatoxin IVA and the omission of Cd2+, the same modified BBS external solution was used as for the above whole-cell voltage-clamp recordings.  Pipettes resistance ranged from 3 to 5 MΩ.  Current was injected to hold the cells at membrane potentials of -60 to -65 mV.  PC imaging was started after at least 30 min of whole-cell dialysis.  Spines were resolved and POIs were placed on spines’ heads and attached dendritic shafts.  Optical transients were subsequently monitored simultaneously in 10 to 50 spines and dendrites at frame rates close to 1 kHz (dwell time 20-50 μs).  In this multiunit recording mode, the POI sequence is sampled repetitively and the signal for each location is displayed online as a continuous or episodic recording and analyzed offline using custom routines written in Igor (Wavemetrics).  Variation of fluorescence was calculated as a ratio of the change in fluorescence (ΔF) of the Ca2+ dye over the fluorescence (R) of the red morphological dye (Alexa 594 ), labeled ΔF/R.  This strategy overcomes movements and loading artifacts and errors due to changes in basal Ca2+ concentration.  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  124 3.5 Acknowledgements We thank Dr. Brian MacVicar for comments on the manuscript.  This work is supported by an operating grant from the Canadian Institutes of Health Research and a Canada Research Tier 1 Chair to T.P.S., by The Centre National pour la Recherche Scientifique, L'Ecole Normale Supérieure, and the L'Agence Nationale pour La Recherche and Human Frontier Science Program, and by Grants-in-Aid for Scientific Research [17023021,17100004] from the Ministry of Education, Culture, Sports, Science and Technology of Japan.  M.E.H. was supported by trainee fellowships from the Natural Sciences and Engineering Research Council of Canada and Michael Smith Foundation for Health Research.  Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  125 3.6 References Aiba, A., Kano, M., Chen, C., Stanton, M.E., Fox, G.D., Herrup, K., Zwingman, T.A., and Tonegawa, S. (1994). Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79, 377-388. Anderson, M.P., Mochizuki, T., Xie, J., Fischler, W., Manger, J.P., Talley, E.M., Scammell, T.E., and Tonegawa, S. (2005). Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc Natl Acad Sci U S A 102, 1743-1748. Araya, R., Jiang, J., Eisenthal, K.B., and Yuste, R. (2006). The spine neck filters membrane potentials. Proc Natl Acad Sci U S A 103, 17961-17966. Araya, R., Nikolenko, V., Eisenthal, K.B., and Yuste, R. (2007). Sodium channels amplify spine potentials. Proc Natl Acad Sci U S A 104, 12347-12352. Arnoult, C., Lemos, J.R., and Florman, H.M. (1997). Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation. Embo J 16, 1593-1599. Baude, A., Nusser, Z., Roberts, J.D., Mulvihill, E., McIlhinney, R.A., and Somogyi, P. (1993). The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11, 771-787. Bloodgood, B.L., and Sabatini, B.L. (2005). Neuronal activity regulates diffusion across the neck of dendritic spines. Science 310, 866-869. Bourinet, E., Alloui, A., Monteil, A., Barrere, C., Couette, B., Poirot, O., Pages, A., McRory, J., Snutch, T.P., Eschalier, A., and Nargeot, J. (2005). Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. Embo J 24, 315-324. Canepari, M., Auger, C., and Ogden, D. (2004). Ca2+ ion permeability and single-channel properties of the metabotropic slow EPSC of rat Purkinje neurons. J Neurosci 24, 3563-3573. Canepari, M., and Ogden, D. (2003). Evidence for protein tyrosine phosphatase, tyrosine kinase, and G- protein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons. J Neurosci 23, 4066-4071. Chadderton, P., Margrie, T.W., and Hausser, M. (2004). Integration of quanta in cerebellar granule cells during sensory processing. Nature 428, 856-860. Chemin, J., Monteil, A., Perez-Reyes, E., Bourinet, E., Nargeot, J., and Lory, P. (2002). Specific contribution of human T-type calcium channel isotypes (alpha1G, alpha1H and alpha1I) to neuronal excitability. J Physiol 540, 3-14. Chen, C.C., Lamping, K.G., Nuno, D.W., Barresi, R., Prouty, S.J., Lavoie, J.L., Cribbs, L.L., England, S.K., Sigmund, C.D., Weiss, R.M., et al. (2003). Abnormal coronary function in mice deficient in alpha1H T-type Ca2+ channels. Science 302, 1416-1418. Christie, B.R., Schexnayder, L.K., and Johnston, D. (1997). Contribution of voltage-gated Ca2+ channels to homosynaptic long-term depression in the CA1 region in vitro. J Neurophysiol 77, 1651-1655. Conquet, F., Bashir, Z.I., Davies, C.H., Daniel, H., Ferraguti, F., Bordi, F., Franz-Bacon, K., Reggiani, A., Matarese, V., Conde, F., and et al. (1994). Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1. Nature 372, 237-243. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  126 Crepel, F., and Daniel, H. (2007). Developmental changes in agonist-induced retrograde signaling at parallel fiber-Purkinje cell synapses: role of calcium-induced calcium release. J Neurophysiol 98, 2550- 2565. Crunelli, V., Cope, D.W., and Hughes, S.W. (2006). Thalamic T-type Ca2+ channels and NREM sleep. Cell Calcium 40, 175-190. Egger, V., Svoboda, K., and Mainen, Z.F. (2005). Dendrodendritic synaptic signals in olfactory bulb granule cells: local spine boost and global low-threshold spike. J Neurosci 25, 3521-3530. Finch, E.A., and Augustine, G.J. (1998). Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites. Nature 396, 753-756. Hartmann, J., Blum, R., Kovalchuk, Y., Adelsberger, H., Kuner, R., Durand, G.M., Miyata, M., Kano, M., Offermanns, S., and Konnerth, A. (2004). Distinct roles of Galpha(q) and Galpha11 for Purkinje cell signaling and motor behavior. J Neurosci 24, 5119-5130. Hildebrand, M.E., David, L.S., Hamid, J., Mulatz, K., Garcia, E., Zamponi, G.W., and Snutch, T.P. (2007). Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors. J Biol Chem 282, 21043-21055. Ichise, T., Kano, M., Hashimoto, K., Yanagihara, D., Nakao, K., Shigemoto, R., Katsuki, M., and Aiba, A. (2000). mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination. Science 288, 1832-1835. Iftinca, M., Hamid, J., Chen, L., Varela, D., Tadayonnejad, R., Altier, C., Turner, R.W., and Zamponi, G.W. (2007). Regulation of T-type calcium channels by Rho-associated kinase. Nat Neurosci 10, 854- 860. Ikeda, H., Heinke, B., Ruscheweyh, R., and Sandkuhler, J. (2003). Synaptic plasticity in spinal lamina I projection neurons that mediate hyperalgesia. Science 299, 1237-1240. Isope, P., and Barbour, B. (2002). Properties of unitary granule cell-->Purkinje cell synapses in adult rat cerebellar slices. J Neurosci 22, 9668-9678. Isope, P., and Murphy, T.H. (2005). Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T-type calcium channels. J Physiol 562, 257-269. Joksovic, P.M., Nelson, M.T., Jevtovic-Todorovic, V., Patel, M.K., Perez-Reyes, E., Campbell, K.P., Chen, C.C., and Todorovic, S.M. (2006). CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus. J Physiol 574, 415-430. Jorntell, H., and Ekerot, C.F. (2006). Properties of somatosensory synaptic integration in cerebellar granule cells in vivo. J Neurosci 26, 11786-11797. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W., and Shin, H.S. (2001). Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31, 35-45. Kim, S.J., Kim, Y.S., Yuan, J.P., Petralia, R.S., Worley, P.F., and Linden, D.J. (2003). Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426, 285-291. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  127 Kishimoto, Y., Fujimichi, R., Araishi, K., Kawahara, S., Kano, M., Aiba, A., and Kirino, Y. (2002). mGluR1 in cerebellar Purkinje cells is required for normal association of temporally contiguous stimuli in classical conditioning. Eur J Neurosci 16, 2416-2424. Kitano, J., Nishida, M., Itsukaichi, Y., Minami, I., Ogawa, M., Hirano, T., Mori, Y., and Nakanishi, S. (2003). Direct interaction and functional coupling between metabotropic glutamate receptor subtype 1 and voltage-sensitive Cav2.1 Ca2+ channel. J Biol Chem 278, 25101-25108. Knopfel, T. (2007). Two new non-competitive mGlu1 receptor antagonists are potent tools to unravel functions of this mGlu receptor subtype. Br J Pharmacol 151, 723-724. Knopfel, T., and Grandes, P. (2002). Metabotropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells. Cerebellum 1, 19-26. Kozlov, A.S., McKenna, F., Lee, J.H., Cribbs, L.L., Perez-Reyes, E., Feltz, A., and Lambert, R.C. (1999). Distinct kinetics of cloned T-type Ca2 + channels lead to differential Ca2 + entry and frequency- dependence during mock action potentials. Eur J Neurosci 11, 4149-4158. Kuo, C.C., Chen, W.Y., and Yang, Y.C. (2004). Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence. J Gen Physiol 124, 27-42. Lee, J.H., Gomora, J.C., Cribbs, L.L., and Perez-Reyes, E. (1999). Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J 77, 3034-3042. Loewenstein, Y., Mahon, S., Chadderton, P., Kitamura, K., Sompolinsky, H., Yarom, Y., and Hausser, M. (2005). Bistability of cerebellar Purkinje cells modulated by sensory stimulation. Nat Neurosci 8, 202-211. Lopez-Bendito, G., Shigemoto, R., Lujan, R., and Juiz, J.M. (2001). Developmental changes in the localisation of the mGluR1alpha subtype of metabotropic glutamate receptors in Purkinje cells. Neuroscience 105, 413-429. Maejima, T., Oka, S., Hashimotodani, Y., Ohno-Shosaku, T., Aiba, A., Wu, D., Waku, K., Sugiura, T., and Kano, M. (2005). Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase Cbeta4 signaling cascade in the cerebellum. J Neurosci 25, 6826-6835. McDonough, S.I., and Bean, B.P. (1998). Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons. Mol Pharmacol 54, 1080-1087. McKay, B.E., McRory, J.E., Molineux, M.L., Hamid, J., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons. Eur J Neurosci 24, 2581-2594. McRory, J.E., Santi, C.M., Hamming, K.S., Mezeyova, J., Sutton, K.G., Baillie, D.L., Stea, A., and Snutch, T.P. (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem 276, 3999-4011. Meacham, C.A., White, L.D., Barone, S., Jr., and Shafer, T.J. (2003). Ontogeny of voltage-sensitive calcium channel alpha(1A) and alpha(1E) subunit expression and synaptic function in rat central nervous system. Brain Res Dev Brain Res 142, 47-65. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  128 Molineux, M.L., McRory, J.E., McKay, B.E., Hamid, J., Mehaffey, W.H., Rehak, R., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Specific T-type calcium channel isoforms are associated with distinct burst phenotypes in deep cerebellar nuclear neurons. Proc Natl Acad Sci U S A 103, 5555-5560. Nakamura, T., Barbara, J.G., Nakamura, K., and Ross, W.N. (1999). Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials. Neuron 24, 727-737. Pereverzev, A., Mikhna, M., Vajna, R., Gissel, C., Henry, M., Weiergraber, M., Hescheler, J., Smyth, N., and Schneider, T. (2002). Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels. Mol Endocrinol 16, 884-895. Perez-Reyes, E. (2003). Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83, 117-161. Petrenko, A.B., Tsujita, M., Kohno, T., Sakimura, K., and Baba, H. (2007). Mutation of alpha1G T-type calcium channels in mice does not change anesthetic requirements for loss of the righting reflex and minimum alveolar concentration but delays the onset of anesthetic induction. Anesthesiology 106, 1177- 1185. Pouille, F., Cavelier, P., Desplantez, T., Beekenkamp, H., Craig, P.J., Beattie, R.E., Volsen, S.G., and Bossu, J.L. (2000). Dendro-somatic distribution of calcium-mediated electrogenesis in purkinje cells from rat cerebellar slice cultures. J Physiol 527 Pt 2, 265-282. Randall, A.D., and Tsien, R.W. (1997). Contrasting biophysical and pharmacological properties of T- type and R-type calcium channels. Neuropharmacology 36, 879-893. Roth, A., and Hausser, M. (2001). Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings. J Physiol 535, 445-472. Sacco, T., and Tempia, F. (2002). A-type potassium currents active at subthreshold potentials in mouse cerebellar Purkinje cells. J Physiol 543, 505-520. Shigemoto, R., Nakanishi, S., and Mizuno, N. (1992). Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J Comp Neurol 322, 121-135. Svoboda, K., Tank, D.W., and Denk, W. (1996). Direct measurement of coupling between dendritic spines and shafts. Science 272, 716-719. Swensen, A.M., and Bean, B.P. (2003). Ionic mechanisms of burst firing in dissociated Purkinje neurons. J Neurosci 23, 9650-9663. Tai, C., Kuzmiski, J.B., and MacVicar, B.A. (2006). Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons. J Neurosci 26, 6249-6258. Takechi, H., Eilers, J., and Konnerth, A. (1998). A new class of synaptic response involving calcium release in dendritic spines. Nature 396, 757-760. Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E., and Bayliss, D.A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19, 1895-1911. Chapter 3: Cav3.1 potentiation by mGluR1 in Purkinje cells  129 Tanaka, J., Nakagawa, S., Kushiya, E., Yamasaki, M., Fukaya, M., Iwanaga, T., Simon, M.I., Sakimura, K., Kano, M., and Watanabe, M. (2000). Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells. Eur J Neurosci 12, 781-792. Tempia, F., Alojado, M.E., Strata, P., and Knopfel, T. (2001). Characterization of the mGluR(1)- mediated electrical and calcium signaling in Purkinje cells of mouse cerebellar slices. J Neurophysiol 86, 1389-1397. Topolnik, L., Azzi, M., Morin, F., Kougioumoutzakis, A., and Lacaille, J.C. (2006). mGluR1/5 subtype- specific calcium signalling and induction of long-term potentiation in rat hippocampal oriens/alveus interneurones. J Physiol 575, 115-131. Welsby, P.J., Wang, H., Wolfe, J.T., Colbran, R.J., Johnson, M.L., and Barrett, P.Q. (2003). A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II. J Neurosci 23, 10116-10121. Williams, S.R., Christensen, S.R., Stuart, G.J., and Hausser, M. (2002). Membrane potential bistability is controlled by the hyperpolarization-activated current I(H) in rat cerebellar Purkinje neurons in vitro. J Physiol 539, 469-483. Williams, S.R., Toth, T.I., Turner, J.P., Hughes, S.W., and Crunelli, V. (1997). The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones. J Physiol 505 ( Pt 3), 689-705. Wolfe, J.T., Wang, H., Howard, J., Garrison, J.C., and Barrett, P.Q. (2003). T-type calcium channel regulation by specific G-protein betagamma subunits. Nature 424, 209-213. Womack, M.D., and Khodakhah, K. (2004). Dendritic control of spontaneous bursting in cerebellar Purkinje cells. J Neurosci 24, 3511-3521. Zamponi, G.W., Bourinet, E., and Snutch, T.P. (1996). Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites. J Membr Biol 151, 77-90.   Chapter 4: Discussion  130 4 DISCUSSION  4.1 Overall significance and strengths 4.1.1 T-type calcium channel modulation in a heterologous system The molecular and physiological analysis of T-type Ca2+ channel modulation is a relatively recent aspect of the Ca2+ channel field.  A major objective of this thesis was to contribute to this area by systematically investigating the effects of two types of neuronal GPCRs, mAChRs (Chapter 2) and mGluR1 (Chapter 3, Figure 1), concerning their ability to modulate the three main types of recombinant T-type Ca2+ channels (Cav3.1, Cav3.2 and Cav3.3).  An initial heterologous approach was chosen in order to eliminate complications associated with the multiple mAChR, mGluR and T-type Ca2+ channel subtypes co-expressed in many native cell types, an issue further complicated by a lack of subtype- specific pharmacological antagonists and possible space-clamping concerns in many native neuronal systems.  Most studies examining intracellular modulators of T-type channel activity, such as Gβγ and CAMKII, have not characterized the effects on all three Cav3 subtypes (Welsby et al., 2003; Wolfe et al., 2003).  Utilizing available channel and receptor clones, this is the first study to characterize the effects of GPCR activation on all three major T-type Ca2+ channel isoforms.  Our demonstration of the selective inhibition of Cav3.3 currents by Gαq/11-coupled mAChR and mGluR1 receptors provides the first evidence for the specific modulation of a T-type isoform other than Cav3.2.  This specific interaction may contribute to Cav3.3 channels playing unique roles in signaling and excitability within the nervous system.  The Gαq-mediated inhibition of Cav3.3 channels is also unique in that it does not involve PLC activity or its downstream signaling elements.  Further study of this pathway may shed light on a novel mode of voltage-independent inhibition that could exist for other classes of Ca2+ channels (see section 4.2.2 for further discussion).  Although the specific modulator that physically binds to Cav3.3 channels has yet to be elucidated, the regions of the Cav3.3 channel that are involved in this interaction have been conclusively identified.  As discussed in Chapter 2, two separate regions of the Cav3.3 are both necessary and sufficient for the inhibition of Cav3.3 by Gαq/11-coupled M1 receptors.  These regions do not contain the domain II-III linker “hotspot” of Cav3.2 modulation (Kim et al., 2006; Welsby et al., 2003; Wolfe et al., 2003), indicating that novel channel regions are involved in this T-type inhibition.  Finally, the potentiation of Cav3.1 and Cav3.2 channels by Gαq/11-coupled M1 and mGluR1 receptors in a percentage of HEK cells indicated that a separate modulatory pathway may exist for these T-type isoforms.  Further exploration into the mechanism and physiological relevance of this potentiation was examined in cerebellar PCs.  Chapter 4: Discussion  131 4.1.2 T-type calcium channel modulation in cerebellar Purkinje cells The basic biophysical properties of the T-type Ca2+ channels expressed in cerebellar PCs has been previously determined (Isope and Murphy, 2005), although their molecular composition, physiological roles and potential modulation by GPCRs remained unknown (reviewed in (Cavelier and Bossu, 2003)).  The present study is the first to demonstrate that cerebellar PC T-type currents consist of Cav3.1 channels.  Furthermore, we show that the physiological stimulation of PF bundles causes the activation of T-type currents at synapses within the distal dendrites of PCs.  In the first example of T- type Ca2+ current modulation within the cerebellum, we found that activation of mGluR1 results in a robust potentiation of T-type currents in both the proximal dendrites and spines of PCs.  This augmentation of T-type current involves both an increase in maximal conductance and a hyperpolarizing shift in the voltage-dependence of activation and occurs through a unique signaling pathway characteristic of mGluR1 signaling in PC dendrites (Canepari and Ogden, 2003).  The effects of the mGluR1-mediated T-type stimulation on local integration and overall excitability within PCs will be discussed in greater detail in section 4.3.1.  Our study on the modulation of PC T-type currents is strengthened by the use of multiple techniques including ultrafast two photon Ca2+ imaging, genetics (Cav3.1 and Cav2.3 KO mice), multiple forms of electrophysiological recordings (voltage clamp, current clamp, and synaptic stimulation), and pharmacological manipulations.  The combination of these strategies allowed us to largely overcome technical challenges such as a lack of specific T-type antagonists and potential space clamping issues.  4.2 Specific inhibition of Cav3.3 channels by Gαq/11-coupled receptors 4.2.1 Working hypothesis In Chapter 2, we demonstrated for the first time that Cav3.3 T-type channels can be specifically inhibited by Gαq/11-coupled mAChRs when expressed in the heterologous HEK system.  The elucidation of a native neuronal system where this modulation might exist is hindered by the lack of identification of Cav3.3 functional expression in the CNS and PNS.  Although many neuronal types have been shown to robustly express Cav3.3 mRNA (Talley et al., 1999) and protein (McKay et al., 2006), the contribution of Cav3.3 channels to functional native T-type currents has not been determined.  Unlike the Cav3.1 and Cav3.2 isoforms, KO mice have yet to be generated for the Cav3.3 isoform, and there are no Cav3.3- specific antagonists that have been identified to date.  The development of these tools along with the use of RNAi knock-down approaches are needed to investigate the impacts of Cav3.3-mediated signaling and excitability in the brain (proposals for future research can be found throughout this discussion chapter). The best candidate for a neuronal population expressing Cav3.3-mediated T-type currents is the thalamic nRT neuron.  It has been known for some time that T-type currents in nRT neurons have much Chapter 4: Discussion  132 slower activation and inactivation kinetics than other “typical” native T-type currents, such as those observed in thalamic TC cells (Huguenard and Prince, 1992).  Along with these unique biophysical properties, the T-type currents in acutely dissociated nRT neurons are only partially blocked by 200 μM Cd2+ and 100 μM Ni2+ (Huguenard and Prince, 1992), suggestive of the presence of Cav3.3 channels rather than Cav2.3, Cav3.1, or Cav3.2 channels (Fox et al., 1987; Lee et al., 1999; Tai et al., 2006; Zamponi et al., 1996).  However, Todorovic and colleagues have recently used an elegant combination of whole-cell patch, nucleated patch, and cell-attached recordings on nRT cells within thalamic slices to demonstrate a small somatic T-type current with fast inactivation kinetics and a larger dendritic T-type current that has significantly slower inactivation kinetics (Joksovic et al., 2005).  Currents in somatic nRT nucleated patches were 10-fold smaller in amplitude and over two-fold faster in inactivation kinetics compared to whole-cell currents (Joksovic et al., 2005).  The same voltage-dependence of activation between T-type currents in nucleated patch and whole-cell recordings indicated that the slower whole- cell currents were not just poorly clamped larger dendritic currents of the same subtype.  Furthermore, cell-attached recordings on proximal dendrites and the soma of nRT cells verified the slower inactivation rates of dendritic T-type currents.  Pharmacology experiments indicated that the faster somatic currents were over two-fold more sensitive to Ni2+ than the slowly inactivating dendritic T-type currents (Joksovic et al., 2005).  This combination of biophysical and pharmacological evidence combined with the detection of Cav3.2 and Cav3.3 mRNA in nRT cells (Talley et al., 1999) suggests that Cav3.3 channels primarily underlie dendritic T-type currents and Cav3.2 currents underlie the smaller somatic currents in nRT cells (Joksovic et al., 2005; Lee et al., 1999; McRory et al., 2001).  The reduction in T- type current amplitude in nRT neurons from Cav3.2 KO mice verifies the contribution of Cav3.2 channels (Joksovic et al., 2006), but the contribution of Cav3.3 channels has yet to be directly demonstrated. However, the resistance to redox potentiation, slower inactivation kinetics, and robust current amplitude of the remaining T-type currents in Cav3.2 KO mice (Joksovic et al., 2006) further implicates the functional presence of Cav3.3 channels in these cells. Ascending cholinergic systems carry sensory information from the PNS to the cortex via relay centers in the thalamus.  The molecular mechanism of relaying this information also regulates the sleep- wake cycle.  When awake, ascending cholinergic systems are activated and cause the resting membrane potential of TC cells to become depolarized.  This depolarization is primarily due to the reduction in a relatively linear, non-voltage-dependent potassium leak current (McCormick, 1992).  The cholinergic- mediated hyperpolarization of nRT neurons also reduces the GABAergic hyperpolarization of TC neurons (reviewed in (Steriade, 2004)).  In the depolarized state, TC T-type currents are no longer de- inactivated and a switch from rhythmic burst firing to tonic firing occurs, preventing the generation of slow sleep spindle waves (McCormick, 1992).  However, the possible direct effect of ascending cholinergic systems on thalamic T-type currents has not been explored.  nRT cells have been shown to Chapter 4: Discussion  133 express high levels of muscarinic acetylcholine M2 and M3 receptors (Levey et al., 1994; Plummer et al., 1999).  Activation of M3 receptors usually results in neuronal depolarization (reviewed in (Jones, 1993)), so their linkage to the hyperpolarizing inhibitory muscarinic response observed in nRT cells is currently unknown (Plummer et al., 1999). Based upon our observations in the heterologous system, activation of Gαq/11-coupled M3 receptors in nRT dendrites may cause the inhibition of native Cav3.3 currents and a concomitant increase in inactivation kinetics.  We also found that M2 receptor activation has no effect on Cav3.3 currents, although the effect of M2 and M3 receptor activation on recombinant Cav3.2 currents has not been tested.  Further experiments are needed to test the effects of activation of these mAChRs on Cav3.1 and Cav3.2 currents expressed in HEK cells.  If M3 receptor activation has the same effects on Cav3.2 currents as M1 receptor activation, then it could be hypothesized that activation of M3 receptors by ascending cholinergic systems would cause a potentiation of Cav3.2-mediated somatic nRT currents and an inhibition of Cav3.3-mediated dendritic nRT currents.  As these modulatory effects could effectively cancel each other out during whole cell recordings, the combined approach of nucleated patch, dendritic cell-attached, and whole-cell recordings needs to be used to test this hypothesis (Joksovic et al., 2005). The slow kinetics of native T-type currents in nRT neurons enables the generation of slow and prolonged bursts that are required for the GABAB-mediated hyperpolarization of TC neurons during rhythmic rebound burst sleep spindle oscillations (Huguenard and Prince, 1992).  An increase in Cav3.3 T-type current kinetics by the activation of M3 muscarinic receptors could cause nRT T-type currents to become more “Cav3.1-like” and potentially shorten burst firing in nRTs, promoting the transition from thalamic rebound burst firing to tonic firing at higher frequencies (Chemin et al., 2002).  Thus, in addition to its depolarizing effects, acetylcholine could act physiologically in a manner similar to exogenous succinimide antiepileptics that reduce burst firing and intrathalamic synchronization through the blockade of nRT T-type currents (Huguenard and Prince, 1992; Pellegrini et al., 1989).  This would support our overall hypothesis that the specific inhibition of Cav3.3-mediated T-type currents by Gαq/11- coupled receptors alters electrical excitability and firing rhythms within the nervous system.  4.2.2 Possible limitations and weaknesses Besides the current lack of demonstration of the inhibition of native Cav3.3 currents by Gαq/11- coupled mAChRs, several other limitations exist in our study on the modulation of T-type channels expressed in HEK cells.  Further experiments are still needed to identify the specific amino acid residues within the Cav3.3 channel that are involved in the Gαq/11-mediated inhibition.  However, before this can be achieved, the intracellular signaling molecule that links Gαq/11 activity to Cav3.3 inhibition needs to be Chapter 4: Discussion  134 identified.  One possibility is that the Gαq/11 molecule itself directly binds to and inhibits Cav3.3 channels. A commonly held view concerning Ca2+ channel modulation occuring through Gαq//11-coupled receptor pathways is that depletion of membrane PIP2 through the activation of PLC underlies the voltage-independent inhibition of Ca channels (and other ion channels; reviewed in (Gamper and Shapiro, 2007; Suh and Hille, 2005)).  However, several studies have now demonstrated that Gαq/11 binding itself can also directly modulate ion channel activity.  Similar to the Gαq/11-mediated inhibition of Cav3.3 channels, the substance P-induced inhibition of Kir3 G-protein coupled inward rectifier K+ channels is rapid, involves Gαq/11-protein signaling, and is independent of membrane PIP2 depletion (Koike-Tani et al., 2005).  Interestingly, Gαq has been shown to co-immunoprecipitate with Kir3.2 (Koike-Tani et al., 2005) and recent GST pull-down assays have conclusively demonstrated that Gαq binds directly and specifically to the N-terminus (likely between amino acids 81 - 90) of Kir3.1, Kir3.2, and Kir3.4 channels (Kawano et al., 2007).  The same direct Gαq-channel binding interaction that is independent of PLC activity has also been shown to underlie the inhibition of two-pore domain K+ leak channels (TASK-1 and TASK-2) by Gαq/11-coupled receptors (Chen et al., 2006). The direct binding of Gαq is not limited to K+ channels; co-immunoprecipitation experiments have revealed a physical interaction between Gαq and the proximal region of the C-terminus of Cav2.2 N-type channels (Simen et al., 2001).  I hypothesize that the inhibition of Cav3.3 channels by Gαq/11- coupled mAChRs is also mediated by the direct binding of activated Gαq/11 protein.  If direct Gαq binding mediates a novel mechanism of voltage-independent inhibition, then identification of specific channel regions and sequences involved in this binding would be essential to the further study of this inhibition pathway for all Ca2+ channel classes.  Specific residues within the amino terminus of a GPCR kinase, GRK2, have been demonstrated to be essential for direct binding to Gαq (Sterne-Marr et al., 2003).  These eight identified residues lay within an RGS homology (RH) domain.  Alignment of this identified Gαq binding region in GRK2 with the Kir3.2 region implicated in Gαq binding (amino acids 81 to 90) and regions within the domain I-II linker of Cav3.1 and Cav3.3 channels demonstrates that several of the residues that are essential for the binding of Gαq to GRK2 are conserved or homologous in both Kir3.2 and Cav3.3, but not in Cav3.1 channels (Fig. 4.1).  Thus, Gαq/11 may directly bind to a region within the domain I-II linker of Cav3.3 channels to cause channel inhibition.  Future experiments that could test this hypothesis are outlined in section 4.4.3.  However, until this Gαq binding inhibitory mechanism is demonstrated, inhibition of Cav3.3 channels by intracellular signals downstream of PLC activation can not be conclusively ruled out, as positive controls to ensure pharmacological activity were not performed on all kinase/ phosphatase antagonists used in Chapter 2. Chapter 4: Discussion  135 Kir3.1: N-terminus LIYVLY-KL LEEYC LIHCVY-QFIEEYC LEKMIYTDFIERSC L-DVL TT-FID--- GRK2: RH domain Cav3.3: I-II linker Cav3.1: I-II linker  Figure 4.1 - Amino acid sequence alignments. Comparison of an amino acid sequence in the domain I-II linker of Cav3.1 and Cav3.3 channels (identified by a Clustal alignment) with a region in the N-terminus of GRK2 that forms a RH domain and has specific residues directly demonstrated to be involved in binding to Gαq (shown in orange) (Sterne- Marr et al., 2003).  Amino acids # 81-90 from the C-terminus of the Kir3.1 channels were also compared to the GRK2 sequence, as these Kir3.1 amino acids are also implicated in direct binding to Gαq (Kawano et al., 2007).  Amino acids that are conserved when compared to the GRK2 sequence are labeled red, while those that are homologous to the GRK2 sequence are labeled yellow.  The Cav3.3 domain I-II linker region has much more sequence homology at critical Gαq binding residues than the Cav3.1 domain I-II linker region.  4.3 Potentiation of Cav3.1 currents by mGluR1 4.3.1 Working hypotheses The potentiation of native Cav3.1 T-type currents by mGluR1 activation could have significant effects on electrical and chemical integration within PC dendritic spines, with the potential to alter plasticity at PF-PC synapses.  Synaptic activation of Ca2+ channels in these distal dendritic spines has been shown to underlie Ca2+ signals that are restricted to individual spines (Denk et al., 1995).  When PF bundles are densely activated, postsynaptic Ca2+ channels can cause a more widespread dendritic depolarization that underlies an mGluR1-independent form of Ca2+ signaling and PF-LTD (Wang et al., 2000).  Other than these two pieces of evidence, almost nothing is known about the composition, localization, modulation and physical interactions of Ca2+ channels within PC distal dendritic spines. Spines are specialized dendritic outgrowths that can compartmentalize both the electrical and biochemical signals that are produced by synaptic inputs (reviewed in (Bloodgood and Sabatini, 2007)). From an electrical perspective, the passive properties of spines include the attenuation of synaptic potentials, depending on the spine neck geometry (narrower neck = higher resistance = higher filtering), as well as the boosting of local EPSPs due to a high spine head input resistance.  The combination of these two opposing passive effects has led many to conclude that spines do not play a significant role as electrical compartments.  However, neuronal spines can also function as active electrical structures, with voltage-gated channels that can amplify synaptic EPSPs to boost signals and generate local APs, or conversely, voltage-gated channels that can filter EPSPs and reduce excitatory inputs (reviewed in (Tsay Chapter 4: Discussion  136 and Yuste, 2004)).  The current state of understanding on this function of spines is that “the electrical role of spines remains open and appears to us a rich field - one in which the key experiments have yet to be done” (Tsay and Yuste, 2004).  Because of the uncertainty regarding their electrical functions, many researchers have concluded that spines act primarily as biochemical compartments.  In support of this notion, the diffusion rates of small substances between spines and their parent dendrites are around 100 times slower than expected for free diffusion of these substances (reviewed in (Nimchinsky et al., 2002)). Cerebellar PCs are an interesting model neuron for studying spine function, as they contain a much higher density of spines (10 spines/μm) than most other neurons and these spines have several unique properties.  Unlike hippocampal and neocortical pyramidal neurons, PC spines are not lost when afferent connections are removed (reviewed in (Nimchinsky et al., 2002)), and induction of LTD does not cause a change in spine size or number (Sdrulla and Linden, 2007).  Thus, changes in synaptic strength in PC dendritic spines likely involve biochemical changes in signaling rather than morphological changes in spiny synapses.  The spines on the distal dendrites of PCs each contain a PF- PC synapse and have more slender necks than the short and stubby spines of proximal CF-PC synapses. The tight regulation of Ca2+ signaling within these distal dendritic spines through high expression of Ca2+ buffers (calbindin and parvalbumin), channels, and pumps also contributes to their biochemical compartmentalization.  PC spines also contain a smooth ER (sER) that continuously extends into the parent dendrite to form a network capable of linking spine biochemical activity to dendrite activity (reviewed in (Nimchinsky et al., 2002)).  In addition to the high biochemical compartmentalization within spines of PC distal dendrites, voltage compartmentalization is several-fold greater in PF-PC spines than hippocampal schaffer collateral-CA1 spines  (reviewed in (Nimchinsky et al., 2002)). Recent experiments have unveiled a complex network of interactions between the transmembrane and intracellular peptides that underlie the highly localized mGluR1 signaling within PC distal dendritic spines (Finch and Augustine, 1998; Takechi et al., 1998).  Within PF-PC spines, mGluR1 has been shown to be physically linked to TRPC1 and Cav2.1 channels in the plasma membrane (Kim et al., 2003; Kitano et al., 2003; Kulik et al., 2004) as well as PLCβ4 and IP3 receptors (IP3Rs) in the intracellular compartment (Nakamura et al., 2004; Sandona et al., 2003; Tu et al., 1998) (Fig. 4.2). Many of these signaling elements are tethered to each other through the binding of Homer cytoskeletal proteins; e.g. - IP3R linkage to TRPC1 and mGluR1 (Tu et al., 1998; Yuan et al., 2003). Other peptides directly interact such as the binding between the carboxy terminals of mGluR1 and Cav2.1 (Kitano et al., 2003).  Functional studies showing an activation of BK K+ currents through a combination of P-type Ca2+ channel activity and mGluR1 activity in PC spines indicates that this channel may also be closely associated with mGluR1 at the PF-PC synapse (Canepari and Ogden, 2006; Edgerton and Reinhart, 2003; Womack et al., 2004).  However, direct evidence has not linked all of these signaling elements Chapter 4: Discussion  137 together within a single PC distal dendritic spine, and the functional consequences of many of these interactions are still unknown. Gαq Gβγ DHPG GTP Tyrosine phosphatase IP3 Ca2+ PLCβ4 PIP2sEPSC (TRPC1) Cav3.1 mGluR1 BK Cav2.1 IP3R PKC * ** * * smooth ER cytoplasm extracellular space  Figure 4.2 - Proposed intracellular signalling microdomains within Purkinje cell spines. Signaling peptides that have been shown to physically couple to mGluR1 (many mediated by Homer linkages) within PC dendritic spines are indicated by * (reviewed in (Hartmann and Konnerth, 2005; Knopfel and Grandes, 2002)).  Activation of mGluR1 in PC spines can increase local intracellular [Ca2+] through IP3-mediated release from the smooth ER and through the activation of the sEPSC, which is partially mediated by TRPC1.  The depolarization-induced activation of Cav3.1 and Cav2.1 Ca2+ channels is also implicated in elevating intracellular [Ca2+].  Very little is known about the modulation of Ca2+ channels by mGluR1 within dendritic spines.  Our pharmacological experiments have indicated that the signaling elements shown in red are not required for the potentiation of T-type currents (Cav3.1) by mGluR1 activation.  For example, blocking phospholipase C (PLC) activity or its downstream signals of protein kinase C (PKC) or IP3 receptor (IP3R) activation had no significant affect on the mGluR1- mediated T-type stimulation.  Dashed lines represent putative signaling pathways that have not been completely explored.  All elements in green are required for the stimulation of T-type currents via mGluR1 activity, which is attenuated when these signals are blocked with pharmacological antagonists. As shown, tyrosine phosphatases are known to also be required for the mGluR1-mediated activation of the sEPSC and the BK K+ current in PCs, but these channels’ activity are not required for the T-type effect.  The calcium-dependency of the T-type stimulation effect is not due to sEPSC or IP3R activity alone, but could be due to calcium influx through a combination of these channels acting within the putative signaling microdomain.  Many other intracellular scaffolding proteins (such as Homer), signaling proteins (such as arachidonic acid), and ion channels (such as AMPA receptors) that are present in PC distal dendritic spines are omitted for simplicity. Chapter 4: Discussion  138 I hypothesize that Cav3.1 is colocalized with mGluR1 and other signaling proteins at PF-PC spines to form signaling microdomains (Fig. 4.2).  Several lines of evidence support an interaction between mGluR1 and Cav3.1 in PC distal spiny microdomains (Chapter 3).  Firstly, the mGluR1- mediated augmentation of T-type Ca2+ transients was observed in PC spines, but not their parent dendrites, indicating that the modulatory interaction was localized to spines.  When blockade of IP3Rs was removed, the T-type potentiation spread to the parent dendrites.  This is consistent with a mechanism whereby robust mGluR1 activation (bath application of DHPG) can cause Ca2+ release from the continuous sER network that propagates the Ca2+-dependent T-type potentiation into adjacent dendrites and spines.  Another key piece of evidence that connects Cav3.1 activity to putative mGluR1 signaling microdomains is the modulation pathway that was identified.  Both sEPSC and Cav3.1 channels are activated/ potentiated by an mGluR1 signaling pathway that is dependent on G-protein and tyrosine phosphatase activity but is independent of the activation of PLC or its downstream effectors (Fig. 4.2). Thus, I propose that activation of this unique mGluR1-mediated pathway can increase intracellular Ca2+ levels within PF-PC spiny microdomains through the activation of both voltage-dependent (Cav3.1) and - independent (sEPSC) membrane Ca2+ channels.  Consistent with this notion, repetitive activation of PF bundles caused an mGluR1-mediated increase in Ca2+ transients that was localized to specific distal dendrites (Chapter 3).  These Ca2+ transients were severely reduced or abolished in Cav3.1 KO mice, indicating that Cav3.1 channels underlie the primary mGluR1-mediated Ca2+ signal in PF-PC spines under our experimental conditions.   The fact that the above experiments were performed at a near physiological temperature (32oC) on older rats (P16 to P30) also demonstrates that the mGluR1- mediation potentiation of Cav3.1 currents occurs during physiological synaptic integration within mature PCs. If the synaptic activation of mGluR1 in PF-PC synapses causes the potentiation of postsynaptic T-type channels, it is also possible that the Ca2+ currents flowing through these channels partially mediate the supralinear Ca2+ signals that underlie PF-LTD.  A role for Ca2+ channels in single spine plasticity has been demonstrated in hippocampal CA1 neurons, where the activity of R-type Ca2+ channels is modulated by postsynaptic depolarizations and impacts EPSP amplitude for tens of minutes (Yasuda et al., 2003).  In our experiments it is interesting to note that the bath application of exogenous DHPG that potentiated T-type currents has recently been shown by Sdrulla and Linden to induce a robust chemically-evoked form of LTD that occurs on a similar timescale as seen for our T-type modulation (Sdrulla and Linden, 2007).  Thus, T-type currents are potentiated under experimental conditions that elicit PF-LTD.  The question that remains is whether this mGluR1-mediated potentiation of T-type currents is necessary to induce any forms of physiological PF-LTD.  A study of PF-LTD induced by the concurrent activation of both PF and CF inputs demonstrated that LTD induced by sparse activation of PFs is restricted to single spines, dependent upon mGluR1 signaling, and greatest when PF activation Chapter 4: Discussion  139 precedes CF activation (Wang et al., 2000).  These observations are consistent with a (simplified) hypothetical mechanism of LTD whereby PF inputs activate mGluR1 to potentiate T-type currents and slowly depolarize the spine through the sEPSC.  This would be followed by further CF-mediated depolarization which allows the membrane potential in the spines to reach threshold for activation of the now-potentiated T-type currents, providing the necessary Ca2+ influx to induce AMPAR internalization. In section 4.4.3, I outline future experiments that could elucidate the contribution of Cav3.1 in integration and plasticity at dendritic spine microdomains of the PF-PC synapse.  Besides these experiments, future experiments using local PF synaptic activation, high resolution two-photon imaging, specific channel/receptor antagonists, conditional KO mice and voltage-sensitive indicators will be required to determine the individual roles that the other signaling elements (e.g. - sEPSC, Cav2.1, PLCβ4, IP3R, etc…) perform within these spines.   Although I hypothesize that all of these elements are co-localized within spine compartments, it is likely that only a subset of these components will be activated for a given physiological response.  Alternatively, it is also possible that mGluR1 is specifically coupled to different effectors and signaling elements in different regions of the PC distal dendritic tree. Besides acting as a putative coincidence detector in PF-LTD, the activation and potentiation of T-type Ca2+ channels in PC dendritic spines could also serve to boost or filter synaptic inputs.  As we showed in Chapter 3, a temporal summation of high frequency EPSPs could result in the opening of Cav3.1 channels in dendritic spines.  This T-type channel opening could cause further depolarization that opens nearby Cav2.1 channels to further boost the depolarizing signal.  Alternatively, the opening of T- type channels could provide the Ca2+ signal necessary to activate nearby BK K+ channels and induce a hyperpolarization that filters the EPSP.  As PCs are spontaneously active, with periods of tonic and burst firing separated by quiescent periods, the potentiation of T-type currents by mGluR1 could be crucial in glutamate-mediated alterations of PC excitability states as well as the timing of firing (Williams et al., 2002; Womack and Khodakhah, 2002; Womack and Khodakhah, 2004). A novel potential role for the stimulation of T-type currents in dendritic spines is the dendritic release of glutamate for both retrograde and autocrine signaling.  T-type currents have been shown to underlie spontaneous synaptic Ca2+ transients that are either localized to individual spines or initiate a more widespread low threshold Ca2+ spike at the large dendrodendritic spiny synapses of olfactory bulb granule cells (Egger et al., 2003, 2005).  Preliminary experiments indicate that these T-type currents underlie a “presynaptic” dendritic Ca2+ signal that results in the AP-independent release of GABA to retrogradely inhibit the adjacent mitral and tufted output cells of the dendrodendritic synapses (Egger et al., 2003).  Exciting new studies indicate that a similar mechanism of dendritic neurotransmitter release may exist in cerebellar PCs.  In PF-PC synapses of nearly mature (P18 to P22) rats, glutamate has been shown to be released from PC spines through an mGluR1-mediated mechanism.  The released glutamate Chapter 4: Discussion  140 binds to kainite receptors on the PF terminals and induces a short-term suppression of excitation that is followed by a potentiation phase (Crepel and Daniel, 2007).  Unlike the suppression of excitation, the potentiation is thought to be mediated by a postsynaptic PC mechanism (Crepel and Daniel, 2007). Another very recent study has shown that low frequency CF stimulation or direct somatic depolarization of PCs causes dendritic glutamate release through a classical SNARE-dependent vesicular release mechanism that is usually observed for neurotransmitter release at presynaptic axons terminals (Duguid et al., 2007).  This dendritic glutamate release from PCs has subsequently been shown to be dependent on Ca2+ channel activity and results in the autocrine activation of mGluR1 receptors, leading to the release of inhibitory endocannabinoids as well as the activation of the mGluR1-mediated sEPSC (Duguid et al., 2007; Shin et al., 2008).  The involvement of mGluR1 in both the release and autocrine action of glutamate combined with the requirement of Ca2+ channel-mediated Ca2+ influx for dendritic glutamate release indicates that the potentiation of T-type Ca2+ channels by mGluR1 could be a critical component for both of these mechanisms.  In fact, the potentiation of T-type currents by autocrine mGluR1 activation might explain the delayed postsynaptic PC potentiation observed after glutamate release (Crepel and Daniel, 2007).  Along these lines, I often observed a run-up in T-type current amplitude during successive test depolarizations under the control conditions of voltage-clamp experiments that may be due to this putative autocrine mGluR1 signaling activity (unpublished observations). The potentiation of T-type currents by mGluR1 is also observed in PC proximal dendrites, suggesting that this modulation could be relevant for CF-PC synaptic functions.  Stimulation of CF inputs can cause the activation of mGluR1 and its associated sEPSC (Dzubay and Otis, 2002). Furthermore, evidence is emerging that indicates that T-type currents underlie a low-threshold spike in the proximal dendrites that can propagate to the soma and may contribute to the complex spike (Cavelier et al., 2008) (reviewed in (Cavelier and Bossu, 2003)).  Isope and colleagues are currently combining experiments involving Ca2+ imaging, current clamp recordings, and CF stimulation to reveal that the CF- mediated Ca2+ signaling in proximal dendritic spines is mainly mediated by Cav3.1 T-type currents in the basal state, while the blockade of a fraction of the IA K+ currents induces a CF-mediated Ca2+ spike that is dependent on P-type channel activity and can propagate throughout the dendritic tree (Philippe Isope, personal communication).  Modulation of T-type currents and other ionic currents by mGluR1 could participate in this “unlocking” of dendritic Ca2+ spikes, possibly through the generation of low-threshold spikes that overcome the inhibitory K+ currents.  As mGluR1 activity is strongly implicated in CF pruning in the proximal dendrites (reviewed in (Hashimoto and Kano, 2005)), the potentiation of T-type currents by mGluR1 could also have a functional role in this process.  Chapter 4: Discussion  141 4.3.2 Possible limitations and weaknesses Because of well-known space clamp concerns, a majority of our experiments were performed on PCs from acute brain slices of young (P8-12) rats and mice.  The dendritic arbourization is limited at this developmental age, allowing us to voltage clamp the PCs and accurately measure the biophysical properties of T-type currents before and after activation of mGluR1 (Chapter 3).  Climbing fiber and PF synapses are still being formed and eliminated during this developmental window, and so the potentiation of T-type currents by mGluR1 was observed during a time of CF pruning and could have a direct physiological role in this process.  Is the observed modulation relevant at mature synapses in fully developed PCs?  We performed a final set of experiments on PCs from older rodents (P16 to P30) and found that the Cav3.1-mediated T-type Ca2+ transients are potentiated by physiological activation of mGluR1 at mature PF-PC spiny synapses (Figure 3.7).  However, indirect contributions of voltage-gated K+ or HVA Ca2+ channels towards the mGluR1-mediated T-type potentiation has not been ruled out in these experiments.  Similar imaging and synaptic activation experiments need to be performed on adult rodents to verify both the functional expression of T-type currents as well as their potentiation by mGluR1.  Cell-attached dendritic recordings with and without DHPG in the pipette could also be used to directly test for the modulation of T-type currents by mGluR1 in adult PCs and to characterize this putative T-type potentiation at the single channel level.  These future experiments are especially important since one study demonstrated a lack of T-type currents in both the soma and dendrites of adult guinea pig PCs (Usowicz et al., 1992). Another limitation of our experiments is that we have not directly demonstrated a specific physiological process involving the mGluR1-induced potentiation of T-type channels.  In order to explore the role of this T-type potentiation in specific physiological processes (discussed in section 4.3.1), a more integrative approach that includes the interactions of mGluR1 with various other effectors and ion channels needs to be utilized.  Most of our experiments characterized the modulation of T-type currents by mGluR1 in isolation so that the results could be most easily interpreted.  Within a physiological context, activation of mGluR1 could involve the simultaneous modulation of various ion channels, including TRPC1 (Kim et al., 2003), BK (Canepari and Ogden, 2006), and P-type channels (Kitano et al., 2003) as well as other modulated channels that remain to be identified (as was the case for T-type channels before our study).  To illustrate the difficulties that arise from this complexity, when all ionic currents were left unblocked for our excitability experiments in Figure 3.4, the role of T-type potentiation in the DHPG-induced changes in firing frequency could not be easily analyzed because multiple ion channels that could be modulated by mGluR1 are involved in this interspike interval parameter (Swensen and Bean, 2003).  In fact, the effect of DHPG on the firing frequency appeared to be highly variable (unpublished observation), which could be due to the antagonistic modulation of various ion channels by mGluR1.  Interpretation of these experiments are also limited by antagonist specificity, Chapter 4: Discussion  142 as both mibefradil and Ni2+  can potentially block R-type currents and other HVA currents at the concentrations used (Lee et al., 1999; Randall and Tsien, 1997; Zamponi et al., 1996).  Thus, even the stated role of mGluR1-mediated potentiation of T-type currents in lowering the AP threshold needs to be further confirmed in Cav3.1 KO mice. The first steps toward understanding these multi-variable processes involve thoroughly characterizing the interactions between mGluR1 and all channels known to be involved in PC physiology.  Although our study is a good step forward in this task, the work is far from complete.  For example, although P-type channels are the predominant HVA Ca2+ channels expressed in PC dendrites and soma (Mintz et al., 1992; Usowicz et al., 1992) and are implicated in the firing of the complex spike (Watanabe et al., 1998), only incomplete biochemical and imaging experiments have tested for a modulatory interaction with mGluR1 (Kitano et al., 2003).  These experiments conclusively showed that mGluR1a physically couples to P-type channels and is co-localized in PC dendrites, making a modulatory interaction both likely and potentially highly relevant (Kitano et al., 2003).  Future voltage clamp experiments are needed to directly characterize the effects of mGluR1 activation on P-type currents in PCs.  To overcome the obvious space clamp issues that arise when dealing with these large dendritic P-type currents, a first step could involve investigating the interaction in acutely dissociated juvenile PCs, where both P-type currents and mGluR1 are expressed (Lopez-Bendito et al., 2001; Mintz et al., 1992).  This could be followed by the more demanding single channel recordings of P-type current modulation in adult PC dendrites.  Once all of these modulatory interactions have been characterized, then KO mice and specific pharmacological antagonists can be used more effectively in combination with Ca2+ imaging and electrophysiological recordings to elucidate the roles of mGluR1-ion channel interactions in physiological processes such as local integration, plasticity, excitability, and synaptic pruning. The intracellular signaling mechanism that directly links mGluR1 activity to the alteration of T- type biophysical properties is also currently unknown.  Our results have shown that the potentiation pathway involves G-protein activation, intracellular Ca2+ signals, and tyrosine phosphatase activity and is independent of the activation of PLC and its downstream signaling pathways (Fig. 4.2).  The first step to further explore this signaling pathway would be to determine whether Gαq/11 or Gβγ is involved in the potentiation.  To test for a role of Gαq/11, a membrane permeable, N-terminal palmitoylated decapeptide (palpeptide) that has been shown to specifically disrupt Gαq/11 function and subsequent modulation of ion channels (Robbins et al., 2006) could be puffed onto the PC before testing for the modulation of T-type currents by DHPG application.  To test for a role of Gβγ binding in the modulation, either purified Gβγ or Gβγ-specific antibodies could be included within the internal pipette to disrupt normal Gβγ-mediated signaling (Zamponi et al., 1997).  However, sufficient time for dialysis of the purified proteins/ antibodies into the distal dendrites would be required before the modulation of DHPG on T-type currents Chapter 4: Discussion  143 could be tested.  These experiments could be followed by co-immunoprecipitation studies to test whether the implicated G-protein subunit is directly involved in a physical interaction with the modulated Cav3.1 channels.  4.4 Conclusions 4.4.1 General conclusions Overall, we have demonstrated that specific T-type Ca2+ channel isoforms are differentially modulated by certain GPCR pathways.   Together, this subtype-specific T-type channel-GPCR coupling likely contributes to the unique cellular functions of each of the various T-type calcium channel subtypes.  We found that the Cav3.3 isoform is specifically inhibited by Gαq/11-coupled receptors with a concomitant robust increase in channel inactivation kinetics.  This Gαq/11-mediated inhibition involves intracellular interactions with two distinct channel regions that are independent of cytoplasmic signals downstream of PLC activation.  I hypothesize that this inhibition of Cav3.3 channels by Gαq/11-coupled mAChRs could be involved in the cholinergic regulation of thalamic nRT firing patterns during sleep- wake transitions.  The Cav3.1 isoform is potentiated by mGluR1 activation within both the heterologous HEK system and cerebellar PCs.  The mGluR1-mediated T-type potentiation within PCs involved both an increase in maximal currents as well as a hyperpolarizing shift in the voltage-dependence of activation.  These effects lead to a decreased AP threshold and resultant increased excitability within PCs.  The potentiation of Cav3.1 by mGluR1 was localized to synaptic locations within PC dendrites, and physiological activation of mGluR1 through a burst of PF stimulation induced a robust augmentation of T-type transients.   I hypothesize that the potentiation of Cav3.1 currents by mGluR1 could contribute to the coincidence detector and supralinear Ca2+ signals required for cerebellar PF-LTD and motor learning. Finally, through our collaboration with Soong and coworkers we have found that Cav3.2 channels are selectively inhibited by corticotropin-releasing factor receptors through a Gβγ-mediated signaling pathway (Appendix 2) that is independent of Gα signaling pathways.  This inhibitory interaction could be a critical component relevant to both cardiac and neuronal physiology.  4.4.2 Possible relevance to human disease Several human movement disorders, such as episodic ataxia type-2 and spinocerebellar ataxia type-6, are caused by point mutations in the Cav2.1 channel that disrupt normal P/Q-type channel function in the cerebellum (Spacey et al., 2004) (Appendix 3) (reviewed in (Adams and Snutch, 2007)). Similar to that for P/Q-type Ca2+ channels, we have now demonstrated that Cav3.1 T-type channels are functionally expressed in PC dendrites and may be involved in similar processes, such as complex spike firing.  From our data I hypothesize that T-type channel dysfunction within PCs could also be involved in Chapter 4: Discussion  144 the pathophysiology of movement disorders such as ataxia.  In an animal model of a severe movement disorder, leaner mice have a spontaneous mutation in the P/Q-type channel gene which results in ataxia and dyskinesia.  Whole-cell and single-channel recordings have demonstrated that the leaner mutation results in a ~ 60% reduction in P-type currents within mature cerebellar PCs by reducing the channel open probability (Dove et al., 1998).  A recent expression study using a combination of laser-capture microdissections, quantitative RT-PCR and in situ hybridization has demonstrated that Cav3.1 expression is significantly increased in mature PCs of leaner mice (Nahm et al., 2005).  In contrast to these results, no change in T-type current is observed in PCs from ataxic tottering mice that have a less severe P-type mutation and phenotype (Erickson et al., 2007).  However, experiments in this second study were done on dissociated PCs that lack the dendritic trees where T-type channels are predominantly expressed and cells were taken from mice at a developmental stage (P6 to P15) that is much earlier than when the motor dysfunction symptoms appear (three to four weeks postnatally) (Erickson et al., 2007).  The correlation between increased Cav3.1 expression in PCs and ataxia and dyskinesia indicates that aberrant potentiation of T-type currents by mGluR1 could also potentially lead to movement disorder phenotypes. It would be of interest to examine the level of augmentation of Cav3.1 T-type currents by mGluR1 in leaner mice to determine whether excessive T-type modulation also contributes to the uncoordinated phenotype in this animal model. The results of recent studies point to the tantalizing possibility that the potentiation of Cav3.1 channels by mGluR1 is also involved in the physiology and pathophysiology of a brain region that is involved in both sleep-wake gating and the genesis of epileptic seizures: the thalamus.  The activation of mGluR1 by cortical inputs is thought to be involved in generating the intrinsic TC oscillations that are critical in slow wave (<1 Hz) sleep rhythms (Hughes et al., 2002).  The underlying mechanism of this slow rhythm induction has been shown to involve a reduction in outward linear leak currents by mGluR1 activation.  This modulation results in an overlap between the current-voltage relationships of the outward leak currents and inward T-type window currents, creating membrane bistability and the resultant UP and DOWN states of firing that underlie rhythmic oscillations (Hughes et al., 2002; Williams et al., 1997) (reviewed in (Crunelli et al., 2006)).  The activation of depolarizing Ih currents during the hyperpolarized DOWN states is what functionally links the two stable resting membrane potential states together to form a continuous slow oscillation (Hughes et al., 2002; Williams et al., 1997).  As Cav3.1 channels form the dominant functional T-type currents in these cells (Kim et al., 2001) and are co-localized with mGluR1 (reviewed in (Alexander and Godwin, 2006)), their potentiation by mGluR1 could lead to an increase in T-type window conductance that directly contributes to these state transitions and network oscillations.  Indeed, one preliminary study indicates that an mGluR1 signaling pathway within TCs regulates T-type channel activity to control the mode of firing (Cheong et al., 2007). Future voltage-clamp experiments should directly test the effects of activating mGluR1 receptors on the Chapter 4: Discussion  145 biophysical properties of T-type channels within TCs.  These experiments could demonstrate a broader impact of Cav3.1 modulation by mGluR1 in the CNS, especially as “T-type Ca2+ channels, therefore, constitute the single most crucial voltage-dependent conductance that permeates all activities of thalamic neurons during NREM sleep” (Crunelli et al., 2006). In addition to an induction of sleep rhythms, the potentiation of Cav3.1 currents by mGluR1 within TCs might also be linked to epileptic SWDs under pathological conditions.  T-type Ca2+ channels have a well-documented role in epileptic absence seizures and their underlying mechanism of thalamocortical 3-4 Hz oscillations (SWDs) (reviewed in (Khosravani and Zamponi, 2006)).  The “cortical focus” theory of absence epilepsy proposes that cortical hyperexcitability initiates these SWDs through the induction of synchronized firing within the thalamus via their glutamatergic connections onto TCs (and nRTs).  This initiation appears to involve mGluR1 receptors, as application of mGluR1 antagonists effectively blocked SWD activity in the “lethargic” mouse model of absence epilepsy (reviewed in (Alexander and Godwin, 2006)).  Thus, excessive activation of mGluR1 receptors in TCs by hyperexcited cortical efferents could induce thalamic SWD activity through the potentiation of T-type currents.  As we have shown that Cav3.2 channels can also be potentiated by mGluR1 activity, this potentiation mechanism could directly increase rhythmic burst firing within both TCs and nRTs. Therefore, future experiments should also test the effects of mGluR1 activation on the biophysical properties of T-type currents within nRTs.  The implication of T-type channel activity and potentiation in movement disorders (Nahm et al., 2005), sleep disorders (Anderson et al., 2005), and epileptic activity (Khosravani and Zamponi, 2006) indicates that specific T-type antagonists have the potential to form novel classes of therapeutic compounds to treat these pathological conditions.  4.4.3 Future directions Throughout this discussion I have outlined several experiments and avenues for future research in the field of T-type modulation.  Here, I will discuss more detailed future experiments on two independent aspects of T-type modulation: 1) the interactions between Gαq/11-mediated intracellular signals and Cav3.3 channels involved in the GPCR inhibitory effect, and 2) the putative physical coupling between mGluR1, Cav3.1, and other ion channels in PC dendritic microdomains and the potential consequences of these interactions. Thus far, we have been unable to directly identify the intracellular signal that links Gαq/11 activity to Cav3.3 channel inhibition.  I hypothesize that Gαq/11 itself may directly bind to and inhibit these Ca2+ channels and propose testing this notion using a constitutively active Gαq construct that has mutations in two key residues to eliminate coupling to PLC (Chen et al., 2006).  Transfection of this PLC activation-deficient Gαq construct into stable Cav3.3-expressing HEK cells is hypothesized to cause Chapter 4: Discussion  146 the same decrease in current amplitude and increase in inactivation kinetics as observed in Figure 3.4 if PLC and its downstream signaling pathways (including PIP2 depletion) are not required for inhibition.  A positive result in this experiment would confirm the results from our pharmacological experiments (Figs. 3.3 and 3.5).  A direct inhibitory interaction involving Gαq could then also be tested by applying purified active Gαq-GTP (and inactive Gαq-GDP as a negative control) to inside-out macropatches of HEK cells overexpressing Cav3.3.  If direct application of active Gαq causes Cav3.3 inhibition in these macropatches then single channel recordings on smaller patches could also be used to characterize what T-type single channel properties are altered in this inhibitory interaction.  Co-immunoprecipitation experiments using specific antibodies on lysates from HEK cells transfected with both constitutively active Gαq proteins and Cav3.3 channels could demonstrate whether or not a direct physical interaction exists between these two proteins.  If successful, GST pull-down assays between specific Cav3.3 regions and Gαq could verify this result and narrow putative interacting regions (Chen et al., 2006; Wolfe et al., 2003).  Combining these pull-down assays with electrophysiological recordings on further Cav3.1-Cav3.3 chimeric channels and then Cav3.3 channels with specific point mutations could demonstrate the channel regions and residues involved in the Gαq-mediated inhibition.  An initial candidate region to be tested would be the domain I-II linker region shown in Figure 4.1.  All of these above experiments would help to understand the molecular underpinnings of a putatively novel form of voltage-independent inhibition of Ca2+ channels. Although mGluR1 activation has been shown to modulate Cav3.1 T-type currents within PCs, the co-localization between these two proteins in PC dendrites has yet to be directly demonstrated. Combining light and electron microscopy with the staining of cerebellar slices using Cav3.1 and mGluR1 antibodies could determine the ultrastructural localization of Cav3.1 channels in PC dendrites and spines as well as their potential co-localization with mGluR1 receptors in the perisynaptic regions of PF-PC and CF-PC synapses.  Subsequent co-immunoprecipitation experiments with these mGluR1 and Cav3.1 antibodies could be used to examine whether the two proteins are physically linked to each other.   The mGluR1 receptor is known to be physically coupled to other ion channel effectors, such as TRPC1, Cav2.1 channels, and IP3Rs, and is proposed here to form signaling microdomains with these proteins in PC dendritic spines (Fig. 4.2).  If mGluR1 is shown to bind to Cav3.1 channels, the potential physical interactions between Cav3.1 channels and other Ca2+ signaling effectors like TRPC1 or Cav2.1 could also be tested using further co-immunoprecipitation experiments.  The relevance of the mGluR1-mediated potentiation of Cav3.1 currents in synaptic plasticity within these spiny microdomains also remains to be tested.  Concurrent activation of PF and CF inputs while recording EPSC amplitude could be used to induce and measure PF-LTD.  If Cav3.1 currents are involved in this depression of EPSC amplitude, then puffing on a T-type antagonist (such as Ni2+) or a specific Cav3.1 T-type antagonist (when one is finally developed) over the PC dendrites should abolish the initiation and/or maintenance of this PF-LTD. Chapter 4: Discussion  147 Alternatively, PF-LTD could be measured and compared between Cav3.1 KO and wt mice.  This genetic model would also enable the determination of whether Cav3.1 channel activity is required for cerebellar motor learning activities, such as the vestibulo-ocular reflex. Chapter 4: Discussion  148 4.5 References Adams, P.J., and Snutch, T.P. (2007). Calcium Channelopathies: voltage-gated calcium channels. In Calcium Signalling and Disease - Molecular Pathology of Calcium, E. Carafoli, and M. Brini, eds. (New York: Springer), pp. 215-251. Alexander, G.M., and Godwin, D.W. (2006). Metabotropic glutamate receptors as a strategic target for the treatment of epilepsy. Epilepsy Res 71, 1-22. Anderson, M.P., Mochizuki, T., Xie, J., Fischler, W., Manger, J.P., Talley, E.M., Scammell, T.E., and Tonegawa, S. (2005). Thalamic Cav3.1 T-type Ca2+ channel plays a crucial role in stabilizing sleep. Proc Natl Acad Sci U S A 102, 1743-1748. Bloodgood, B.L., and Sabatini, B.L. (2007). Ca(2+) signaling in dendritic spines. Curr Opin Neurobiol 17, 345-351. Canepari, M., and Ogden, D. (2003). Evidence for protein tyrosine phosphatase, tyrosine kinase, and G- protein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons. J Neurosci 23, 4066-4071. Canepari, M., and Ogden, D. (2006). Kinetic, pharmacological and activity-dependent separation of two Ca2+ signalling pathways mediated by type 1 metabotropic glutamate receptors in rat Purkinje neurones. J Physiol 573, 65-82. Cavelier, P., and Bossu, J.L. (2003). Dendritic low-threshold Ca2+ channels in rat cerebellar Purkinje cells: possible physiological implications. Cerebellum 2, 196-205. Cavelier, P., Lohof, A.M., Lonchamp, E., Beekenkamp, H., Mariani, J., and Bossu, J.L. (2008). Participation of low-threshold Ca2+ spike in the Purkinje cells complex spike. Neuroreport 19, 299-303. Chemin, J., Monteil, A., Perez-Reyes, E., Bourinet, E., Nargeot, J., and Lory, P. (2002). Specific contribution of human T-type calcium channel isotypes (alpha(1G), alpha(1H) and alpha(1I)) to neuronal excitability. J Physiol 540, 3-14. Chen, X., Talley, E.M., Patel, N., Gomis, A., McIntire, W.E., Dong, B., Viana, F., Garrison, J.C., and Bayliss, D.A. (2006). Inhibition of a background potassium channel by Gq protein alpha-subunits. Proc Natl Acad Sci U S A 103, 3422-3427. Cheong, E., Lee, S., Choi, B.J., Sun, M., Lee, C.J., and Shin, H.S. (2007). Concomitant control of T-type and L-type Ca2+ channels in thalamocortical neurons by the mGluR1-PLCβ4 cascade sets the thalamic sensory gating. In Society for Neuroscience Meeting (San Diego). Crepel, F., and Daniel, H. (2007). Developmental changes in agonist-induced retrograde signaling at parallel fiber-Purkinje cell synapses: role of calcium-induced calcium release. J Neurophysiol 98, 2550- 2565. Crunelli, V., Cope, D.W., and Hughes, S.W. (2006). Thalamic T-type Ca2+ channels and NREM sleep. Cell Calcium 40, 175-190. Denk, W., Sugimori, M., and Llinas, R. (1995). Two types of calcium response limited to single spines in cerebellar Purkinje cells. Proc Natl Acad Sci U S A 92, 8279-8282. Dove, L.S., Abbott, L.C., and Griffith, W.H. (1998). Whole-cell and single-channel analysis of P-type calcium currents in cerebellar Purkinje cells of leaner mutant mice. J Neurosci 18, 7687-7699. Chapter 4: Discussion  149 Duguid, I.C., Pankratov, Y., Moss, G.W., and Smart, T.G. (2007). Somatodendritic release of glutamate regulates synaptic inhibition in cerebellar Purkinje cells via autocrine mGluR1 activation. J Neurosci 27, 12464-12474. Dzubay, J.A., and Otis, T.S. (2002). Climbing fiber activation of metabotropic glutamate receptors on cerebellar purkinje neurons. Neuron 36, 1159-1167. Edgerton, J.R., and Reinhart, P.H. (2003). Distinct contributions of small and large conductance Ca2+- activated K+ channels to rat Purkinje neuron function. J Physiol 548, 53-69. Egger, V., Svoboda, K., and Mainen, Z.F. (2003). Mechanisms of lateral inhibition in the olfactory bulb: efficiency and modulation of spike-evoked calcium influx into granule cells. J Neurosci 23, 7551-7558. Egger, V., Svoboda, K., and Mainen, Z.F. (2005). Dendrodendritic synaptic signals in olfactory bulb granule cells: local spine boost and global low-threshold spike. J Neurosci 25, 3521-3530. Erickson, M.A., Haburcak, M., Smukler, L., and Dunlap, K. (2007). Altered functional expression of Purkinje cell calcium channels precedes motor dysfunction in tottering mice. Neuroscience 150, 547- 555. Finch, E.A., and Augustine, G.J. (1998). Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites. Nature 396, 753-756. Fox, A.P., Nowycky, M.C., and Tsien, R.W. (1987). Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones. J Physiol 394, 149-172. Gamper, N., and Shapiro, M.S. (2007). Regulation of ion transport proteins by membrane phosphoinositides. Nat Rev Neurosci 8, 921-934. Hartmann, J., and Konnerth, A. (2005). Determinants of postsynaptic Ca2+ signaling in Purkinje neurons. Cell Calcium 37, 459-466. Hashimoto, K., and Kano, M. (2005). Postnatal development and synapse elimination of climbing fiber to Purkinje cell projection in the cerebellum. Neuroscience research 53, 221-228. Hughes, S.W., Cope, D.W., Blethyn, K.L., and Crunelli, V. (2002). Cellular mechanisms of the slow (<1 Hz) oscillation in thalamocortical neurons in vitro. Neuron 33, 947-958. Huguenard, J.R., and Prince, D.A. (1992). A novel T-type current underlies prolonged Ca(2+)-dependent burst firing in GABAergic neurons of rat thalamic reticular nucleus. J Neurosci 12, 3804-3817. Isope, P., and Murphy, T.H. (2005). Low threshold calcium currents in rat cerebellar Purkinje cell dendritic spines are mediated by T-type calcium channels. J Physiol 562, 257-269. Joksovic, P.M., Bayliss, D.A., and Todorovic, S.M. (2005). Different kinetic properties of two T-type Ca2+ currents of rat reticular thalamic neurones and their modulation by enflurane. J Physiol 566, 125- 142. Joksovic, P.M., Nelson, M.T., Jevtovic-Todorovic, V., Patel, M.K., Perez-Reyes, E., Campbell, K.P., Chen, C.C., and Todorovic, S.M. (2006). CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus. J Physiol 574, 415-430. Jones, S.V. (1993). Muscarinic receptor subtypes: modulation of ion channels. Life Sci 52, 457-464. Chapter 4: Discussion  150 Kawano, T., Zhao, P., Floreani, C.V., Nakajima, Y., Kozasa, T., and Nakajima, S. (2007). Interaction of Galphaq and Kir3, G protein-coupled inwardly rectifying potassium channels. Mol Pharmacol 71, 1179- 1184. Khosravani, H., and Zamponi, G.W. (2006). Voltage-gated calcium channels and idiopathic generalized epilepsies. Physiol Rev 86, 941-966. Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W., and Shin, H.S. (2001). Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+) channels. Neuron 31, 35-45. Kim, J.A., Park, J.Y., Kang, H.W., Huh, S.U., Jeong, S.W., and Lee, J.H. (2006). Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A. J Pharmacol Exp Ther 318, 230-237. Kim, S.J., Kim, Y.S., Yuan, J.P., Petralia, R.S., Worley, P.F., and Linden, D.J. (2003). Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1. Nature 426, 285-291. Kitano, J., Nishida, M., Itsukaichi, Y., Minami, I., Ogawa, M., Hirano, T., Mori, Y., and Nakanishi, S. (2003). Direct interaction and functional coupling between metabotropic glutamate receptor subtype 1 and voltage-sensitive Cav2.1 Ca2+ channel. J Biol Chem 278, 25101-25108. Knopfel, T., and Grandes, P. (2002). Metabotropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells. Cerebellum 1, 19-26. Koike-Tani, M., Collins, J.M., Kawano, T., Zhao, P., Zhao, Q., Kozasa, T., Nakajima, S., and Nakajima, Y. (2005). Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel. J Physiol 564, 489-500. Kulik, A., Nakadate, K., Hagiwara, A., Fukazawa, Y., Lujan, R., Saito, H., Suzuki, N., Futatsugi, A., Mikoshiba, K., Frotscher, M., and Shigemoto, R. (2004). Immunocytochemical localization of the alpha 1A subunit of the P/Q-type calcium channel in the rat cerebellum. Eur J Neurosci 19, 2169-2178. Lee, J.H., Gomora, J.C., Cribbs, L.L., and Perez-Reyes, E. (1999). Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H. Biophys J 77, 3034-3042. Levey, A.I., Edmunds, S.M., Heilman, C.J., Desmond, T.J., and Frey, K.A. (1994). Localization of muscarinic m3 receptor protein and M3 receptor binding in rat brain. Neuroscience 63, 207-221. Lopez-Bendito, G., Shigemoto, R., Lujan, R., and Juiz, J.M. (2001). Developmental changes in the localisation of the mGluR1alpha subtype of metabotropic glutamate receptors in Purkinje cells. Neuroscience 105, 413-429. McCormick, D.A. (1992). Cellular mechanisms underlying cholinergic and noradrenergic modulation of neuronal firing mode in the cat and guinea pig dorsal lateral geniculate nucleus. J Neurosci 12, 278-289. McKay, B.E., McRory, J.E., Molineux, M.L., Hamid, J., Snutch, T.P., Zamponi, G.W., and Turner, R.W. (2006). Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons. Eur J Neurosci 24, 2581-2594. McRory, J.E., Santi, C.M., Hamming, K.S., Mezeyova, J., Sutton, K.G., Baillie, D.L., Stea, A., and Snutch, T.P. (2001). Molecular and functional characterization of a family of rat brain T-type calcium channels. J Biol Chem 276, 3999-4011. Chapter 4: Discussion  151 Mintz, I.M., Adams, M.E., and Bean, B.P. (1992). P-type calcium channels in rat central and peripheral neurons. Neuron 9, 85-95. Nahm, S.S., Jung, K.Y., Enger, M.K., Griffith, W.H., and Abbott, L.C. (2005). Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy. Journal of neurobiology 62, 352-360. Nakamura, M., Sato, K., Fukaya, M., Araishi, K., Aiba, A., Kano, M., and Watanabe, M. (2004). Signaling complex formation of phospholipase Cbeta4 with metabotropic glutamate receptor type 1alpha and 1,4,5-trisphosphate receptor at the perisynapse and endoplasmic reticulum in the mouse brain. Eur J Neurosci 20, 2929-2944. Nimchinsky, E.A., Sabatini, B.L., and Svoboda, K. (2002). Structure and function of dendritic spines. Annu Rev Physiol 64, 313-353. Pellegrini, A., Dossi, R.C., Dal Pos, F., Ermani, M., Zanotto, L., and Testa, G. (1989). Ethosuximide alters intrathalamic and thalamocortical synchronizing mechanisms: a possible explanation of its antiabsence effect. Brain Res 497, 344-360. Plummer, K.L., Manning, K.A., Levey, A.I., Rees, H.D., and Uhlrich, D.J. (1999). Muscarinic receptor subtypes in the lateral geniculate nucleus: a light and electron microscopic analysis. J Comp Neurol 404, 408-425. Randall, A.D., and Tsien, R.W. (1997). Contrasting biophysical and pharmacological properties of T- type and R-type calcium channels. Neuropharmacology 36, 879-893. Robbins, J., Marsh, S.J., and Brown, D.A. (2006). Probing the regulation of M (Kv7) potassium channels in intact neurons with membrane-targeted peptides. J Neurosci 26, 7950-7961. Sandona, D., Scolari, A., Mikoshiba, K., and Volpe, P. (2003). Subcellular distribution of Homer 1b/c in relation to endoplasmic reticulum and plasma membrane proteins in Purkinje neurons. Neurochemical research 28, 1151-1158. Sdrulla, A.D., and Linden, D.J. (2007). Double dissociation between long-term depression and dendritic spine morphology in cerebellar Purkinje cells. Nat Neurosci 10, 546-548. Shin, J.H., Kim, Y.S., and Linden, D.J. (2008). Dendritic glutamate release produces autocrine activation of mGluR1 in cerebellar Purkinje cells. Proc Natl Acad Sci U S A 105, 746-750. Simen, A.A., Lee, C.C., Simen, B.B., Bindokas, V.P., and Miller, R.J. (2001). The C terminus of the Ca channel alpha1B subunit mediates selective inhibition by G-protein-coupled receptors. J Neurosci 21, 7587-7597. Spacey, S.D., Hildebrand, M.E., Materek, L.A., Bird, T.D., and Snutch, T.P. (2004). Functional implications of a novel EA2 mutation in the P/Q-type calcium channel. Ann Neurol 56, 213-220. Steriade, M. (2004). Acetylcholine systems and rhythmic activities during the waking--sleep cycle. Progress in brain research 145, 179-196. Sterne-Marr, R., Tesmer, J.J., Day, P.W., Stracquatanio, R.P., Cilente, J.A., O'Connor, K.E., Pronin, A.N., Benovic, J.L., and Wedegaertner, P.B. (2003). G protein-coupled receptor Kinase 2/G alpha q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits. J Biol Chem 278, 6050-6058. Chapter 4: Discussion  152 Suh, B.C., and Hille, B. (2005). Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15, 370-378. Swensen, A.M., and Bean, B.P. (2003). Ionic mechanisms of burst firing in dissociated Purkinje neurons. J Neurosci 23, 9650-9663. Tai, C., Kuzmiski, J.B., and MacVicar, B.A. (2006). Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons. J Neurosci 26, 6249-6258. Takechi, H., Eilers, J., and Konnerth, A. (1998). A new class of synaptic response involving calcium release in dendritic spines. Nature 396, 757-760. Talley, E.M., Cribbs, L.L., Lee, J.H., Daud, A., Perez-Reyes, E., and Bayliss, D.A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19, 1895-1911. Tsay, D., and Yuste, R. (2004). On the electrical function of dendritic spines. Trends in neurosciences 27, 77-83. Tu, J.C., Xiao, B., Yuan, J.P., Lanahan, A.A., Leoffert, K., Li, M., Linden, D.J., and Worley, P.F. (1998). Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21, 717-726. Usowicz, M.M., Sugimori, M., Cherksey, B., and Llinas, R. (1992). P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells. Neuron 9, 1185-1199. Wang, S.S., Denk, W., and Hausser, M. (2000). Coincidence detection in single dendritic spines mediated by calcium release. Nat Neurosci 3, 1266-1273. Watanabe, S., Takagi, H., Miyasho, T., Inoue, M., Kirino, Y., Kudo, Y., and Miyakawa, H. (1998). Differential roles of two types of voltage-gated Ca2+ channels in the dendrites of rat cerebellar Purkinje neurons. Brain Res 791, 43-55. Welsby, P.J., Wang, H., Wolfe, J.T., Colbran, R.J., Johnson, M.L., and Barrett, P.Q. (2003). A mechanism for the direct regulation of T-type calcium channels by Ca2+/calmodulin-dependent kinase II. J Neurosci 23, 10116-10121. Williams, S.R., Christensen, S.R., Stuart, G.J., and Hausser, M. (2002). Membrane potential bistability is controlled by the hyperpolarization-activated current I(H) in rat cerebellar Purkinje neurons in vitro. J Physiol 539, 469-483. Williams, S.R., Toth, T.I., Turner, J.P., Hughes, S.W., and Crunelli, V. (1997). The 'window' component of the low threshold Ca2+ current produces input signal amplification and bistability in cat and rat thalamocortical neurones. J Physiol 505 ( Pt 3), 689-705. Wolfe, J.T., Wang, H., Howard, J., Garrison, J.C., and Barrett, P.Q. (2003). T-type calcium channel regulation by specific G-protein betagamma subunits. Nature 424, 209-213. Womack, M., and Khodakhah, K. (2002). Active contribution of dendrites to the tonic and trimodal patterns of activity in cerebellar Purkinje neurons. J Neurosci 22, 10603-10612. Womack, M.D., Chevez, C., and Khodakhah, K. (2004). Calcium-activated potassium channels are selectively coupled to P/Q-type calcium channels in cerebellar Purkinje neurons. J Neurosci 24, 8818- 8822. Chapter 4: Discussion  153 Womack, M.D., and Khodakhah, K. (2004). Dendritic control of spontaneous bursting in cerebellar Purkinje cells. J Neurosci 24, 3511-3521. Yasuda, R., Sabatini, B.L., and Svoboda, K. (2003). Plasticity of calcium channels in dendritic spines. Nat Neurosci 6, 948-955. Yuan, J.P., Kiselyov, K., Shin, D.M., Chen, J., Shcheynikov, N., Kang, S.H., Dehoff, M.H., Schwarz, M.K., Seeburg, P.H., Muallem, S., and Worley, P.F. (2003). Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell 114, 777-789. Zamponi, G.W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T.P. (1997). Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit. Nature 385, 442-446. Zamponi, G.W., Bourinet, E., and Snutch, T.P. (1996). Nickel block of a family of neuronal calcium channels: subtype- and subunit-dependent action at multiple sites. J Membr Biol 151, 77-90.   Appendix 1: T-type channels in pain signaling  154 APPENDIX 1: CONTRIBUTIONS OF T-TYPE CALCIUM CHANNELS TO THE PATHOPHYSIOLOGY OF PAIN SIGNALING*    * A version of this appendix has been published.  Hildebrand, M.E., and Snutch, T.P. (2006). Contributions of T- type calcium channels to the pathophysiology of pain signaling. Drug Discovery Today: Disease Mechanisms 3, 335-341. Appendix 1: T-type channels in pain signaling  155  Appendix 1: T-type channels in pain signaling  156   Appendix 1: T-type channels in pain signaling  157  Appendix 1: T-type channels in pain signaling  158  Appendix 1: T-type channels in pain signaling  159  Appendix 1: T-type channels in pain signaling  160  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  161 APPENDIX 2: ACTIVATION OF CORTICOTROPIN-RELEASING FACTOR RECEPTOR 1 SELECTIVELY INHIBITS CAV3.2 T-TYPE CALCIUM CHANNELS*    * A version of this appendix has been published.  Tao, J., Hildebrand, M.E., Liao, P., Liang, M.C., Tan, G., Li, S., Snutch, T.P., and Soong, T.W. (2008). Activation of corticotropin-releasing factor receptor 1 selectively inhibits CaV3.2 T-type calcium channels. Mol Pharmacol. 73, 1596-1609.  Reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics.  All rights reserved.  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  162    Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  163  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  164  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  165  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  166  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  167  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  168  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  169  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  170  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  171  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  172  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  173  Appendix 2: Activation of CRFR1 selectively inhibits CaV3.2  174 Appendix 3: Other Publications  175 APPENDIX 3: OTHER PHD PUBLICATIONS  Besides my above publications that relate directly to T-type modulation and physiology, I also have contributed to several other publications during my PhD studies (see below).  Most significantly, I designed and performed all electrophysiology experiments and participated in the writing of the paper by Spacey et al. (2004).  This paper examined the functional impact of a missense mutation in the P/Q-type Cav2.1 channel that was identified in a patient with type-2 episodic ataxia.  The H1736L mutation caused a significant reduction in Cav2.1 current density, an increase in inactivation kinetics, and a depolarizing shift in the voltage-dependence of activation, consistent with the loss of P/Q-type channel function that is hypothesized to underlie this form of ataxia.  I also performed all experiments and data analysis and participated in the writing of Hildebrand et al. (2004).  This study demonstrated that a commonly used househould insecticide (allethrin) potently blocked all major subfamilies of Ca2+ channels (Cav1.2, Cav2.1, and Cav3.1) by accelerating their inactivation kinetics and shifting their voltage-dependence of inactivation to more hyperpolarized potentials.  As the physiological effects of pyrethroid pesticides like allethrin are thought to be mediated by the prolonged opening of voltage-gated Na+ channels, our study identified voltage-gated Ca2+ channels as a novel molecular target for pyrethroid action.  Additional publications during my PhD studies:  Spacey, S.D., Hildebrand, M.E., Materek, L.A., Bird, T.D., and Snutch, T.P. (2004). Functional implications of a novel EA2 mutation in the P/Q-type calcium channel. Ann Neurol 56, 213-220.  Hildebrand, M.E., McRory, J.E., Snutch, T.P., and Stea, A. (2004). Mammalian voltage-gated calcium channels are potently blocked by the pyrethroid insecticide allethrin. J Pharmacol Exp Ther 308, 805- 813.  McRory, J.E., Hamid, J., Doering, C.J., Garcia, E., Parker, R., Hamming, K., Chen, L., Hildebrand, M., Beedle, A.M., Feldcamp, L., et al. (2004). The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution. J Neurosci 24, 1707-1718.  Vieira, L.B., Kushmerick, C., Hildebrand, M.E., Garcia, E., Stea, A., Cordeiro, M.N., Richardson, M., Gomez, M.V., and Snutch, T.P. (2005). Inhibition of high voltage-activated calcium channels by spider toxin PnTx3-6. J Pharmacol Exp Ther 314, 1370-1377. Appendix 4: Certificates  176 APPENDIX 4: UBC RESEARCH CERTIFICATES OF APPROVAL       Appendix 4: Certificates  177 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0066474/manifest

Comment

Related Items