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A PDZ-3 mediated physical and functional interaction between the CaV3.2 T-type calcium channel and neuronal… Mulatz, Kirk James 2013

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A PDZ-3 MEDIATED PHYSICAL AND FUNCTIONAL INTERACTION BETWEEN THE CaV3.2 T-TYPE CALCIUM CHANNEL AND NEURONAL NITRIC OXIDE SYNTHASE by Kirk James Mulatz B.Sc., University of Saskatchewan, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2013  © Kirk James Mulatz, 2013  Abstract: T-type voltage-gated calcium channels are expressed throughout the central and peripheral nervous systems as well as in several non-neuronal tissues and contribute to variety of functions such as neuronal excitability, intracellular calcium influx, shaping action potentials, pace-making activity, hormone secretion, and neurotransmitter release. Of the three T-type channel isoforms, Cav3.2 is uniquely sensitive to redox modulation with oxidizing reagents inhibiting and reducing compounds enhancing channel activity. This modulation has been shown to alter firing patterns of reticular thalamic neurons and to affect the nociceptive threshold in vivo suggesting that redox modulation of Cav3.2 may play an important role in regulating neuronal activity. A potential source of oxidizing molecules in vivo is neuronal nitric oxide synthase (nNOS), a calcium dependent enzyme which synthesizes nitric oxide (NO) from arginine. Interestingly, the carboxyl terminus of Cav3.2 possesses a putative PDZ-3 binding ligand which is compatible with the PDZ-3 domain of nNOS. I hypothesize that Cav3.2 and nNOS physically interact via the PDZ-3 binding ligand of Cav3.2 and that this physical interaction mediates a functional interaction whereby Cav3.2 activity stimulates nNOS to produce NO which, in turn, inhibits Cav3.2 activity. Cav3.2 and nNOS were expressed in a heterologous system which allowed us to examine the putative PDZ-3 mediated interactions between the two proteins. Immunoprecipitation experiments using Cav3.2 specific antibodies demonstrate that Cav3.2 and nNOS can interact via the carboxyl PDZ-3 ligand of Cav3.2 and that this interaction is disrupted when the PDZ-3 ligand is mutated. Utilizing a NO sensitive fluorometric assay we show that Cav3.2 activity can stimulate nNOS to produce NO and that disruption the PDZ-3  ii  interaction precludes nNOS activation. We also demonstrate that the PDZ-3 mediated physical interaction facilitates the inhibition of Cav3.2 by nNOS derived NO. Disruption of the Cav3.2/nNOS interaction in vivo using intraperitoneal injection of membrane permeable peptides designed to competitively disrupt the PDZ-3 interaction produces an exaggerated respiratory response to changes in available oxygen and a blunted response in the hyperoxic response test. These results indicate that Cav3.2 and nNOS physically and functionally interact to contribute to normal physiological processes.  iii  Preface: I designed all experiments and analyzed all experimental results with input and feedback from other researchers. I performed all experiments except for the PSD-95 coimmunoprecipitation and the glossopharyngeal nerve immunohistochemistry experiments. The PSD-95 co-immunoprecipitation experiments were performed by Zeina Waheed who also assisted in the preparation for the Cav3.2/nNOS/PSD-95 current density experiments. I performed the dissections to harvest the glossopharyngeal nerves and Karen Jones performed the staining, mounting and imaging for the immunohistochemistry experiments. Dr. Anamika Singh collected the initial current density measurements and provided valuable guidance in optimizing the electrophysiological experiments. 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 (Animal care certificate number: A10-0286). I participated in the molecular portion of Appendix 1 (Hildebrand, M.E., David, L.S., Hamid, J., Mulatz, K., Garcia, E., Zamponi, G.W., Snutch, T.P. 2007. Selective inhibition of Cav3.3 T-type calcium channels by Gq/11-coupled muscarinic acetylcholine receptors. Journal of Biological Chemistry. 282(29), 21043-21055) and Appendix 2 (Adams, P.J., Garcia, E., David, L.S., Mulatz, K.J., Spacey, S.D., Snutch, T.P. 2009. Cav2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: Implications for calcium channelopathies. Channels (Austin). 3(2):110121) generating clones which were used for the experimental procedures. In Appendix 3 (Gonzalez, L.E., Kotler, M.L., Vattino, L.G., Conti, E., Reisin, R.C., Mulatz, K.J., Snutch, T.P., Uchitel, O.D. 2011. Amyotrophic lateral sclerosis-immunoglobulins selectively interact iv  with neuromuscular junctions expressing P/Q-type calcium channels. Journal of Neurochemistry. 119(4),826-38) I participated in the design and analysis of the Cav2.1 immunoprecipitation experiments.  v  Table of Contents: Abstract  ii  Preface  iv  Table of Contents  vi  List of Tables  xii  List of Figures  xiii  List of Abbreviations  xv  Acknowledgements  xxi  Dedication  xxii  Chapter 1  1  1. Introduction  1  1.1. Calcium channel overview  1  1.1.1. Brief history of calcium channels – Discovery of calcium currents  1  1.1.2. Brief history of calcium channels – Classes and nomenclature  2  1.1.3. Molecular structure and composition  9  1.2. Cav3 calcium channels  12  1.2.1. Cav3 calcium channel expression  12  1.2.2. Biophysical properties of Cav3 channels  16  1.2.3. Physiological roles of T-type Ca2+ channels  20  1.2.4. Pharmacological properties of T-type Ca2+ channels  27  1.2.5. Modulation of T-type Ca2+ channels  30  1.3. PDZ ligands and domains: partnering regulatory enzymes to their targets  36  1.3.1. Structure and function  36  1.3.2.PDZ domains of nNOS  38 vi  1.4. Functional relevance of nitric oxide and nitric oxide synthases  39  1.4.1. Molecular structure and composition  39  1.4.2. Synthesis of nitric oxide  41  1.4.3. Mechanisms of modulation by nitric oxide  44  1.4.4. Degradation of nitric oxide  45  1.4.5. Pharmacological properties of neuronal nitric oxide synthase  46  1.4.6. Expression of nNOS and roles of nNOS derived NO  47  1.5. Nitric oxide and nNOS in the ventilatory response  50  1.5.1. Neuronal pathways involved in respiration  50  1.5.2. Role of NOS in respiration  53  1.5.3. Properties and role of nNOS expressing microganglia in the glossopharyngeal nerve (GPN) 1.6. Cav3.2 channels and nociception  55 56  1.6.1. Neuronal pathways involved in nociception  56  1.6.2. Role of Cav3.2 channels in nociception  57  1.6.3. Role of nNOS in nociception  58  1.7. Thesis hypothesis and objectives  60  1.7.1. Hypothesis  60  1.7.2. Objectives  61  Chapter 2  62  2. Cav3.2 and nNOS interact physically and functionally to form a putative negative feedback regulatory loop  62  2.1. Introduction  62  2.2. Materials and methods  65 vii  2.2.1. Generation of fusion peptides  65  2.2.2. Generation and transformation of chemically competent DH5 cells  69  2.2.3. Mutation of the Cav3.2 PDZ-3 binding ligand  70  2.2.4. nNOS and PSD-95 clones  71  2.2.5. Cell culture and transfection  74  2.2.6. Generation of stable cell lines  76  2.2.7. GST pull down  78  2.2.8. Co-immunoprecipitation  79  2.2.9. Western blot analysis  80  2.2.10. Nitric oxide detection assay  82  2.2.11. Whole cell patch clamp analysis  83  2.3. Results  86  2.3.1. Cav3.2 and nNOS interact via the PDZ-3 binding ligand located at the carboxyl terminus of Cav3.2  86  2.3.2. The PDZ-3 domain of the PDZ-2 beta finger binding ligand of nNOS can facilitate the formation of larger protein complexes  91  2.3.3. The physical interaction between Cav3.2 and nNOS in required for Cav3.2 activity to stimulate the production of nitric oxide by nNOS  93  2.3.4. nNOS activity inhibits Cav3.2 channels in a heterologous expression system  98  viii  2.4. Discussion  109  2.4.1. Cav3.2 and nNOS physically and functionally interact in a heterologous system 2.4.2. nNOS can facilitate the formation of larger protein complexes  109 113  2.4.3. Inhibition of Cav3.2 does not involve the activation of soluble guanylyl cylase 114 2.4.4. Potential implication of a Cav3.2/nNOS complex in vivo  115  Chapter 3 117 3.  Disruption of the Cav3.2 and nNOS interaction in vivo alters the respiratory response in Sprague Dawley rats  117  3.1. Introduction  117  3.2. Materials and methods  120  3.2.1. Animal care  120  3.2.2. GPN dissection  120  3.2.3. Quantitative real-time-PCR  122  3.2.4. Immunohistochemistry  122  3.2.5. Design of the TAT fusion peptides  124  3.2.6. Respirometry  125  3.2.7. Analysis of respirometry data  129  ix  3.3. Results  132  3.3.1. Cav3.2 is present in nNOS expressing microganglia within the GPN of Sprague Dawley rats  132  3.3.2. Disruption of the Cav3.2/nNOS interaction in vivo results in an augmented steady state response to hypoxic challenges  136  3.3.3. The hyperoxic response may be attenuated in animals where the Cav3.2/nNOS interaction is disrupted 3.4. Discussion  146 149  3.4.1. Disruption of the Cav3.2/nNOS complex has a significant effect on the respiratory rate in response to available oxygen  149  3.4.2. Implications of the observed increase in tidal volume and total ventilation following the administration of a mutant PDZ-3 TAT fusion peptide 3.4.3. Potential relevance to respiration  154 155  Chapter 4  156  4.  Discussion  156  4.1. Overall significance of the thesis  156  4.1.1. Identification of a physical and functional interaction between Cav3.2 and nNOS 157 4.1.2. Potential relevance for nociception  158  4.1.3. Relevance to the respiratory system  159  x  4.2. The Cav3.2 T-type calcium channel and neuronal nitric oxide synthase physically and functionally interact forming a putative negative feedback regulatory loop  159  4.2.1. Working hypothesis  159  4.2.2. Limitations and weaknesses  160  4.3. Intraperitoneal injection of a TAT fusion peptide to disrupt the Cav3.2 and nNOS interaction in vivo produces an augmented respiratory response to changes in available oxygen in rats  162  4.3.1. Working hypothesis  162  4.3.2. Limitations and weaknesses  163  4.4. Conclusions 4.4.1. General conclusion  164 164  4.4.2. Relevance to modulation of neuronal networks co-expressing Cav3.2 and nNOS 165 4.4.3. Relevance to the respiratory system  166  4.4.4. Future directions  167  Bibliography  170  Appendix 1: Selective Inhibition of Cav3.3 T-type Calcium Channels by Gq/11-coupled Muscarinic Acetylcholine Receptors  204  Appendix 2: CaV2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations  217  Appendix 3: Amyotrophic lateral sclerosis-immunoglobulins selectively interact with neuromuscular junctions expressing P/Q-type calcium channels  229  xi  List of Tables: Table 1.1: Evolution of the voltage-gated Ca2+ channel nomenclature.  7  Table 1.2: Summary of the biophysical characteristics of the low threshold Cav3 channels 19 Table 1.3: Summary of Cav3.1 and Cav3.2 knockout mice phenotypes  26  Table 2.1: Primers used to generate the clones utilized in this thesis  68  Table 2.2: Agents utilized to maintain selection pressure for the transfected constructs  76  Table 3.1: Summary of the respirometry responses from TAT-control, TAT-DEPV and TAT-DEPE injected animals 140  xii  List of Figures: Figure 1.1: Phylogeny of the voltage-gated Ca2+ channel family  8  Figure 1.2: Structural features of voltage-gated Ca2+ ion channels  11  Figure 1.3: Summary of the known modulators of Cav3 channel activity  35  Figure 1.4: Domain structure of nitric oxide synthases  41  Figure 1.5: Reaction schematic and electron flow diagram of the conversion of arginine to citrulline and nitric oxide 43 Figure 2.1: Alignment of voltage-gated Ca2+ channels demonstrating a PDZ-3 motif unique to Cav3.2 65 Figure 2.2: Cav3.2 and nNOS can physically interact via the carboxyl PDZ-3 binding ligand of Cav3.2  88  Figure 2.3: The physical interaction of Cav3.2 and nNOS can be competitively disrupted using a peptide containing the PDZ-3 binding ligand 90 Figure 2.4: The Cav3.2/nNOS complex can further interact with PSD-95 to form a larger complex  92  Figure 2.5: The PDZ-3 binding interaction between Cav3.2 and nNOS is required for Cav3.2 activity to stimulate nNOS to produce nitric oxide  96  Figure 2.6: nNOS activity inhibits Cav3.2  101  Figure 2.7: A physical interaction between Cav3.2 and nNOS is required for the basal inhibition of Cav3.2 activity by nNOS  103  Figure 2.8: The inhibition of Cav3.2 by nNOS activity is independent of soluble guanylyl cyclase activation 105 Figure 2.9: Co-expression of PSD-95 with nNOS and Cav3.2 results in increased inhibition of Cav3.2  107  Figure 2.10: The nNOS-mediated changes in peak current density are not the result of the direct action of L-NAME, TAT-DEPV or PSD-95 on Cav3.2 109 Figure 2.11: Schematic summarizing the proposed physical and functional interaction between Cav3.2 and nNOS  112  Figure 3.1: Schematic summarizing the location and innervation of the carotid body  119  Figure 3.2: Experimental setup used to acquire the respirometry and metabolic data  127 xiii  Figure 3.3: Graphical representation of the respirometry protocol  128  Figure 3.4: qRT-PCR analysis illustrating the Ca2+ channel expression profile in the glossopharyngeal nerve (GPN) in Sprague Dawley rats  134  Figure 3.5: Immunohistological staining of Cav3.2, nNOS and ChAT in the microganglia of the glossopharyngeal nerve of Sprague Dawley rats 135 Figure 3.6: Disruption of the Cav3.2/nNOS interaction in vivo results in an elevated respiratory response to changes in oxygen concentration  142  Figure 3.7: Average tidal volume and total ventilation in TAT-DEPE injected Sprague Dawley rats differs significantly from Cav3.2/nNOS disrupted (TAT-DEPV) and TAT-control 144 Figure 3.8: Disruption of the Cav3.2/nNOS interaction alters the response in the hyperoxic response test 147 Figure 3.9: Schematic illustrating the hypothesized contribution of the Cav3.2/nNOS interaction to the NO mediated inhibition of the carotid body  153  xiv  List of Abbreviations: 7-NI: 7-Nitro indazole AChR: acetylcholine receptor ACR: air convection requirement ADP: after depolarizing potential AM: acetomethylester AngII: angiotensin-II receptor II ANOVA: analysis of variance Arg: arginine Ba2+: barium BH4: tetrahydrobiopterin BotC: Botzinger complex Ca2+: calcium CaM: camodulin CaMKII: camodulin dependent protein kinase kinase II cAMP: cyclic monophosphate CAPON: carboxy-terminal PDZ ligand of nNOS ccm-1: cubic centimeters per minute cGK: cGMP dependent kinases cGMP: cyclic guanosine monophosphate CNS: central nervous system CO2: carbon dioxide -COOH: carboxyl terminus CRF-1: corticotropin-releasing factor receptor-1 xv  CSN: carotid sinus nerve CtBP; carboxyl-teminal binding protein Cu2+: copper (II) ion cVRG: caudal ventral respiratory group DAG: diacyl glycerol DAR-4M: diaminorhodamine-4M AM Dlg1: Drosophila disc large tumor suppressor DMEM: Dulbecco's Modified Eagle Medium DMSO: dimethyl sulfoxide DNA: deoxyribonucleic acid dNTP: deoxyribonucleotide triphosphate DRG: dorsal root ganglion DTNB: 5,5-dithio-bis-(2-nitrobenzoic acid) DTT: dithiothreitol EDTA: ethylenediaminetetraacetic acid ETA: endothelin type A receptor eNOS: endothelial nitric oxide synthase FAD: flavin adenine dinucleotide FBS: fetal bovine serum FMN: flavin adenine mononucleotide GAERS: Genetic Absence Epilepsy Rat from Strasbourg GDB: Human Gene Database GFP: green fluorescent protein GPN: glossopharyngeal nerve xvi  GST: glutathione S-transferase GTP: guanosine triphosphate HEK: human embryonic kidney HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HUGO: Human Gene Organization HVA: high voltage activated Hz: hertz IC50: half maximal inhibitory concentration iNOS: inducible nitric oxide synthase IPTG: isopropyl -D-1 thiogalactopyranoside IUPHAR: International Union of Pharmacology IV curve: current-voltage relationship kHz: kilohertz KLHL1: kelch-Like 1 LB: Lauria broth L-NAME: methyl ester of N-Nitro-L-arginine L-NMA: N-Methyl-L-arginine L-NNA: N-Nitro-L-arginine LPA: lysophosphatidic acid LTD: long term depression LTP: long term potentiation LTS: low threshold spike LVA: low voltage activated M: megaOhm  xvii  mg: milligram ml: millilitre mM: millimolar MOPS: 3-(N-morpholino)propanesulfonic acid mOsm/kg: milliosmole per kilogram mRNA: messenger ribonucleic acid ms: millisecond mV: millivolts NADPH: nicotinamide adenine dinucleotide phosphate ng: nanograms nm: nanometer nM: nanomolar NHA: L-arginine to N-hydroxy-L-arginine Ni2+: nickel NiCl2 : nickel chloride NKR1: neurokini 1 receptor nM: nanomolar NMDAR: N-methyl-D-aspartic acid receptor nNOS: neuronal nitric oxide synthase NO: nitric oxide NO2-: nitrites NO3-: nitrates NOS: nitric oxide synthase nRT: thalamic reticular nucleus xviii  NST: nucleus of the solitary tract O2: oxygen ODQ: 1H-[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one OONO-: peroxynitrites pA: picoamperes PBS: phosphate buffered saline pCO2: partial pressure of carbon dioxide PCR: polymerase chain reaction PDE: phosphodiesaterase PDZ: post synaptic density protein (PSD-95), Drosophila disc large tumor suppressor (Dlg1), and the zonula occludens-1 protein (Zo-1) pF: picofarad pFRG: parafacial respiratory group qRT-PCR: quantitative real-time polymerase chain reaction PIP2: phosphatidylinositol 4,5-bisphosphate PKA: protein kinase A PLCphospholipase C  pO2: partial pressure of oxygen pre-BotC: pre-Botzinger complex pS: picosiemens PSD-95: post synaptic density protein – 95kDa ROCK: Rho-associated protein kinase RTN: retrotrapeziod nuleus rVRG: rostral ventral respiratory group xix  sGC: soluble guanylyl cyclase Sr2+: strontium TAT: human immunodeficiency Virus 1 transactivator of transcription TRPV1: transient receptor potential vanilloid-1 g: microgram l: microlitre M: micromolar VGCC: voltage-gated calcium channel V: volts V50act: half maximal activation voltage V50inact: half maximal inactivation voltage Vol: volume x g: relative centrifugal force Zn2+: zinc Zo-1: zonula occludens-1 protein  xx  Acknowledgements: I would like to thank my supervisor, Terry Snutch, for the patience, guidance and encouragement he gave me during my PhD studies. A special thanks to Esperanza Garcia and her patience and advice; in addition to teaching me the theory and techniques of electrophysiology she taught me that a tool and technique is infinitely more useful when you understand why it works in addition to how it works. I thank John Tyson who provided much needed advice and encouragement when my immunoprecipitations were less than stellar. Many thanks to my supervisory committee: Steven Vincent, Yu Tian Wang, and William Milsom for their direction and advice. I would like to extend additional acknowledgement to Steven Vincent for proposing that I explore the potential Cav3.2/nNOS interaction for my PhD and to William Milsom for providing a crash course in respirometry, knowledgeable advice, and for allowing me access to his laboratory and equipment. I would also like to thank Stella Lee from William Milsom’s lab for her guidance in getting the respirometry equipment to behave and for being so accommodating when we were scheduling the equipment usage. To all past and present members of the Snutch Lab, thank you for making the lab a great place to learn and work; the atmosphere was always welcoming and supportive. My appreciation goes to the Michael Smith Foundation for Health Research, the BC Epilepsy Society and the UBC graduate fellowship program for providing me with trainee funding during my graduate studies. A heartfelt thank you goes to my parents and grandparents for your support and for teaching me the value of hard work and persistence. Finally, I would like to express my endless gratitude to my wonderful wife Raagini for her support throughout all stages of my PhD studies. xxi  Dedication: To my wife Raagini, my son Dylon, my parents Ralph and Elaine, and my grandparents Joe and Clara, and Frank and Katherine.  xxii  Chapter 1. 1. Introduction 1.1. Calcium channel overview 1.1.1. Brief history of calcium channels – Discovery of calcium currents Evaluating the field of voltage-gated calcium (Ca2+) channels (VGCCs) in 2012, one can confidently make the statement that VGCCs play numerous crucial roles in neuronal activity. A search for “calcium channels” using the online PubMed (US National Library of Medicine/National Institutes of Health) search engine reveals a plethora of over 60,000 peer reviewed articles and additional queries for the specific subtypes of VGCCs uncover even more articles indicating the considerable interest these ion channels have garnered in the research community. Sixty years ago this was not the case. In the early 1950s PubMed and online searches were elements in the realm of science fiction at best and the idea that a Ca2+ specific voltage dependent ion channels even existed was only beginning to take shape. The study of voltage dependent Ca2+ currents was slow to gain momentum, at the time many researchers in the field were focused on the sodium and potassium currents underlying the action potential (Hodgkin and Huxley, 1945; Hodgkin and Katz, 1949; Nastuk and Hodgkin, 1950; Draper and Weidmann, 1951; Huxley and Stampfli, 1951). However, amid a field of exciting discoveries including new findings on excitatory and inhibitory neuronal communication (Fatt and Katz, 1951, 1953a) and the quantal basis of neurotransmitter release (Del Castillo and Katz, 1954) arose guarded conclusions regarding the possible presence of Ca2+ currents in crustacean muscle fibers (Fatt and Katz, 1953b). These guarded conclusions did not immediately pique interest in Ca2+ channels and four years lapsed before additional evidence was put forward by Hodgkin and Keynes suggesting a potential role for Ca2+ in neuronal processes such as vesicle release and excitation1  contraction coupling (Hodgkin and Keynes, 1957). A year later, in 1958, Fatt and Ginsborg published a paper demonstrating that the movement of divalent ions (including Ca2+) across the membrane could support action potentials (Fatt and Ginsborg, 1958) providing additional evidence to support their 1953 conclusions regarding the presence of a Ca2+ current in crustacean muscle fibers. Still, interest in the role Ca2+ currents in neuronal activity was slow to evolve. It was another five years before Fleckenstein discovered that verapamil, and later nifedipine (a dihydropyridine) inhibited excitation-contraction coupling by disrupting Ca2+ currents, highlighting the significance of Ca2+ in this process (reviewed in (Fleckenstein, 1983)). A few years afterwards, Reuter and coworkers used the voltage clamp technique to identify a voltage-dependent Ca2+ current in the purkinje fibers of sheep and calf hearts which further strengthened the link between Ca2+ currents and excitation-contraction coupling (Reuter, 1967). In the same year, Katz and Miledi put forward the Calcium Hypothesis of synaptic communication which stated “that inward movement of a positively charged Ca compound, or the Ca2+ ion itself, constitutes one of the essential links in the ‘electro-secretory’ coupling process of the axon terminal ” (Katz and Miledi, 1967). Further elaboration and evidence of the Calcium Hypothesis was published in 1970 (Katz and Miledi, 1970). The evidence demonstrating the critical role Ca2+ currents play in synaptic activity was building and Ca2+ currents could no longer be ignored. A full review of the history of Ca2+ channels can be found in a superbly written albeit not entirely unbiased book chapter by Tsein and Barret (Tsien and Barrett, 2005).  1.1.2. Brief history of Ca2+ channels – Classes and nomenclature The initial perception was that all Ca2+ currents were derived from a single channel type; a notion that had support up until the early 1980s (Brown et al., 1982) despite early 2  evidence from Hagiwara and coworkers which identified two distinct types of Ca2+ current (what are now known as high voltage activated (HVA) and low voltage activated (LVA)) (Hagiwara et al., 1975). However, by the mid-1980s due to the contributions from a number of groups there was general consensus that, based primarily on their threshold of activation, there were at least two distinct types of Ca2+ channels (Llinás and Yarom, 1981; Carbone and Lux, 1984a, 1984b; Armstrong and Matteson, 1985; Bean, 1985; Fedulova et al., 1985; Matteson and Armstrong, 1986). Upon further analysis, Nowycky and Fox identified a third type of Ca2+ channel and put forward criteria for defining the three types of channels (Nowycky et al., 1985). The new criteria identified the L-type channel as a high voltageactivated channel with a large unitary conductance and a long lasting Ba2+ current whereas the T-type channel has a low threshold of activation, a tiny unitary conductance and a transient current whole cell waveform with Ba2+ as the charge carrier. N-type currents were neither exactly L-type nor T-type, although like L-type channels the N-type were high voltage-activated and similar to T-type channels they required a significantly hyperpolarizing potential to be available for opening. Additionally, the N-type currents were found to be insensitive to the L-type dihydropyridine agonist Bay K8644 (Nowycky et al., 1985). Acceptance of the new classification was not universal (Swandulla and Armstrong, 1988). Fortunately, a peptide toxin (-conotoxin GVIA) identified and isolated by Olivera and Yoshikami from venomous cone snails (Yoshikami et al., 1989; Olivera et al., 1991) proved to be a specific inhibitor of N-type channels (McCleskey et al., 1987; Plummer et al., 1989; Boland et al., 1994). This antagonist in conjunction with the L-type dihydropyridine antagonists identified by Flekenstein allowed for the isolation of the L-type and N-type HVA currents and was crucial in the further identification of various Ca2+ currents in peripheral neurons (Plummer et al., 1989). 3  A fourth Ca2+ channel class was identified in cerebellar Purkinje neurons. The noninactivating, dihydropyridine insensitive currents were designated P-type because of their localization (Llinás et al., 1989, 1992) and were found to be uniquely sensitive to peptide toxins isolated from funnel web spider venom ( -Aga-IVA and -Aga-IVB )(Mintz et al., 1992). This fourth class of Ca2+ channel soon after became known as the P/Q-type when it was discovered that the Q-type currents identified based upon their distinct inactivation kinetics and relative insensitivity to -Aga-IVA (Randall and Tsien, 1995) were highly similar in both distribution and biophysical properties to P-type currents (Stea et al., 1994). This notion was subsequently confirmed by Bourinet and coworkers who determined that the P- and Q-type currents result from alternative splicing of the same Cav gene (Bourinet, 1999). While performing a pharmacological dissection of the Ca2+ currents found in cerebellar granule neurons, Randall and Tsien identified a fifth class of native Ca2+ channels resistant to the pharmacological compounds known to specifically block the other HVA channels and was given the designation R-type. The identification of R-type Ca2+ channel was met with some criticism as the currents recorded could potentially be explained by a number of mechanisms including incomplete pharmacological blockade or splice variation of one of the previously identified classes. Once again, peptide toxins provided confirmatory insights in the form of SNX-482, an apparent R-type specific antagonist isolated from tarantula (Hysterocrates gigas) venom (Newcomb et al., 1998). It should be noted here that Snutch and colleagues had previously identified and cloned a novel fifth type of Ca2+ channel that exhibited biophysical and pharmacological properties distinct from both HVA and LVA subtypes (Soong et al., 1993) and that Randall and Tsien’s R-type current was largely confirmatory of this new Ca2+ conductance first described by molecular cloning.  4  Concurrent with the biophysical and pharmacological identification of the different classes of native Ca2+ currents, efforts were being made to identify and clone the underlying genes responsible for native Ca2+ currents. The first Ca2+ channel clones to be identified were from the dihydropyridine-sensitive L-type channel isolated from skeletal muscle (Tanabe et al., 1987) and subsequently the homologous L-type channels from cardiac and smooth muscles (Mikami et al., 1989; Biel et al., 1990; Koch et al., 1990). The primary structures of the L-type channels from the cardiac/smooth muscle tissue were more related to each other than to the L-type channel from skeletal muscle suggesting that they were encoded by different genes and further presented the possibility that multiple genetic isoforms for each pharmacological class may exist. This notion was subsequently confirmed in that two Ca2+ channel isoforms identified in the brain were found to be closely related to the cardiac/smooth muscle L-types (called 1C and 1D) while other brain cDNAs encoded quite distinct Ca2+ channel types (called 1A and 1B, and shown to encode P/Q-type and N-type channels, respectively) (Snutch et al., 1990; Starr et al., 1991; Dubel et al., 1992; Stea et al., 1994). Additionally, splice variants of the neuronal Ca2+ channel isoforms were shown to be expressed both in the brain and other tissues (Snutch et al., 1991). Taken together, the considerable structural and functional diversity of Ca2+ channels was becoming apparent. By 1994, with the wealth Ca2+ channel cloned isoforms identified, it was becoming increasingly difficult to identify which exact combination of channel and auxiliary subunits was being used by the various researchers in the field; a very real problem considering that co-expression of auxiliary subunits can significantly affect channel biophysical properties. As such, the field adopted the letter naming scheme initiated by Snutch and co-workers to define the main pore forming 1 subunits and numbers to refer to the , 2, and  auxiliary subunit variants (Snutch et al., 1990, 1991; Starr et al., 1991; Birnbaumer et al., 1994). The 5  standardization of nomenclature was taken a step further in 1997 to include the gene names under a format approved by the Human Genome Organization/ Human Gene Database (HUGO/GDB) nomenclature committee (Lory et al., 1997). Finally, the current nomenclature was proposed in 2000 and approved by the International Union of Pharmacology (IUPHAR) committee in 2003; it is based on the amino acid sequence similarity to represent the phylogenic relationship of the channels (Ertel et al., 2000; Catterall et al., 2003). Table 1.1 provides a summary of the evolution of the Ca2+ channel nomenclature and Figure 1.1 illustrates how the most recent nomenclature better suits phylogenic alignment of VGCCs.  6  Table 1.1: Evolution of the voltage-gated Ca2+ channel nomenclature. Historical  HUGO/GDB  Snutch  gene  IUPHAR  First  Group  Class  nomenclature 1  nomenclature 2  nomenclature 3  described:  HVA  L-type  1S  CACNA1S  Cav1.1  4  1C  CACNA1C  Cav1.2  5  1D  CACNA1D  Cav1.3  6  1F  CACNA1F  Cav1.4  7  Type  1A  CACNA1A  Cav2.1  8  N-type  1B  CACNA1B  Cav2.2  9  R-type  1E  CACNA1E  Cav2.3  10  T-type  1G  CACNA1G  Cav3.1  11  1H  CACNA1H  Cav3.2  12  1I  CACNA1I  Cav3.3  13  P/Q-  LVA  Table 1.1: Evolution of the voltage-gated Ca2+ channel nomenclature. Summary of the evolution of the nomenclature used to identify the main pore forming unit of voltage-gated Ca2+ channels. HVA: high voltage-activated, LVA: low voltage-activated. 1  (Birnbaumer et al., 1994). 2 (Lory et al., 1997). 3 (Ertel et al., 2000; Catterall et al., 2003). 4 (Tanabe  et al., 1987). 5 (Mikami et al., 1989; Biel et al., 1990; Koch et al., 1990; Starr et al., 1991). 6 (Hui et al., 1991; Williams et al., 1992b). 7 (Strom et al., 1998; Naylor et al., 2000; McRory et al., 2001). 8 (Mori et al., 1991; Starr et al., 1991). 9 (Dubel et al., 1992; Williams et al., 1992a; Fujita et al., 1993). 10  (Niidome et al., 1992; Soong et al., 1993). 11 (Perez-Reyes et al., 1998; Zhuang et al., 2000). 12  (Cribbs et al., 1998; Williams et al., 1999). 13 (Lee et al., 1999a).  7  Figure 1.1: Phylogeny of the voltage-gated Ca2+ channel family  Figure 1.1: Phylogeny of the voltage-gated Ca2+ channel family Phylogenic tree displaying the percent identity between the members of the voltage-gated  Ca2+ channel family. Predicted amino acid sequences of each channel were compared pairwise and the percentage similarity plotted. The historical 1994 nomenclature followed by the IUPHAR nomenclature is used to identify each channel illustrating how the standardized IUPHAR nomenclature better reflects the phylogenic relationship of the channels. GenBank Accession Numbers of the VGCCs used: rat α1A, M64373; rat α1B, M92905; rat α1C, M67515; rat α1D, AF370009; rat α1E, L15453; human α1F, AJ224874; rat α1G, AF290212; rat α1H, AF290213; rat α1I, AF290214; rabbit α1S, M23919. Adapted from Snutch et al. (Snutch et al., 2005)  8  1.1.3. Molecular structure and composition Functional HVA VGCCs are multi-protein structures comprised of a main pore forming unit (1), together with several auxiliary subunits (, 2, and ) (Takahashi et al., 1987; Campbell et al., 1988; Catterall et al., 1988; Ruth et al., 1989; Jay et al., 1990). The main pore forming unit is structurally similar to that of sodium channels (Tanabe et al., 1987), comprised of four homologous domains each with six hydrophobic transmembrane segments (S1-S6) (Campbell et al., 1988). The fourth segment (S4) of each domain acts as a voltage sensor due to the regularly repeating pattern of positively charged residues within this segment ((Lam et al., 2005; Kurejová et al., 2007) and reviewed in (Catterall, 2005, 2011; Snutch et al., 2005; Yu et al., 2005)). Between segments S5 and S6 in each domain a pore forming loop allows for the transport of ions through the channel and contains a conserved locus which confers Ca2+ ion specificity. The key residues within this conserved locus are four glutamates in HVA channels (EEEE locus) and a combination of glutamate and aspartic acid residues (EEDD locus) in LVA channels ((Kim et al., 1993; Talavera et al., 2001; Cens et al., 2007) and reviewed in (Sather, 2005)). The 1 unit generally confers most of the biophysical and pharmacological properties of each Ca2+ channel subtype although various properties such as trafficking, voltage dependence, kinetics and second-messengerdependent modulation have all been shown to be significantly altered by the auxiliary subunits (Hofmann et al., 1994; Stea et al., 1994; Hosey et al., 1996; Letts et al., 1998; Arikkath and Campbell, 2003; Cens et al., 2005; Herlitze and Mark, 2005; Snutch et al., 2005). The S1-S6 segments and pore forming loops are well conserved across the VGCC family with approximately 80% amino acid similarity. However, when considering the entire channel the sequence similarity drops to about 70% within subfamilies and to less than 40% 9  between subfamilies (Catterall, 2000; Ertel et al., 2000). This dissimilarity arises primarily from the amino termini, cytosolic loops and carboxyl termini of the channels and is a major source of diversity within the VGCC family as they contain the  subunit binding site as well as many regulatory domains (Dolphin et al., 1999; Ivanina et al., 2000; Soong et al., 2002; Wolfe et al., 2002; Welsby et al., 2003; Chaudhuri et al., 2004; Dubel et al., 2004; DePuy et al., 2006). The Cav3 (T-type) VGCCs are the most divergent of the VGCCs with only 40% similarity with the other classes and they do not retain such features such as the  subunit binding site in the cytosolic domain I-II linker and the E-F hand at the carboxyl terminus. As such, unlike the HVA channels, the auxiliary subunits are unlikely to play an important role in the regulation and function of the LVA channels (Dolphin et al., 1999; Dubel et al., 2004). An illustration of the typical topology of the pore forming unit of VGCCs can be seen in Figure 1.2.  10  Figure 1.2: Structural features of voltage-gated Ca2+ ion channels  Figure 1.2: Structural features of voltage-gated Ca2+ ion channels The main pore-forming unit of voltage-gated Ca2+ channels consists of four domains with six transmembrane segments each. Within each domain the fourth transmembrane segment (coloured red) contains positively charged residues which act a voltage sensor and respond to membrane depolarizations to confer a structural change in the channel which opens the pore. Between the fifth and sixth transmembrane segments of each domain there is a re-entrant extracellular loop that forms the pore of the channel and confers selectivity for Ca2+ ions. The domains are connected by intracellular loops, the amino acid sequence of which is highly variable between the channel types.  11  1.2. Cav3 calcium channels 1.2.1. Cav3 calcium channel expression Cav3 channels (T-type) are expressed in various neuronal cell types of the central and peripheral nervous system as well in non-neuronal tissues throughout the body (reviewed in (Perez-Reyes, 2003)). Studies utilizing Northern blot analysis, immunohistochemistry, and reverse transcript PCR in conjunction with electrophysiological characterizations were effective in mapping the distribution of Cav3.1 (Perez-Reyes et al., 1998; Craig et al., 1999; Monteil et al., 2000a; McRory et al., 2001), Cav3.2 (Cribbs et al., 1998; McRory et al., 2001; Lee et al., 2002), and Cav3.3 (Lee et al., 1999a; Monteil et al., 2000b; McRory et al., 2001). Talley et al. were the first to characterize the distribution of the three Cav3 subtypes within the CNS using in situ hybridization (Talley et al., 1999). The mRNA expression profiles of the Cav3 subtypes display a significant amount of overlap which is most apparent in the hippocampus and the olfactory bulb where all three subtypes are expressed in moderate to high levels. In most regions however, expression is dominated by one (and sometimes two) subtype(s). Cav3.1 is the most extensively expressed subtype with at least some degree of in situ mRNA staining observed in nearly every region of the CNS, whereas Cav3.2 and Cav3.3 expression within the CNS are relatively more isolated. Cav3.1 is the predominant subtype in many regions such as the bed nucleus of stria terminalis and claustrum of the basal forebrain, in most areas of the amygdala, the cerebral cortex, the thalamus (with the exception of the reticular nucleus) and hypothalamus, the medulla and spinal cord, the posterior granule cell and Purkinje cell layers of the cerebellum, and the inferior olive (Talley et al., 1999). The expression of Cav3.2 is highest in the olfactory bulb and the hippocampus and it is the predominant subtype in the olfactory tubercle, the indusium griseum of the basal 12  forebrain, the granule cell layer of the dentate gyrus. Of note, the expression Cav3.2 mRNA is limited in the neocortex to layer 5 and to the reticular thalamic nucleus (nRT) of the thalamus suggesting that specific features of the Cav3.2 channel are important in these neurons. In the PNS, Cav3.2 expression is particularly significant in the dorsal root ganglia (Talley et al., 1999). Similar to Cav3.2, the highest levels of Cav3.3 expression are found in the olfactory system and the hippocampus. The glomerular layer of the olfactory system is the only area identified in which Cav3.3 mRNA expression dominates over the other T-subtypes. In most cases Cav3.3 expression is similar to, or less than, the other Cav3 channels in other regions of the CNS and PNS (Talley et al., 1999). Only a few of regions analyzed lacked or had limited expression of any of the Cav3 subtypes. For example, the globus pallidus of the basal forebrain, the substantia nigra and the reticular fields of the spinal cord appeared devoid of Cav3 expression using in situ hybridization (Talley et al., 1999). It is important to note that while the T-type channel mRNA expression patterns overlapped in many areas, in situ hybridization does not provide information as to whether the T-type channels themselves are expressed in similar or distinct subcellular regions of neurons or if they are isolated to distinct populations of cells within each region. In this regard, efforts have also been made to determine the subcellular localization of the T-type channel proteins. Immunohistochemical staining has shown that Cav3 expression can vary from primarily somatic expression (dentate gyrus granule cells and neurons of the midline thalamic nuclei) to include expression in the proximal dendrites (Layer 1-4 of the cortex and thalamic neurons) and extend as far as the distal dendrites (Purkinje cells, deep cerebellar neurons and pyramidal cells of cortical Layer 5 and the hippocampal formation) (Craig et al., 1999; McKay, 2006). Expression of the Cav3.1 and 13  Cav3.2 isoforms was found to be limited to the soma and the proximal dendrites in most instances with Cav3.1 expression in the deep cerebellar neurons extending to the distal dendrites being the exception. Conversely, expression of the Cav3.3 isoform extended to the distal dendrites in several of the neuronal cell types analyzed (Craig et al., 1999; McKay, 2006). A detailed review of Cav3 expression in non-neuronal tissues such as the heart, kidney, smooth muscle tissue, skeletal muscle, germ cells, and endocrine tissues was written by Perez-Reyes in 2003 (Perez-Reyes, 2003). Examples of non-neuronal Cav3 expression include Cav3.1 and Cav3.2 in germ cells (Jagannathan et al., 2002; Son et al., 2002; Darszon et al., 2006), Cav3.1 in the renal tubules of the kidney (Andreasen et al., 2000), and in healthy mature cardiac tissue (Cribbs et al., 2001), Cav3.2 in the smooth muscle tissue in the kidney (Williams et al., 1999; Hansen et al., 2001), in embryonic and newborn skeletal muscle (Berthier et al., 2002), in the adrenal glands (Mlinar et al., 1993; Schrier et al., 2001; Levitsky and López-Barneo, 2009), and in the pituitary and pineal gland (Talley et al., 1999). It is interesting to note that Cav3.3 expression outside of the CNS is notably lacking (PerezReyes, 2003). Developmental regulation of Cav3 expression has been demonstrated in several tissues. Overall expression of Cav3.1 in the rat brain is up-regulated during the early postnatal period from low levels at birth to higher levels comparable to that of adult mice by post natal day 11 whereas Cav3.3 expression is initially high and decreased substantially by post natal day 15 (Yunker, 2003). The splice variants of the Cav3 isoforms have also been shown to be developmentally regulated in rat brain (Emerick et al., 2006) and heart tissue (David et al., 2010). During embryogenesis there is a spatio-temporal regulation of Cav3.2 throughout many tissues, for example postnatal up-regulation of Cav3.2 in the testis (Son et al., 2002). 14  The developmental regulation is not limited to up regulation. Chromaffin cells display a developmental loss of Cav3.2 expression during maturity (Levitsky and López-Barneo, 2009), and the expression of both Cav3.1 and Cav3.2 in embryonic and newborn skeletal muscle decreases to undetectable by postnatal day 22 (Berthier et al., 2002). As a final example of the dynamic regulation of Cav3 channels during development, a switch in Cav3 expression occurs in cardiac tissue wherein Cav3.2 expression is down-regulated and Cav3.1 is up-regulated so that in the adult heart the predominant T-type channel isoform is Cav3.1 (Niwa et al., 2004; Cribbs, 2010). In addition to developmental regulation, Cav3 expression is significantly altered in several pathological conditions/models. In rodents, an increase in Cav3.2 current density has been linked to diabetic neuropathic pain (Jagodic et al., 2007) and following status epilepticus (seizure activity lasting greater than thirty minutes or more than two seizures between which the subject does not regain consciousness (Heafield, 2000)), a transcriptional up-regulation of Cav3.2 has been suggested to contribute to the chronic epileptic condition (Su, 2002; Vitko et al., 2007; Becker et al., 2008). In cardiac tissue re-expression of the Cav3.2 channel has been shown to accompany the induction of hypertrophy (Nuss and Houser, 1993; Martínez et al., 1999; Huang et al., 2000; Chiang et al., 2009; Cribbs, 2010; David et al., 2010). Further, Cav3.1 expression is down-regulated and Cav3.2 up- regulated following hypoxia in acutely dissociated ventricular myocytes (Pluteanu and Cribbs, 2009) while in chronic hypoxia both Cav3.1 and Cav3.2 expression are up-regulated in adrenal chromaffin cells (Carabelli et al., 2007b; Levitsky and López-Barneo, 2009; Souvannakitti et al., 2010). Also in rat chromaffin cells, exposure to cAMP or -adrenergic stimulation results in an up-regulation of Cav3.2 T-type channels which has been shown to be mediated by a PKA independent pathway involving the cAMP receptor Epac, a cAMP-guanine nucleotide 15  exchange factor (Novara et al., 2004). This is not an exhaustive list of examples of Cav3 dynamic regulation; although it does illustrate that each of the Cav3 isoforms likely plays key roles in many tissues during development and in various pathological conditions.  1.2.2. Biophysical properties of Cav3 channels Initial attempts to characterize T-type channels were complicated by seemingly conflicting results and at first it was proposed that the discrepancies observed were due to differences in the recording solutions (Huguenard, 1996). However, with the identification of the three distinct Cav3 molecular subtypes (Cav3.1, Cav3.2 and Cav3.3) and subsequent determination of their expression profiles, it became apparent that many of the apparent discrepancies could be attributed to the differential expression of the Cav3 channels across cell types and tissues. With the availability of Cav3 cDNA clones the T-type channels were studied in isolation in heterologous systems enabling investigators to determine their biophysical, pharmacological and modulatory properties. The LVA (T-type) class of VGCCs originally derived its name from three key features: a relatively negative threshold of activation, a small single channel conductance, and a rapidly inactivating waveform compared to HVA VGCCs. The activation threshold of Cav3 channels is generally between -75 and -60 mV with half maximal activation (V50 act) approximately -45 and -40 mV (McDonald et al., 1994; Huguenard, 1996; Perez-Reyes et al., 1998; Klöckner et al., 1999; Perez-Reyes, 2003; Talavera and Nilius, 2006). Half maximal inactivation (V50 inact) of the Cav3 channels is also quite negative, occurring between -80 and -60 mV (Huguenard, 1996; Klöckner et al., 1999; Talavera and Nilius, 2006). The overlap in activation and inactivation profiles results in a narrow range of potentials in which a small percentage of Cav3 channels can be open at the resting potential of many cell types. Known 16  as the “window current” (Coulter et al., 1989; Perez-Reyes, 2003; Cain and Snutch, 2010), it has been proposed to contribute to both intracellular Ca2+ homeostasis and to the shaping of neuronal firing patterns (Carbone and Lux, 1984a; Coulter et al., 1989; Williams et al., 1997; Hughes et al., 1999; Crunelli et al., 2005; Contreras, 2006; Dreyfus et al., 2010). Cav3 channels are permeable to multiple divalent ions including Ca2+, Ba2+ and Sr2+. Utilizing heterologous expression systems is has been determined that Cav3.1 channels are slightly more permeable to Ca2+ over Ba2+ while Cav3.2 channels are slightly more permeable to Ba2+ over Ca2+ and Cav3.3 channels being equally permeable to Ca2+ and Ba2+ (McRory et al., 2001; Kaku, 2003). The single channel conductance of Cav3 channels is dependent on the external concentration of divalent cation with reports of 5-9 pS using extracellular recording solutions containing 10 to 110 mM Ca2+ or Ba2+ and a projected conductance of 1 pS in 2 mM Ca2+ (reviewed in (Huguenard, 1996)). The “transient” characteristic of native Cav3 currents is attributable to their relatively fast inactivation kinetics. Comparison of the cloned Cav3 isoforms has revealed that Cav3.1 and Cav3.2 channels exhibit comparable inactivation kinetics to those generally observed for native T-type; a relatively low tau of inactivation and that is strongly voltage-dependent. Cav3.3 channels however, inactivate at a significantly lower rate resulting in more prolonged currents during depolarization. A similar distinction is observed concerning activation kinetics with Cav3.1 and Cav3.2 activation being faster than Cav3.3 channels. (Huguenard, 1996; Perez-Reyes et al., 1998; Klöckner et al., 1999; Kozlov et al., 1999; Lee et al., 1999a; Williams et al., 1999; McRory et al., 2001; Perez-Reyes, 2003). Consistent with native Ttype currents, the cloned Cav3 channels exhibit a voltage-dependence of activation with activation kinetics increasing with further depolarization which generates a characteristic  17  crisscrossing pattern of the whole cell current traces (Huguenard, 1996; Randall and Tsien, 1997; Perez-Reyes, 2003) . Following a depolarizing stimulus, Cav3 channels deactivate (close) comparatively slower than HVA channels resulting in a significant influx of Ca2+ as the driving force rapidly increases upon membrane potential repolarization. Cav3.3 channels deactivate the quickest followed by Cav3.2 and finally with Cav3.1 exhibiting the slowest deactivation kinetics (McCobb and Beam, 1991; Klöckner et al., 1999; Kozlov et al., 1999; Serrano et al., 1999; Monteil et al., 2000b). Additionally, there are differences in the rate of recovery from inactivation between the Cav3 channel isoforms with Cav3.1 recovering the fastest and Cav3.2 the slowest (Klöckner et al., 1999). A summary of the biophysical properties of the cloned Cav3 channels is presented in Table 1.2.  18  Table 1.2: Summary of the biophysical characteristics of the low threshold Cav3 channels Cav3.1  Cav3.2  Cav3.3  Threshold of activation  ~ -60mV  ~ -60mV  ~ -60mV  Half peak activation (V50act)  -45.5 mV  -45.8 mV  -43.8 mV  Peak activation  -29.2 mV  -29.4 mV  -27.6 mV  Tau of activation (depolarization potential = -45mV)  6 ms  8 ms  30 ms  Voltage dependent activation  Yes  Yes  Yes  Tau of deactivation (repolarization potential = -120 mV)  1.7 ms  2.7 ms  1.0 ms  Voltage-dependent deactivation  Yes  Yes  Yes  Half Inactivation (V50inact)  -72.8 mV  -72.0 mV  -72.5 mV  30 ms  47 ms  137 ms  18.7 mV  13.5 mV  13.9 mV  11 ms  16 ms  69 ms  at negative potentials  at negative potentials  at negative potentials  117 ms  395 ms  359 ms  1806 ms  2587 ms  611ms  7.5 pS  9 pS  11 pS  Tau of inactivation (depolarization potential = -45 mV) Potential difference for e-fold change in Tau of inactivation Tau of inactivation (positive depolarization potentials) Voltage-dependent inactivation Recovery from short term inactivation Recovery from long term inactivation Conductance (barium)  Reference (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) (Klöckner et al., 1999) Reviewed in (Perez-Reyes, 2003)  Table 1.2: Summary of the biophysical characteristics of the low threshold Cav3 channels Biophysical characteristics of the Cav3 channels cloned from rat (Cav3.1 and Cav3.3) and human (Cav3.2) and stably expressed in human embryonic kidney cells. The recordings were made using 1.25 mM Ca2+ as the charge carrier (Klöckner et al., 1999).  19  1.2.3.  Physiological roles of T-type Ca2+ channels  T-type Ca2+ channels make important contributions to many neuronal and nonneuronal cell types. The low thresholds of activation and inactivation, window current, and the prolonged deactivation permit T-type channels to participate in burst firing, rhythmic oscillatory activity, and regulate intracellular Ca2+ levels and second-messenger-dependent pathways. Together, these unique properties have been shown to play significant roles in signal amplification (Williams et al., 1997; Hughes et al., 1999), sensory coding (Krahe and Gabbiani, 2004), rhythmic pace making (Park et al., 2010), hormone secretion (reviewed in (Perez-Reyes, 2003)), neurotransmitter release (Pan et al., 2001; Carbone et al., 2006a; Jacus et al., 2012), and the generation and maintenance of neuronal firing patterns which underlie physiologically important states such as sleep (review in (Steriade, 2005)). The low threshold of activation of T-type channels permits depolarizing potentials that are insufficient to elicit an action potential to produce low threshold Ca2+ spikes (LTSs) which are critical for the low threshold burst-firing observed in many neuronal cell types. If the density of Cav3 channels is high enough, the LTS can depolarize the membrane sufficiently to surpass the action potential threshold required to trigger an action potential. LTSs may also be generated following hyperpolarization (e.g. following an action potential) as the Cav3 channels are reactivated during the restoration of the resting membrane potential. Depending upon the intensity and duration of the LTS, multiple action potentials can crest the LTS in a “burst” of activity. A LTS following an action potential burst is known as an after depolarization potential (ADP) which is capable of stimulating additional action potentials if the intensity and duration of the ADP is sufficient (Deschenes et al., 1982; Llinás and Jahnsen, 1982; Jahnsen and Llinás, 1984; Crunelli et al., 1989; White et al., 1989; Huguenard and Prince, 1992; Aptel et al., 2007; Cain and Snutch, 2010; Dreyfus et al., 2010). 20  T-type channels can participate in several distinct neuronal firing patterns depending on the resting membrane potential. For example, overlap of the steady-state activation and inactivation properties of the Cav3 channels (window current) contributes to a bi-stability in neuronal firing patterns (Williams et al., 1997; Hughes et al., 1999). If membrane potentials are sufficiently hyperpolarized to remove inactivation the T-type channels can drive a repetitive low threshold burst-firing pattern. As membrane potentials approach the window current a bi-phasic firing pattern can become apparent resulting in a slow (<1 Hz) oscillatory firing pattern (Coulter et al., 1989; Williams et al., 1997; Hughes et al., 1999; Crunelli et al., 2005; Cain and Snutch, 2010). At more depolarized resting potentials T-type channels are almost completely inactivated and appear to contribute little to neuronal activity in tonic firing states. Overall, amongst other contributions, this bi-stability plays an important role in establishing and maintaining the distinct neuronal firing patterns observed during sleep and wakefulness (reviewed in (Steriade, 2005)). The low activation threshold and prolonged deactivation time course of T-type channels contributes to the intracellular Ca2+ increase that occurs in response to sub-threshold stimuli. In this regard, a T-type channel-mediated intracellular Ca2+ increase has been shown to be critically involved in hormonal secretion and more recently neurotransmitter release. Ttype channels have been linked with stimulus-secretion coupling in the release of aldosterone and cortisol from cells in the adrenal glomerulosa (Cohen et al., 1988), in insulin secretion from -cells of the pancreas (reviewed in (Perez-Reyes, 2003)), and in the cyclic release of gonadotropin releasing hormone (Zhang et al., 2009). Additionally, T-type currents have been identified and are believed to be involved in the hormone release in lactotropes, corticotropes, gonadotropes, and thyrotropes of the pituitary gland (reviewed in (PerezReyes, 2003)). 21  Recently, Cav3.2 channels have been found to be necessary for the release of catecholamines from chromaffin cells of the adrenal medulla in response to acute hypoxia. In the early post-natal period chromaffin cells possess oxygen sensing capabilities, which play a vital role in the transition to extrauterine life. This oxygen sensitivity is lost in early development along with an accompanying decrease in Cav3.2 expression in chromaffin cells. Under chronic hypoxia however, Cav3.2 channels are re-expressed and chromaffin cells regain oxygen sensitivity (Carbone et al., 2006b; Carabelli et al., 2007a, 2007b; Levitsky and López-Barneo, 2009). Acute sympathetic stress has also been found to recruit Cav3.2 channels resulting in a fast, low-threshold-dependent release of catecholamines as part of the fight or flight response (Novara et al., 2004; Giancippoli et al., 2006; Currie, 2010; Hill et al., 2011). T-type channels have also been found to play roles in neurotransmitter release in several neuronal cell types such as retinal bi-polar cells (Pan et al., 2001), dendro-dendritic reciprocal synapses between granule cells and mitral/tufted cells of the olfactory bulb (reviewed in (Carbone et al., 2006a)), and in populations of nociceptive neurons in the superficial laminae of the dorsal horn (Bao et al., 1998; Jacus et al., 2012). The low threshold of activation of T-type channels allows for fast, low threshold exocytosis permitting retinal bipolar cells to respond to graded depolarizations (Pan et al., 2001). In the inhibitory synapses found between the granule and mitral cells of the olfactory bulb, T-type channelmediated neurotransmitter release provides a means for low threshold inhibition which has been proposed to play a role in the gating of olfactory information to cortex (reviewed in (Carbone et al., 2006a)). Finally, more recently Cav3.2 channels have been identified as regulating the frequency of spontaneous micro excitatory postsynaptic currents in nociceptive  22  neuron within the dorsal horn and demonstrating a role for Cav3.2 in the regulation of the excitability associated with peripheral pain sensitization (Jacus et al., 2012). T-type channels also play roles in pathological conditions such as cancer, epilepsy and cardiac hypertrophy. Recent studies suggest that T-type channels are important in the cell proliferation associated with some cancer cell lines (Li et al., 2009; Santoni et al., 2012; Zhang et al., 2012) . While the precise pathway(s) remain unclear, it has been suggested that T-type channels contribute to proliferation through the regulation of intracellular Ca2+ levels. In these studies the inhibition of T-type channel activity significantly reduces cell proliferation and migration in select cancer cell lines presenting the possibility that specific T-type channel isoforms may be potential therapeutic targets in for certain cancers (reviewed in (Santoni et al., 2012)). T-type channels are abundantly expressed in the thalamus and participate in oscillatory neuronal activity within a reciprocally connected circuit comprised of thalamocortical, thalamic reticular and cerebral cortical neurons. Under normal physiological conditions this oscillatory activity contributes to naturally occurring processes such as sleep, while in the pathological conditions associated with certain types of epilepsy abnormal oscillatory activity within the thalamocortical circuit contributes to seizure activity (reviewed in (Zamponi et al., 2009; Santoni et al., 2012)). Several lines of evidence demonstrate that specific Cav3 channel subtypes are involved in various forms of epilepsy. In temporal lobe epilepsy an increase in Cav3.1 expression has been observed in thalamic cells and an accompanying increased low threshold activity is believed to contribute to the abnormal thalamocortical activity. Support for this can be found in Cav3.1 gene knock-out mice which are resistant to chemically induced spike and wave discharges which underlie seizure activity (Kim et al., 2001). Furthermore, naturally occurring mutations which alter the physiological 23  properties or expression of Cav3.2 channels have been associated with childhood absence epilepsy (Crunelli and Leresche, 2002; Chen et al., 2003b; Peloquin et al., 2006; Vitko et al., 2007). Additional evidence for a role of Cav3.2 channels in absence epilepsy has come from the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) and the WAG/Rijj models of absence epilepsy wherein increased expression of Cav3.2 has been identified in the thalamic reticular nucleus. In the GAERS model, a mutation in the Cav3.2 gene has been identified that associates with the epileptic phenotype and that contributes to a gain-of-function in Ttype channel activity in GAERS compared to control animals (Powell et al., 2009). Further convincing evidence for the involvement of T-type channels to epileptic phenotypes comes from a recent study demonstrating that selective Cav3 blockers significantly reduce the number and duration of seizures in the GAERS model (Tringham et al., 2012). In cardiac tissue there is a developmental switch in Cav3 expression from predominantly Cav3.2 in neonate and early post-natal rats to almost exclusive Cav3.1 expression in the adult rat. Under normal physiological conditions the Cav3.1 subtype appears to be involved in the regulation of the pace making current in the sinoatrial node and excitation-contraction coupling (Mangoni et al., 2006; Vassort et al., 2006) while Cav3.2 is involved in the NO mediated vasodilation of the circulatory system (Chen et al., 2003a). In the pathological condition following hypertrophy induction or following myocardial infarction a remodeling of Cav3 expression occurs with Cav3.2 being re-expressed in cardiac tissue (reviewed in (Vassort et al., 2006)). Although the exact consequences of this remodeling of Cav3 expression remains unclear, it is interesting to note that the ratio of the Cav3.2 exon 25(+) vs. Cav3.2 exon 25(-) splice variants expressed after remodeling is significantly different that found in the neonatal heart. As shown by David and coworkers, the 25(+) splice variant confers a distinct voltage-dependent facilitation characteristic to 24  Cav3.2 channels thus the change in relative expression between splice variants may affect excitability and/or intracellular Ca2+–mediated signaling (David et al., 2010). In discussion of the physiological roles of T-type channels it is important to mention the phenotypes of Cav3.1 (Kim et al., 2001) and Cav3.2 (Chen et al., 2003a) knock-out mice strains. In the absence of subtype-specific antagonists the knock-out mice have been an invaluable tool in assessing the physiological consequences of complete T-type channel lossof-function. Outwardly, the global loss of either Cav3.1 or Cav3.2 expression is not lethal and the knock-out mice generally appear phenotypically normal with the only reported noticeable difference being a decreased body size for the Cav3.2 knock-out mice. Further investigation however, has revealed differences in the cardiac and circulatory systems, nociceptive responses to peripheral and visceral pain, learning and memory, and in the threshold of seizure induction. Table 1.3 provides a summary of the phenotypes observed in the Cav3.1 and Cav3.2 knock-out mice. As of this writing there has been no report of the phenotypes associated with Cav3.3 gene knock-out.  25  Table 1.3: Summary of Cav3.1 and Cav3.2 knockout mice phenotypes Cav3.2 Knock-out 1 General phenotype - viable and fertile 1 - small body size 1 - General behaviour (motor associated and emotion-related functions) normal as determined by: 3 - open box test - horizontal and vertical activities - rotarod - light/dark transition Cardiac/Circulatory system - heart rate normal 1 - ECG waveforms normal 1 - no cardiac arrhythmias 1 Ventricles - 10 week - cardiac fibrosis 1 - 1 year - increases cardiac fibrosis, necrosis, lymphocyte infiltration 1 Vessels - constricted and irregular 1 Coronary arteries - NO mediated dilation is defective 1 Nociception - decreased LVA current in DRGs 1 - HVA currents unaffected 1 - decreased nociceptive response to: 3 - mechanical stimuli (tail clip) - thermal stimuli (hot plate) - chemical stimuli (capsaicin) - noxious chemical stimuli (both phases)  Cav3.1 Knock-out 2 - viable and fertile 2 -normal growth 2 - Not reported  -decreased heart rate 5 - bradycardia 5 - atrioventricular dysfunction 5 - no histological defects in organs such as heart, intestine, kidney, pancreas, liver 2  - normal nociceptive response to 6 - mechanical stimuli (tail clip) - thermal stimuli (hot plate)  26  Table 1.3: Summary of Cav3.1 and Cav3.2 knockout mice phenotypes - continued  Cav3.2 Knock-out 1 Neuropathic pain - normal response 3  Learning and Memory - late phase LTP lost in hippocampal slices 4 - normal memory formation 4 - decreased retrieval in context cued trace fear learning 4 - decreased retrieval in context cued passive avoidance task 4 Epilepsy - not reported - not reported - not reported  - not reported  Cav3.1 Knock-out 2 - decreased spontaneous pain 7 - decreased mechanical hyperalgesia 7 - decreased thermal hyperalgesia 7 - not reported - not reported - not reported - not reported - brain morphology normal 2 - burst rebound action potentials lacking in thalamocortical neurons 2 - resistance to pharmacological initiation of spike and wave discharges (absence epilepsy) 2 ~ -Baclofen ~  -Butyrolactone ~normal response to Bicuculline -normal susceptibility to tonic-clonic seizures (determined pharmacologically) 2  Table 1.3: Summary of Cav3.1 and Cav3.2 knockout mice phenotypes Summary of the phenotypes observed for Cav3.1 and Cav3.2 knock-out mice strains compared to wild type littermate controls. References: 1. (Chen et al., 2003a), 2. (Kim et al., 2001), 3. (Choi et al., 2007), 4. (Chen et al., 2012), 5. (Mangoni et al., 2006), 6. (Kim et al., 2003), 7. (Na et al., 2008).  1.2.4. Pharmacological properties of T-type Ca2+ channels One of the most significant hurdles in studying the LVA VGGCs in vivo has been the lack of T-type channel-specific antagonists to differentiate LVA currents from HVA currents and also Cav3 isoforms from each other. While the availability of Cav3 cDNAs has enabled 27  the characterization of their biophysical properties, there are limitations as to how far this information can translate to in vivo preparations. Specific antagonists would be particularly useful in determining the contribution of T-type channel isoforms to neuronal firing patterns which are comprised of multiple ionic conductances. Additionally, the involvement of specific Cav3 channel subtypes in pathological conditions such as pain (Bourinet et al., 2005), cardiac hypertrophy (Chiang et al., 2009) and epilepsy (Khosravani and Zamponi, 2006) makes them potential therapeutic targets. Towards these objectives efforts are ongoing to develop high affinity Cav3 specific antagonists (Shipe et al., 2008; Yang et al., 2008; Smith et al., 2010; Woo et al., 2011). Nickel (Ni2+) was one of the first T-type channel antagonists to be identified (reviewed in (Yunker, 2003)). While Ni2+ generally inhibits T-type currents, the affinity of this divalent cation was found to be highly variable across preparations (Kaneda et al., 1990; Satoh et al., 1991). Analysis of the Ni2+ sensitivity of cloned Cav3 channels subsequently revealed the differential inhibition of the three subtypes with Cav3.2 being the most sensitive (~ IC50 =12M) and with Cav3.1 and Cav3.3 being less sensitive to Ni2+ blockade (~ IC50s 250 M and 216 M, respectively; (Lee et al., 1999b). The differential sensitivity to Ni2+ has allowed for the selective block of Cav3.2 channels and provided a useful pharmacological tool although other considerations remain such as the partial blockade of the other T-type isoforms (Zamponi et al., 1996; Lee et al., 1999b). The structural determinant for the high Ni2+ sensitivity of Cav3.2 has been identified as a histidine residue at position 191 (H191) located on the putative extracellular loop linking segments S3-S4 linker of domain I (Kang et al., 2006). Interestingly, this same residue also confers Cav3.2 sensitive to zinc and copper (Kang et al., 2006; Traboulsie et al., 2006; Sun et al., 2007) which poses a problem if recording solutions or the tissue specimen are not free of these trace elements. A potential 28  solution to this issue is the use of divalent cation chelators (Nelson et al., 2007b). . A final note regarding divalent cation blockade is that while T-type channels are sensitive to Ni2+, Zn2+ and Cu2+ they are resistant to cadmium (Cd2+) at certain concentrations. Taking advantage of differences in the ion selectivity of the pore forming loop of HVA and LVA channels, Cd2+ may be used at low concentrations (20-100 M) to block HVA currents while preserving Cav3 currents. Another well-known T-type channel antagonist is mibefradil, an effective therapeutic antihypertensive that was subsequently withdrawn from the market due to potentially fatal side reactions (reviewed in (Welker et al., 1998)). Although mibefradil has proven useful as a relatively selective T-type channel antagonist at low concentrations (<1 M (Martin et al., 2000), at higher greater concentrations it also inhibits HVA channels which has resulted in this agent generally being classified as a non-specific antagonist (Randall and Tsien, 1997; Viana et al., 1997). The non-specific inhibition of T-type channels by various pharmacological agents has been reviewed extensively (Perez-Reyes, 2003; Yunker, 2003) and continues to be addressed as additional compounds are identified (Joksovic et al., 2005; Freeze et al., 2006; Kraus et al., 2007). The general non-specific inhibition of the T-type channels is a common theme across an impressive list of pharmacological agents and highlights the real need for the development of Cav3-specific agents in order to better dissect their relative contributions to various pathological conditions. While peptide toxins have proven invaluable in the study of HVA channels there has not been a similar description of these agents towards T-type channels. For example, kurtoxin isolated from scorpions (Parabuthus transvaalicus) was originally found to block Cav3.1 and Cav3.2 channels although unfortunately later shown to also block sodium channels as well as L-type, N-type and R-type Ca2+ channels (Chuang et al., 1998; Sidach and Mintz, 2002). More recently, two 29  peptide toxins, ProTx-I and ProTx-II have been isolated from tarantula venom (Thrixopelma pruriens). ProTX-I differentially blocks Cav3.1 over Cav3.2 with IC50s of 0.2 M and 32 M respectively and ProTX-II blocks Cav3.3 channels with an IC50 of 0.8 M (Edgerton et al., 2010; Ohkubo et al., 2010). As such, these peptide toxins hold some promise as they can either be used in conjunction to completely block Cav3 currents or be used separately to isolate Cav3.2 or Cav3.1 currents in experimental preparations. TTA-P2, a small synthetic piperidine-based molecule (Shipe et al., 2008; Yang et al., 2008) has been reported to selectively block T-type currents of the thalamocortical and reticular thalamic neurons with a high degree of potency (IC50 = 22 nM) while leaving HVA Ca2+ and sodium currents intact (Dreyfus et al., 2010). Development of this series of molecules is ongoing (Smith et al., 2010; Woo et al., 2011) although as TTA-P2 induces sleep upon administration to animals it remains to be seen if this agent/class of molecule will prove generally useful. Recently two new compounds, Z941 and Z944, have been demonstrated to block T-type channels with IC50s in the nanomolar range. These novel small molecule blockers are highly selective for the T-type channels while leaving sodium and potassium currents relatively unscathed. Furthermore administration of the Z941 and Z944 has been found to reduce both the duration and frequency of seizures in the GAERS absence epilepsy model (Tringham et al., 2012).  1.2.5. Modulation of T-type Ca2+ channels There is an accumulating amount of evidence regarding the physiological modulation of Cav3 channel isoforms. Some mechanisms appear conserved across all three Cav3 isoforms, such as current enhancement by protein kinase C (PKC) and protein kinase A (PKA), while other mechanisms such as the potentiation of Cav3.2 by Ca2+/calmodulin30  dependent kinase II (CaMKII) appear selective for particular Cav3 subtypes (reviewed in (Iftinca, 2011)). As this thesis focuses on Cav3.2 channels the emphasis of this section will be on the Cav3.2 subtype. The cytoplasmic loop linking domains II and III is an important regulatory domain for all Cav3 channels (reviewed in (Iftinca and Zamponi, 2009; Iftinca, 2011)). In Cav3.2 the modulatory sites for CaMKII, G protein 2 and  subunits (G2), PKA and ROCK have all been localized to the domain II-III linker (Welsby et al., 2003; Kim et al., 2006; Iftinca et al., 2007). Lu and coworkers demonstrated that T-type currents in adrenal glomerulosa cells could be enhanced by CaMKII activity (Lu et al., 1994) and subsequently this enhancement was demonstrated to be specific to Cav3.2 channels (Wolfe et al., 2002). Of note, the underlying mechanism occurs through direct phosphorylation of a serine residue at position 1198 in the domain II-III linker (Yao et al., 2006) which results in a shift in activation to more hyperpolarized potentials (Lu et al., 1994; Wolfe et al., 2002). In adrenal glomerulosa cells, stimulation of the angiotensin-II receptor II (AngII) has been shown to activate CaMKII to phosphorylate serine 1198 of Cav3.2 suggesting a possible role for this regulation in the progression of cardiac disease (Fern et al., 1995; Yao et al., 2006). PKC was shown to enhance Cav3.2 (Park et al., 2003) and later Cav3.1 and Cav3.3 (Park et al., 2006) currents without affecting the surface expression of the channels in Xenopus oocytes. A crucial site for PKC modulation of Cav3.1 channels was localized to the domain II-III linker suggesting that possibility the interaction site may be similarly localized for Cav3.2 channels (Park et al., 2006; Rangel et al., 2010). Recently, Rangel and coworkers have described a signaling pathway involving neurokinin 1 receptors (NKR1) whereby stimulation of NKR1 activates Gq/11 which in turn activates phospholipase C  (PLC)  31  converting phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) to activate PKC and ultimately enhance Cav3.2 currents (Rangel et al., 2010). The domain II-III linker was also found to be involved with the augmentation of Cav3.2 currents by PKA in Xenopus oocytes (Kim et al., 2006). Interestingly, the modulation of Cav3.2 currents by PKA and PKC was only reproducible in mammalian cells at physiological temperatures (Chemin et al., 2007) which emphasizes an important point to consider when investigating modulatory interactions outside of normal physiological parameters. In mouse neonatal ventricular myocytes PKA augmentation of Cav3.2 currents was found to be regulated by caveolin-3 with siRNA silencing of caveolin-3 occluding the effect of PKA activation (Markandeya et al., 2011). On the other hand, PKA has also been found to be essential in mediating an inhibitory effect of G2(Hu et al., 2009). It is possible that PKA plays a dual role it modulation of Cav3.2 channels and that its effect depends upon the specific interactions available within the cell with some interactions resulting in augmentation and others in inhibition. In 1997 it was discovered that T-type currents in the adrenal glomerulosa could be inhibited by activation of the dopamine D1 G-protein-coupled receptor and to occur specifically via G (Drolet et al., 1997). Later, Wolfe and coworkers used heterologous expression and chimeric Cav3 constructs to determine that G-protein G2 subunits selectively inhibit Cav3.2 currents through a direct interaction with the domain II-III linker (Welsby et al., 2003). It was further determined that the inhibition was due to a decrease in the open probability of Cav3.2 channels and not to changes in channel expression or waveform kinetics (DePuy et al., 2006). Utilizing human adrenocortical carcinoma cell line Hu and coworkers found that the inhibition of Cav3.2 currents via the activation of dopamine  32  receptors was more complicated than originally envisioned; requiring the activation of D1 and D2 dopamine receptors and the presence of activated PKA (Hu et al., 2009). In HEK293T cells, stimulation of an exogenously expressed corticotropin-releasing factor receptor-1 (CRF-1) inhibited Cav3.2 currents through a cholera-toxin sensitive, G pathway which was dependent on G (Tao et al., 2008). In the MN9D cell line CRF-1 inhibition of Cav3 currents was shown to occur through a PKC-dependent signaling pathway (Kim et al., 2007). The difference in findings between groups may be attributable to the different cell types utilized in each study. Considering that in other studies, PKC was found to generally enhance Cav3 currents, the results from Kim et al. suggest that PKC modulation of Cav3.2 may involve additional cellular mechanisms that are as yet unidentified (Iftinca, 2011). Lysophosphatidic acid (LPA) was found to differentially modulate the three Cav3 channels with inhibition of Cav3.1 and Cav3.3 and enhancement of Cav3.2 currents occurring through the phosphorylation of conserved ROCK phosphorylation sites in the domain II-III linker. Enhancement of Cav3.2 currents by LPA/ROCK was shown to be the result of a hyperpolarizing shift in voltage-dependent activation and inactivation whereas these parameters were unaffected in the Cav3.1 and Cav3.3 channels (Iftinca et al., 2007). Recently, it has been shown that the presence of Kelch-Like 1 (KLHL1), a neuronal actin binding protein, both increases the deactivation time of Cav3.2 and causes in increase in the number of Cav3.2 channels in the membrane (Aromolaran et al., 2009, 2010). It would be interesting to know whether the effect is constitutive or further regulated through KLHL1 expression levels or through unidentified pathways yet to be described. Among the Cav3 channels, Cav3.2 is unique in that it is exquisitely sensitive to redox modulation. Redox sensitivity of Cav3.2 has been demonstrated in both acutely dissociated 33  dorsal root ganglia (DRG) neurons and in nRT neurons in thalamic slices. Cav3.2 currents are attenuated in the presence of oxidizing agents such as 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and enhanced by reducing compounds such as dithiothretiol (DTT) and the endogenous amino acid L-cysteine (Todorovic et al., 2001b; Joksovic et al., 2006). Nitric oxide (NO), an important chemical messenger present throughout the body, also inhibits Cav3.2 currents (Joksovic et al., 2007). The same residue which bequeaths Ni2+ sensitivity to Cav3.2, histidine at position 191 (H191), has also been identified as essential for the redox sensitivity of Cav3.2 currents. Nelson and coworkers have provided evidence that reducing compounds enhance Cav3.2 currents through the chelation of metal ions (Zn2+) from the histidine residue thereby relieving a tonic inhibition of the Cav3.2 channel (Nelson et al., 2007b). In the presence of certain metals (Zn2+ for example) ascorbate can oxidize histidine residues through metal catalyzed oxidization (Samuni et al., 1983; Stadtman, 1991; Schöneich, 2000). After observing that ascorbate inhibited Cav3.2 it was concluded that inhibition was attributable to the oxidation of H191 (Nelson et al., 2007a). Selective inhibition of Cav3.2 currents by nitrous oxide was also found to be the result of metal catalyzed oxidization of H191 (Todorovic et al., 2001a; Joksovic et al., 2007; Bartels et al., 2009; Orestes et al., 2011) . A summary of the known modulators of Cav3 activity is presented in Figure 1.3.  34  Figure 1.3: Summary of the known modulators of Cav3 channel activity  35  Figure 1.3: Summary of the known modulators of Cav3 channel activity The square boxes represent various receptors known to initiate modulation of Cav3 channels. Ovals represent the reagents or effector molecules that modulate the channels. A question mark indicates the mode of action and/or site of action is undetermined. Blue indicates Cav3.2 specific modulation, green is Cav3.1 specific, orange is Cav3.2 specific and white is non-specific. Abbreviations: LPA1-5 = Lysophosphatidic acid receptors 1 to 5, MaPKII = mitogen associated protein kinase II, CaMKII = calmodulin-dependent protein kinase II, NKR1 = neurokinin 1 receptor, CRF-1 = corticotropin releasing hormone receptor 1and KLHL-1 = kelch-like 1.  1.3. PDZ ligands and domains: partnering regulatory enzymes to their targets 1.3.1. Structure and function The carboxyl terminus of Cav3.2 possesses a motif that corresponds with the consensus sequence of class 3 PDZ binding ligands which could potentially facilitate interactions with proteins containing PDZ-3 binding domains. PDZ domains were first identified as modular repeats of ~90 amino acids in several proteins including post synaptic density protein (PSD-95), Drosophila disc large tumor suppressor (Dlg1) and the zonula occludens-1 protein (Zo-1); hence the PDZ acronym. As one of the most common protein interaction domains identified in sequenced genomes, PDZ domains are known to play significant roles in facilitating interactions between both membrane bound and cytosolic proteins to form macro and micro domains of signaling complexes (reviewed in (Sheng and Sala, 2001; Hung and Sheng, 2002)). Numerous proteins have been identified that possess one or more PDZ domains, of particular note are scaffolding proteins such as PSD-95 and glutamate receptor interacting proteins (GRIPs) which allows them to multimerize and form protein scaffolds (Srivastava et al., 1998; Dong et al., 1999), cluster membrane bound channels (Hsueh et al., 1997; Hsueh and Sheng, 1999), participate in the trafficking of 36  receptors to the membrane (Lu and Ziff, 2005) and bring together signaling complexes (Christopherson et al., 1999). More detail on the functional diversity of PDZ domains can be found in excellent reviews on this topic (Sheng and Sala, 2001; Hung and Sheng, 2002). Structurally, PDZ domains are comprised of five to six  strands and two  helices. This structure forms a binding groove with a terminal hydrophobic pocket which contains a carboxylate-binding loop and signature Glycine-Leucine-Glycine-Phenylalanine (GLGF) motif. The first reports of PDZ specific interactions were between the carboxyl terminus of Shaker-type potassium channels, the NR2 subunit of N-methyl-D-aspartic acid receptors (NMDAR) and the PDZ domains of PDS-95 ((Kim et al., 1995; Kornau et al., 1995; Niethammer et al., 1996) and reviewed in (Sheng and Sala, 2001)). Carboxyl terminal binding has since been found to be typical of PDZ interactions although internal PDZ binding structures have also been reported; a specific example of which is the -hairpin structure found near the amino-terminus of neuronal nitric oxide synthase (nNOS) (Hillier et al., 1999; Tochio et al., 1999; Trejo, 2005). There is considerable variation in amino acids sequence amongst PDZ domains resulting in domains with different specificities and binding affinities (Gee et al., 2000). Three classes of PDZ domains have been identified based on specificity to the last four amino acids of the carboxyl tail of the binding peptide. The residues at position 0 (last carboxyl amino acid) and, proceeding towards the amino terminus, position -2 are the most crucial for PDZ binding (reviewed in (Sheng and Sala, 2001)). A hydrophobic residue is invariably found at position 0 which, when bound, is aligned to fit within the hydrophobic pocket of the PDZ domain (Cohen et al., 1996; Songyang et al., 1997; Niethammer et al., 1998). Specificity for the residue at position -2 defines the PDZ domain class. Class I PDZ domains are characterized by serine or threonine residues at position -2 and which may be 37  phosphorylated to regulate binding affinity as in the case of the interaction between the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 and the PKC interacting protein (PICK1) ((Lu and Ziff, 2005) and reviewed in (Sheng and Sala, 2001)). A hydrophobic residue at position -2 is characteristic of class II domains (Songyang et al., 1997) and negatively charged residue defines class III domains. The residues at position -1 and -3 generally do not define specificity, however they have been found to interact within the PDZ domain and certain PDZ interactions prefer some residues over others (Doyle et al., 1996; Songyang et al., 1997; Sheng and Sala, 2001).  1.3.2. PDZ domains of nNOS Residues 11 to 133 of nNOS contains both PDZ ligand and PDZ domains immediately adjacent each other (Tochio et al., 1999). An internal class II PDZ (PDZ-2) binding ligand with a -finger structure (residues 100 to 133) (Hillier et al., 1999; Tochio et al., 1999) mediates interactions with proteins such as PDS-95, PSD-93, and syntrophin (Brenman et al., 1996a, 1996b) whereas a class III PDZ (PDZ-3) binding domain (residues 11 to 100) (Stricker et al., 1997; Tochio et al., 1999)permits PDZ interactions with proteins such as CAPON (carboxyl-terminal PDZ ligand of nNOS) and carboxyl-terminal binding protein (CtBP) (Jaffrey et al., 1998; Riefler and Firestein, 2001). Key residues within the PDZ domain are required to stabilize the -finger binding ligand of nNOS which suggests that the formation internal -finger ligands depends on the proximity to PDZ domains for stabilization (Hillier et al., 1999; Tochio et al., 2000). Consequently, it has been proposed that the head-to-tail arrangement of PDZ domain/ligand organization may not be uncommon as alignment of several known PDZ sequences has revealed sequences similar to nNOS (Hillier et al., 1999). Contrastingly, an internal PDZ 38  ligand that is not adjacent to a PDZ domain was identified within the endothelin type A receptor (ETA) protein demonstrating that the head-to-tail arrangement of PDZ domain/ligand is not essential for the formation of an internal -finger PDZ binding ligand (reviewed in (Trejo, 2005)). The presence of both a PDZ ligand and a domain within the amino terminus of nNOS could potentially facilitate the formation of larger complexes utilizing both structures; however evidence indicates that binding of CAPON to the PDZ domain of nNOS disrupts the interaction with PSD-95 which is mediated by the -finger PDZ binding ligand of nNOS (Jaffrey et al., 1998). The PDZ-2 mediated interaction between nNOS and PSD-95 allows nNOS to be functionally coupled to NMDA receptors. This functional interaction has been shown to mediate glutamate neurotoxicity by linking nitric oxide (NO) production with NMDA receptor activity (Dawson et al., 1991; Sattler et al., 1999). Disruption of the nNOS/PSD-95 interaction also disturbs the functional interaction between nNOS and NMDA receptors which has been demonstrated to reduce NMDA receptor-dependent excitotoxicity (Aarts et al., 2002).  1.4. Functional relevance of nitric oxide and nitric oxide synthases 1.4.1. Molecular structure and composition A physiological role for NO was first identified in the early 1980s when an endogenous factor released from the endothelium was found to play an important role in blood vessel relaxation. Throughout the 1980s efforts to identify this endothelium-derived relaxation factor culminated in the identification of NO as the active substance (Ignarro, 1989). During this period additional roles for NO were unveiled, including a role in the 39  macrophage response to both infection and tumor cells (Nathan and Hibbs, 1991; Moilanen and Vapaatalo, 1995), in the stimulation of soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP) (Böhme et al., 1984), in the modulation of NMDA receptor activity in brain tissue (Manzoni et al., 1992).Purification of nNOS, one of the enzymes responsible for NO synthesis, in 1990 and subsequent cloning in 1991 allowed for further characterization and understanding of the physiological function of the highly reactive and unstable NO molecule (reviewed in (Bredt, 1995)). The eventual cloning of neuronal (nNOS) (Bredt et al., 1991; Nakane et al., 1993; Ogura et al., 1993), endothelial (eNOS) (Janssens et al., 1992; Lamas et al., 1992; Sessa et al., 1992) and inducible (iNOS) forms (Lyons et al., 1992; Lowenstein et al., 1993; Xie et al., 1993) revealed a structurally similar but physiologically diverse family of enzymes. Structurally, NOS can be divided into two functional regions, an amino terminal oxygenase domain and a carboxyl terminal reductase domain which bears significant similarity to cytochrome P450 reductase. The oxygenase domain forms binding sites for the substrate arginine as well as for cofactors haem and tetrahydrobiopterin (BH4). The cytochrome P450 reductase-like carboxyl terminus possesses binding sites for cofactors reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). Between the two functional regions lies a calmodulin (CaM) binding site. The molecular weight of NOS in monomeric form ranges from ~130 kDa for eNOS and iNOS to 160 kDa for nNOS. Functional NOS enzyme consists of a NOS protein dimer with attached cofactors: haem, BH4, NADPH, FAD, FMN, and CaM. nNOS is the only isoform which possesses PDZ ligand and binding domains (reviewed in (Bredt, 1995; Mayer, 1995; Andrew and Mayer, 1999; Alderton et al., 2001; Li and Poulos, 2005). Figure 1.4 illustrates the domain structure of the NOS isoforms. 40  Figure 1.4: Domain structure of nitric oxide synthases  Figure 1.4: Domain structure of nitric oxide synthases The structure of the three nitric oxide synthases is very similar with the oxygenase domain (red boxes) containing the arginine, haem and BH4 binding sites situated towards the amino half of the enzyme and the reductase domain (green and blue boxes) containing the FMN, FAD and NADPH binding sites positioned towards the carboxyl portion of the enzyme. The calmodulin binding domain (CaM) is positioned between the oxygenase domain and the reductase domain. The amino acid number of the start and stop of each domain is indicated to illustrate that the distribution of the domains is similar for all three isoforms. Notable structural differences between the structures are the presence of a PDZ-3 domain and the PDZ-2 internal binding ligand at the amino terminus of nNOS (black box), and the lack of an auto-inhibitory loop within the FMN region of the iNOS enzyme. Figure adapted from Alderton et al. (Alderton et al., 2001)  1.4.2. Synthesis of nitric oxide Activation of the neuronal and endothelial isoform of NOS requires CaM binding. Increases in intracellular Ca2+ activate CaM which in turn activates nNOS/eNOS thus these isoforms are considered Ca2+/CaM-dependent. Further, NO production is only stimulated by 41  an intracellular increase in Ca2+ thereby allowing constitutive expression and they are often referred to as constitutive NOS isoforms. Conversely, iNOS binds CaM with an affinity range such that iNOS remains active even in the presence of Ca2+ chelators and instead NO production via iNOS is regulated by transcriptional and translational mechanisms. Binding of CaM to its recognition site induces changes in structural conformation. These conformational changes result in a displacement of auto-inhibitory control element within NOS to permit the flow of electrons from the flavin cofactors to the haem domain. Evidence indicates that the conformational shift also propagates to the carboxyl tail of NOS which is believed to remove a conformational constraint on the FMN subdomain allowing for more efficient activity (reviewed in (Li and Poulos, 2005)). The conversion of arginine is a two-step reaction: 1) conversion of L-arginine to Nhydroxy-L-arginine (NHA) and 2) conversion of NHA to the final products: NO and citrulline (Figure 1.5). Three electrons are required to drive this conversion with the flow of electrons being donated by NADPH. The electrons are passed to FAD and then to FMN within the reductase domain. The haem cofactor within the oxygenase domain, where arginine binds, is then reduced by electrons received from FMN in the reductase domain. An oxygen molecule binds to the reduced haem which then receives an electron from the BH4 cofactor. Two protons are used to cleave the haem-bound oxygen to generate a reactive haem-oxygen species which transfers the oxygen to one of the chemically equivalent guanidino-nitrogens of arginine to produce NHA. Once the electron complement of BH4 is restored, courtesy of an electron from the flavin cofactors, the process is repeated to convert NHA into NO and L-citrulline. Each NADPH can provide two electrons meaning that for each mole of L-citrulline and NO produced 1.5 moles of NADPH are consumed (Mayer, 1995; Alderton et al., 2001; Hurshman et al., 2003; Li and Poulos, 2005). In the absence of 42  arginine the reaction is incapable of producing NO; instead, two highly reactive oxygen species, superoxide anions and hydrogen peroxide, are produced which can interact with NO to form peroxynitrite (reviewed in (Mayer, 1995)).  Figure 1.5: Reaction schematic and electron flow diagram of the conversion of arginine to citrulline and nitric oxide  43  Figure 1.5: Reaction schematic and electron flow diagram of the conversion of arginine to citrulline and nitric oxide A schematic illustrating the two-step process involved in the production of nitric oxide from arginine (A). Step 1 illustrates the hydroxylation of arginine (Arg) to produce the intermediate Nhydroxy-L-arginine (NHA) and Step 2 illustrates the oxidization of N-hydroxy-L-arginine to produce NO and citrulline. Three electrons are required for the production of NO, one for the hydroxylation of arginine, one to reduce H3B to H4B prior to the initiation of step two and a third for the oxidization of N-hydroxy-L-arginine. Each NADPH can supply two electrons therefore three NADPH are required for the synthesis of two nitric oxide molecules. The electron transfer flow is illustrated in panel B. Electrons from NAPDH are received by FAD, passed to FMN and then to haem in the course of nitric oxide synthesis. The schematic in panel A was originally published by Hurshman et al. (Hurshman et al., 2003) and the electron flow diagram is adapted from Alderton et al. (Alderton et al., 2001)  1.4.3. Mechanisms of modulation by nitric oxide NO and its reaction products can influence cellular physiology through several different mechanisms. The best characterized nNOS/NO pathway involves the activation of sGC which converts guanosine triphosphate to cyclic guanosine monophosphate (cGMP). Downstream targets of cGMP include two types of phosphodiesterase (PDE) isoforms (PDE2 and PDE3) (Bender and Beavo, 2006; Zaccolo and Movsesian, 2007), cyclic nucleotidegated ion channels ((Kaupp and Seifert, 2002), and cGMP dependent protein kinases (cGK). cGKs have been found to affect a diverse range of downstream targets (Hofmann et al., 2009); consequently, NO has the potential to influence a wide array of cellular processes through the activation of sGC (Wang and Robinson, 1997; Domek-Łopacińska and Strosznajder, 2005; Guix et al., 2005; Bender and Beavo, 2006; Kleppisch and Feil, 2009). A 44  description of the extensive array of processes affected by cGMP is outside the scope of this thesis however, the topic has been reviewed extensively in the citations listed above. Under physiological conditions NO undergoes various reactions, the products of which include nitrites (NO2-), nitrates (NO3-) and peroxynitrites (OONO-). Peroxynitrites can react with tyrosine residues to produce 3-nitrotyrosine (Beckman, 1996; Galiñanes and Matata, 2002). Protein nitrotyrosination can alter the protein function through conformational changes (Cassina et al., 2000; Amici et al., 2003) and through inhibition of phosphorylation events (Newman et al., 2002). Nitroxyl anions (NO-), a byproduct of breakdown of NO (Murphy and Sies, 1991) and the arginine intermediate NHA (Fukuto et al., 1992) can react with thiol group of cysteine residues to produce S-nitrothiols. The main targets for protein Snitrosylation appears to be enzymes and kinases (Broillet, 1999) however the modulation of proteins such as NF- (Marshall and Stamler, 2001) and NMDA receptors (Choi et al., 2000; Jaffrey et al., 2001) by S-nitrosylation has also been demonstrated.  1.4.4. Degradation of nitric oxide NO diffuses freely from the source of production and readily crosses the lipid cell membrane. Given the reactive nature of NO and its reaction products it is logical that a method of deactivation must exist to constrain the sphere of influence of NO and its conversion products to prevent cytotoxicity. The sphere of influence of NO refers to how far NO diffuses before it is degraded to the concentration where it is no longer physiologically active. Degradation of NO occurs through several pathways including simple diffusion away from the site (Lancaster, 1994; Wood and Garthwaite, 1994) and reaction with oxyhaemoglobin in nearby blood vessels (Lancaster, 1994; Liu et al., 1998), with superoxide (O2-) (Kissner et al., 1997) and lipid peroxyl radicals (O’Donnell et al., 1997). 45  The half-life of NO was previously reported to be 3-5 seconds (Palmer et al., 1987; Ignarro, 1989), however these studies assumed physiological NO concentrations on the micro molar scale. More recent studies indicate that nanomolar concentrations of NO are more probable (Garthwaite, 2005). Using a rat brain tissue Hall and Garthwaite developed a model to investigate NO degradation and found that at nanomolar concentrations the half-life of NO may be as low as 10 ms (Hall and Garthwaite, 2006). With such a brief half-life the sphere of influence of NO is likely tightly regulated and implies that the intended targets must be localized near the source of NO production for optimal efficiency.  1.4.5. Pharmacological properties of neuronal nitric oxide synthase A number of compounds have been shown to disrupt the NOS activity, however due to similarities with cytochrome P450 many of these agents are not considered NOS specific (reviewed in (Mayer, 1995). As such, the search for pharmaceutical agents capable of selectively antagonizing the specific isoforms of NOS at therapeutically relevant dosages is ongoing (reviewed in (Granik and Grigor’ev, 2002)). Here, I discuss three NOS specific inhibitors which have proven invaluable in research and which many of the newer compounds are derived from. The first compound used for NOS inhibition was N-MethylL-arginine (L-NMA). This arginine derivative is a competitive inhibitor for the binding site of L-arginine and inhibits all NOS isoforms. L-NMA inhibits NO synthesis but it does not block the reduction of oxygen in first phase of the conversion process which results in the production of superoxide and hydrogen peroxide. Additionally, L-NMA itself can be slowly metabolized by NOS to produce NO. Other L-arginine derived inhibitors are N-Nitro-Larginine (L-NNA) and its methyl ester, (L-NAME). The main difference between L-NNA and L-NAME is that L-NAME remains inactive until it is hydrolyzed by esterases within the 46  cell. Inhibition of NOS by L-NNA or L-NAME occurs through competition with the Larginine binding site and it is presumed that occupation of the site results in a conformational change in NOS. The effects of L-NNA and L-NAME are reversible with the addition of excess L-arginine. With these inhibitors the formation of hydrogen peroxide and superoxide is also blocked and further, neither L-NNA nor L-NAME can be metabolized by NOS. The constitutive isoforms are preferentially inhibited by L-NNA and L-NAME. The third class of compound, 7-Nitro indazole (7-NI), blocks NO synthesis by competing with both L-arginine and HB4 binding through a distinct functional domain preventing arginine binding and oxygenase activity. A drawback to 7-NI is its insolubility in water requiring aprotic solvents such as dimethylformamide or dimethyl sulfoxide which could adversely affect cellular and tissue preparations (reviewed in (Mayer, 1995; Granik and Grigor’ev, 2002)).  1.4.6. Expression of nNOS and roles of nNOS derived NO nNOS is the predominant NOS isoform and its expression in the brain has been studied extensively (Kowall et al., 1985; Mizukawa et al., 1988; Kinjo et al., 1989; Mizukawa, 1989; Leigh, 1990; Davis, 1991; Meyer et al., 1991; Vincent, 1992; Vincent and Kimura, 1992; DeFelipe, 1993; Meyer and González-Hernández, 1993; Sugaya and McKinney, 1994). Fortunately, the extensive body of information regarding nNOS has been consolidated in comprehensive reviews by (Vincent, 1994, 1995b). nNOS is expressed in varying degrees throughout the brain in a variety of neuronal cell types which will be summarized here. In the telencephalon of the rat brain NOS expressing neurons are scattered through layers II through VI of the cortex, and in the striatum and the amygdala. The basket cells and interneurons of the dentate gyrus express nNOS, as do magnocellular neurons of the diagonal band of Broca and the medial septum. 47  nNOS is found in the interneurons of the hippocampus however and its presence in pyramidal cells is controversial. Additionally, many neurons within the olfactory bulb contain nNOS with the granular layer displaying a high percentage of expressing cells. In the diencephalon populations of magnocellular neurosecretory neurons within the hypothalamus are nNOS positive and nNOS is present in gonadotrophs and folliculo-stellate cells of the anterior pituitary. In the retina nNOS can be found in rod cells (cone cells in humans), interneurons and the ganglion cell layer. In the midbrain NOS is found in neurons within the inferior colliculus, intercollicular commissure, posterior commissure and superior colliculus. There are also nNOS expressing neurons in the dorsolateral periaqueductal grey, laterodorsal and pedunculopontine tegmental nuclei. In the medulla, many neurons in the nucleus of the solitary tract and the ventrolateral medulla and scattered neurons in the reticular formation express nNOS. The cerebellum exhibits high nNOS expression in the granule cells as well as basket cells in the molecular layer. Finally, in the spinal cord nNOS is expressed in a subset of neurons of laminae I through IV and in ganglia of the dorsal root. Although across species nNOS expression has been generally found to be highly similar, differences have been noted; for example, in humans only a small percentage of neurons in the subcortical white matter have been found to be NOS positive whereas in the rat nNOS is found in most of the these neurons (Mizukawa et al., 1988; Springall et al., 1992; Vincent and Kimura, 1992; DeFelipe, 1993; Vincent, 1995a). nNOS expression has been demonstrated outside of the central nervous system as well. A splice variant of nNOS, nNOS, has been identified in the skeletal muscle (Silvagno et al., 1996), smooth muscle (Boulanger et al., 1998; Schwarz et al., 1999), cardiac muscle (Xu et al., 1999) and the corpora cavernosa of the penis and the lower urinary tract (Magee et al., 1996).  48  Given the wide range of nNOS expression in the brain it would be impractical to review here all of the physiological roles of nNOS-derived NO in each area. Here, I will touch upon two areas of research which have received considerable attention in the research community because of the potential therapeutic applications. Long term potentiation (LTP) and long term depression (LTD) have long been the proposed mechanisms underlying the synaptic plasticity required for learning and memory. NO has been reported to be important in the induction of LTP in the cerebellum (Jacoby et al., 2001; Qiu and Knöpfel, 2007), hippocampus (Phillips et al., 2008; Taqatqeh et al., 2009) and neocortex (Hardingham and Fox, 2006). Additionally, NO has been identified to play a role in the induction of LTD in the cerebellum (Shin and Linden, 2005; Ogasawara et al., 2007). Many in vivo studies using NOS inhibitors have shown that NO plays an important role in learning and memory ((Böhme et al., 1993; Hölscher et al., 1996; Richards et al., 2003; Koylu et al., 2005) and reviewed in (Susswein et al., 2004)). It has become apparent, however, that the contributions of NO to learning and memory varies with the type of learning (classical conditioning vs. habitual learning for example) and the stage of memory formation (short vs. long term memory) (reviewed in (Susswein et al., 2004)). This variation is perhaps not surprising given that nNOS is expressed in many of the neuronal populations involved in learning and memory and since NO can have differential effects among distinct populations of neurons depending on the available neuronal mechanism. For example, in one population of neurons NO may have a direct effect on synaptic plasticity through the nitrosylation of NMDA receptors to suppress LTP whereas in another population synaptic plasticity may be affected through the activation of cGMP and its downstream effectors (reviewed in (Susswein et al., 2004)). Understanding the mechanisms that are involved in  49  learning and memory is crucial in developing effective strategies to facilitate the learning process and to increase memory formation and retention. Neuronal cell death following a stroke can have debilitating and often deadly consequences depending on the size and location of the infarct. In ischemia there is an initial increase followed by a decline in NO levels and after reperfusion NO levels are elevated compared to pre-ischemic conditions suggesting that NO may contribute to neuronal damage following an ischemic event (Malinski et al., 1993). A NMDA receptor/NO signaling cascade has been identified as a major contributor towards neurotoxicity suggesting that NMDA and nNOS play a role in excitotoxicity and the damaging effects of ischemia (Dawson et al., 1993). In support of this is evidence from nNOS knockout mice; in these animals neuronal tissue displays resistance to ischemic insult (Dawson et al., 1996; Ferriero et al., 1996; Huang et al., 2000). Additionally, disrupting the PSD-95 mediated interaction between NMDA receptors and nNOS using membrane permeable TAT-peptides reduce the ischemic damage (Aarts et al., 2002). Taken together, evidence suggests that nNOS and/or the NMDA/nNOS interaction may be potential targets for the development of pharmaceuticals to minimize damage due to stroke.  1.5. Nitric oxide and nNOS in the ventilatory response 1.5.1. Neuronal pathways involved in respiration Respiration is the exchange of O2 and CO2 through rhythmic alternating cycles of inspiration and expiration. A sophisticated network of neuronal circuits sustains this involuntary process while maintaining the flexibility to adapt to changes in metabolic demand and ambient conditions as well as conscious control and interruptions such as sneezing, coughing, vomiting and vocalization. Despite the substantial amount of research 50  that has been devoted to the respiratory system, our understanding of the neuronal networks that comprise this system is far from complete. Indeed, over the years numerous models have been proposed and either subsequently rejected or significantly refined as new evidence is put forward. Current models propose a series of intricately connected circuits that are spatially distributed within the pons and the caudal region of the medulla. Together these circuits generate rhythmic patterns creating a breathing pattern that consists of 3 phases: inspiration, post-inspiration and expiration (Ramirez and Richter, 1996). A detailed review of the emerging views regarding the neuronal circuits of the respiratory network was published by Smith and coworkers (Smith et al., 2009). Here, I will introduce these circuits and summarize their contributions to respiration. The components required for the initiation, maintenance and termination of inspiration are all present in the pre-Botzinger complex (pre-BotC) making it a critical component of the respiratory system. Within this circuit there is a heterogeneous mix of neurons with different electrophysiological profiles and which form an auto rhythmic circuit which projects excitatory connections to the premotor inspiratory neurons to initiate and maintain inspiration. Also present in the pre-BotC are inhibitory neurons which are activated by the increase in inspiratory drive and facilitate both the termination of the inspiratory phase and coordinate the onset of the expiration. Activity within the pre-BotC circuit is under a number of modulatory influences including tonic excitatory inputs and rhythmic inhibitory signals which regulate and shape the stimulation pattern of the premotor inspiratory neurons. Activity within the rostral ventral respiratory group (rVRG) is driven largely through inputs from the pre-BotC. This circuit does not possess rhythmogenic capability; instead activation of this circuit produces an ascending ramp of stimulation that shapes the 51  inspiratory motor output pattern. A number of respiratory drive signals converge on the rVRG to shape the characteristic ramping pattern, fine tuning the inspiratory pattern to satisfy the demands placed on the system. The expiratory counterpart to the rVRG is considered to be the caudal ventral respiratory group (cVRG). While the rVRG is inhibited during expiration, the cVRG receives excitatory input from several sources including the Botzinger complex to modulate the pattern of excitatory drive to pre-motor neurons. The Botzinger complex (BotC) provides oscillating inhibitory input to most of the other circuits of the respiratory network including the pre-BotC and the rVRG to facilitate the transition from inspiration and engage expiration in the post-inspiration and expiration phases. This auto rhythmic inhibitory circuit is the main source of expiratory activity within the respiratory network. Similar to the circuits discussed previously, activity within the BotC is modulated through inputs from other circuits which helps maintain the alternating inspiration/expiration pattern of respiration. Almost all of the respiratory circuits receive excitatory drive signals which have been postulated to maintain the rhythmogenic oscillations of the various circuits. A major source of this excitatory drive is the retrotrapeziod nucleus/parafacial respiratory group (RTN/pFRG). The RTN has chemosensing capability, responding to CO2 levels and providing the excitatory drive necessary to meet metabolic demand. Additionally, the RTN integrates excitatory drive input from other chemoreceptors such as the carotid and arterial bodies and forwards the necessary response to the core respiratory networks enabling the entire network to adapt to the metabolic state of the system. Other sources of respiratory drive come from the PONS, raphe nucleus and the NST which project to one or more circuits within the respiratory network and provide information regarding the metabolic state of the body. 52  The RTN and pFRG are mentioned as a single group because there is spatial overlap between the two circuits. The contribution of the pFRG circuit is currently debated. Evidence has been put forward that oscillatory activity of pFRG provides a significant drive for the rhythmic activity of the pre-BotC. There is also experimental evidence suggesting that the pFRG facilitates inspiration/expiration phase transitions. Future investigations should provide the insight necessary to better determine the contributions of this circuit to the respiratory network. While much has been done over the past century to understand the seemingly simple activity of respiration we are only now identifying the neuronal populations and interactions involved in this process that is absolutely essential for life.  1.5.2. Role of NOS in respiration NO plays a significant role in the ventilatory response during hypoxia. Systemic inhibition of NOS significantly attenuates the ventilatory response to hypoxia suggesting that NO promotes neuronal activity either directly by increasing excitability of the neurons involved, or indirectly through the inhibition of the incoming inhibitory connections (Haxhiu et al., 1995; Gozal et al., 1996b; Antoniou and Murariu, 1999). NOS expressing neurons are found in several modulatory structures/circuits involved in respiration such as the NTS, the carotid body, the petrosal ganglion and microganglia within the glossopharyngeal nerve (Vincent and Kimura, 1992; Wang et al., 1993; Grimes et al., 1994; Haxhiu et al., 1995). Inhibition of NOS within the NTS using microinjection results in a decrease of neuronal activity and an attenuated hypoxic response which coincides with the results from the systemic NOS inhibition studies (Ma et al., 1995; Ogawa et al., 1995) and suggests a stimulatory role for NO in respiration. Additional support for a stimulatory role for NO 53  during hypoxia comes from eNOS knock-out mice which, in general, display a blunted hypoxic response (Kline et al., 2000). Conversely, in the carotid body, localized administration of NOS inhibitors which preferentially inhibit nNOS results in an increase of activity in neurons of the carotid sinus nerve and an augmented hypoxic response in vivo (Wang et al., 1994; Gozal et al., 1996a; Valdés et al., 2003) implying that NO can play either an inhibitory or excitatory role depending on the source of NO and the respiratory circuits affected. Furthermore, the ventilatory response to hypoxia in nNOS knock-out mice is augmented and these animals also display attenuation of short term facilitation and long term potentiation of ventilation demonstrating that nNOS derived NO plays a significant role in regulating the respiratory response (Kline et al., 1998, 2002). Clearly, the contribution of NO to respiration is significant however; our understanding of the contribution of the eNOS and nNOS isoforms within the various structures involved in respiration remains limited. The systemic inhibition of NOS resulting in inhibition of both eNOS and nNOS provides valuable information regarding the global contribution of NO but little concerning the contribution of either isoform or of the role of NO within the various structures involved in respiration. Knock-out mice provide an excellent resource for determining the contribution of NO derived from either isoform but again, the effects are systemic and there is a lack of studies investigating the specific respiratory structures involved utilizing these animals. Investigations utilizing the microinjection of relatively specific NOS isoform inhibitors have been performed but due to the lack of truly specific NOS inhibitors, inhibition of the other NOS informs remains a possibility in these investigations. An additional complication arises when we consider that NOS inhibitors can differently alter ventilatory behaviour in different strains of animals and which can make comparison between research groups problematic  54  (Subramanian et al., 2002). Finally, little is known about the cellular mechanisms within these structures that mediate the production of NO in the respiratory pathway.  1.5.3. Properties and role of nNOS expressing microganglia in the glossopharyngeal nerve (GPN) Two populations of nNOS positive microganglia have been identified within the GPN, one proximal to the bifurcation to the carotid sinus nerve (CSN) and another located distally along the GPN, which are a major source of efferent fibers to the carotid body (Wang et al., 1993, 1995a, 1995b; Prabhakar, 1999; Campanucci et al., 2003; Campanucci and Nurse, 2005). The carotid bodies are peripheral chemoreceptors that sense pH, pCO2 and pO2 levels in the blood and provide afferent communication via the CSN and the GPN to confer information regarding the state of the body and modulate respiratory activity (reviewed in (Gonzalez et al., 1994)). It has been demonstrated that the microganglia of the GPN are involved in the NO mediated efferent inhibition of the carotid body and that the inhibition of nNOS results in increase of activity in Type I glomerus cells of the carotid body and ultimately in an increase in phrenic nerve activity (Wang et al., 1994; Gozal et al., 1996a; Valdés et al., 2003; Campanucci et al., 2006). These findings are congruent with those of Kline and coworkers who observed increased CNS activity and an augmented hypoxic response in nNOS knock-out mice compared to wild type controls (Kline et al., 1998) suggesting that the exaggerated hypoxic response in these mice may be due to the lack of efferent inhibition of the carotid body. Campanucci and Nurse have made significant contributions to our understanding of the NO mediated efferent inhibition of the carotid body. They identified a novel O2 sensing mechanism (Campanucci et al., 2003), demonstrated the expression of purinergic P2X receptors (Campanucci et al., 2006) and 55  characterized the whole cell ionic currents of the GPN microganglia (Campanucci and Nurse, 2005). From these observations a model was put forward outlining the mechanism of NOmediated efferent inhibition of the carotid body (Campanucci and Nurse, 2007). In the model, the efferent inhibition of the carotid body is peripheral in origin. Under hypoxic conditions the O2 sensing mechanism of the microganglia causes a depolarization resulting in an increase in excitability and consequently increased activity. This increased activity results in the activation of the multiple VGCCs types, including a nickel sensitive Ttype Ca2+ channel, which has been proposed to increase intracellular Ca2+ levels sufficient to stimulate nNOS. Upon activation, nNOS produces NO which diffuses across the plasma membrane to the Type I glomerus cells of the carotid body where inhibition of the L-type (Cav1) VGCCs has been proposed to result in the reduction of carotid body activity (Summers et al., 1999). It is further proposed that activation of ionotropic purinergic P2X receptors in the microganglia by ATP released from the carotid body while under hypoxia may also contribute to the intracellular Ca2+ levels and facilitate nNOS stimulation.  1.6. Cav3.2 channels and nociception 1.6.1. Neuronal pathways involved in nociception Mechanical, thermal and chemical noxious stimuli are detected by A and C nociceptive fibers in the peripheral nervous system. Receptors within these fibers transduce the noxious stimuli into a sensory potential and, if the potential is great enough, an action potential is triggered within the dorsal root ganglion (DRG). The action potential travels along the axon to the dorsal horn of the spinal cord where it synapses onto nociceptivespecific neurons within the superficial laminae. The nociceptive neurons within the spinal cord can be part of either the ascending tract or interneurons that are involved with reflex 56  pathways. Signals propagating along the ascending tract into the brain activate the thalamocortical system. The lateral thalamocortical system, which is composed of relay nuclei in the lateral thalamus and areas SI and SII of the post central gyrus, interprets the incoming information to determine intensity, duration and localization of the pain and conveys this information to the consciousness as acute pain. The affective aspects of pain are subsequently mediated by the medial thalamocortical system and efferent signals from the periaqueductal grey and nucleus raphe magnus descend the dorsolateral funiculus of the spinal cord and synapse onto interneurons within the dorsal horn to suppress the nociceptive signal (reviewed in (Kandel et al., 2000; Schaible and Richter, 2004; Hildebrand and Snutch, 2006)).  1.6.2. Role of Cav3.2 channels in nociception T-type Ca2+ currents have been identified in small (20-27 m) and medium (33-38 m) sized DRG neurons suggesting a potential nociceptive role (Carbone and Lux, 1984a; Nowycky et al., 1985; Scroggs and Fox, 1992). The systemic administration of mibefradil has been demonstrated to have an analgesic effect on mechanical and thermal stimuli (Todorovic et al., 2002; Dogrul et al., 2003; Kim et al., 2003) providing pharmacological evidence for a nociceptive role for T-type channels. However, given the questionable specificity of mibefradil (Viana et al., 1997; Jimenez et al., 2000), these results are not conclusive proof. In one study in rats, Todorovic and colleagues described the redox modulation of Cav3.2 currents in small (21-27 m), capsaicin-sensitive DRGs; in this study the oxidizing agent DTNB inhibited and reducing agents DTT and L-cysteine enhanced Cav3.2 currents. They further showed that microinjection of DTNB into the paw produced analgesic responses 57  to mechanic and thermal stimuli and that reducing agents produced hyperalgesic responses suggesting that Cav3.2 channels play a role in peripheral nociception (Todorovic et al., 2001b). Subsequently, a novel population of small (26-31 m) DRGs with substantial T-type currents and virtually no HVA Ca2+ currents that responded to capsaicin and stained positive for the nociceptive marker IB4 was described (Nelson et al., 2005). Utilizing these “T-rich” DRGs, Nelson and coworkers provided evidence that the main nociceptive role of Cav3.2 channels may be a contribution to regulating the excitability of DRGs. In these experiments the application of the endogenous reducing agent L-cysteine enhanced an ADP generated by Cav3.2 currents and which increased the likelihood of triggering action potential firing during the ADP. The most conclusive evidence yet of a nociceptive role for Cav3.2 channels was presented by Bourinet and colleagues who showed that the selective antisense-mediated knock-down of Cav3.2 channel RNA and protein in the L5 and L6 dorsal spinous processes both had an analgesic effect in healthy rats and relieved the hyperalgesia associated with mechanical and thermal stimuli in a chronic constriction injury model of neuropathic pain (Bourinet et al., 2005).  1.6.3. Role of nNOS in nociception A significant body of pharmacological evidence demonstrates that NOS is involved in the mechanical and thermal hyperalgesia observed in neuropathic models of pain. Reduction of mechanical and thermal hyperalgesia has been observed with systemic (Hao and Xu, 1996; Yoon et al., 1998; Dableh and Henry, 2011) and intrathecal (Meller et al., 1992; Roche et al., 1996; Guan et al., 2007; Chacur et al., 2010) administration of NOS inhibitors. A comparison 58  of nNOS and eNOS knock-out mice reveals that a reduction in neuropathic hyperalgesia is present only in the nNOS knock-out mice ( a 50% reduction in thermal hyperalgesia and a total reversal of mechanical hyperalgesia) (Boettger et al., 2007; Guan et al., 2007). More recently, it has been shown that nNOS is up-regulated in small and medium sized DRGs following nerve injury and is likely a contributing factor towards hyperalgesia (Chacur et al., 2010; Kim et al., 2011). Support for nNOS up-regulation underlying peripheral hyperalgesia was also provided using antisense knock-down of the various nNOS isoforms. Examining the formalin induced pain model Kolesnikov and coworkers showed that intrathecal administration of siRNA targeting the  and  variants of nNOS inhibited hyperalgesia during the tonic phase of the formalin response. Interestingly, only the siRNA targeting the  variant inhibited hyperalgesia when delivered supraspinally or peripherally (Kolesnikov et al., 2009). One of the most significant structural differences between the two nNOS isoforms is the lack of the PDZ-3 binding domain and internal PDZ-2 ligand in the  isoform which suggests localization and/or trafficking of nNOS may be important for the contribution of nNOS to nociception in the supraspinal and peripheral regions. The mechanism and effects of nNOS up-regulation are poorly understood although it has been suggested that an NMDA receptor/nNOS interaction is involved (Kim et al., 2011) since MK-801, a NMDA antagonist, delays the onset of neuropathic pain (Jang et al., 2004). Support for a role of a NMDA receptor/nNOS interaction in the development of hyperalgesia has been found in the mechanism behind morphine tolerance. A NMDA/NO/PKC pathway in involved in the development of morphine tolerance and systemic administration of L-NNA prevents and reverses morphine tolerance indicating that NOS plays an important role in this process (Kolesnikov et al., 1993). This evidence suggests that the interaction between  59  NMDA and nNOS may facilitate neuronal excitability resulting in increased sensitivity to mechanical and thermal stimuli.  1.7. Thesis hypothesis and objectives 1.7.1. Hypotheses I originally noted that the carboxyl terminus of the Cav3.2 channel possesses a putative PDZ binding ligand which is conserved across human, rat and mouse. Further, the amino acid sequence of the PDZ binding ligand corresponds with the consensus sequence for the PDZ-3 biding domain of nNOS suggesting the possibility that the two proteins may physically interact in vivo. As nNOS is a Ca2+-dependent enzyme which produces NO, and given that NO has been shown to inhibit Cav3.2 currents, I hypothesized that a functional interaction may also occur. While the external application of redox agents has been show to modulate Cav3.2 activity, an in vivo mechanism for redox modulation of Cav3.2 channels has not been previously identified. The expression profiles of Cav3.2 and nNOS overlap in several regions of the central and peripheral nervous systems including two structures involved with the ventilatory response, the NTS and microganglia within the GPN, presenting a potential role for a putative Cav3.2/nNOS interaction in the ventilatory response. In this thesis I specifically hypothesized: 1) that Cav3.2 and nNOS physically interact via the carboxyl PDZ-3 ligand of Cav3.2 channels 2) that the Cav3.2 and nNOS interaction results in the production of NO allowing for a functional feedback regulation wherein Cav3.2 activity stimulates nNOS to produce NO which in turn feeds back to inhibit Cav3.2 channels  60  3) that systemic disruption of the putative Cav3.2/nNOS interaction in vivo will have a measurable effect on the ventilatory response to hypoxia  1.7.2. Objectives In order to address these hypotheses the following scientific objectives were generated: 1) Determine whether Cav3.2 and nNOS can physically interact via the carboxyl PDZ-3 binding ligand of Cav3.2 through co-immunoprecipitation and western blot analysis 2) Determine whether Cav3.2 activity is sufficient to stimulate nNOS to produce NO in a heterologous expression system 3) Determine whether Cav3.2 activity is inhibited when co-expressed with functional nNOS in a heterologous expression system 4) Investigate a potential role for the putative Cav3.2/nNOS interaction in vivo by disrupting the PDZ-3 mediated interaction and measuring parameters of the ventilatory response of rats under hypoxic conditions.  61  Chapter 2. 2. Cav3.2 and nNOS interact physically and functionally to form a putative negative feedback regulatory pathway 2.1. Introduction T-type VGCCs play important roles in regulating neuronal activity. In addition to contributing to neuronal excitability, the shaping of action potentials and mediating intracellular Ca2+ influx, the unique biophysical properties of T-type channels supports a range of firing patterns from rhythmic low threshold burst-firing to slow oscillations (reviewed in (Perez-Reyes, 2003; Cain and Snutch, 2010)). This versatility allows T-type channels to play important roles in functions as diverse as aldosterone and cortisol secretion, boosting of nociceptive signals, and mediating thalamocortical rhythms (reviewed in (Park et al., 2003; Steriade, 2005; Jevtovic-Todorovic and Todorovic, 2006)). A number of cellular mechanisms such as G-protein coupled receptors (Wolfe et al., 2003; Hildebrand et al., 2007), secondary messengers PKC and PKA (Park et al., 2003; Kim et al., 2006; Chemin et al., 2007), tyrosine kinase (Arnoult et al., 1997), Rho-associated protein kinase (Iftinca et al., 2007) and reducing/oxidizing agents (Todorovic et al., 2001b; Nelson et al., 2007a, 2007b) have been shown to modulate Cav3 activity and affect the excitability and firing patterns of neurons in which they are expressed (Nelson et al., 2005, 2007a, 2007b; Joksovic et al., 2006, 2007; Iftinca et al., 2007; Hildebrand et al., 2009) indicating that Cav3 modulation may be an important in vivo mechanism for regulating neuronal activity. Of the T-type channels, Cav3.2 is uniquely sensitive to redox modulation. Todorovic et al. have demonstrated that reducing agents such as DTT and L-cysteine enhance Cav3.2 activity by removing the tonic Zn2+ inhibition at the histidine residue at position 191 whereas 62  oxidizing agents such as DNTB, ascorbate and NO inhibit Cav3.2 activity through metal catalyzed oxidization (Nelson et al., 2007a, 2007b). Furthermore, enhancement of Cav3.2 activity with the application of reducing agents increases the likelihood of stimulating a second action potential in acute preparations of nociceptive DRGs and increases burst firing in nRT neurons of thalamic slices while the inhibition of Cav3.2 by oxidizing agents reduces the amplitude and duration of the after depolarizing potential in DRGs and inhibits burst firing in nRT neurons (Nelson et al., 2005, 2007a, 2007b; Joksovic et al., 2006, 2007). Phenotypically, Todorovic et al. show that in the neuropathic pain model, administration of reducing agents intrathecally or by localized microinjection exacerbates the nociceptive response to noxious stimuli whereas the hyperalgesic response is minimized with the administration of oxidizing agents (Todorovic et al., 2001b, 2004; Nelson et al., 2007b). The effect of the endogenous compounds L-cysteine, ascorbate and NO on Cav3.2 activity and consequently DRG and nRT neurons suggests that redox modulation of Cav3.2 may occur in vivo to regulate neuronal activity. In particular, the inhibitory effects of NO are intriguing as nNOS has been found to be expressed in nociceptive DRGs and up-regulated in the neuropathic pain model (Steel et al., 1994; Choi et al., 1996; Luo et al., 1999).. nNOS is a Ca2+ -dependent enzyme which catalyzes the synthesis of NO and L-citrulline from Larginine; co-expression of the Cav3.2 channel and nNOS within the same neuron would provide a convenient source of NO for the regulation of Cav3.2 activity. We have identified a putative PDZ-3 binding ligand at the carboxyl terminus of the Cav3.2 channel (Figure 2.1A). Alignment of the carboxyl amino acids of the channels within the Ca2+ family demonstrates that the putative PDZ-3 binding motif is unique to Cav3.2 and that it is conserved across human, rat and mouse species (Figure 2.1B). The sequence of the PDZ-3 motif corresponds with the consensus sequence known to interact with the PDZ-3 63  domain of nNOS suggesting that a physical interaction between the two proteins is possible (Schepens et al., 1997; Stricker et al., 1997). Furthermore, a physical interaction between Cav3.2 and nNOS may facilitate a functional interaction by anchoring nNOS within effective range of the localized Ca2+ influx generated during activation of Cav3.2(Augustine et al., 2003). I hypothesize that Cav3.2 and nNOS physically interact via the carboxyl PDZ-3 ligand of Cav3.2 allowing for the functional feedback regulation wherein Cav3.2 activity stimulates nNOS to produce nitric oxide which in turn further inhibits Cav3.2 channels. In this section I put forward evidence that Cav3.2 and nNOS can physically and functionally interact via the carboxyl PDZ-3 motif of Cav3.2 forming a novel feedback loop. Utilizing a heterologous expression system I demonstrate that a physical interaction between Cav3.2/nNOS is feasible and that this interaction facilitates a functional interaction whereby Cav3.2 mediated Ca2+ entry stimulates nNOS activity which in turn inhibits Cav3.2 activity. This putative negative feedback loop could potentially play an important role in regulating neuronal activity in DRGs involved in nociception under normal and pathological conditions and in nRT neurons during development.  64  Figure 2.1: Alignment of voltage-gated Ca2+ channels demonstrating a PDZ-3 motif unique to Cav3.2  Figure 2.1: Alignment of voltage-gated Ca2+ channels demonstrating a PDZ-3 motif unique to Cav3.2 Alignment of the last 30 residues from the amino acid sequences of human voltage-gated Ca  2+  channels (A). The Cav3.2 is the only VGCC with a carboxyl sequence that corresponds with the PDZ3 binding motif consensus sequence x-D/E-x-V/I/L-COOH (Schepens et al., 1997; Stricker et al., 1997). Alignment of the carboxyl human, rat and mouse Cav3.2 amino acid sequences shows that the consensus sequence for the PDZ-3 binding ligand is conserved across species (B).  2.2. Materials and methods 2.2.1. Generation of fusion peptides Plasmids encoding the glutathione S-transferase (GST) fusion protein constructs were generated by inserting the carboxyl terminal coding sequence of Cav3.2 or Cav3.1 in frame and downstream of the GST coding sequence. Polymerase chain reactions (PCRs) consisting of Taq DNA polymerase, HotStarTaq Master Mix with Q solution (Qiagen, Toronto, ON, 65  Canada), 200M dNTPs (Invitrogen, Grand Island, NY, USA), and 0.4M forward and reverse oligos (refer to Table 2.1 for oligo sequences) were performed to amplify the coding sequence of the carboxyl 100 amino acids of pcDNA3.1 zeo (+) plasmid clones of human Cav3.2 (NCBI accession number: NM_001005407) and human Cav3.1 (NCBI accession number: NM_018896). For the Cav3.2 GST fusion proteins, oligonucleotides (oligos) were designed to introduce BamHI and XhoI restriction endonuclease recognition sites at the 5’ and 3’ ends, respectively. Cav3.1 oligos introduced an EcoRI restriction endonuclease site at the 5’ end and an XhoI restriction endonuclease site at the 3’ end. Further, a GST/Cav3.2 fusion peptide lacking a PDZ-3 binding ligand was generated using a downstream oligo designed to introduce a stop codon at the start of the DDPV-COOH motif of Cav3.2. A PTC200 gradient thermocycler (MJ Research/ BioRad, Mississauga, ON, Canada) was utilized to perform a 25 cycle profile of 98oC for 30 seconds for denaturation, 50 to 65 oC for 1 minute for annealing, and 65 oC to 78 oC for 1 minute for extension. Once amplified, the Cav3.2 fragments were digested with BamHI (New England Biolabs, Ipswich, MA, USA) and XhoI (New England Biolabs, Ipswich, MA, USA) restriction endonucleases and the Cav3.1 fragment with EcoRI (New England Biolabs, Ipswich, MA, USA) and XhoI restriction endonucleases before ligation using T4 ligase (New England Biolabs, Ipswich, MA, USA) into a pGEX4T1 vector (GE Healthcare, Baie d’Urfe, QC, Canada) which was digested using the same endonulceases. The ligated fragments were transformed into chemically competent DH5 cells (Invitrogen, Grand Island, NY, USA) using the heat shock protocol detailed in Section “2.2.2. Generation of transformation and chemically competent DH5 cells” and plated on lysogeny broth (LB) agar plates (1% (w/v) tryptone, 1% (w/v) NaCl, 0.5% (w/v) yeast extract, 1.5 w/v agar) (Bertani, 1951, 2004) with 50 g/ml ampicillin to select for transformed cells. After incubating the LB agar plates with ampicillin overnight at 37oC 66  individual colonies were cultured overnight at 37oC in 5ml of LB with 50 g/ml ampicillin, and the DNA was extracted using a commercial DNA miniprep kit (Invitrogen, Grand Island, NY, USA). The GST fusion protein clones were sequenced (NAPS, UBC) to ensure the sequence was correct and encoded the desired fusion protein. BL21DE (Invitrogen, Grand Island, NY, USA) cells were utilized to express the GST fusion proteins. The cells were transformed using the described heat shock protocol and then plated on LB agar plates with 50 g/ml ampicillin to select for transformed cells. After incubating the LB ampicillin plates overnight at 37oC an individual colony was cultured at 37oC in 50ml of LB for use as a started culture. After 18 hours in a shaking incubator the optical density (A600) of the starter culture was measured using a spectrophotometer and used to seed 500ml of LB at an optical density of 0.2. The 500ml culture was monitored and when the optical density reached 0.6 induction of the GST fusion protein expression was initiated with the addition of 300M isopropyl -D-1 thiogalactopyranoside (IPTG) and incubating for 3 hours at 37oC with shaking. The induced cells were pelleted by centrifugation at 7000 x g for 10 minutes, resuspended in GST lysis buffer (25 mM Tris, 2 mM EDTA, pH 7.6 with HCl) and lysed with 1mg/ml lysozyme (GE Healthcare, Baie d’Urfe, QC, Canada) for 20 minutes at room temperature. A ten minute sonication in a sonicating water bath was used to assist the lysing of the cells and the lysate was centrifuged for 20 minutes at 18,000 x g at 4 oC to remove the cell debris before the lysate was transferred to new 1.5ml tubes and frozen at -20oC until required for subsequent experiments. Cell-penetrating TAT-fusion peptides were designed and ordered from Gensrcipt (Piscataway, NJ, USA). The peptides contained the protein transduction domain of the Human Immunodeficiency Virus 1 transactivator of transcription (TAT) protein (YGRKKRRQRRR) fused with the last 9 amino acids of the rat Cav3.2 sequence 67  (PDDSGDEPV) which possess a carboxyl terminal PDZ-3 binding ligand. A second TATfusion peptide with a mutation in the Cav3.2 PDV-3 binding ligand (PDDSGDEPE) was also designed ordered. The peptides were used at a concentration of 50 nM.  Table 2.1: Primers used to generate the clones utilized in this thesis Purpose  Target  Primer orientation  GST fusion protein  Cav3.2  Forward  GST fusion protein  Cav3.2  Reverse  GST fusion protein  Cav3.2  Reverse  GST fusion protein  Cav3.1  Forward  GST fusion protein  Cav3.1  Reverse  Full length mutant Removal of GST from nNOS clone Tagging of PSD95 with DsRED2 Tagging of PSD95 with DsRED2  Primer sequence 5' to 3' CGGATCTGGTTCCGC GTGGATCCTCCTGC CGGGCTGAGCACCT GACC TCAGTCACGATGCGG CCGCTCGAGCTACA CGGGGTCATCTGCA CCAC CAGTCACGATGCGGC CGCTCGAGCTATGC ACCACCCCCTGGGG CAGGGGT GCCTGGACGAATTC TCCCAACCCCACCTG GGCACAGACCCCTC T AAGTGGGGCTCGAG TCAGGGGTCCAGGT CTGCTGGGTCAGAG G  Amino acid substitution details  Restriction Sites  No amino acid substitution  Introduces BamHI Restriction endonuclease site  No amino acid substitution  Introduces XhoI Restriction endonuclease site  Introduces stop codon to remove last 4 amino acids (deletes DDPV sequence)  Introduces XhoI Restriction endonuclease site  No amino acid substitution  Introduces EcoRI Restriction endonuclease site  No amino acid substitution  Introduces XhoI Restriction endonuclease site  DEPV to DAPA mutation  Native Cav3.2 PflMI and pcDNA3.1 vector XhoI Restriction endonuclease sites utilized  Cav3.2  Duplex oligo  CTGCCCCAGGGGGT GGTGCAGATGCCCC CGCGTAGCTCGGTC TAGAGTGC  nNOS  Duplex oligo  CTTAGTAAGCGGCC GCTTACTAAG  Introduce native stop codon at carboxyl terminus of nNOS  nNOS NotI Restriction endonuclease site utilized  DsRED 2  Forward  GGAATTCGGATGGC CTCCTCCGAGAACGT CATCA  No amino acid substitution  Introduces EcoRI Restriction endonuclease site  DsRED 2  Reverse  GGAATTCTAGAGTC GCGGCCGCTACAGG AACA  No amino acid substitution  Introduces EcoRI Restriction endonuclease site  68  Table 2.1: Primers used to generate the clones utilized in this thesis To generate the plasmid constructs used in the study various oligos were designed that introduced restriction endonuclease recognition sites (bold blue bases) to facilitate the in frame cloning of the PCR fragments. For a few of the constructs the oligos were also used to introduce base pair mutations into the coding sequence (bold red bases) to alter the amino acid sequence. The bases in normal black type are those that match and bind to the target, those in black italics are a random assortment of bases required for to optimize the digestion of the PCR fragments with the restriction enzymes.  2.2.2. Generation and transformation of chemically competent DH5 cells DH5 cells (Invitrogen, Grand Island, NY, USA) were maintained as glycerol stocks at -150oC. To prepare chemically competent cells a scraping of the DH5glycerol stock was used to seed a 5ml starter culture of SOC++ media (2% w/v tryptone, 0.5% w/v yeast extract, 0.05% w/v NaCl, 250 mM KCl, 2 M MgCl2, 1 M glucose, pH 7.4 with NaOH) which was incubated overnight at 37 oC with shaking. The next day 1ml of the started culture was used to seed a 75 ml of SOC++ media culture which was incubated at 19oC with shaking until the optical density (A600) was 0.6 at which time the cell were pelleted with a 10 minute 3,000 x g centrifugation at 4oC. One wash using 80ml of ice cold TB buffer (10 mM PIPES, 15 mM CaCl2*2H2O, 250 mM KCl, 55 mM MnCl2*4H2O, pH 6.7 with KOH) was completed and the cells were re-suspended in 20 ml of TB buffer. 1.5 ml dimethyl sulfoxide (DMSO) (SigmaAlrich, St.Louis, MO, USA) was added to the re-suspended cells to assist in cryoprotection and 200 l aliquots were flash frozen in liquid nitrogen followed by long term storage at -80 o  C. Transformation of the chemically competent DH5 cells was accomplished using a  heat shock protocol. The plasmid DNA (the entire ligation mixture OR 500ng of purified 69  plasmid) was gently mixed with a 200l aliquot of DH5 cells which were allowed to thaw on ice. After a 10 minute incubation on ice the cells were placed in a 42oC water bath for 60 seconds and then immediately transferred to an ice bath for 5 minutes. The heat shock treated cells were transferred to 1ml of pre-warmed (37oC) LB and incubated at 37oC with shaking for 1 hour and then plated on LB agar plates with the appropriate selection agents.  2.2.3. Mutation of the Cav3.2 PDZ-3 binding ligand Key amino acids of the carboxyl PDZ-3 motif of Cav3.2 were mutated to determine whether full length Cav3.2 and nNOS interact via the PDZ-3 domain of nNOS. Using the 3’ terminus as a template, a 50-mer duplex oligo was designed to introduce two single base pair mutations near the stop codon of Cav3.2. The mutations encode for two amino acid substitutions; one at the -2 position where the second Aspartic acid of the –DDPV-COOH motif is changed to Alanine, and the other at position 0 where the hydrophobic valine residue is substituted with Alanine. The 5’ end of the duplex oligo spanned a native PflMI restriction endonuclease site and the 3’ region was designed to include an XbaI restriction endonuclease recognition site downstream of the Cav3.2 stop codon. Restriction endonulceases PflMI (New England Biolabs, Ipswich, MA, USA) and XbaI (New England Biolabs, Ipswich, MA, USA) were used to digest the full length Cav3.2 clone in pcDNA3.1 zeo (+).The duplex oligo and the digested fragments were separated by size on a 0.8% low melting point agarose (SigmaAlrich, St.Louis, MO, USA) gel in TAE buffer (40 mM Tris, 5.71% glacial acetic acid, 1mM EDTA, pH 8.0) at a constant voltage of 100 V and a commercial gel extraction kit (Qiagen, Toronto, ON, Canada) was used to recover individual fragments from the gel. A single fragment of ~50 base pairs was recovered from the duplex oligo digest. There are two PflMI restriction endonulcease recognition sites within the Cav3.2 clone, therefore two fragments of 70  4306 and 7665 base pairs in size were recovered from the digest; the third fragment of 123 base pairs was discarded. The larger 7665 base pair fragment was dephosphorylated by adding 2 l each of alkaline phosphatase and buffer (Roche, Laval, QC, Canada) to 20 l of the fragment sample and incubating at 37oC for 1 hour. Ligation of the plasmid clone was completed in two steps. The 4306 base pair fragment from the Cav3.2 digest was first ligated to the digested duplex oligo using T4 DNA ligase at 16 oC. After 40 minutes the dephosphorylated 7665 base pair fragment was added to the ligation mixture and all 3 fragments were ligated overnight at 16 oC. The ligated fragments were transformed into chemically competent DH5 cells using the described heat shock protocol and plated on LB agar plates with 50 g/ml ampicillin to select for transformed cells. After incubating the LB ampicillin plates overnight at 37oC an individual colonies were cultured overnight at 37 oC in 5 ml of LB with 50 g/ml ampicillin and the DNA was extracted using a DNA miniprep kit (Invitrogen, Grand Island, NY, USA). A test restriction endonulcease digest using the BstXI restriction endonulcease (New England Biolabs, Ipswich, MA, USA) was performed to confirm the proper insertion of the duplex oligo fragment. After confirming that the oilgo fragment was ligated successfully, a sample of the transformed bacterial stock was cultured overnight at 37oC in 200 ml of LB with 50 g/ml ampicillin and the DNA was extracted using a commercial midiprep kit (Invitrogen, Grand Island, NY, USA). The clones were sequenced (NAPS, UBC) to ensure the single base pair mutations were present in the clone and to ensure sequence errors were not introduced during the cloning process.  2.2.4. nNOS and PSD-95 clones The original full length neuronal nitric oxide synthase (nNOS) (NCBI accession number: NM_052799) was cloned into a pCIS vector by D.S.Bredt (Johns Hopkins 71  University School of Medicine) (Bredt et al., 1991). A green fluorescent protein (GFP) tagged version of the full length nNOS clone in the pCIS vector was generously provided by Dr. S.R. Vincent (University of British Columbia). This GFP tagged nNOS clone was utilized in the electrophysiology experiments so that transfected cells could be readily identified. A second nNOS construct was generated to remove the GFP tag to eliminate the possibility of background fluorescence from the GFP in the nitric oxide detection assay; this construct was used for all experiments other than electrophysiology. To remove the GFP tag the pCIS nNOS-GPF clone was digested with XbaI for to linearize the clone a immediately 5’ of where the stop codon is positioned in the wild type nNOS sequence. Mung Bean Nuclease (New England Biolabs, Ipswich, MA, USA) was used to digest the XbaI 5’ overhang to create a blunt ended linear fragment. A second digestion using the NotI restriction endonulcease (New England Biolabs, Ipswich, MA, USA), which recognizes a restriction site 3’ of the GFP sequence of the nNOS/GFP plasmid was performed to excise the GFP coding sequencing from the linearized plasmid. The digested fragments were separated by size on a low melting point gel and the large fragment containing the nNOS coding sequence and vector backbone was recovered using a commercial gel extraction kit (Qiagen, Toronto, ON, Canada). To reconstitute the native nNOS sequence a phosphorylated palindromic oligonucleotide was generated (Integrated DNA Technologies: San Diego, California) which contained the final five nucleotides of the native nNOS coding sequence and a NotI restriction endonuclease recognition site 3’ of the native nNOS stop codon. Digestion of the palindromic oligonucleotide with NotI generated two identical fragments containing a blunt 5’ end possessing the last five nucleotides of the native nNOS sequence and a NotI 5’ overhang at the 3’ end. The digested oligo fragments were ligated with the nNOS vector 72  fragment using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) for 2 hours at room temperature and then transformed into chemically competent DH5chemically competent cells using the described heat shock protocol and plated on LB agar plates with 50 g/ml ampicillin to select for transformed cells. After incubating the LB agar plates overnight at 37 oC individual colonies were cultured overnight at 37 oC in 5 ml of LB with 50 g/ml ampicillin for selection, and the DNA was extracted using a DNA miniprep kit. A test digest was performed using the KpnI restriction endonuclease (New England Biolabs, Ipswich, MA, USA) to determine proper excision of the GFP tag and the re-ligated clone was sequenced to ensure the restored sequence corresponded to the native nNOS sequence. The Post Synaptic Density Protein 95kD (PSD-95) (NCBI accession number: NM_019621) clone was originally generated by Dr. D. S. Bredt (University of California) (Topinka and Bredt, 1998) and a green fluorescent protein (GFP) tagged version of this full length PSD-95 clone in a GW1 vector was received from Dr. Y.T.Wang (University of British Columbia). Since the nNOS clone already contained a GFP tag, the GFP tag of the PSD-95 clone was substituted with a DsRED tag from the pDsRED2 vector. This DsRED tagged PSD-95 clone was used for all experiments requiring expression of PSD-95. PCR primers were designed to amplify the coding sequence of the DsRED tag from the pDsRED2 vector (CloneTech: Mountain View, CA, USA) and to introduce EcoRI restriction endonuclease recognition sites at the 5’ and 3’ ends of the PCR fragment. EcoRI (New England Biolabs, Ipswich, MA, USA) restriction endonulcease was used to digest both the PCR fragment and the PSD-95 clone to excise the GFP tag. The digests were separated by size on a low melting point agarose gel and the vector backbone as well as the PCR fragment was recovered using a commercial gel extraction kit. The vector backbone was dephosphorylated and the PCR fragment was ligated into the backbone using T4 DNA ligase. 73  The ligated fragments were transformed into chemically competent DH5 cells using the described heat shock protocol and plated on LB agar plates with 50 g/ml ampicillin to select for transformed cells. After incubating the LB agar plates with ampicillin overnight at 37 oC individual colonies were cultured overnight at 37 oC in 5 ml of LB with 50 g/ml ampicillin, and the DNA was extracted using a commercial DNA miniprep kit. A restriction endonuclease test digest using SbfI (New England Biolabs, Ipswich, MA, USA) and BamHI was performed to confirm the insertion of the PCR fragment in the proper orientation. The newly generated DsRed2 tagged PSD-95 clone was sequenced to ensure that DNA sequence errors were not introduced during the cloning process.  2.2.5. Cell culture and transfection Human embryonic kidney (HEK293) cells were grown in Dulbecco's Modified Eagle Medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY, USA) which was heat inactivated at 55oC for 30 minutes, 50 U/ml penicillin/50 g/ml streptomycin (Invitrogen, Grand Island, NY, USA) and 1% non-essential amino acids (Invitrogen, Grand Island, NY, USA). A humidified chamber was utilized to incubate the cells at 37oC in 5% CO2 balanced with room air. The reagents used to maintain selection pressure for the various cell lines stably expressing Cav3.2 are listed in Table 2.2. Selection pressure was maintained during cell growth and the selection reagents were refreshed with each media change. A Ca2+ phosphate transfection procedure was utilized to transiently transfect HEK293cells. The day before transfection the cells were dissociated using 0.25% trypsinEDTA (Invitrogen, Grand Island, NY, USA) and diluted in growing media without selection reagents so that the confluency of the re-seeded T-25 flasks (Fisher Scientific, Ottawa, ON, 74  Canada) was between 20-25%. On the day of transfection the cells were typically between 35-45% confluent. For each T-25 to be transfected, 150 l Ca2+ chloride (250 mM) and 150 l 2x HEPES Buffered Saline (HeBS) (274 mM NaCl, 40 mM HEPES, 12 mM D-glucose, 10 mM KCl, 1.4 mM Na2HPO4*7H2O, pH 7.04) were dispensed into separate 5 ml tubes. 1.8 g of the pCIS nNOS clone and/or 2 g of the GW1 PSD-95 clone were gently pipetted into the Ca2+ chloride solution and empty pBlueScript SK (-) vector was added to bring the total amount of DNA up to 6 g. The mixture was then added drop-wise to the 2xHEBS solution to initiate the formation of phosphate precipitate. Precipitates were allowed to form for 20 minutes at room temperature during which time the media in the flasks was aspirated and replaced with fresh media without selection reagents. The transfection mixture was added drop wise to the cell media and allowed to incubate in a humidified chamber at 37oC with 5% CO2 balanced with room air for 12 hours before the media was replaced with fresh growing media. Transfections which were to be used for immunoprecipitation experiments were incubated an additional 30-36 hours before lysis. Transfections intended for electrophysiology experiments were dissociated 24 hours after transfection using 0.25% Trypsin/EDTA, diluted using fresh growing media and seeded into 35mm petri dishes at 10% confluency. The nitric oxide assay required the transfections to be dissociated and diluted in phenol free DMEM (Invitrogen, Grand Island, NY, USA) and seeded into 12 well cell culture plates (Costar: 3336) at 30% confluency. For experiments utilizing the HEK293TREX cell line stably expressing Kir2.3 and Cav3.2 under an inducible promoter (a gift from Neuromed Pharmaceuticals: Vancouver, BC, Canada), was induced with 1.5 g/ml doxycycline to initiate Cav3.2 expression at this time. The newly seeded 35 mm dishes and the 12 well culture plates were incubated for an additional 24 hours before use.  75  Table 2.2: Agents utilized to maintain selection pressure for the transfected constructs Cell Type HEK293H  Type of Expression -Stable  HEK293H  -Stable  HEK tsa201 HEK tsa201  -Stable  HEK293TREX  -Stable -Inducible (1.5g/ml Doxycyclin) -Stable  Construct Cav3.2 (Human) Cav3.2 (Human) with PDZ-3 mutation  Selection Agent(s) 50 g/ml Zeocin  -Stable  Cav3.2 (Human)  25 g/ml Zeocin  -Stable  Kir 2.1 (Human) Cav3.2 (Human) with PDZ-3 mutation Kir 2.1 (Human)  300 g/ml Geneticin  Cav3.2 (Human) Kir 2.3(Human)  50 g/ml Zeocin  25 g/ml Zeocin  Mutation Details DDPV to DAPA  DDPV to DAPA  300 g/ml Geneticin 150 g/ml Hygromycin 600 g/ml Geneticin 10 g/ml Blasticidin  Table 2.2: Agents utilized to maintain selection pressure for the transfected constructs Several cell lines stably expressing Cav3.2 were used in this study. To maintain the stable expression of transfected constructs the various cell lines were cultured in the presence of selection agents. The concentration of the selection agents used for each cell line is listed in table. Doxycycline was used to induce the expression of the Cav3.2 construct in the HEK-TRex cell line.  2.2.6. Generation of stable cell lines The wild type Cav3.2 clone and the Cav3.2 clone with the mutant PDZ-3 ligand were transfected into HEK293 cells and into tsa201 cells, a HEK293 derivative, already stably expressing the inward rectifying potassium channel Kir2.1 (a gift from Neuromed Pharmaceuticals: Vancouver, Canada) to generate cell lines which stably express either the wild type or mutant Cav3.2 channel. The cells were maintained and transfected using the protocol described in Section “2.2.5. Cell Culture and Transfection”. Total DNA used for the 76  transfection was 1.8 g of either the wild type or PDZ-3 mutant Cav3.2 clone and 4.2 g of an empty pBlueScript SK (-) vector. The pcDNA3.1 zeo (+) plasmid contains a zeocin resistance gene therefore transfected HEK293 cultures were treated with 50 g/ml zeocin (Invitrogen, Grand Island, NY, USA) to select for cells transfected with the pcDNA3.1 zeo (+) Cav3.2 clone. A Kir2.1 inward rectifier clone which confers geneticin resistance was previously transfected and was stably expressed in tsa201 cells, therefore 25 g/ml zeocin and 300 g/ml geneticin (Invitrogen, Grand Island, NY, USA) were used to select for cells expressing both the Cav3.2 clone and the Kir2.1 clone. A media change was performed 12 hours after transfection and did not include selection reagents; however, subsequent media changes contained the selection agents and were performed every 24 hours. After 1 week the cells were dissociated with 0.25% Trypsin-EDTA and diluted in fresh selection media so that the re-seeded confluency in the fresh T-25 flask was 20%. The selection media was replaced daily until the number of cells perishing daily due to lack of the antibiotic resistance was substantially reduced, at which time the cells were dissociated and re-seeded into 24-well cell culture plates (BD Biosciences, Mississauga, ON, Canada) at 5% confluency. Two additional selection media changes were performed 24 hours apart and then the frequency of the media changes was reduced to once every 72 hours. In the following weeks, colonies developed from individual cells (1-2 per well) which were gently removed with a 1000 l pipette tip, mechanically dissociated in fresh selection media and re-seeded into 24 well culture plates. Every 72 hours the selection media in the 24 well plates was replaced and as the cultures in various wells reached confluency they were dissociated and transferred to 12 well culture plates (BD Biosciences, Mississauga,ON, Canada), then 6 well culture plates (Fisher Scientific, Ottawa, ON, Canada) and finally T-25 flasks. Each new cell line was tested for Cav3.2 expression using Western blot analysis and whole cell patch clamp electrophysiology. 77  Acceptable cell lines were expanded and cryogenically frozen in freezing media containing 7.5% heat inactivated FBS, 12.5% DMSO and 0.5% non-essential amino acids in DMEM.  2.2.7. GST pull down Glutathione sepharose 4B beads (GE Healthcare, Baie d’Urfe, QC, Canada) were used to precipitate the wild type Cav3.2, wild type Cav3.1 and Cav3.2 with the deleted PDZ-3 ligand GST fusion proteins. The beads were washed once with 10 volumes of phosphate buffered saline solution (PBS) (136 mM NaCl, 2.6 mM KCl, 1 mM Na2HPO4*7H2O, 1.8 mM KH2PO4, pH 7.4), twice with 10 volumes of GST lysis buffer and re-suspended in GST lysis buffer immediately before use. 200 l of GST fusion protein extract was incubated with a 40l bed volume of washed beads for 30 minutes with gentle agitation at room temperature. A two minute centrifugation step 500 x g was applied to sediment the sepharose beads so that the lysate could be aspirated and the beads retained. The bead bound GTS fusion proteins were then used to probe 50 l of lysate extracted from human embryonic kidney cells (HEK293) (ATCC, Manassas, VA, USA) which were transiently transfected with the nNOS clone and the total sample volume was brought to 250 l with GST lysis buffer. After one hour incubation the sepharose beads were washed once with 10 volumes of PBS and twice with higher stringency washes of 10 volumes of PBS with an additional 100 mM NaCl added. Two washes of 80ul glutathione elution buffer (10 mM reduced glutathione (Sigma-Alrich, St.Louis, MO, USA) and 50mM Tris, pH 8.0 with HCl) were used to release the GST fusion and associated proteins from the beads. The eluate was denatured with the addition of an equal volume of Laemmli buffer (0.5 M Tris, 10% SDS, 20% glycerol, 2% b-mercaptoethanol, 0.004% bromophenol blue) and 10 minute incubation at 80oC. 78  2.2.8. Co-immunoprecipitation To ensure that the ratio of Cav3.2, nNOS and PSD-95 proteins were consistent between samples cell lysate from HEK293 cells expressing Cav3.2, nNOS and PSD-95 was prepared individually and the lysates were mixed immediately prior to incubation with the Cav3.2 specific polyclonal antibody used to precipitate Cav3.2. Cav3.2 lysate was harvested from HEK293 cells stably expressing either wild type Cav3.2 clone or the Cav3.2 clone with the mutant PDZ-3 ligand. nNOS and PSD-95 lysate was harvested from HEK293 cells that were transiently transfected with the respective plasmid clones using the methodology described in section “2.2.5. Cell Culture and Transfection”. To harvest the lysate the cells were grown to confluency in a T-25 flask, washed twice with 1ml of PBS and scraped using a sterile cell scraper into 300 l of lysis buffer (50 mM Tris, 150 mM NaCl, 50 mM EDTA, pH 7.5) with 0.5% NP-40 and Complete EDTA-free protease inhibitor (Roche, Laval, QC, Canada) added. The lysis solution was repeatedly passed through a 26 gauge syringe followed by a 29 gauge insulin syringe to mechanically disrupt the cell membrane and then centrifuged at 18,000 x g for 30 minutes at 4oC to pellet to cell debris so the supernatant could be transferred to a fresh 1.5 ml tube. The nNOS lysate was pre-cleared at 4oC for 30 minutes on a rotating mixer with 50 l/ml Protein G PLUS Agarose (Santa Cruz Biotechnology: Santa Cruz, CA, USA) which was washed 3 times with 10 volumes of lysis buffer and re-suspended in lysis buffer with 0.5% NP-40 added. Following the pre-clear, the nNOS lysate was centrifuged at 1,000 x g for 5 minutes at 4oC and the supernatant was retained. A Qubit Protein Assay Kit (Invitrogen, Grand Island, NY, USA) was utilized to determine the concentration of the lysates. Only fresh lysate was used to in the immunoprecipitation samples and, unless otherwise indicated, the amount of each lysate used was 200g of Cav3.2 lysate, 150g of nNOS lysate and 100g of PSD-95. In addition to the 79  cell lysate, the immunoprecipitation samples consisted of, 0.4 g of Cav3.2 specific polyclonal antibody (Santa Cruz Biotechnology: Santa Cruz, CA, USA: Cat-SC-16261), 30 l of washed protein G PLUS agarose, and were brought up to a final volume of 200 l using lysis buffer without NP-40. After incubating the samples overnight at 4oC on a rotating mixer the samples were washed once with 1.5 ml lysis buffer with 0.5% NP-40, twice with 1.5ml of lysis buffer without NP-40 and re-suspended in 35 l of 2x Laemmli buffer. The resuspended samples were denatured at 80oC for 10 minutes and stored at -20oC.  2.2.9. Western blot analysis NuPAGE® 4–12% Bis-Tris 20 well Gels (Invitrogen, Grand Island, NY, USA) and a Novex NuPAGE® SDS-PAGE Gel System were used to separate the proteins in the samples by molecular weight. MOPS running buffer (50 mM MOPS, 50 mM Tris, 0.1% SDS, 1 mM EDTA, pH 7.7) was used to provide denaturing and reducing conditions during separation. 15 l of sample was loaded in each well and a constant voltage of 120 V was applied for 80 minutes to separate the proteins. Protein transfer to a membrane consisted of tightly sandwiching the gel containing the proteins between the Hybond ECL Nitrocellulose membrane (GE Healthcare, Baie d’Urfe, QC, Canada) and filter paper, submersing in western blot transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol (vol/vol) and 0.001% SDS) and applying a constant 100 V potential across the surface of the gel for 1 hour so that the proteins migrate towards the positively charged pole of the apparatus and bind to the nitrocellulose membrane. A thirty minute incubation on a shaker with 2% skim milk in PBS was used to block the membrane and prevent non-specific antibody binding. Incubation with primary antibodies was performed at room temperature with shaking for 2 hours followed by three 15 minute 80  washes with 25 ml 2% skim milk in PBS. Antibodies specific to Cav3.2 (Santa Cruz Biotechnology: Santa Cruz, CA, USA: Cat- SC-16261), nNOS (Santa Cruz Biotechnology: Santa Cruz, CA, USA: Cat-SC5302) or PSD-95 (Fisher Scientific, Ottawa, ON, Canada: CatMA1-046) were diluted in 2% skim milk in PBS at 1:2,000, 1:5,000 and 1:2,000 respectively. Peroxidase conjugated secondary antibodies, donkey anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA,USA:Cat-SC-2020) for use against the Cav3.2 primary antibody and goat anti-mouse (Fisher Scientific, Ottawa, ON, Canada: PI31444) for use against the nNOS and PSD-95 primary antibodies were diluted 1:10,000 in 2% skim milk and incubated with the membrane for 1 hour. Three 15 minute washes of 25ml PBS were used to remove excess blocking agent and excess antibodies from the membrane. The SuperSignal West Pico Chemiluminescent Substrate (Fisher Scientific, Ottawa, ON, Canada) and Amersham Hyperfilm ECL film(GE Healthcare, Baie d’Urfe, QC, Canada) were utilized to visualize the protein bands. Equal volumes of the peroxide solution and the luminol solution of the chemiluminescent substrate were mixed as needed and 3ml of the mixture was incubated on the membrane for 5 minutes. Excess chemiluminescent substrate was poured off and the membrane was gently pulled across the lip of the incubation tray to remove the remaining substrate before the membrane was wrapped in plastic film. A dark room was utilized to expose the membranes to the film. Typically, three exposures were made with durations of 5 seconds, 30 seconds and 2 minutes. Additional exposures of longer or shorter duration were made if necessary. A Kodak X-Omat M35 processor was used to develop the film.  81  2.2.10. Nitric oxide detection assay A flourometric assay was developed using Diaminorhodamine-4M AM (DAR-4M AM) (Millipore, Billerica, MA, USA) to detect nitric oxide production within HEK293 cells transiently transfected with nNOS. Cells were transfected and prepared for the assay using the methodologies described in Section “2.2.5. Cell Culture and Transfection”. On the day of the experiment the cells were loaded with DAR-4M by replacing the media with loading buffer (18.4 mM HEPES, 118 mM NaCl, 11.7 mM D-glucose, 1.2 mM K2HPO4, 0.5 mM MgSO4*7H2O, 4 mM KCl, 10 M DAR-4M AM, 295 mOsm/kg (adjusted with mannitol), pH 7.2) (adapted from (Kim et al., 2004; Belardetti et al., 2009)) and incubated for 25 minutes at 37oC and 5% CO2/room air atmosphere. The acetomethylester (AM) component allows the dye to pass through the cell membrane where cellular esterases cleave the AM component leaving the non-permeable DAR-4M fluorophore confined within the cell. The loading buffer was aspirated and excess dye was removed with a single wash using 600ul wash buffer (140 mM choline chloride, 16mM HEPES, 10 mM D-glucose, 1mM MgCl2*6H2O ,1mM KCl, 295 mOsm/kg, pH 7.4 (adjusted with N-Methyl-D-glucamine)) (adapted from (Belardetti et al., 2009)). A 20 minute incubation at 37oC and 5% CO2/room air atmosphere in 600ul of assay buffer (140 mM choline chloride, 16 mM HEPES, 10 mM D-glucose, 1 mM MgCl2*6H2O ,1 mM KCl, 2 mM CaCl2, 25 M N[Tris(hydroxymethyl)methyl]glycine (tricine), 295 mOsm/kg, pH 7.4 (adjusted with NMethyl-D-glucamine)) (adapted from (Belardetti et al., 2009)) prepared the cells for the depolarization step. Calcium entry was facilitated by either introducing ionomycin, a Ca2+ ionophore, at a concentration of 7 M or by activating the Cav3.2 channels through chemical depolarization which involved rapidly increasing the KCl concentration of the assay buffer by 65 mM (66 mM final concentration). Ionomycin treated cells were incubated for 1 hour 82  and the chemically depolarized cells were incubated for 20 minutes at 37oC and 5% CO2/room air atmosphere before the relative fluorescence was measured using a GeminiXS fluorometer using the SoftMAX Pro software version 4 (Molecular Devices, Sunnyvale, CA, USA). The fluorometer was set to excite the sample at 530 nm and read the emission at 580 nm with a cutoff value of 570 nm and with the photomultiplier set to automatic. A total of 69 data points were per well were analyzed to obtain average relative fluorescence for each well. Each data set was normalized to the average relative fluorescence of the wells of the same set which were stimulated but treated with an inhibitor. For example: the data from the nNOS transfected cells stimulated with KCl was normalized to nNOS transfected cell stimulated with KCl stimulated but inhibited with L-NAME (Sigma-Alrich, St.Louis, MO, USA). nNOS activity was inhibited with the application of L-NAME at a concentration of 300 M which was added to both the loading buffer and the assay buffer of the appropriate wells. The Cav3.2 channels were inhibited with 25 M NiCl2 which was added to the appropriate wells when the assay buffer was applied. All data were exported in text format using the SoftMAX pro software and imported into MicroSoft Excel 2010 to calculate the average signal for each well and to perform the normalization calculations. Statistical analysis, ANOVA followed by Tukey’s Multiple Comparison test, was performed using the GraphPad Prism software version 5.  2.2.11. Whole cell patch clamp analysis Whole cell electrophysiological recordings of the exogenous Ca currents due were obtained from HEK293 cells stably expressing either the wild type Cav3.2 or Cav3.2 with the mutant PDZ-3 ligand. GFP tagged nNOS and DsRED tagged PSD-95 were individually or co-transfected into the stable cell lines using the Ca2+ phosphate protocol outlined in Section 83  “2.2.5. Cell Culture and Transfection” and electrophysiological recordings were obtained using the whole cell patch clamp technique 48 hours after transfection. The cells were bathed in external recording solution consisting of 92 mM cesium chloride, 40 mM tetraethylammonium chloride, 10 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, 10 mM Dglucose, 150 M L-arginine, 25 M tricine, 300 mOsm/kg, pH 7.4 and electrically grounded with a 3M KCl agar bridge. Borosilicate glass patch pipettes (Sutter Instrument Company, Novato, CA, USA: Cat-BF150-86-10) with 3-5  resistance were made using a horizontal puller (Sutter Instrument Company, Novato, CA,USA), fire polished with a mircoforge (Narishige, East Meadow, NY, USA) and filled with internal solution (130mM cesium methanesulphonate, 10 mM HEPES, 2 mM MgCl2*6H2O, 0.1 mM EDTA, 5 mM Mg-ATP, 0.3 mM Na-GTP, 10 M L-arginine, 100 nM flavin adenine dinucleotide disodium salt hydrate, 100 nM (6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride, 295 mOsm/kg, pH 7.4). An Axopatch 200A amplifier and CV203BU head stage were utilized to record the whole cell currents (Molecular Devices, Sunnyvale, CA, USA). The recordings were filtered at 2 kHz using the low pass Bessel filter on the amplifier, digitized with a Digitdata 1322A (Molecular Devices, Sunnyvale, CA, USA), sampled at 10 kHz and stored using a personal computer running the pClamp version 9 software package (Molecular Devices, Sunnyvale, CA, USA). Currents were measured during 80ms depolarizing pulses from a holding membrane potential of -110 mV to membrane potentials ranging from -80 to 50 mV in 10 mV steps with 5mV steps between -50 and -10 mV and plotted as a function of the depolarizing potential. To avoid accumulation of inactivation there was a three second pause at a holding potential of -110mV between test pulses. Current-voltage curves were fitted using a modified Boltzmann equation: I=(Gmax*(Vm-Er))/(1+exp((Vm-V50)/k)), where Gmax is the maximum 84  slope conductance, Vm is the depolarizing potential, Er is the reversal potential, V50 is the half-activation potential and k is the slope of the activation curve (goodness of fit had R2 values ≥ 0.993). The conductance was calculated from the current-voltage curves and plotted as a function of membrane potential to generate activation curves. The data was fit with the Boltzmann equation: G/Gmax=A1+(A2-A1)/(1+exp((Vm-V50)k))), where A2 is the maximum normalized conductance, A1 is the minimum normalized conductance, Vm is the test potential, V50 is the half-activation potential and k is the slope of the activation curve (goodness of fit had R2 values ≥ 0.993). Current density due to Ca2+ ion flow was determined for each cell using the whole cell recordings used to generate the current voltage relationship and the membrane capacitance compensation values from the amplifier (Iwasaki et al., 2008). The recordings were analyzed to determine the absolute value for the peak current (pA) due to Ca2+ ion flow. The peak current values were then divided by the capacitance of the cell (pF), which correlates with cell size, to determine the current density due to Ca2+ ion flow (pA/pF) for each cell. Normalization of the current density data consisted of dividing the current density for each cell by the average current density of similarly transfected cells treated with LNAME. For example: [cell current density (Cav3.2+nNOS)] / [average current density (Cav3.2+nNOS+L-NAME)] Inactivation curves were generated by calculating the maximum normalized current from currents measured during a 60 ms membrane depolarization to a membrane potential of -40mV from a series of holding potentials ranging from -120 to -20 mV in 10 mV steps and plotting as a function of membrane potential. The holding potentials were held for 10 seconds to allow inactivation of the channel to reach a steady state. The data was fit using the 85  Boltzmann equation: I/Imax=A1+(A2-A1)/(1+exp((Vm-V50)k))), where A2 is the maximum normalized conductance, A1 is the minimum normalized conductance, Vm is the test potential, V50 is the half-activation potential and k is the slope of the activation curve (goodness of fit had R2 values ≥ 0.993). Axon instruments’ pClamp version 9 software was utilized to extract the data from the recorded currents, Excel 2010 (Microsoft) was used to perform the normalization calculations and Origin software version 8 (OriginLabs, Northampton, MA, USA) was used to graph the data and perform statistical analysis.  2.3. Results 2.3.1. Cav3.2 and nNOS interact via the PDZ-3 binding ligand located at the carboxyl terminus of Cav3.2 Protein precipitations utilizing tagged fusion proteins are particularly useful in determining whether a particular domain of one protein interacts with another protein. Precipitations utilizing tagged proteins are generally more robust than immunoprecipitation as concerns regarding antibody affinity and specificity under the precipitation conditions are mitigated. Therefore, to initiate the investigation of whether the putative PDZ-3 binding ligand of CaV3.2 is capable of interacting with nNOS, GST fusion proteins which contain the carboxyl 100 amino acids of the human Cav3.2 isoform were generated and used to probe lysate from HEK293H cells transiently transfected with nNOS. Western blot analysis (Figure 2.2A) shows that the full-length nNOS co-precipitates with the Cav3.2 carboxyl-GST fusion protein but not with a GST fusion protein possessing the 100 carboxyl residues of Cav3.1 channel which lacks the putative carboxyl PDZ-3 motif. Further, deletion of the carboxyl residues that constitute the PDZ-3 binding motif of Cav3.2 (DDPV) resulted in complete loss of nNOS binding demonstrating that the putative binding 86  motif is required for nNOS binding. Non-transfected HEK293H cell lysate (H labeled lanes) was also probed and run alongside the nNOS transfected lysate (N labeled lanes) as negative controls to show that the antibody used in the Western blot analysis was specific for nNOS. Coomassie staining (Figure 2.2A) shows that the amount of GST fusion protein that precipitated was relatively equal across all samples. The full-length Cav3.2 protein is a sizable 255 kDa which may introduce steric effects which could potentially shield the PDZ-3 binding motif of Cav3.2 and inhibit an interaction with nNOS. To address this, full length Cav3.2 was immunoprecipitated from lysate of cells stably expressing Cav3.2 with a Cav3.2 specific antibody in the presence of lysate from cells transiently expressing nNOS. Western blot analysis (Figure 2.2B) shows that nNOS coimmunoprecipitates with full length Cav3.2 but not when the key amino acids of the Cav3.2 PDZ-3 binding motif at positions 0 and -2 (relative to the carboxyl terminus) are substituted with residues not found in the PDZ-3 consensus sequence (Schepens et al., 1997; Stricker et al., 1997). An antibody that does not recognize the Cav3.2 protein was used in immunoprecipitations and run in parallel to demonstrate that the results were not due to the non-specific precipitation of Cav3.2 and nNOS. The Cav3.2/nNOS interaction could be competitively inhibited with synthetic peptides possessing a PDZ-3 binding motif. Figures 2.3A and 2.3B that when a peptide containing a PDZ-3 binding ligand (-DEPV) is introduced the co-immunoprecipitation of nNOS is substantially reduced compared to immunoprecipitations in the presence of a peptide with a mutated PDZ-3 binding ligand (-DEPE).  87  Figure 2.2: Cav3.2 and nNOS can physically interact via the carboxyl PDZ-3 binding ligand of Cav3.2  88  Figure 2.2: Cav3.2 and nNOS can physically interact via the carboxyl PDZ-3 binding ligand of Cav3.2 (A) GST fusion proteins generated from the carboxyl 100 amino acids of human Cav3.2 and human Cav3.1 were incubated with full length nNOS from lysate of nNOS transfected (“N” labeled lanes) and non-transfected (“H” labeled lanes) HEK293H cells and a nNOS specific monoclonal antibody was used to probe the membrane in the Western analysis (top panel). nNOS co-precipitated with the GST-Cav3.2 fusion protein but not with the GST or GST-Cav3.1 fusion protein controls which lack the PDZ-3 binding ligand (-DDPV-COOH). Additionally, nNOS did not co-precipitate when the last four amino acids (-DDPV-COOH), which comprise the PDZ-3 binding ligand, were removed from the carboxyl terminus of the GST-Cav3.2 fusion protein (GST-Cav3.2mut del. DDPV). Coomassie staining (bottom panel) shows that the amount of GST fusion peptide recovered in the precipitations was comparable to amount recovered with the control precipitations where no HEK293 lysate was added (“P” labeled lanes). (B) Co-immunoprecipitation of full length Cav3.2 and nNOS. nNOS co-immunoprecipitated with full length human Cav3.2 using a polyclonal Cav3.2 specific antibody but did not co-immunoprecipitate when the key residues of the PDZ-3 binding ligand of Cav3.2 were mutated from -DDPV-COOH to –DAPA-COOH (Cav3.2mut (-DAPA-COOH). An antimGluR1 antibody was used as a control against non-specific precipitation.  89  Figure 2.3: The physical interaction of Cav3.2 and nNOS can be competitively disrupted using a peptide containing the PDZ-3 binding ligand.  Figure 2.3: The physical interaction of Cav3.2 and nNOS can be competitively disrupted using a peptide containing the PDZ-3 binding ligand. A TAT fusion peptide consisting of the transactivator of transcription (TAT) coding sequence (GRKKRRQRRRPQ) and the PDZ-3 binding ligand of the rat Cav3.2 channel (-DEPV-COOH) was designed to compete for the PDZ-3 domain of nNOS and disrupt the Cav3.2/nNOS interaction. As a control, a second peptide was designed with an amino acid substitution at key residue of the PDZ-3 binding ligand (-DEPE-COOH). The Cav3.2/nNOS (A) and Cav3.2/nNOS/PSD-95 (B) samples were prepared using lysate from separate HEK293 cultures individually transfected with Cav3.2, nNOS or PSD-95. A Cav3.2 specific antibody was utilized to precipitate Cav3.2 and the associated proteins in the presence of the TAT-DEPV or the TAT-DEPE peptides. Cav3.2 specific and nNOS specific antibodies were used to detect the precipitated proteins in Western analysis. It can be seen that coincubation of TAT-DEPV reduces the amount of nNOS co-immunoprecipitation in both samples whereas the TAT-DEPE peptide does not. In the presence of PDS-95 (B) a noticeable increase in the amount of nNOS that co-precipitated with Cav3.2 was observed. The band intensity was 4.95 times more intense (when normalized to Cav3.2 staining) in the presence of PSD-95 even though the nNOS concentration in the Cav3.2/nNOS/PSD-95 lysate mix was less than half the concentration used in the Cav3.2/nNOS lysate mix. 90  2.3.2. The PDZ-3 domain and the PDZ-2 beta finger binding ligand of nNOS can facilitate the formation of larger protein complexes In addition to possessing a PDZ-3 binding domain, nNOS also possesses an internal PDZ-2 binding ligand in a head-to-tail arrangement (Hillier et al., 1999; Tochio et al., 2000). This head-to-tail arrangement stabilizes the internal PDZ-2 binding ligand so that it can interact with proteins possessing PDZ-2 domains such as PSD-95 and syntrophin (Brenman et al., 1996a, 1996b). The presence of both a PDZ-3 domain and an internal PDZ-2 binding ligand presented the possibility that nNOS can facilitate the formation of larger protein complexes. To test this possibility, co-immunoprecipitation experiments were performed in the presence of PSD-95. Figure 2.4A shows that when nNOS is present PSD-95 is able to coimmunoprecipitate with Cav3.2 indicating that nNOS can interact with both Cav3.2 and PSD95 simultaneously. The co-immunoprecipitation of PSD-95 in the presence of a competing peptide containing a PDZ-3 binding ligand is markedly reduced compared to a peptide with a mutation in the PDZ-3 motif providing further evidence that the interaction between Cav3.2 and PSD-95 is facilitated by nNOS (Figure 2.3B). Interestingly, it was observed that the apparent efficiency of the nNOS coimmunoprecipitation increased substantially when PSD-95 was introduced. To explore this, Cav3.2, nNOS and PSD-95 lysate was harvested separately and specific amounts of each lysate were mixed such that the amount of Cav3.2 and nNOS was kept constant while the amount of PSD-95 varied. Western blot analysis of the immunoprecipitations shows that as the concentration of PSD-95 in the mixture increased, the amount of nNOS that coimmunoprecipitated with Cav3.2 also increased (Figure 2.4B). To address the possibility that the additional cell lysate containing the PSD-95 protein may have contributed to the increased nNOS immunoprecipitation, possibly through the stabilization of the proteins 91  and/or the interaction site, Cav3.2 and nNOS were immunoprecipitated with either an equal concentration of non-transfected HEK cell lysate or 2% glycerol in order to help stabilize the interaction (Vagenende et al., 2009). As observed in Figure 2.4C, neither untransfected HEK cell lysate nor 2% glycerol increased the amount of nNOS co-immunoprecipitation, and in fact the treatments appeared to have had a negative effect.  Figure 2.4: The Cav3.2/nNOS complex can interact with PSD-95 to form a larger complex.  92  Figure 2.4: The Cav3.2/nNOS complex can interact with PSD-95 to form a larger complex. Adjacent to the PDZ-3 binding domain of nNOS is a PDZ-2 beta-finger binding ligand which is known to interact with proteins that possess a PDZ-2 binding domain such as PSD-95 and Syntrophin. (A) Western blot analysis of the Cav3.2 immunoprecipitation from HEK cell lysate containing Cav3.2, nNOS and PSD-95 shows that PSD-95 co-precipitates with Cav3.2 only when nNOS is present in the lysate mixture. To ensure a consistent ratio of Cav3.2, nNOS and PSD-95 protein in the lysate samples the Cav3.2, nNOS and PSD-95 plasmids were transfected individually into separate HEK293H cell cultures and cell lysate from each culture was mixed immediately prior to incubation with the Cav3.2 specific polyclonal antibody. (B) The amount of nNOS that coimmunoprecipitates with Cav3.2 increases as the concentration of PSD-95 in the lysate mixture is increased. The Cav3.2 and nNOS concentrations remained the same for all samples and the concentration of PSD-95 was increased by adding additional PSD-95 transfected HEK lysate. All samples were brought to equal volumes using the same buffer used to prepare the lysates. To determine if the increase in nNOS co-precipitation was due to a stabilization of the Cav3.2/nNOS interaction due to the overall increase in HEK protein levels the immunoprecipitations were repeated using non-transfected HEK lysate and with 2% glycerol. (C) The addition non-transfected HEK293H cell lysate or 2% glycerol failed to increase nNOS co-precipitation and actually reduced the amount of nNOS precipitated with Cav3.2.  2.3.3. A physical interaction between Cav3.2 and nNOS is required for Cav3.2 activity to stimulate the production of nitric oxide by nNOS A NO sensitive fluorometric assay was developed in order to determine if Cav3.2 channel activity can result in the stimulation of nNOS to produce NO. nNOS is considered to be Ca sensitive in that binding with activated calmodulin is required to stimulate the enzymatic activity. HEK cells have been reported to express calmodulin however in 93  heterologous expression systems the endogenous levels of Ca2+ may be a limiting factor (Persechini and Stemmer, 2002; Dai et al., 2010; Tian et al., 2011). To determine if there is sufficient calmodulin present in HEK cells to activate nNOS the ionophore ionomycin was used to facilitate Ca2+ influx. Figure 2.5A illustrates that NO can be detected nNOS transfected cells following the application of ionomycin. nNOS transfected cells display a significant increase (p<0.001) in fluorometric signal compared to cells treated with the nNOS inhibitor L-NAME and to non-ionomycin treated cells. No increase in fluorometric signal was observed in sham transfected cells treated with ionomycin. For each group the data were normalized to the L-NAME treated cells. These results indicate that endogenous levels of calmodulin are sufficient for the activation of nNOS. tsa201 HEK cells stably expressing both Cav3.2 and an inwardly rectifying potassium channel (Kir2.1) were utilized in this assay to investigate whether Cav3.2 activity can activate nNOS (Figure 2.5B). Expression of Kir2.1 allowed for the manipulation of the resting membrane potential into a more hyperpolarized state and removing Cav3.2 channel inactivation. The cells could then be depolarized chemically by a rapid increase in the external KCl concentration. In the nNOS co-transfected cells chemical depolarization to a calculated membrane potential of ~-30 mV produced a significant increase (p<0.001) in the fluorometric signal compared to the L-NAME inhibited and non-stimulated cells. Contrastingly, KCl-induced depolarization had no effect on non-transfected control cells. For each group the data was normalized to the L-NAME treated cells. To demonstrate that the increase in NO sensitive fluorometric signal was due to the activation of Cav3.2 channels, a cell line stably expressing Cav3.2 under an inducible promoter as well as constitutive Kir2.3 expression (an inward rectifying potassium channel) was examined. The cells were transfected with nNOS and stimulated under Cav3.2 induced 94  and non-induced conditions. Chemical depolarization produced a significant increase (p<0.001) in NO fluorometric signal in the induced cells compared to non-induced cells (Figure 2.5C). Block of Cav3.2 channels with 25 M Ni2+ application (a concentration known to inhibit Cav3.2) (Lee et al., 1999b) prevented the ability of KCl depolarization to induce NO production. In these experiments the data were normalized to the noninduced/Ni2+ treated cells. A cell line stably expressing Kir2.1 and Cav3.2 with point mutations introduced into the PDZ-3 binding ligand was further generated to determine whether the Cav3.2/nNOS physical interaction is required for the stimulation of nNOS (and NO production) following Cav3.2 activation. When selecting an appropriate stable cell line the Cav3.2 current density was measured and matched to a cell line stably expressing Kir2.1 and wild type Cav3.2 to ensure that Ca2+ influx was relatively similar in both groups. Both cell lines were transiently transfected with nNOS and chemically depolarized with KCl. The wild type Cav3.2 cell line produced a significantly greater (p<0.05) fluorometric signal in the KCl-stimulated nNOS transfected cells compared to both non-stimulated cells and to stimulated cells treated with LNAME (Figure 2.5D). Of note, no significant difference was observed in the NO fluorometric signal between the stimulated, non-stimulated and L-NAME treated stimulated groups of the cells expressing the Cav3.2 channel with point mutations in the PDZ-3 binding ligand (-DAPA). For each group the data were normalized to the L-NAME treated cells.  95  Figure 2.5: The PDZ-3 binding interaction between Cav3.2 and nNOS is required for Cav3.2 activity to stimulate nNOS to produce nitric oxide.  96  Figure 2.5: The PDZ-3 binding interaction between Cav3.2 and nNOS is required for Cav3.2 activity to stimulate nNOS to produce nitric oxide. A NO-sensitive fluorometric assay was developed to detect NO production following stimulation of Cav3.2 channels. (A) Demonstration of the NO detection capabilities of the assay. The ionophore ionomycin was used to allow Ca2+ to permeate the membrane of HEK293H cells transfected with nNOS or empty vector (sham) and preloaded with the NO-sensitive fluorophor DAR4M AM. Ionomycin treatment produced a significant increase in the fluorescent signal in cells transfected with nNOS and this increase in signal was inhibited when the cells were pre-incubated with the nNOS inhibitor L-NAME (p<0.001 [ANOVA followed by Tukey’s multiple comparison test], n=4). No significant difference in the fluorometric signal was observed in the sham transfected cells. (B) HEK293H cells stably expressing a potassium inward rectifier (Kir2.3) and Cav3.2 under an inducible promoter were transfected with nNOS or an empty vector and chemically depolarized with a rapid increase in the external KCl concentration from 1 mM to 66 mM. Cav3.2 expression was induced in both nNOS transfected and sham transfected cells and chemical depolarization produced a significant increase in fluorometric signal in the nNOS transfected cells (p<0.001 [ANOVA followed by Tukey’s multiple comparison test], n=8). The increase in signal was not observed in nontransfected cells and was inhibited in the transfected cells by pre-incubation with 300 M L-NAME. (C) To demonstrate that the increase in the NO fluorescent signal is due to the activation of Cav3.2, induced cells were compared to non-induced cells using nNOS transfected stable Kir2.3/inducible Cav3.2 cells. Depolarization with KCl produced a significant increase in fluorescent signal in the Cav3.2 induced cells and this signal was inhibited with the application of Ni2+ at a concentration known to inhibit Cav3.2 (25 M) (p<0.001) [ANOVA followed by Tukey’s multiple comparison test], n=27). D: A physical interaction between Cav3.2 and nNOS is required for activation of nNOS by Cav3.2. Cells stably expressing Kir2.1 and either Cav3.2 with wild type carboxyl PDZ-3 binding ligand (-DDPV-COOH) or Cav3.2 with mutant carboxyl PDZ-3 binding ligand (-DAPA-COOH) were transfected with nNOS and then chemically depolarized. An increase in 97  NO fluorescent signal was observed when the Cav3.2 PDZ-3 binding ligand was intact (p<0.05 [ANOVA followed by Tukey’s multiple comparison test], n=12) whereas no increase in signal was observed when the PDZ-3 binding interaction was interrupted (p>0.05 [ANOVA followed by Tukey’s multiple comparison test], n=12). In panels A, B and D each experimental data group was normalized to the L-NAME inhibited data set within the group. In panel C the data was normalized to the non-induced, KCl-stimulated group. Error bars represent standard error. The n’s indicate the number of cultures tested for each condition in the experiment.  2.3.4. nNOS activity inhibits Cav3.2 channels in a heterologous expression system The redox modulation of Cav3.2 channels by NO results in a reduction in the maximum amplitude of macroscopic currents (Joksovic et al., 2007). The maximum macroscopic current that can be recorded from a cell depends on several factors including the physical size of the cell and the channel expression level. The physical size of the cell can be accounted for by determining the current density which is a measure of the whole cell current divided by the capacitance of the cell membrane which is reflective of the size of the cell (Haedo and Golowasch, 2006; Khorkova and Golowasch, 2007; Iwasaki et al., 2008; Golowasch et al., 2009). Cell lines stably expressing Cav3.2 were utilized to minimize the effects of varying expression levels on the current density measurements. With the aforementioned considerations, a reduction in the macroscopic Cav3.2 current due to nNOS activity would be expected to present as a reduction in T-type current density. It was noted that the maximal T-type current density of cells stably expressing Cav3.2 and transiently expressing nNOS was approximately 25% lower (statistical significance p<0.01) than similar cells treated with the nNOS inhibitor L-NAME suggesting that nNOS activity might basally inhibit Cav3.2 channels (Figure 2.6A). The application of L-NAME 98  had no significant effect on T-type current density in non-nNOS transfected cells further supporting the notion that the effect results from the inhibition of nNOS activity and not as a result of a non-specific enhancement of Cav3.2 currents/expression by the NOS inhibitor. No significant differences in the current/voltage relationship (IV curve), steady state inactivation and steady state activation of Cav3.2 currents were observed between non-transfected and nNOS transfected cells (Figure 2.6B) or between L-NAME inhibited and non-inhibited cells (Figure 2.6C). Additionally, no obvious differences were observed in Cav3.2 current kinetics under the various experimental conditions (Figure 2.10B). nNOS co-expression has no significant effect on T-type current density when expressed in cells stably expressing Cav3.2 containing point mutations in its PDZ-3 binding ligand site (Figure 2.7A). This result suggests that the Cav3.2/nNOS physical interaction is required for the nNOS/NO-mediated inhibition of Cav3.2. To support these findings, the current density was measured in cells expressing Cav3.2 and nNOS and treated with membrane permeable TAT fusion peptides containing either an intact PDZ-3 binding ligand and or a non-functional mutant PDZ-3 ligand (Figure 2.7B). Competitively disrupting the Cav3.2/nNOS interaction with a TAT fusion peptide possessing an intact PDZ-3 ligand relieved the reduction in T-type current density to the same extent as that for L-NAME treatment (p<0.05). Contrastingly, in cells treated with the TAT fusion peptide containing the mutant PDZ-3 ligand T-type current density remained suppressed, similar to that observed for L-NAME treatment. The TAT fusion peptide itself did not directly affect Cav3.2 currents as application of the peptide had no effect on the current density (see Figure 2.10A). Figure 2.7C shows that the mutation of the PDZ-3 binding ligand of Cav3.2 did not alter the current voltage relations of the channel compared to wild type.  99  A well characterized target of NO is sGC, an enzyme which synthesizes cGMP from GTP (reviewed in (Krumenacker et al., 2004; Potter, 2011)). cGMP-dependent protein kinases (PKG) have been demonstrated to affect T-type currents in newt olfactory receptor cells (Kawai and Miyachi, 2001) and although direct modulation of Cav3.2 by PKG has not been reported, there is a possibility that downstream effectors may influence the channel. To exclude sGC activity and subsequent activation of secondary messenger pathways as the mechanism of Cav3.2 inhibition in this study, the well documented sGC inhibitor [1H[1,2,4]oxadiazolo-[4,3-]quinoxalin-1-one] (ODQ) was utilized at a concentration of 20 M (Zhao et al., 2000). Figure 2.8A illustrates that the application of ODQ or the solvent (DMSO) both failed to relieve the reduction in T-type current density indicating that the inhibition of Cav3.2 currents does not involve the activation of sGC or its downstream effectors. ODQ did have a significant effect on channel activation at membrane potentials of -50 mV and -45 mV, however, the differences at these potentials has minimal contribution to the current density measurements since the current density was calculated from the peak of the IV (~-30 mV) (Figure 2.8B). As the presence of PSD-95 was found to increase the efficiency of the coimmunoprecipitation of nNOS, it was of interest to also examine the effect of nNOS and PSD-95 co-expression on T-type current density in Cav3.2 stably expressing cells. Of note, co-expression of PSD-95 resulted in a further reduction of the T-type current density compared to the cells transfected with nNOS alone (Figure 2.9A; p<0.005 [Student’s t-test]). In cells co-expressing nNOS and PSD-95 the current density was reduced to ~50% of the LNAME treated control cells and current density could be restored by treatment with either LNAME or the competitively disrupting TAT-DEPV fusion peptide (p<0.001). Co-expression of PSD-95 did not have a significant effect on either the Cav3.2 current-voltage relation 100  (Figure 2.9B) or on current kinetics (Figure 2.10B). Additionally, PSD-95 did not have a significant effect on T-type current density of Cav3.2 stably expressing cells in which nNOS was also not co-expressed (Figure 2.10A) indicating that nNOS is required for the increased inhibition of basal Cav3.2 macroscopic currents.  Figure 2.6: nNOS activity inhibits Cav3.2  101  Figure 2.6: nNOS activity inhibits Cav3.2 The T-type current density due to Ca2+ influx was calculated for HEK293H cells stably expressing Cav3.2 with and without transient transfection of nNOS. (A) To determine current density [pA/pF] the peak recorded Ca2+ current [pA] was divided by the capacitance of the cell [pF] which correlates with cell size. The data for each experimental group was normalized to the L-NAME inhibited data. In nNOS transfected cells, the current density due to Ca2+ influx was significantly lower (p<0.01) than when the cells were pre-incubated with the nNOS inhibitor L-NAME. This difference was not observed in the non-transfected cells. (B) Normalized current/voltage (IV) curves and the steady state activation/inactivation curves comparing HEK293H cells stably expressing Cav3.2 and transfected with nNOS to the non-transfected cells. No significant difference was observed between the two groups. (C) Normalized current/voltage (IV) curves and the steady state activation/inactivation curves comparing HEK293H cells stably expressing Cav3.2 and transfected with nNOS under nNOS inhibited (L-NAME treated) and uninhibited conditions. The application of L-NAME did not significantly affect the IV or steady-state inactivation profiles. (D) IV curve comparing HEK293H cells stably expressing Cav3.2 and transfected with nNOS under nNOS inhibited (L-NAME treated) and uninhibited conditions. The error bars represent standard error. (cell numbers: nNOS transfected + L-NAME=18, nNOS transfected no L-NAME=32, non-transfected + L-NAME= 6, non-transfected no L-NAME=6)  102  Figure 2.7: A physical interaction between Cav3.2 and nNOS is required for the basal inhibition of Cav3.2 activity by nNOS  103  Figure 2.7: A physical interaction between Cav3.2 and nNOS is required for the basal inhibition of Cav3.2 activity by nNOS (A) A HEK293H cell line was generated that stably expresses a mutant form of Cav3.2 where key Cav3.2 carboxyl terminal amino acids of the PDZ-3 binding ligand are mutated (-DDPV to DAPA). The Cav3.2 mutant expressing cell line was transfected with nNOS and the current density due to Ca2+ influx was calculated. To determine current density [pA/pF] the peak recorded Ca2+ current [pA] was divided by the capacitance of the cell [pF]. No significant difference of peak current density was observed between the nNOS transfected Cav3.2 mutant expressing cell line under LNAME inhibited and uninhibited conditions. (p>0.05 [Student’s t-test], n=[Cav3.2+nNOS+LNAME=18, Cav3.2+nNOS no L-NAME=32, Cav3.2mut+nNOS+L-NAME=7, Cav3.2mut+nNOS no L-NAME=8]) (B) Normalized current/voltage (IV) curves and the steady state activation/inactivation curves comparing HEK293H cells stably expressing Cav3.2 and transfected with nNOS to HEK293H cells stably expressing the Cav3.2 PDZ-3 mutation (DAPA-COOH) and transfected with nNOS. No significant difference was observed between the two groups. (C) A HEK293H cell line stably expressing Cav3.2 and transiently expressing nNOS was pre-incubated with TAT-fusion peptides designed to competitively disrupt the Cav3.2/nNOS interaction (tat-DEPV). A second peptide similar to the first but with a mutation in a key residue of the PDZ-3 binding ligand (tat-DEPE) was used as a vehicle control. The current density of the TAT-DEPV peptide treated cells was significantly greater than the cells treated with the non-disrupting tat-DEPE control peptide (p<0.05 [Student’s t-test]). The relief in the inhibition of current density was similar to that observed with the application of the nNOS inhibitor L-NAME. (p<0.05 [Student’s t-test], n=[L-NAME=18, no L-NAME=32, tat-DEPV=9, tat-DEPE=9]) (D) Normalized current/voltage (IV) curves from nNOS transfected HEK293H cells stably expressing Cav3.2 with nNOS under normal and tat-DEPV treated conditions. In the current-voltage profile the application of the TAT-DEPV peptide did have one point at -50 mV where the normalized current was significantly less than the non-treated cells (p<0.05 [Student’s t-test]) however, for all other voltages there was no significant difference and since the current density was calculated using the peak current at -30 mV the difference at -50 mV does not 104  affect the current density results. The data in the L-NAME experimental group was normalized to the L-NAME inhibited data set and the data in the TAT peptide data set was normalized to the TATDEPV data set so that the degree of Cav3.2 inhibition could be visualized. The error bars represent standard error.  Figure 2.8: The inhibition of Cav3.2 by nNOS activity is independent of soluble guanylyl cyclase activation  105  Figure 2.8: The inhibition of Cav3.2 by nNOS activity is independent of soluble guanylyl cyclase activation A potential downstream target of NO is soluble guanylyl cyclase (sGC) which, when activated, catalyzes the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). (A) HEK293H cells stably expressing Cav3.2 and transfected with nNOS were treated with either the sGC inhibitor ODQ [1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one] (20M) or dimethyl sulfoxide (DMSO) as the vehicle control. ODQ and DMSO were ineffective at relieving the nNOS-mediated inhibition of Cav3.2 currents as current density remained significantly lower than that for L-NAME treated cells (p<0.001 [ANOVA followed by Tukey’s Multiple Comparison test], n=[L-NAME=18, no L-NAME=32, DMOS=8, ODQ=10). The data for all data sets was normalized to the L-NAME inhibited data set. (B) Normalized current-voltage profiles from HEK293H cells stably expressing Cav3.2 and transfected with nNOS show that overall ODQ had little effect on the IV relationship. At -50 mV and -45 mV there the normalized current was significantly less in the ODQ treated cells (p<0.05 [Student’s t-test]) however, there was no significant difference at the current use to calculate T-type current density (-30 mV). To determine current density (pA/pF) the peak recorded Ca  2+  current (pA) was divided by the cell capacitance  (pF). The error bars represent standard error.  106  Figure 2.9: Co-expression of PSD-95 with nNOS and Cav3.2 results in increased inhibition of Cav3.2  107  Figure 2.9: Co-expression of PSD-95 with nNOS and Cav3.2 results in increased inhibition of Cav3.2 (A) HEK293H cells stably expressing Cav3.2 were co-transfected with nNOS and PSD-95 and the T-type current density calculated. To determine current density [pA/pF] the peak Ca2+ current [pA] was divided by the cell capacitance [pF]. In the presence of PSD-95 and nNOS (n=7) the current density was significantly lower than nNOS only transfected cells (n=32) (p<0.005 [Student’s t-test]). Incubation of the PSD-95/nNOS transfected cells with L-NAME (n=7) relieved the inhibition similar to that observed in the nNOS only transfected cells. (p<0.001 [ANOVA followed by Tukey’s multiple comparison test]) Additionally, pre-incubation with the TAT-DEPV peptide (n=9) was effective in relieving the Cav3.2 inhibition (p<0.001 [ANOVA followed by Tukey’s multiple comparison test]) whereas the current density of TAT-DEPE treated cells (n=9) remained significantly lower than the non-treated nNOS only cells (p<0.005 [Student’s t-test]). The data in each experimental group was normalized to the L-NAME inhibited data set except for the data in the TAT-peptide data set which was normalized to the TAT-DEPV data set so that the degree of Cav3.2 inhibition could be compared to the L-NAME experimental group. (B) Normalized current-voltage (IV) profiles from HEK293H cells stably expressing Cav3.2 and transfected with nNOS and PSD-95 compared to nNOS transfection alone shows that PSD-95 did not significantly alter the IV relation. The error bars represent standard error.  108  Figure 2.10: The nNOS-mediated changes in peak current density are not the result of the direct action of L-NAME, TAT-DEPV or PSD-95 on Cav3.2  (A) To ensure that the various experimental conditions examined (L-NAME, TAT-DEPV and PSD95) did not alterCav3.2 channel activity directly, the T-type current density experiments were performed on non-transfected HEK293H cells stably expressing Cav3.2. Pre-incubating cells with LNAME or TAT-DEPV peptide had no significant effect on the current density of the cells and neither did the transfection of PSD-95. All data sets were normalized to the L-NAME treated data set for comparison to the previous figures. The error bars represent standard error. (B) Representative traces of macroscopic Cav3.2 currents evoked at -30 mV. The traces are normalized and overlaid to demonstrate that the kinetics of the channel was not noticeably affected by the various treatments. (p>0.05 [Student’s t-tests], n=[L-NAME=18, no l-NAME=32, tat-DEPV=9,PSD-95=5])  2.4. Discussion 2.4.1. Cav3.2 and nNOS physically and functionally interact in a heterologous system This study presents the first evidence that the Cav3.2 T-type channel and nNOS can physically and functionally interact in a heterologous system. The results from the GSTfusion/immunoprecipitation experiments demonstrate that Cav3.2 channels and nNOS can physically interact. In support, deletion or mutation of the PDZ-3 binding motif located at the 109  carboxyl terminal of the Cav3.2 channel prevents the co-precipitation of nNOS. Together, these results indicate that the interaction occurs through the PDZ-3 binding ligand at the carboxyl terminus of Cav3.2. Intracellular Ca2+ concentrations are highly regulated, generally ranging from 10-100 nM under basal conditions. This tight regulation of intracellular concentration imposes limitations on Ca2+ signaling with the speed and magnitude of the signal being inversely proportional to the distance between the Ca2+ source and its target (reviewed in (Bootman and Berridge, 1995; Berridge et al., 1998; Augustine et al., 2003; Clapham, 2007)). A physical interaction between Cav3.2 and nNOS which anchors the Ca2+ dependent nNOS near a Ca2+ source could have numerous important implications in vivo. Functionally, the results from the NO-sensitive fluorometric assay show that depolarization of cells co-expressing Cav3.2 and nNOS stimulates the production of NO. Utilizing cells expressing Cav3.2 under an inducible promoter we further showed that chemical depolarization produced an increase in NO levels only when Cav3.2 channel expression had been induced and that the application of Ni2+ to block Cav3.2, precluded the production of NO. This result demonstrates that Cav3.2 activity is sufficient to stimulate NO production from co-expressed nNOS. Furthermore, and relevant to a negative feedback type of regulation, the NO produced by nNOS is sufficient to inhibit Cav3.2 activity as revealed by analysis of the T-type current density in cells co-expressing Cav3.2 and nNOS. The PDZ-3 mediated interaction between Cav3.2 and nNOS is required for a functional interaction to occur; disrupting the Cav3.2/nNOS interaction through point mutations in the Cav3.2 PDZ-3 binding ligand prevented an increase in fluorometric signal in the NO assay following depolarization. This result suggests that nNOS must be properly anchored to Cav3.2 for Ca2+ entering through the channel to effectively activate the enzyme. Consequently, nNOS expression has no effect on the T-type current density in Cav3.2 expressing cells when the 110  interaction is disrupted either through point mutation of the PDZ-3 binding ligand or after application of a peptide that competes for the PDZ-3 binding domain of nNOS. Taken together the evidence demonstrates that a physical interaction between the Cav3.2 T-type channel and nNOS can occur in a heterologous system. Furthermore, a functional interaction wherein Cav3.2 activity stimulates nNOS to produce NO and which, in turn, feeds back inhibit Cav3.2 activity has also been shown to occur. A schematic summarizing the Cav3.2/nNOS interaction is presented Figure 2.11. This putative negative feedback circuit may potentially play an important role in regulating neuronal activity through the rapid modulation of Cav3.2 activity.  111  Figure 2.11: Schematic summarizing the proposed physical and functional interaction between Cav3.2 and nNOS  Cav3.2 and nNOS interact via a PDZ-3 binding ligand located at the carboxyl terminus of the Cav3.2 channel (1). Calcium influx from Cav3.2 activity is proposed to increase the local intracellular Ca2+ concentration and activate calmodulin (2) which binds to, and activates, nNOS. Once active, nNOS converts L-arginine to nitric oxide (NO) (3) which inhibits further Cav3.2 activity (4) through metal catalyzed oxidization of the H191 residue. nNOS possesses both a PDZ-3 domain and an internal PDZ-2 -finger which can both be utilized to potentially facilitate the formation of larger complexes via interactions with PDZ-2 containing scaffold proteins such as PSD-95 (5).  112  2.4.2. nNOS can facilitate the formation of larger protein complexes The results of this study suggest that the PDZ-3 domain and the PDZ-2 internal binding ligand of nNOS can be occupied simultaneously. The presence of PSD-95 facilitates the co-immunoprecipitation of nNOS which is in contrast to the work of Jaffery et al. who showed that a PDZ-2 mediated interaction between nNOS and PSD-95 is disrupted by the binding of CAPON to the PDZ-3 domain of nNOS (Jaffrey et al., 1998). It is possible that the binding of CAPON to nNOS introduces a specific allosteric inhibition or conformational change in the PDZ-2 internal binding ligand which disrupts the interaction between PSD-95 and nNOS. Support for our immunoprecipitation data can be found in the current density experiments wherein PSD-95 and nNOS were co-expressed in Cav3.2 expressing cells. We observed an additional inhibition of Cav3.2 activity in the presence of PSD-95 and nNOS which was evident by a significant reduction in T-type current density compared to effects of nNOS alone. It is possible that the binding of nNOS induces a conformational change in nNOS which increases the affinity of the Cav3.2/nNOS interaction and might explain the immunoprecipitation results. An increase in the affinity of the Cav3.2/nNOS interaction may also explain the increased Cav3.2 inhibition as a greater percentage of Cav3.2 channels could bind nNOS resulting in more Cav3.2 channels being inhibited by NO and resulting in reduced macroscopic currents. An alternative explanation may come from the multimerization of PSD-95 (Hsueh et al., 1997; Hsueh and Sheng, 1999; Keith and El-Husseini, 2008). In theory, a multimerized PSD-95 complex would be able to recruit several nNOS proteins which could potentially co-immunoprecipitate as a large unit. Additionally, the recruitment of multiple nNOS proteins to Cav3.2 could potentially increase the local NO concentration following Ca2+ influx; e.g., an increased NO level may increase the likelihood of metal  113  catalyzed oxidation of Cav3.2 resulting in a higher fraction of Cav3.2 channels being inhibited. The internal PDZ-2 ligand of nNOS may facilitate the anchoring of Cav3.2 to scaffolding proteins such as PSD-95. The PDZ-2 domain is found in many scaffolding proteins (for review see (Hung and Sheng, 2002; Lee and Zheng, 2010)) as well other proteins such as -syntrophin (Brenman et al., 1996a). A PDZ-2 mediated interaction may play a role in vivo to anchor the Cav3.2/nNOS complex to regions of the cell where it would be functionally relevant.  2.4.3. Inhibition of Cav3.2 does not involve the activation of soluble guanylyl cyclase Soluble guanylyl cyclase is a well-documented target of NO. When activated, sGC catalyzes the conversion of GTP to cGMP, an important secondary messenger which has a number of downstream effectors including PKG, phosphodiesterases (PDE) and cyclic nucleotide-gated channels (Krumenacker et al., 2004; Domek-Łopacińska and Strosznajder, 2005; Kleppisch and Feil, 2009). In our current density analysis experiments utilizing ODQ we found that the inhibition of Cav3.2 currents through nNOS activity does not involve the activation of sGC. More likely, the mechanism of inhibition of Cav3.2 is through the metal catalyzed oxidization of the H191 residue of the Cav3.2 channel (Joksovic et al., 2007; Nelson et al., 2007a). Even though sGC appears not to be involved in the NO-mediated inhibition of Cav3.2, it is possible that the NO produced via the Cav3.2/nNOS complex may still activate this secondary messenger pathway. The consequences of activating sGC are difficult to predict as this depends upon the downstream effectors and targets expressed within a given cell type; however, it has been shown that the catalytic activity of nNOS can 114  be inhibited by PKG (Dinerman et al., 1994) which could function as a regulatory component in interaction between Cav3.2 and nNOS. Activation of the sGC/cGMP pathway has also been demonstrated to modulate several members of the VGCC family. L-type Ca2+ channels have been shown to be inhibited in Bovine chromaffin cells, rat insulinoma cells, and glomus cells of the rat carotid body (Grassi et al., 1999; Summers et al., 1999; Carabelli et al., 2002), N-type Ca2+ channels have been found to be inhibited in DRGs innervating the bladder of rats and in human neuroblastoma cells (Yoshimura et al., 2001; D’Ascenzo et al., 2002), whereas P/Q-type Ca2+ channels were demonstrated to be inhibited in rat insulinoma cells (Grassi et al., 1999) and augmented in the mouse medial nucleus of the trapezoid body (Tozer et al., 2012).  2.4.4. Potential implications of a Cav3.2/nNOS complex in vivo Redox modulation of Cav3.2 channels has been shown to affect the excitability of nociceptive DRG neurons and to alter the response threshold to noxious stimuli in the rat neuropathic pain model. Of note, nNOS has also been found to be expressed in nociceptive DRGs suggesting that a Cav3.2/nNOS complex might contribute to the transmission and/or regulation of nociceptive signaling. Conceivably, activation of the Cav3.2/nNOS complex would allow for a rapid NO-driven response to excitatory input until the NO-mediated feedback inhibition of Cav3.2 reduced channel activity and either attenuated or dampened the signal. In this model, an initial nociceptive stimulus would invoke a rapid maximal response by activation of Cav3.2 channels, affecting both DRG excitability directly via Ca2+ influx and secondarily through nNOS activation to produce NO to then regulate various target effectors relevant to pain signaling.  115  Gain of function point mutations in the Cav3.2 channel have been associated with childhood epilepsy and other forms of idiopathic generalized epilepsy (Chen et al., 2003b; Heron et al., 2004; Khosravani and Zamponi, 2006; Peloquin et al., 2006; Vitko et al., 2007; Zamponi et al., 2009). Recently, a gain-of-function mutation in the Cav3.2 gene has been shown to segregate with the seizure phenotype in the Genetic Absence Epilepsy Rats of Strasbourg (GAERS). Further, the mutation affects the firing of nRT neurons in such a way that favours synchronous firing within the thalamocortical circuit (Powell et al., 2009). nNOS has been found to be developmentally expressed in the nRT of the ferret (McCauley et al., 2002) and it is possible that the transient expression of nNOS during development serves to add an inhibitory influence to the developing circuitry. Cav3.2 and nNOS are co-expressed in a number of populations of neurons in the brain thus there are many potential areas of functional interaction. One notable region where both proteins are expressed is the olfactory bulb which is of interest since there is considerable plasticity involved in both learning new odours and in becoming desensitized to them over prolonged exposure (Kaba and Huang, 2005; Gao and Strowbridge, 2009). It is possible that a Cav3.2 and nNOS interaction may contribute to the synaptic plasticity of neurons in the olfactory bulb, although further investigation is required to fully understand any contribution of a potential Cav3.2/nNOS complex in vivo. The future consideration of the consequences of Cav3.2 inhibition on neuronal activity towards NO-regulated downstream effectors of cGMP would also be important to examine. In this regard, it may be necessary to first determine the contribution, if any, of sGC and to then study the effects of silencing both Cav3.2 and nNOS expression in relevant signaling pathways.  116  Chapter 3. 3. Disruption the Cav3.2 and nNOS interaction in vivo alters the respiratory response in Sprague Dawley rats 3.1. Introduction Respiratory drive plays a crucial role in the initiation of inspiration in the respiratory cycle. A major source of respiratory drive comes from the carotid body in the peripheral respiratory network. Located at the bifurcation of the carotid artery, this major chemoreceptor is composed of two cell types: sustentacular type II glomus cells and chemosensory type I glomus cells that respond to changes in O2 and CO2 levels in the blood and sends modulatory signals via the carotid sinus nerve to the central respiratory network contributing to the regulation of overall respiratory activity (Figure 3.1A and B)The importance of this peripheral chemosensor has long been acknowledged and substantial progress has been made in the understanding of the transduction pathways and neurotransmitters involved in the processing of the afferent signals from the carotid body. Less is understood however, of the efferent modulation of the carotid body. Even though efferent innervation of this major peripheral chemoreceptor has long been identified, the complexities of this process have only recently begun to unfold (reviewed in (Campanucci and Nurse, 2007)). Campanucci and coworkers have put forward a model for the NO-mediated efferent inhibition of carotid bodies by the microganglia of the GPN. In this model two mechanisms were proposed for the stimulation of nNOS activity: 1) that under hypoxic conditions the intrinsic O2 sensing capability conferred by a halothane inhibitable K+ channel related to the 2-pore domain inwardly rectifying TWIK potassium channels results in neuronal depolarization and increased neuronal excitability which in turn results in the activation of 117  the various VGCCs in the microganglia and consequently the stimulation of nNOS activity, and 2) that ATP released from type I glomus cells of the carotid body during hypoxia stimulates ionotropic purinergic P2X receptors of the microganglia to permit Ca2+ influx necessary to stimulate nNOS activity (Campanucci and Nurse, 2007). Our data together with evidence put forward by others suggests that for optimal activation of nNOS, it should be localized near the source of Ca2+ responsible for stimulating NO production (Aarts et al., 2002). Whole cell Ca2+ currents in the microganglia of the GPN have been described including a Ni2+ sensitive T-type current which may represent Cav3.2 activity. These findings, in conjunction with our own regarding the Cav3.2 and nNOS interaction, present the possibility that Cav3.2 and nNOS may physically and functionally interact within the GPN microganglia and contribute the NO mediated inhibition of the carotid body. I hypothesized that an interaction between Cav3.2/nNOS contributes to the NO mediated inhibition of the carotid body and that disruption of this interaction would result in an increase in the hypoxic response similar that observed both after the localized administration of NOS inhibitors (Gozal et al., 1996a) and in nNOS knockout mice (Kline et al., 1998). In the current study we investigate the effect of intraperitoneal administration of membrane permeable peptides designed to disrupt the Cav3.2/nNOS interaction on the hypoxic and hyperoxic response of Sprague Dawley rats.  118  Figure 3.1: Schematic summarizing the location and innervation of the carotid body  119  Figure 3.1: Schematic summarizing the location and innervation of the carotid body (A)The carotid body is located in the bifurcation of the carotid artery and is innervated by the carotid sinus nerve which branches from the glossopharyngeal nerve. The nNOS expressing proximal (black arrow) and distal (blue arrow) microganglia both innervate the carotid body (Wang et al., 1993; Campanucci et al., 2003) and are connected through reciprocal projections. (B) Two types of cells make up the carotid body, the sustentacular type II glomus and the chemosensory type I glomus cells. Type I glomus cells receive efferent inhibition from microganglia in the GPN and send modulatory signals via the carotid sinus nerve to the CNS in response to changes in O2 and CO2 levels in the blood.  3.2. Materials and methods 3.2.1. Animal care 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 (Animal care certificate number: A10-0286). The 18 to 25 day old male Sprague Dawley rats utilized in this study were obtained from the University of British Columbia Animal Care Center, Vancouver, BC, Canada.  3.2.2. GPN dissection The glossopharyngeal nerves (GPN) of 18 to 25 day old Sprague Dawley rats were isolated for quantitative real-time-PCR and immunohistochemical analysis. Isoflurane (Sigma-Alrich, St.Louis, MO, USA) was used to anaesthetize the animals and was delivered with an isoflurane vapourizer (VetEquip, Pleasanton, CA, USA) connected to a 120  polycarbonate chamber. Once sufficiently sedated (indicated by a failure to respond to the Toe-Pinch reflex test), the animal was painlessly sacrificed by decapitation using a guillotine. The head and neck were mounted ventral side up in a Sylgard filled petri dish and an incision was made in the skin to expose the trachea and hyoid bone which were carefully removed along with the surrounding muscle tissue to reveal the carotid arteries and vagus nerves. Muscle and fatty tissue were also removed to expose the tympanic bulla and the posterior lacerated foramen. To isolate the GPN the connective tissue along the carotid artery and vagus nerve was carefully removed as the structures were gently lifted and pulled anteriorly. Once the carotid bifurcation and nodose ganglion were free of the connective tissue they were lifted away from the posterior lacerated foramen by gently pulling posteriorly on the severed ends of the carotid artery and vagus nerve. Microscissors were used to cut blood vessels and nerves as distal from the carotid bifurcation and the nodose ganglion as possible and a scalpel was used to sever the nerves where they exited the posterior lacerated foramen. Once isolated, the carotid bifurcation, nodose ganglion and proximal tissues were placed in a dissecting dish filled with L-15 media (Invitrogen, Grand Island, NY, USA) where any remaining connective tissue was removed to expose the GPN. In dissections destined for quantitative real-time-PCR the GPN was cut from the petrosal ganglion and immediately frozen in liquid nitrogen for later processing. For immunohistochemical analysis, the GPN was left connected to the nervous tissue immediately adjacent which included the petrosal ganglion, the nodose ganglion and the proximal regions of the vagus, pharyngeal and superior laryngeal nerves.  121  3.2.3. Quantitative real-time-PCR Quantitative real-time-PCR analysis was performed on a pooled sample of 26 GPNs dissected from 13 animals. The tissue was flash frozen in liquid nitrogen at the time of dissection and stored at -80 oC until needed for cDNA synthesis. RNA extraction was performed using the MagMAX-96 total RNA isolation kit (Invitrogen, Grand Island, NY, USA). The tissue was thawed and homogenized in 200 l of TRI-reagent using a Dounce homogenizer. 20 l of 1-bromo-3-chloro-propane was added to the homogenate and the mixture was allowed to incubate for 5 minutes at room temperature before being centrifuged at 12,000 x g for 10 minutes at 4 oC. After centrifugation, 100 l of the aqueous phase was transferred to a 1.5 ml tube with 50 l of isopropanol and mixed for one minute before being bound to 10l of RNA binding beads for 3 minutes. The beads were washed twice with 150l of wash solution and allowed to air dry before the RNA was eluted with 50 l of elution buffer. cDNA synthesis was performed using a High-Capacity reverse transcriptase kit from Applied Biosystems/Invitrogen (Invitrogen, Grand Island, NY, USA). The relative cDNA levels of each VGCC present in the GPN were determined using an Applied Biosystems 7500 Real-Time PCR System. Primers and Taman probes designed to amplify and quantitate the respective cDNA targets were purchased from Applied Biosystems/Invitrogen (Invitrogen, Grand Island, NY, USA). Analysis of the cDNA expression levels using each primer/probe set was run in triplicate and the expression levels were normalized to the housekeeping gene -actin.  3.2.4. Immunohistochemistry Cav3.2 (Santa Cruz Biotechnology: Santa Cruz, CA, USA: Cat-SC-16261), and nNOS (Santa Cruz Biotechnology: Santa Cruz, CA, USA: Cat-SC-5302), specific antibodies were 122  used to probe the GPN for immunohistochemical analysis. The GPN was dissected with the petrosal ganglion, nodose ganglion and proximal sections of the vagus, pharyngeal, and superior laryngeal nerves intact to provide a frame of reference for the mounted material. Additionally the nuclei were stained with DAPI and the preparation was probed with a choline acetyltransferase (ChAT) specific polyclonal antibody (Abcam, Toronto, ON, Canada: Cat-AB34419) as previous work has shown that the nNOS positive neurons of the GPN are cholinergic; expressing ChAT and vesicular acetylcholine transporter (Wang et al., 1993; Campanucci et al., 2003). Immediately after dissection the tissue was submersed in a 4% paraformaldehyde/PBS solution. After 4 hour incubation the nerves were washed for 5 minutes in 1ml PBS, flash frozen in liquid nitrogen for 3 seconds and thawed in PBS. The nerves were permeabilized and pre-blocked for 1 hour with a PBS solution containing 0.5% Triton X-100, 1% normal donkey serum and 0.25% BSA before application of the primary antibodies. Primary antibodies were diluted 1:400 in PBS with 1% normal donkey serum and 0.25% BSA and incubated with the nerves for 48 hours at 4o C on a rotator. After three 5 minute washes with 1 ml PBS, Alexa Fluor (Invitrogen, Grand Island, NY, USA) or DyLight Fluor (Fisher Scientific, Ottawa, ON, Canada) conjugated secondary antibodies were diluted 1:400 and 4',6-diamidino-2-phenylindole (DAPI) was diluted 1:300 in PBS with 1% normal donkey serum and 0.25% BSA and incubated with the tissue for four hours at room temperature. A final three washes with PBS were performed and the nerves were whole mounted on slides with Vectashield mounting media (Fisher Scientific, Ottawa, ON, Canada) and sealed with nail polish. An Olympus Fluoview 10i confocal microscope was utilized to acquire images from the preparations. A 60x oil immersion objective with a numerical aperture of 1.35 was used to acquire the images. Within the software image quality was set to  123  8x with 1024x1024 pixels and an aperture setting of 2.5x. Typically the laser intensity was kept at 25% or lower and the sensitivity maintained at approximately 45%.  3.2.5. Design of the TAT fusion peptides The protein transduction domain of the Human Immunodeficiency Virus 1 TAT protein (Green and Loewenstein, 1988) is able to mediate the transport of heterologous proteins across the cell membrane (Fawell et al., 1994). TAT fusion proteins have been shown to be able to transduce functional proteins into a variety of tissues including neuronal tissue (Schwarze et al., 1999; Cao et al., 2002). Additionally TAT fusion peptides have been used in vivo to competitively disrupt protein interactions such as the interaction between the NMDA receptor and PSD-95 (Aarts et al., 2002). The TAT fusion peptides were ordered from Genscript (Piscataway, NJ, USA) and contained the protein transduction domain of the human immunodeficiency Virus 1 transactivator of transcription (TAT) protein (YGRKKRRQRRR) fused with the last 9 amino acids of the rat Cav3.2 sequence (PDDSGDEPV) which possess a carboxyl terminal PDZ-3 binding ligand. A TAT-fusion peptide with a mutation in the Cav3.2 PDV-3 binding ligand (PDDSGDEPE) and a peptide with just the TAT sequence were also ordered. The peptides were injected at a concentration of 9 mg/kg (~3.6moles/kg for the TAT fusion peptides and 5.6 moles/kg for the TAT control) as it was the highest level of TAT peptide injection that we could find in the literature that did not appear to have adverse effects (Cao et al., 2002).  3.2.6. Respirometry The experimental setup is illustrated in Figure 3.2. A Cameron Instruments gasmixing flow meter (Cameron Instruments, Brownsville, TX) was used to mix pure gases and 124  deliver 100%, 21% and 12% oxygen (O2) with the balance comprised of nitrogen. The air mixtures were bubbled through water to humidify the mixture and sampled continuously to monitor the O2 concentration with a galvanic O2 sensor. The single mixed airline was split to provide air to two identical 500 ml plethysmograph chambers; an experimental chamber where the animal was placed and a reference chamber. A differential pressure transducer (DP103) (Valydine, Northridge, CA,USA) with a #06 diaphragm and a CD16 carrier demodulator (Valydine, Northridge, CA,USA) was used to measure the pressure differential between the two chambers generated by the ventilation of the animal (Malan, 1973; Jacky, 1978). A Gould DC amplifier was used to amplify the signal from the pressure transducer and to provide low pass filtering at 5 Hz. The post-chamber gases were analyzed for carbon dioxide (CO2) using a non-dispersing infrared sensor and a second O2 sensor was plumbed so that same sensor could be utilized to obtain post-chamber oxygen values and pre-chamber values to be used to calculate oxygen consumption. The air flow measurements were obtained using a Fleisch Pneumotachograph connected to a differential pressure transducer with a #14 diaphragm and a CD16 carrier demodulator (Valydine, Northridge, CA, USA). A Gould integrator was utilized to provide signal gain. All data were digitized with a DATAQ model DI-205 digitizer and acquired with Windaq software version Pro+ DI-720 at a sample rate of 166.66 Hz per channel using a personal computer running Microsoft Windows XP. The animals were kept in the transport container undisturbed for 30 minutes after delivery to allow for acclimatization to the laboratory before proceeding with the experiments. An intraperitoneal injection of one of the TAT-fusion peptides (TAT-DEPV, TAT-DEPE or TAT-0) was administered at a dose of 9 mg/kg immediately prior to placing the animal within the plethysmograph chamber. A minimum of 15 minutes was provided for acclimatization to the chamber with a gas mixture of 100% O2 with a flow rate of 400 125  ml/min; if the respiratory parameters did not stabilize within the 15 minute period additional time was allowed. Before each respiratory response test the chamber was purged at 2000 ml/min for 7 minutes with the O2 percentage required for the subsequent test. For example: in the 100% O2 test, the chamber was purged for 7 minutes with 100% O2 and then the respiratory response of the animal was measured at 100% O2 with 400 ml/min flow for 7 minutes. The testing procedure consisted of measuring the respiratory responses at 100%, 21% and 12% O2. This was followed by a hyperoxic transition test (Dejour’s test) which consisted of a 10 minute purge of 100% O2, followed by a conditioning phase of 12% O2 at 2000 ml/min for 10 minutes and a test phase of 100% O2 at 2000 ml/min for 5 minutes. A second round of tests was performed after a 10 minute purge at 100% O2. A graphical representation of the protocol can be seen in Figure 3.3. After completion of the test procedures the animals were anaesthetized and painlessly sacrificed by decapitation per 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 (Animal care certificate number: A10-0286)  126  Figure 3.2: Experimental set-up used to acquire the respirometry and metabolic data  127  Figure 3.2: Experimental set-up used to acquire the respirometry and metabolic data Pure oxygen and nitrogen were mixed using a gas mixer to produce 100%, 21% or 12% O2 with balance nitrogen. The mixed gas was humidified by bubbling through water in an Erlenmeyer flask and the flow was split to feed both sides of the plethysmograph chamber. The respiratory rate and tidal volume were calculated from the pressure difference in the air between the animal half and the reference half of the chamber. A pneumotach connected to a differential pressure transducer was used to monitor the flow rate of the gas. To ensure the appropriate O2 concentrations were being mixed an oxygen sensor sampled the gas mixture before the chamber. A second oxygen sensor was utilized to take readings pre-chamber and post-chamber to determine O2 consumption. CO2 production was measured post-chamber using a CO2 sensor. All the data was digitized, recorded and stored on a computer for analysis.  Figure 3.3: Graphical representation of the respirometry protocol  128  Figure 3.3: Graphical representation of the respirometry protocol The respirometry protocol represented as percent oxygen over time. Thick lines represent a flow rate of 2000 ml/min and thin lines represent 400 ml/min flow rate.  3.2.7. Analysis of respirometry data For each respiratory response test a 15 second window of data from as late in the testing period as possible was obtained from the acquired data using the Windaq software and saved in CSV format. Microsoft Excel was used to open the CSV file and transfer the data into Origin software version 8. Care was taken to ensure that the animal was settled but not sleeping during the period taken for analysis. The ventilation pattern during sleep could be determined visually and compared to notes taken during the experiment to determine if the animal was sleeping. Indicators that the animal was unsettled were sniffing (sharp repetitive spikes in the ventilation data) and fluctuations in the CO2 recordings indicative of animal movement such as pacing or scratching. The respiratory rate (inspirations/min) was calculated by counting the number of inspirations on the graph, multiplying by 60 (seconds/min) and dividing by the number of seconds between the peak of the first inspiration and the last inspiration. Tidal volume was calculated using the following formula (Jacky, 1980): Vt =  {(Pm x Vcal x Tb(Pb - PcH2O)) / (Pcal x [Tb(Pb - Pch2o) - Tc(Pb - PaH2O)])} / {[1-Ti/Ttot(1  - Ga/Gn)]} Vt=  Tidal volume corrected for partial pressure of water at the given barometric  pressure Vcal= Volume used to calibrate the equipment Pm=  Observed pressure reading  Pb=  Barometric pressure (mm Hg) 129  Pcal=  Pressure reading observed from input of calibrated volume  PcH2O= Water vapour pressure in chamber based on ambient temperature from table (Dejours, 1988) PaH2O= Water vapour pressure based on alveolar temperature of animal (Dejours, 1988) Tb=  Absolute temperature of alveoli (310.5K) (Jacky, 1980)  Tc=  Absolute temperature of the chamber  Ti=  Duration of inspiration  Ttot=  Total breath duration  Ga=  Constant defined as = [Tb * (Pb – PcH2O)] / [Tb * (Pb-PcH2O) – Tc * (Pb-PaH2O)]  Gn=  Constant defined as = [Tb * (Pb – PnH2O)] / [Tb * (Pb – PnH2O) – Tn * (Pb –  PaH2O)] PnH2O= Water vapour pressure based on nasal temperature from table (Dejours, 1988) Tn=Absolute temperature at the nose (301.8K) (Jacky, 1980) During acquisition the baseline tended to drift from center depending on movements from the animal, therefore the inspiration time and total breath duration had to be calculated manually. To facilitate data collection I compiled a short script for Origin 8 that allows the manual selection of data points and the automatic recording of the x and y coordinates to a temporary spreadsheet. The script has been submitted to the file exchange on the Originlab website (http://www.originlab.com/fileexchange/details.aspx?fid=137) and to date it has been downloaded 585 times. The full script is as follows: del r1; del r2; del datatemp; 130  create datatemp -c 150; define EndToolBox { create tempor -wn 2 Xaxis Yaxis; range [tempor]Sheet1 r1=Col(Xaxis), r2=Col(Yaxis); r2 = datatemp; r1 = datatemp_A; }; getpts -n datatemp 60; Using this script the peak values and the baseline for each inspiration were identified and recorded. The time points of initiation and cessation of inspiration as well as the conclusion of expiration were recorded and used to determine the inspiration duration and the total breath duration. The data for the hyperoxic response test was transferred to Origin 8 software in the same manner as the respiratory response data. The data collection script mentioned above was used to locate the peak of every inspiratory event within a 2 minute window starting from the switch from 12% O2 to 100% O2. The data was filtered for sniffs and animal movement such and scratching using the two criteria. Periods of sniffing and scratching were compared to quiescent periods to determine the criteria to filter sniff and scratching events. The nominal respiratory rate of rats is approximately 90 breaths per minute and the maximum respiratory rate approximately 170 breaths per minute (Gozal et al., 1996b; Subramanian et al., 2002). We observed that the peak to peak interval during sniff and scratch event was considerably less than regular breathing and setting a maximum of 200 breaths per minute was sufficient to filter sniffs and scratching events lasting longer than 600 131  ms. For sniff and scratch events lasting less than 600 ms it was determined that sniffs and scratches could be identified by a disproportionate decrease in the peak to peak interval compared to the preceding three peaks; a factor of 1.6x was sufficient to exclude the majority of sniffs and scratches lasting less than 600ms from the data. Once collected and filtered a rolling average of four events was calculated and normalized to the respiratory rate at the 10 second mark. The ten second mark was chosen as the normalization point as it was at this point that O2 concentration started to increase; the delay in the increase was due to the dead space volume present in the tubing and the chamber of the experimental setup. For each experimental group the timelines were synchronized, divided in to 0.75 second intervals and the average respiratory rate for each animal within every interval calculated. Finally, the respiratory rates of each 0.75 s interval for all the animals in the experimental group were averaged together and plotted.  3.3. Results 3.3.1. Cav3.2 is expressed in nNOS expressing microganglia within the GPN of Sprague Dawley rats Campanucci and colleagues originally characterized whole cell currents in acute preparations of microganglia in the GPN of rats (Campanucci and Nurse, 2007). Pharmacological dissection of the VGCC currents revealed that all VGCC classes are represented in these neurons including those for T-type channels. Since antagonists capable of differentiating the three Cav3 channels are not available, the specific T-type isoforms expressed within the microganglia of the GPN were not identified. However, in finding that Ni2+ at 100 M resulted in current blockade suggests that the T-type currents are most likely due to the Ni2+-sensitive Cav3.2 channel (rather than the Cav3.1 and Cav3.3 channels which 132  have Ni2+ IC50s of ~250 M and ~215 M, respectively). Figure 3.4 shows that based on quantitative real-time PCR, mRNAs for the Cav2.1 (P/Q-type) and Cav2.2 (N-type) channels are the most abundant occurring at significantly higher levels (p<0.05) than those for the other VGCCs. Cav1.2, Cav1.3 (L-types), Cav2.3 (R-type) and Cav3.2 (T-type) mRNAs were also detected; however the levels were low compared to those of Cav2.1 and Cav2.2. Together, all of the VGCCs giving rise to the currents detected by Campanucci and colleagues (Campanucci and Nurse, 2007) are represented in the quantitative Real-Time PCR analysis. In comparing Cav3.1, Cav3.2 and Cav3.3 T-type mRNA expression, Figure 3.4B shows that the level of mRNA for Cav3.2 is significantly greater (p<0.05) than either that for Cav3.1 or Cav3.3 and which corresponds with well with the pharmacological data (Campanucci and Nurse, 2007). Further evidence for the presence of the Cav3.2 T-type channel in the microganglia of the GPN is revealed using immunohistochemical staining. Whole nerve preparations of the GPN were probed with Cav3.2, nNOS and ChAT specific antibodies. A low magnification image of a whole nerve mount is displayed in Figure 3.5A. The petrosal ganglion, GPN and carotid sinus nerve are labeled and the locations of the high magnification confocal images are indicated with arrows (1. = Proximal, 2. = Distal). Figure 3.5B shows high magnification confocal images of neurons within the microganglia of the proximal (1.) and distal (2.) regions of the GPN. In these representative images it can be seen that Cav3.2 staining can be found in the neurons that comprise the microganglia of the GPN and the merged images show that a subset of these neurons also express nNOS. A ChAT-specific antibody was used as a marker for cholinergic neurons as previous work indicated that the nNOS positive microganglia also stained positive for the vesicular acetylcholine transporter and ChAT (Wang et al., 1993; Campanucci et al., 2003). All neurons within the regions of interest 133  stained positive for ChAT indicating that they are the microganglia identified by Camanucci and colleagues. The co-expression of Cav3.2 and nNOS in these microganglia presents the possibility that a Cav3.2/nNOS functional interaction exists within these cells.  Figure 3.4: qRT-PCR analysis illustrating the Ca2+ channel expression profile in the glossopharyngeal nerve (GPN) in Sprague Dawley rats  A 3.5 mm to 4 mm segment encompassing the proximal and distal microganglia of the GPN was harvested from 16 Sprague Dawley rats (P18 to P25). qRT-PCR oligos specific for each Ca2+ channel were used to amplify cDNA generated from RNA extracted from the pooled GPNs. The values were normalized to actin B expression. (A) Full Ca2+ channel expression profiles for the GPN of Sprague Dawley rats shows the L-, P/Q-, N-, R- and T-type isoforms expressed in rat GPN. Expression levels for the Cav2.1 and Cav2.2 are both significantly higher than the other VGCCs (p<0.01 [ANOVA followed by Tukey’s multiple comparison test]) (B) Scaled up view of the T-type channel transcript profile illustrating that Cav3.2 is the predominant T-type channel isoform present in the glossopharyngeal nerve with approximately 8x greater abundance compared to the other T-types (p<0.05 [ANOVA followed by Tukey’s multiple comparison test]). The error bars represent standard error.  134  Figure 3.5: Immunohistological staining of Cav3.2, nNOS and ChAT in the microganglia of the glossopharyngeal nerve of Sprague Dawley rats  135  Figure 3.5: Immunohistological staining of Cav3.2, nNOS and ChAT in the microganglia of the glossopharyngeal nerve of Sprague Dawley rats The glossopharyngeal nerve of Sprague Dawley rats was isolated, probed with Cav3.2, nNOS, ChAT specific antibodies and DAPI and whole mounted on a slide for confocal analysis. (A) 10x phase contrast scan of the mounted nerve with the petrosal ganglion (PG), glossopharyngeal nerve (GPN) and carotid sinus nerve (CSN) labeled. Images were acquired of the microganglia located proximal (1.) and distal (2.) to the bifurcation to the CSN. (B) representative 60x confocal images of neurons within the proximal (1.) and distal (2.) microganglia of the GPN showing nNOS (green), Cav3.2 (red) and ChAT (purple) expression. The choline acetyltransferase was used as a marker for the microganglia (purple) (Wang et al., 1993; Campanucci et al., 2003). Many of the microganglia express both Cav3.2 and nNOS although some express one and not the other (e.g., those illustrated in set 2 in panel B).  3.3.2. Disruption of the Cav3.2/nNOS interaction in vivo results in an augmented steady state response to hypoxic challenges Systemic administration of a TAT-fusion peptide designed to bind to the PDZ-3 domain of nNOS and competitively disrupt the Cav3.2/nNOS interaction resulted in an augmented steady state response to hypoxic challenges. Figures 3.4, 3.5 and Table 3.1 illustrate the effect of intrathecal injection of the PDZ-3 binding peptide TAT-DEPV or the mutant non-binding peptide TAT-DEPE versus control experiments consisting of administration of a peptide containing only the TAT sequence. No significant difference in the respiratory rate under hyperoxic (100% O2) or normoxic (21% O2) conditions was observed between any of the experimental groups (Figure 3.6A and Table 3.1). Under hypoxic (12% O2) conditions the respiratory rate of all groups was significantly higher (p<0.05) compared to the hyperoxic and normoxic conditions. The 136  increase in the respiratory rate in the TAT-DEPV treated animals was modestly, but not significantly, higher than that in the TAT peptide control animals and the non-disrupting peptide treated animals (Figures 3.4B, 3.4C and 3.4D). No significant change in the respiratory rate was observed between the hyperoxic and normoxic conditions in any group (Figure 3.6B), which is expected as neither condition is anticipated to increase respiratory drive. In contrast, there was a considerable increase in the respiratory rate of the TAT-DEPV treated animals compared to the controls when comparing the normoxic to hypoxic steady state respiratory rate (Figure 3.6C). This increase was also significantly greater (p<0.05) than that seen in the non-disrupting TAT-DEPE treated group but not compared to the TAT-only control group. This result is comparable to that seen in nNOS inhibited (Gozal et al., 1996b) and nNOS knockout mice (Kline et al., 1998) wherein significant differences in respiratory response were observed in response to changes in the available oxygen when nNOS activity was absent. When comparing the two extremes, hyperoxia to hypoxia (Figure 3.6D), the response in the TAT-DEPV treated group was significantly greater than that in the TAT-only and the TAT-DEPE treated animals (p<0.05) and this is also in agreement with the data from the nNOS knockout mouse study of Kline et al. (1998). Hypoxia produced a significant increase in tidal volume in the TAT and TAT-DEPV treated animals but not in the TAT-DEPE treated group (Figure 3.7A). Correspondingly, the total ventilation increased significantly (p<0.05) in the TAT peptide and TAT-DEPV groups in 12% O2 compared to 100% O2 and 21% O2 but not in the TAT-DEPE treated animals (Figure 3.7B). Given the slightly greater increases observed in the respiratory rate (Figure 3.6A) and tidal volume (Figure 3.7A) of the TAT-DEPV treated animals under hypoxic conditions (summarized in Table 3.1) it was anticipated that the total ventilation under hypoxia 137  (Respiratory rate * Tidal Volume) would be significantly greater in the TAT-DEPV treated group compared to TAT treated control animals. As observed from Figures 3.5D, however, even though the total ventilation on 12% O2 was higher in the TAT-DEPV treated group, it still was not significantly greater than the responses of the TAT and TAT-DEPE treated groups (Figure 3.7D and Table 3.1). In contrast to data from nNOS knockout mice which display a significantly higher tidal volume under normoxic conditions compared to wild type mice (Kline et al., 1998), in the current study no significant difference was observed in the tidal volume or total ventilation under normoxic conditions between the TAT-DEPV treated group and the TAT control group. Interestingly, the TAT-DEPE injected animals displayed a significantly greater (p<0.05) tidal volume in 100% O2 and 21% O2 compared to the TAT and TAT-DEPV animals (Figure 3.7C and Table 3.1) and this translated into a significantly greater total ventilation (p<0.05) (Figure 3.7D and Table 3.1). The increased tidal volume at 100% and 21% O2 measured in the TAT-DEPE animals likely explains why no significant difference was observed in this group when the total ventilation under hyperoxic, normoxic and hypoxic conditions were compared. Oxygen consumption and carbon dioxide release are directly related to metabolic rate. When considering the hyperoxic, normoxic, and hypoxic conditions we observed that expired CO2 was greatest under hypoxic conditions and least under normoxic conditions with expired CO2 levels under hyperoxic conditions falling in between; a pattern that was consistent across all three groups. However, the differences were not statistically significant between the TAT, TAT-DEPV, and TAT-DEPE groups (Table 3.1). These results contrast with the nNOS knockout mice of Kline’s study which report a significant decrease in the expired CO2 under hypoxic conditions compared to hyperoxic conditions. Oxygen consumption at 100% O2 was slightly, but not significantly increased in the TAT-DEPV group which is comparable 138  to the Kline et al. study. Unfortunately, observed O2 consumption at 21% and 12% O2 concentrations was deemed inaccurate since the values were physiologically improbable, a finding discovered during analysis after the data had been collected. The most probable explanation for the erroneous O2 values is a flaw in the chamber design which allowed the 100% O2 purge air to become trapped which then escaped sporadically during the testing phase which artificially inflated the O2 readings for the 21% and 12% O2 tests. The air convection requirement (ACR) was calculated assuming a respiratory quotient of 1.0. For the TAT-DEPV and TAT groups the ACR was significantly greater (p<0.05) at 12% O2 compared to 21% and 100% O2. No significant difference was observed between the groups when comparing the ACR at 100%, 21% and 12% O2.  139  Table 3.1: Summary of the respirometry responses from TAT-control, TAT-DEPV and TAT-DEPE injected animals %O2  Injection  (balanc e N 2)  100%  21%  12%  tat injected (n=12) tatDEPV injected (n=12) tat-DEPE injected (n=10) tat injected (n=12) tatDEPV injected (n=12) tat-DEPE injected (n=10) tat injected (n=12) tatDEPV injected (n=12) tat-DEPE injected (n=10)  Respiratory Rate  Tidal Volume (Vt)  Total Ventilation (V'E)  CO2 production  O2 Consumpt ion  Air Convection Requirement (ventilation / metabolism)  (breath/ min)  (l/g[BW])  (l/g[BW]/ min)  (ml/min/ g[BW])  (ml/min/ g[BW])  (ml/ml)  113.3 +/3.43  7.83 +/0.35  882.7 +/39.13  0.0334 +/0.0023  0.0379 +/0.0033  28336 +/- 2612  111.3 +/4.55  8.08 +/0.55  909.2 +/85.39  0.0329 +/0.0013  0.0480 +/0.0072  29478 +/- 2354  117.0 +/3.46  9.79 +/0.61 2  1141 +/72.28 4  0.0351 +/0.0025  0.0342 +/0.0216  33937 +/- 3198  115.4 +/4.36  7.35 +/0.37  853.2 +/62.69  0.0320 +/0.0016  N/A  27350 +/- 3184  116.3 +/4.56  7.27 +/0.50  855.6 +/81.47  0.0301 +/0.0018  N/A  29376 +/- 2338  121.1 +/3.23  8.96 +/0.67 2  1090 +/92.07 4  0.0331 +/0.0022  N/A  34527 +/- 3350  137.3 +/4.65 *  8.61 +/0.36 1  1173 +/45.77 3  0.0309 +/0.0020  N/A  39724 +/- 20375  147.8 +/4.01 *  9.06 +/0.59 1  1350 +/109.1 3  0.0297 +/0.0017  N/A  50052 +/- 41245  137.4 +/3.71 *  9.53 +/0.55  1305 +/75.43  0.0324 +/0.0020  N/A  40447 +/- 23325  140  Table 3.1: Summary of the respirometry responses from TAT-control, TAT-DEPV and TAT-DEPE injected animals The average respiratory rate, tidal volume, O2 consumption and CO2 production were measured directly during the experiment. Total ventilation (respiratory rate * tidal volume) and the respiratory quotient (CO2 produced/ O2 consumed) were calculated from the measured variables. (*) The average respiratory rate on 12% O2 was significantly higher than the rate at 100% or 21% O2 for all three groups (p<0.001 [ANOVA followed by Tukey’s multiple comparison test]). (1) Average tidal volume in the TAT-0 and TAT-DEPV treated animals was significantly greater at 12% O2 however the TAT-DEPE treated animals were not (p<0.05 [Student’s t-test]). (2) The tidal volume of the TATDEPE treated animals was significantly greater than the TAT-0 and TAT-DEPV treated animals at 100% and 21% O2 (p<0.05 [Student’s t-test]). (3) Total ventilation of the TAT-0 and TAT-DEPV injected animals was significantly greater at 12% compared to 21% and 100% O2 (p<0.05 [Student’s t-test]). (4) Total ventilation for the TAT-DEPE treated animals was significantly greater than the TAT-0 injected animals at 100% and 21% O2 (p<0.05 [Student’s t-test]). (5) The air convection requirement in the TAT-0 and TAT-DEPV treated animals was significantly greater at 12% O2 however the TAT-DEPE treated animals were not (p<0.05 [[ANOVA followed by Tukey’s multiple comparison test]) Across the O2 concentrations and between the treatment groups there was no significant difference in CO2 production and O2 consumption. All data are represented +/- standard error.  141  Figure 3.6: Disruption of the Cav3.2/nNOS interaction in vivo results in an elevated respiratory response to changes in oxygen concentration  142  Figure 3.6: Disruption of the Cav3.2/nNOS interaction in vivo results in an elevated respiratory response to changes in oxygen concentration Sprague Dawley rats (P18 to P25) received an intraperitoneal injection in the abdomen of the Cav3.2/nNOS disrupting TAT-DEPV peptide (n=12) or the non-disrupting TAT-0 (n=12) or TATDEPE peptides (n=10) and respirometry and metabolic data was collected at 100% O2, 21% O2 and 12% O2 (balance nitrogen). The peptides were injected at dose of 9 mg/kg which equates to a blood concentration of approximately 720 M (assuming a blood volume of approximately 5 ml) (A) All three treatment groups displayed a normal physiological response to hypoxia with a significantly higher respiratory rate at 12% O2 compared to 21% and 100% O2. The animals injected with the Cav3.2/nNOS disrupting TAT-DEPV peptide showed a slightly elevated respiratory rate over the TAT-0 and TAT-DEPE controls however the difference was not significant. (B, C and D) Graphs illustrating the percent change in respiratory rate at steady state comparing 100% to 21% O2. There was no significant difference between the groups in the percent change in the respiratory rate comparing 100% O2 to 21% O2 (B). When comparing the respiratory rates at 21% O2 and 12% O2 (C) the TAT-DEPV treated animals displayed a significantly higher (p<0.05 [Student’s t-test], n= TATDEPV=12, TAT-DEPE=10) response compared to the TAT-DEPE treated animals but no significant difference compared to the TAT-0 treated animals. (D) At 100% O2 to 12% O2 the difference in respiratory rates between the groups becomes more apparent and the TAT-DEPV injected animals display a significantly higher percentage change compared to the TAT-0 and TAT-DEPE controls (p<0.05 [ANOVA followed by Tukey’s multiple comparison test] , n=[TAT=12, tat-DEPV=12, tatDEPE=10). The error bars represent standard error and the asterisk indicate significance at the p value indicated.  143  Figure 3.7: Average tidal volume and total ventilation in TAT-DEPE injected Sprague Dawley rats differs significantly from Cav3.2/nNOS disrupted (TAT-DEPV) and TATcontrol animals  144  Figure 3.7: Average tidal volume and total ventilation in TAT-DEPE injected Sprague Dawley rats differs significantly from Cav3.2/nNOS disrupted (TAT-DEPV) and TATcontrol animals. Sprague Dawley rats (P18 to P25) received an intraperitoneal injection of either the Cav3.2/nNOS disrupting TAT-DEPV peptide (n-12) or the non-disrupting TAT-0 (n=12) or TATDEPE peptides (n=10) and respirometry and metabolic data were collected at 100% O2, 21% O2 and 12% O2 (balance nitrogen). (A) Average tidal volume of TAT and TAT-DEPV animals was significantly higher (p<0.05 [Student’s t-test]) at 12% O2 compared to 21% O2 whereas no significant difference was observed in TAT-DEPE injected animals. (B) Consequently, the total ventilation (respiratory rate * tidal volume) in the TAT-0 and TAT-DEPV treated animals was also significantly greater at 12% O2 compared to 21% and 100% O2 (p<0.001 [ANOVA followed by Tukey’s multiple comparison test]). The TAT-DEPV treated animals also demonstrated higher total ventilation at 12% O2 compared to 21% and 100% O2 but the difference was not significant. (C) Comparing the average tidal volumes of the TAT-0, TAT-DEPV and TAT-DEPE treated animals shows that the total ventilation of the TAT-DEPE injected animals is significantly greater than the TAT-0 and TATDEPV injected animals at 100% and 21% O2 (p<0.05 [Student’s t-test]). The total ventilation (D) of the TAT-DEPE treated animals was also elevated at 100% O2 and 21% O2 compared to the TAT-0 and TAT-DEPV injected rats but only reached significance compared to the TAT-0 injected animals (p<0.05 [Student’s t-test]. The error bars represent standard error and the asterisks indicate significance at the p value indicated.  145  3.3.3. The hyperoxic response may be attenuated in animals where the Cav3.2/nNOS interaction is disrupted The goal of the hyperoxic transition test is to observe the respiratory response of the animal when the concentration of available oxygen is rapidly changed from normal or hypoxic conditions to a hyperoxic condition. If the exchange is sufficiently rapid the initial response is largely mediated by the peripheral chemoreceptors (Dejours, 1962). TAT peptide and TAT-DEPV injected animals were pre-conditioned under hypoxic conditions (12% O2) before exposure to 100% O2 at the maximum flow rate allowed by the experimental equipment (2000 ml/min). Due to the dead space in the air lines, approximately 10 seconds elapsed before the O2 concentration began to rise (indicated by the green line in Figure 3.8 which represents the O2 concentration over time). Initially, the respiratory rate of the TAT peptide group (black squares) and the TAT-DEPV group fell with the same trajectory, however at approximately the 40 second time point, the respiratory rate of the TAT-DEPV treated animals stabilized whereas respiratory rate of TAT injected animals continued to decline for an additional 10 seconds before rebounding and stabilizing at respiratory rate similar to the TAT-DEPV treated animals. During the short period that the two traces diverged 6 of the 12 TAT-DEPV data points were significantly higher (p<0.05) than the nearest TAT peptide data point. These results suggest that the TAT-DEPV treatment may be affecting the peripheral chemoreceptors resulting in a blunted hyperoxic response. The TATDEPE animals were also tested and it was anticipated that these animals would display a response similar to the TAT injected animals, however there was insufficient data after filtering for sniffs and scratches to accurately plot the respiratory response.  146  Figure 3.8: Disruption of the Cav3.2/nNOS interaction alters the response in the hyperoxic response test  147  Figure 3.8: Disruption of the Cav3.2/nNOS interaction alters the response in the hyperoxic response test The respiratory rate of Sprague Dawley rats (p18 to P25) during a rapid change in available oxygen was analyzed to determine the effect of disrupting the Cav3.2/nNOS interaction in vivo. The animals received an intraperitoneal injection of either TAT-DEPV (n=6) or TAT-0 (n=7) and 12% O2 was administered at a flow rate of 2000 ccm-1 until the respiratory rate stabilized. 100% O2 was then administered at 2000 ccm-1 and the respirometry data was recorded. The green line represents the oxygen concentration in the chamber (right scale). There was approximately a 10 second delay from the initiation of the 100% O2 gas flow until the O2 concentration within the chamber began to increase which is the point the analysis began. Sniffs and animal movement were excluded from the analysis using two criteria: 1) If the rate between two data points was 60% greater than the average of the previous four data points it was excluded. 2) If the rate between two data points was greater than the equivalent of 200 breaths per minute (20% faster than the maximum rate observed under all conditions in this study) it was excluded. A rolling average using a window of five successive data points was used to calculate the change in the respiratory rate over time for each animal. The rolling averages for each animal were normalized to the steady state respiratory rate of the animal at 12% O2 and then aligned with a standard time course and each experimental group was averaged at 0.75 second intervals and plotted. The response of the two groups is similar until the oxygen concentration surpasses 80% at approximately the 40 second mark. At this time the respiratory rate of the TATDEPV treated animals stabilized whereas respiratory rate of TAT injected animals continued to decline for an additional 10 seconds before rebounding and stabilizing at rate similar to the TATDEPV treated animals. At the point where the two traces diverged the respiratory rate at 6 of the 12 TAT-DEPV data points was significantly greater (p<0.05 [Student’s t-test]) than the nearest TAT-0 peptide data point. The TAT-DEPE animals were also tested however there was insufficient data after filtering for sniffs and scratches to accurately plot the respiratory response.  148  3.4. Discussion 3.4.1. Disruption of the Cav3.2/nNOS complex has a significant effect on the respiratory rate in response to available oxygen The central and peripheral chemoreceptors play a crucial role in adjusting respiration in response to both changes in metabolic demand and environmental cues such as oxygen availability. In this study we provide preliminary evidence that Cav3.2/nNOS interactions may be involved with the modulation of respiratory activity, potentially through negative feedback modulation of carotid glomus cell chemosensitivity by the microganglia of the GPN. In animals treated with the TAT-DEPV peptide there was a greater percent increase in breathing frequency, tidal volume, total ventilation and air convection requirement when switching from 21% to 12% O2 compared to the other two groups. While the increase in these parameters is congruent with the original hypothesis they are not statistically greater than the controls. The percent increase in the respiratory rate was significantly greater in the TAT-DEPV animals when going from 100% O2 to 12% O2 which also supports the original hypothesis. Considering that a definite trend towards an increased percent change in the aforementioned parameters, the most likely explanations as to why significance was observed in only one parameter and only under artificial extremes is the physiological range of the parameters. If the respiratory rate has a larger range than the tidal volume then the smaller changes observed in the tidal volume could potentially be lost in the variation between the animals. With some refinement of the experimental technique and additional repetitions it is possible that a significant increase may be seen in tidal volume, total ventilation, and air convection requirement as well. These results are comparable to those observed in previous studies examining the effects of NOS inhibitors or nNOS knockout animals (Gozal et al., 1996b; Kline et al., 1998; Subramanian et al., 2002) indicating that disruption of the 149  Cav3.2/nNOS interaction inhibits NO production and suggests a role in attenuating the hypoxic respiratory response; potentially through the NO mediated inhibition of carotid glomus cell chemosensitivity. The attenuated hyperoxic response in the TAT-DEPV treated animals supports this notion as this experimental challenge is designed to test the contribution of the peripheral receptors involved in the respiratory response (Dejours, 1962). The quantitative real-time PCR results show that Cav3.2 is the predominant T-type Ca2+ channel expressed in the GPN supportive of the notion that the T-type currents recorded by Campanucci and coworkers in the microganglia of the GPN result from Cav3.2. We also find that Cav3.2 and nNOS can both be found in the neurons which comprise the microganglia of the GPN, suggesting that a Cav3.2/nNOS functional interaction is possible in these cells. The microganglia of the GPN are an integral part of the peripheral chemoreceptor network which project efferent fibers to the carotid body, a major peripheral chemosensor. These efferent projections have been shown to provide NO mediated inhibition of the O2 sensing type I glomus cells in the carotid body and it has been proposed that the microganglia of the GPN function as part of a peripheral negative feedback circuit to modulate the hypoxic reflex response. The microganglia possess some O2 sensing capability and are depolarized to become more excitable under hypoxic conditions. In the current proposed model, this increase in excitability in conjunction with the reciprocal activation of purinergic receptors of the microganglia by type I cells of the carotid body provides a condition specific stimulus for NO production necessary for the inhibition of the hypoxic response of the carotid body (Campanucci and Nurse, 2007). A caveat in this model is that the source of Ca2+ for nNOS activation has not been demonstrated. The data presented in Chapter 2 of this thesis, together with the work of others, suggests that for optimal stimulation of nNOS the source of Ca2+ 150  should be local. A functional interaction between Cav3.2 and nNOS melds nicely with the proposed model linking nNOS with a source of Ca2+ influx. In a revised model incorporating the data from the present study, increased microganglia activity would activate the Cav3.2 channels that drives Ca2+ influx sufficient to activate nNOS and to produce NO which in turn inhibits the hypoxic response of the carotid body. A schematic of the revised hypothetical model is presented in Figure 3.9. This new model may account for the increased hypoxic response observed in the animals treated with the Cav3.2/nNOS disrupting TAT-DEPV peptide. Potentially, dissociating nNOS from the Ca2+ source (Cav3.2 channels) in the microganglia could severely inhibit NO synthesis and as a result the NO-mediated efferent inhibition of the carotid body would be compromised and the hypoxic reflex response would proceed unabated. Our results demonstrating an attenuated hyperoxic response conflicts with the data obtained from nNOS knockout mice that display an exaggerated hyperoxic response (Kline et al., 1998). It is possible that since nNOS expression is eliminated globally in the knockout mice, the exaggerated response observed by Kline et al. is indicative of the total contribution nNOS activity on respiration which includes nNOS activated by other Ca2+ sources at other sites in the chemoreflex pathway. Only a few proteins have been shown to interact with the PDZ-3 domain of nNOS with Cav3.2 being the only protein identified to date that is capable of providing a source of Ca2+ for the activation of nNOS. Utilizing a TAT fusion peptide demonstrated to disrupt the Cav3.2/nNOS interaction in a heterologous system the results of the present study suggest that Cav3.2 activity may contribute to the activation of nNOS and the modulation of respiratory activity. The hypothesized revisions to the model proposed by Campanucci and co-workers would accommodate our data suggesting an attenuated hyperoxic response in the following way. It is possible that, in the control animals exposed to 151  hypoxic conditions, NO mediated inhibition moderates the reflex response from the carotid body and, with the sudden introduction of a hyperoxic environment, this inhibition may remain for a short period until the NO and any secondary messenger signals are degraded to relieve the inhibition of the carotid body. This would explain the more pronounced decrease and the subsequent rebound observed in the respiratory rate in the TAT control animals. In animals treated with the disrupting TAT-DEPV peptide, the NO-mediated inhibition of the carotid body is repressed and as a result there would be little delay in the response of the carotid body to the hyperoxic conditions and the respiratory rate would follow a trajectory that corresponds directly with the O2 environment without the slight overshoot observed in the control animals. Considering the TAT-DEPV peptide was administered through intraperitoneal injection and therefore acting systemically, it is possible that the peptide is disrupting Cav3.2/nNOS or other PDZ-3 mediated interactions in other neuronal regions involved in respiration such as the NST. Further investigations are required to provide the evidence required to distinguish between the scenarios hypothesized above. One potential experiment would be to use a co-culture of acutely dissociated microganglia and type I glomus cells of the carotid body (Campanucci et al., 2006) to demonstrate the effect of disrupting the Cav3.2/nNOS interaction on the NO-mediated inhibition of type I glomus cells. Another potential experiment would be to obtain electrophysiological recordings of carotid sinus nerve activity during the hyperoxic response test under normal and Cav3.2/nNOS disrupted conditions. This approach would have the advantage of in vivo recording and offer the possibility of applying TAT peptides locally rather than systemically. This, however, would require sedating the animals which has been shown to affect respiratory responses. In addition to providing a potential source of NO for the inhibition of the carotid body response, the Cav3.2/nNOS interaction may also contribute to the regulation of microganglia activity 152  wherein the NO produced feeds back to inhibit Cav3.2 channels potentially reducing the excitability of the microganglia. Electrophysiological recordings of acutely dissociated microganglia to examine the contribution of a Cav3.2/nNOS interaction would address this possibility.  Figure 3.9: Schematic illustrating the hypothesized contribution of the Cav3.2/nNOS interaction to the NO-mediated inhibition of the carotid body  153  Figure 3.9: Schematic illustrating the hypothesized contribution of the Cav3.2/nNOS interaction to the NO-mediated inhibition of the carotid body 2+  Hypoxia causes an inhibition of TASK-like and large conductance Ca -activated (BK) potassium channels in type I cells (1) resulting in depolarization of the cell and leading to Ca2+ influx which results in the release of acetylcholine and ATP. The neurotransmitters bind to purinergic P2X receptors (P2XR) and nicotinic acetylcholine receptors (AChR) in the sensory nerve endings which relay the chemosensory signal to the central nervous system. Hypoxic conditions also activate TWIKrelated halothane inhibitable K+ channels (THIK) in the microganglia (2) which results in depolarization and increases neuronal excitability. Potentially, the increased excitability would lead to the activation of Cav3.2 channels in the membrane and the Ca2+ dependent activation of nNOS to produce nitric oxide (NO) (3). NO diffuses freely across the neuronal membranes to inhibit Cav1 channels in the type I glomus cells and increase BK channel activity (4) resulting in decreased depolarizing activity and hyperpolarization of the cell. In addition to the NO mediated inhibition of type I cells of the carotid body, NO could potentially feedback to inhibit further Cav3.2 (5) to limit the degree of inhibition imposed on the type I cell.  3.4.2. Implications of the observed increase in tidal volume and total ventilation following administration of a mutant PDZ-3 TAT fusion peptide Interestingly, administration of the TAT peptide with a mutated PDZ-3 motif (DEPE) resulted in a significant increase in the tidal volume and consequently in the total ventilation under hyperoxic and normoxic conditions. In Chapter 2 of this thesis we showed that this peptide does not affect either the co-immunoprecipitation or the functional interaction of Cav3.2 and nNOS. We were unable to find any reference in the literature of the mutated motif interacting with other proteins, however, given that the peptides are administered systemically it is possible that the mutant peptide could disrupt protein-protein interactions 154  and/or act as an agonist/antagonist on as yet unidentified proteins involved in the respiratory network. Considering that the mutant TAT peptide had an effect on the tidal volume and not the respiratory frequency, it is unlikely to affect the central respiratory oscillator but probably affects one or more of the inhibitory and excitatory circuits that directly modulate the inspiratory motor neurons. Identifying which protein(s) the mutant peptide interacts with and/or the potential interactions that it disrupts would be a first step in identifying the mode of action of this peptide on the respiratory system. Precipitation studies using the TAT-DEPE peptide as the bait followed by mass spectrometry analysis would be an interesting way to begin this investigation.  3.4.3. Potential relevance to respiration The data obtained from the present experiments suggest that a functional interaction between Cav3.2 and nNOS contributes to the regulation of the respiratory response to oxygen, potentially through a NO mediated inhibition of type I glomus cells of the carotid body by the microganglia in the GPN. These results contribute to our understanding of the regulatory mechanisms that modulate the respiratory response under hyperoxic, normoxic and hypoxic conditions. The interactions that underlie the neuronal activity providing respiratory drive to the central respiratory circuits are complex and a greater understanding of these interactions is required to enable critical evaluation and treatment of respiratory pathologies such as sleep apnea (reviewed in (Bradley, 2002; Eckert et al., 2007)). This knowledge would also help us develop better strategies to acclimate the body to relatively extreme conditions such the decreased oxygen levels found at higher altitudes which has  155  been found to have a pronounced effect on the respiratory system and the body as a whole (reviewed in (Eckert et al., 2007)).  156  Chapter 4. 4. Discussion 4.1. Overall significance of the results 4.1.1. Identification of a physical and function interaction between Cav3.2 and nNOS We presented data from immunoprecipitation, NO sensitive fluorometric assay and electrophysiology experiments demonstrating a novel physical and functional interaction between the Cav3.2 T-type Ca2+ channel and nNOS. This interaction closely localizes nNOS to a source of Ca2+ influx and further links NO production to neuronal activity. We show that the NO produced following the activation on nNOS is sufficient to inhibit Cav3.2 activity forming a putative negative feedback pathway which could contribute to the regulation of neuronal activity in cells where both proteins are expressed. Additionally, since NO is able to freely diffuse throughout the cell, the NO produced from the Cav3.2/nNOS interaction may also influence other cellular functions either through direct action or through second messenger pathways via the activation of soluble guanylyl cyclase. Furthermore we demonstrate that nNOS may facilitate the formation of larger complexes by utilizing both the PDZ-3 binding domain and the internal PDZ-2 binding ligand at the amino terminus of the protein. This capability could potentially associate the Cav3.2/nNOS complex with other neuronal proteins which respond to increases in local Ca2+ or NO concentrations such as large conductance Ca2+-dependent potassium channels (BK channels) (Bolotina et al., 1994; George and Shibata, 1995). Such interactions would significantly increase the contribution of the Cav3.2/nNOS interaction to neuronal activity; potentially linking sub-threshold stimulation and low threshold spikes to NO production and the regulation of numerous  157  cellular events through Ca2+ influx, NO production or the activation of secondary messenger pathways. 4.1.2. Potential relevance for nociception Cav3.2 channels and nNOS have both been demonstrated to play a significant role in nociception. Mice lacking the Cav3.2 channel display a significant reduction in the response to noxious stimuli (Choi et al., 2007) and redox modulation of Cav3.2 has been shown to modulate the nociceptive response (Todorovic et al., 2004). nNOS knockout mice have also been shown to display analgesia to noxious mechanical, thermal and chemical stimuli (Guan et al., 2007). The Cav3.2/nNOS interaction identified in this study could explain how such similar phenotypes could arise from silencing either protein. It is feasible that the Cav3.2/nNOS complex would permit the stimulation of DRGs to activate Cav3.2 to produce a localized increase in intracellular Ca2+ and stimulate nNOS to produce NO which would then proceed to influence other cellular processes involved in producing the nociceptive response. Given that the nNOS knockout mice display analgesia to noxious stimuli, a similar response would be anticipated following disruption of the Cav3.2/nNOS interaction with the TATDPEV fusion peptide. Additionally, the negative feedback of Cav3.2 by NO may serve to regulate the production of NO to limit the nociceptive response and possibly to avoid the potential cytotoxic buildup of NO within the cell. The potential for nNOS to facilitate the formation of larger protein complexes may also contribute to the modulation of nociception. Recently, activation of transient receptor potential vanilloid-1 (TRPV1) channels in rat DRG neurons have been demonstrated to inhibit Cav3.2 channels which may be a potential mechanism underlying capsaicin-induced analgesia (Comunanza et al., 2011). Several members of the TRPV family have been shown to interact with proteins possessing PDZ domains (Islam, 2011) suggesting that nNOS could 158  potentially mediate a functional interaction between Cav3.2 and TRPV1 by facilitating the co-localization of the two channels. 4.1.3. Relevance to the respiratory system The data presented in this study suggests that a functional interaction between Cav3.2 and nNOS contributes towards the regulation of the respiratory rate in response to available oxygen. The hyperoxic response data suggests that this contribution occurs in the peripheral nervous system. I propose the hypothesis that the Cav3.2/nNOS interaction associates nNOS with a source of Ca2+ influx in the microganglia of the GPN which directly links NO production with the intrinsic O2 sensing capability of the microganglia to provide efferent inhibition of the carotid body. NO mediated inhibition of the respiratory response by the microganglia within the GPN has been suggested to occur independently of the central respiratory system which is interesting as it presents the possibility of enhancing the respiratory drive to stimulate increased respiration without negatively impacting the complex circuits which comprise the central respiratory network. This may have an impact on conditions where a decreased respiratory drive contributes to pathological conditions such as central sleep apnea.  4.2. The Cav3.2 T-Type calcium channel and neuronal nitric oxide synthase physically and functionally interact forming a putative negative feedback regulatory loop 4.2.1. Working hypothesis The evidence presented in Chapter 2 demonstrates that Cav3.2 and nNOS can physically and functionally interact when co-expressed in a heterologous system. The physical interaction is mediated by the PDZ-3 binding ligand situated at the carboxyl 159  terminus of the Cav3.2 which interacts with the PDZ-3 domain of nNOS. This interaction facilitates the Ca2+ dependent activation of nNOS by Cav3.2 activity which results in the production of NO. The NO produced by nNOS in turn inhibits further Cav3.2 activity thereby completing the negative feedback circuit. In addition to inhibiting Cav3.2 the NO produced as a result of this circuit could potentially mediate other cellular events through the direct interaction with other proteins or through the activation of sGC to produce the secondary signaling molecule cGMP. We also demonstrate that nNOS can facilitate the formation of larger complexes through the internal PDZ-2 binding ligand adjacent to the PDZ-3 domain. In this study PSD95 was utilized to show that both PDZ structures of nNOS can be occupied simultaneously. PSD-95 is a scaffolding protein well known to associate nNOS with other proteins, such as NMDA receptors, to facilitate NO-mediated modulation of various targets. Additionally, sGC has been shown to bind to PSD-95 which could potentially link T-type channel activity with the production of cGMP. Furthermore, we show that in the presence of PSD-95 the inhibitory effects of nNOS on Cav3.2 are significantly increased suggesting that the utilization of the internal PDZ-2 ligand of nNOS may increase the affinity of the Cav3.2/nNOS interaction thereby supporting the possibility of this interaction occurring in vivo.  4.2.2. Limitations and weaknesses The inhibition of Cav3.2 by NO has been demonstrated to occur through metal catalyzed oxidization of the histidine residue at position 191 of the channel. Inhibition of Cav3.2 channels in this study was assumed to occur through this process as well; however experiments to confirm that the H191 residue is involved in the inhibition were not 160  examined. A solution would be to generate a Cav3.2 mutant substituting the H191 for glutamine as was done by Kang et al., Bartels et al., and Orestes et al. and repeat the current density experiments utilizing this construct (Kang et al., 2006; Bartels et al., 2009; Orestes et al., 2011). A second potential weakness is that L-NAME introduces a slight structural conformation change in the nNOS protein which is believed to contribute to the inhibition of nNOS (Mayer, 1995). While unlikely, it is possible that the reduction in T-type current density observed is due to the physical interaction of nNOS and Cav3.2 and that application of L-NAME relieves the inhibition due to the shift in the structural conformation of nNOS. To test this possibility the current density experiments could be repeated in the presence of a NO scavenger such as CPTIO in order to remove NO as it is produced. In these experiments we would expect the application of the NO scavenger to restore the current density to levels similar to those observed with application of the NOS inhibitor. Alternatively, the current density experiments could be repeated in the presence of barium rather than Ca2+. Barium should not activate nNOS therefore we would not expect to see a difference in current density with the application of L-NAME. A potential limitation of this study is that while we have evidence to show that the Cav3.2/nNOS interaction is required for the activation of nNOS, we do not have evidence to determine whether the interaction is required for the NO produced from nNOS to inhibit Cav3.2. A possible solution would be to co-transfect constitutively active calmodulin with nNOS into the stable cell line expressing the Cav3.2 with the mutant PDZ-3 motif and perform current density analysis. If the interaction is not required for Cav3.2 inhibition by nNOS derived NO then we would expect a decrease in the current density with the constitutively active calmodulin. We recognize that these weaknesses and limitations exist and do not believe that they would significantly alter the conclusions presented here. 161  4.3. Intraperitoneal injection of TAT fusion peptide to disrupt the Cav3.2 and nNOS interaction in vivo produces an augmented respiratory response to changes in available oxygen in rats 4.3.1. Working hypothesis We present evidence that a membrane permeable peptide designed to disrupt the Cav3.2/nNOS interaction has a significant effect on the respiratory response to hypoxia. The data from the hyperoxic response experiments suggests that the disrupting peptide is affecting the peripheral network of the respiratory system. We also show that Cav3.2 and nNOS are both expressed in the microganglia of the GPN which has been demonstrated to contribute to the efferent inhibition of the carotid body, a major chemoreceptor of the peripheral respiratory system. Our working hypothesis is that a physical/functional interaction between Cav3.2 and nNOS contributes to the NO-mediated efferent inhibition of the carotid body by the microganglia of the GPN. Building on the model put forward by Campanucci et al. we propose that the intrinsic O2 sensing capability of the microganglia of the GPN increases the excitability of the microganglia which activates Cav3.2 channels and consequently stimulates NO to produce NO in response to hypoxic conditions. The NO produced then participates in the inhibition of the reflex hypoxic response generated by the carotid body thereby imposing a cap on the increase in the respiratory drive which contributes to the modulation of the respiratory rate. Disruption of the interaction between Cav3.2 and nNOS would prevent the generation of NO therefore inhibition of the response of the carotid body would not occur, effectively removing the cap from the increase of the respiratory drive and which may reflect the observed increase in respiratory rate under hypoxic conditions. 162  4.3.2. Limitations and weaknesses The metabolic data in this respiratory response study is incomplete due to a likely design limitation in the chamber allowing minute quantities of pure oxygen to become trapped and slowly escape during normoxic and hypoxic challenges. This was discovered during the analysis of the data after the U.B.C. Animal Care license expired therefore additional data could not be collected. Since the amount of O2 trapped was small and only resulted in a partial percentage change in O2 concentration it was not likely that this limitation significantly affected the respiratory rate, tidal volume or the CO2 measurements. Another potential limitation is the maximum possible air flow allowed by the equipment (2000 ml/min). Ideally, for the hyperoxic response test, it is desirable to exchange to O2 concentration as rapidly as possible to invoke the maximum peripheral response with minimal input from the central respiratory circuits. The flow rate used in this study was acceptable but not ideal therefore we cannot state with certainty that the observed effects are purely peripheral in origin as modulatory efferent signals from the central respiratory circuits may be present. Systemic administration of the peptide presents a few possible experimental limitations. The peptide is not limited to the GPN therefore the data may also include effects of the peptides in other regions of the body involved in the respiratory response and that express both Cav3.2 and nNOS and which might either mask or exaggerate the data. The proposed model is speculative based on the available data; further investigations into the effect of disrupting the Cav3.2/nNOS interaction within the GPN are required to substantiate the model. While the PDZ-3 domain has thus far been found to be unique to nNOS and few proteins have been identified as having compatible PDZ-3 binding ligands, there is the possibility that the peptides can disrupt interactions that have yet to be identified. Co163  precipitation using the peptides as the bait and utilizing high-pressure liquid chromatography to analyze proteins that co-precipitate may be one way to determine the specificity of the peptides. Additionally, it has been shown that the protein CAPON possesses a PDZ-3 binding ligand and can bind to nNOS which disrupts the PDZ-2 interaction between PSD-95 and nNOS. It is possible that the peptide designed to disrupt the Cav3.2/nNOS interaction may also disrupt PDZ-2 interactions with other proteins in vivo. Localized administration of the TAT-fusion peptides is one method which may minimize possible effects of non-specific disruption of protein interactions. Another potential option would be to use Cav3.2 knockout mice to generate knock-in mice with a mutant Cav3.2 channel lacking the PDZ-3 binding ligand. With these knock-in mice concerns regarding the non-specific effects of a systemically administered compound would be elevated. Degradation of the injected peptides after injection is a possible limitation. We administered the peptides at a dose of 9 mg/kg as it was the highest level of TAT peptide injection that we could find in the literature that did not appear to have adverse effects. Lower doses have been utilized in the literature with the same degree of efficacy which minimized our concerns regarding the degradation of the peptide. Furthermore, animals underwent two rounds of testing with approximately one hour between the start of the first round and the start of the second, thus if significant degradation occurred in that period a difference between the observed responses should have been apparent.  4.4. Conclusions 4.4.1. General conclusion Here we present evidence of a novel physical and functional interaction between Cav3.2 and nNOS. We show that Cav3.2 possesses a carboxyl PDZ-3 binding ligand that can 164  interact with the PDZ-3 domain of nNOS and that this integration facilitates the activation of nNOS by Cav3.2 mediated Ca2+ influx. We also demonstrate that NO produced by nNOS can in turn feedback to inhibit Cav3.2 activity. To our knowledge this is the first description of an interaction between these proteins and the first evidence of a self-inhibiting circuit involving the Cav3.2 channel. We further demonstrate the potential for Cav3.2 and nNOS to form larger complexes through the utilization of both the internal PDZ-2 binding ligand and the PDZ-3 domain of nNOS. Evidence for a role for the Cav3.2/nNOS interaction in vivo is also presented. We found that administration of membrane permeable peptides designed to disrupt the Cav3.2/nNOS interaction has an effect on the respiratory response to hypoxia suggesting a possible role for this complex in the modulation of respiratory drive.  4.4.2. Relevance to modulation of neuronal networks co-expressing Cav3.2 and nNOS A functional interaction between Cav3.2 and nNOS may contribute to the regulation of neuronal activity in networks where both proteins are expressed. Tight control over neuronal activity ensures the efficient and proper function of the brain as a whole and disruption of the modulatory mechanisms can, under the right circumstances, lead to abhorrent activity such as seizures. The contribution of Cav3.2 activity to neuronal excitability and the generation of specific firing patterns such as burst firing have been well documented. A self-inhibiting circuit consisting of Cav3.2 and nNOS would potentially impose an upper limit to the contribution of Cav3.2 channel activity to neuronal activity in certain populations of cells. This restriction could assist in preventing the over stimulation of neuronal networks and affect the establishment of firing patterns that underlie certain pathological conditions such as epilepsy. 165  Additionally, the interaction between Cav3.2 and nNOS provides a link between neuronal depolarization and the production of NO which has been demonstrated to play a role in the modulation of neuronal activity and tuning of neuronal networks. In this scenario the contribution of Cav3.2 and nNOS could extend well beyond the modulation of cell excitability and the contribution of Cav3.2 to neuronal firing patterns to include the cell-tocell signaling events and possibly have longer lasting effects such as short term facilitation and long term potentiation.  4.4.3. Relevance to the respiratory system Our understanding of the complexity of the neuronal networks that comprise the respiratory system is steadily improving, however there is still much that remains a mystery. The evidence presented here showing that Cav3.2 and nNOS are potentially involved in the regulation of the hypoxic respiratory response contributes to the overall understanding of the respiratory system, particularly the modulation of respiratory drive. Modulation of respiratory drive allows for rapid responses to changes in metabolic demand and the environment. In some pathological conditions the respiratory drive or the respiratory response to this drive is insufficient resulting in potentially life threating conditions. For example, a decreased respiratory drive is one of the key factors in the pathological condition of central sleep apnea (Bradley, 2002; Eckert et al., 2007). Additionally, significant alterations in cardiorespiratory responses have been observed in patients with obstructive sleep apnea syndrome. These respiratory adaptations are a result of chronic intermittent hypoxia exposure that has been shown to invoke adaptive changes in the peripheral chemosensory reflex (Kline et al., 2007). The peripheral chemosensory reflex has also been demonstrated to be critical for the acclimation of the respiratory response to the 166  decreased O2 concentrations at high altitude (hypoxic ventilatory acclimation). Furthermore, at altitudes greater than 2,500 m, permanent residents are at risk of developing chronic mountain sickness, a condition associated with the reduced sensitivity of the peripheral chemoreceptors (reviewed in (Joseph and Pequignot, 2009)). As our understanding of the factors that underlie and modulate the respiratory drive improve we can likely develop better options for the clinical treatment and prevention of conditions such as central sleep apnea, chronic intermittent hypoxia exposure and chronic mountain sickness. Additionally, pharmaceuticals may be developed to speed high altitude acclimation that would improve the safety of those needing to ascend to high altitude rapidly such as alpine rescue teams.  4.4.4. Future directions The results presented in this thesis demonstrate that a physical and functional interaction between Cav3.2 and nNOS can occur in a heterologous expression system. Further investigations are required, however, to elucidate the role(s) of this interaction in vivo. Data have been presented which indicate that an interaction between Cav3.2 and nNOS may play a role in the regulation of the respiratory response; presumably involving the efferent inhibition of the carotid body. To further investigate the contribution of Cav3.2 channels and nNOS to the efferent inhibition of the carotid body it would be necessary to identify whether the two proteins truly interact in microganglia of the GPN. Electrophysiological recordings of acutely dissociated cultures of the microganglia of the GPN to determine if disruption of the Cav3.2/nNOS interaction has an effect on the Cav3.2 currents would be one indicator of an interaction. Another possible indicator would be demonstration that the synthesis of NO is significantly reduced when the Cav3.2/nNOS interaction is disrupted. From there, it would be interesting to investigate the contribution of the Cav3.2 and nNOS to the efferent inhibition of 167  the carotid bodies utilizing a co-culture of acutely dissociated microganglia from the GPN and type I glomus cells from the carotid bodies. The results of the electrophysiological recordings obtained from cells of the co-culture could then be used to justify further in vivo experiments, particularly electrophysiological recordings of the carotid sinus nerve in TATDEPV treated animals. As mentioned in the previous chapter, in these experiments the peptide could be applied locally to the region of interest, alleviating the problems inherent with systemic administration of the peptide. The NO assay data presented in Chapter 1 has been forwarded to Dr. E. Bourinet as part of collaboration between Dr.T. P. Snutch’s laboratory and Dr. Bourinet’s laboratory (Université de Montpellier, France) into the role of Cav3.2 and nNOS in nociception. Cav3.2 and nNOS have both been identified in the nociceptive DRGs and given that Cav3.2 knockout mice display an increased nociceptive threshold would expect the disrupting the Cav3.2/nNOS interaction would produce a hyperalgesic response. On the other hand, nNOS knockout mice also display an increased nociceptive threshold suggesting that the role of the Cav3.2/nNOS interaction may not be via the direct regulation of Cav3.2 but rather via facilitation of the production of NO by anchoring nNOS to a source of Ca2+ related to other activities of the cell. The NO produced as a result of Cav3.2 activity may play a role in the nociceptive response through the activation of second-messenger systems and/or the direct action on other cellular signaling pathways. Using cultures of acutely dissociated DRGs it should be possible to determine if Cav3.2 and nNOS interact within these neurons. This information could then be used to justify in vivo experiments examining the effect of peptides designed to disrupt the Cav3.2/nNOS interaction on nociceptive responses. Furthermore, it would be interesting to investigate the effects on the nociceptive response of inhibiting the downstream targets of NO such as soluble guanylyl cyclase. 168  Mutations in the Cav3.2 channel have been associated with the pathological condition of childhood absence epilepsy. Interestingly, this condition generally resolves itself as the patient reaches maturity suggesting that the Cav3.2 mutations are able to contribute to seizure activity for a limited period during development. It has been found that nNOS is developmentally expressed in the nRT of ferrets and the same developmental expression may occur in the animal models of childhood absence epilepsy. It is possible that a Cav3.2/nNOS interaction may play a role in the regulation of the neuronal networks during development and that the Cav3.2 mutations disrupt the balance of neuronal activity thereby contributing to seizure activity. Investigations into this possibility would involve identifying nNOS expression in the nRT of the absence epilepsy models followed by electrophysiological recordings from acute brain slices through the nRT at different stages of development to determine if disrupting the Cav3.2/nNOS interaction has a significant impact on the neurological activity in that region. Here, only a few suggestions have been made for possible avenues of investigation into the role of Cav3.2 and nNOS in vivo. There are many other potential projects that could also be considered including the role of a Cav3.2 and nNOS interaction in learning and memory and also in the olfactory response. It is apparent that investigations concerning the roles of Cav3.2 channels in neuronal circuits should also include a possible interaction with nNOS. 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