UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Structural and genetic modulators of voltage-gated potassium channel activation kinetics Rezazadeh-Roudsari, Saman 2007

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

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-ubc_2007-319147.pdf [ 13.55MB ]
Metadata
JSON: 831-1.0100738.json
JSON-LD: 831-1.0100738-ld.json
RDF/XML (Pretty): 831-1.0100738-rdf.xml
RDF/JSON: 831-1.0100738-rdf.json
Turtle: 831-1.0100738-turtle.txt
N-Triples: 831-1.0100738-rdf-ntriples.txt
Original Record: 831-1.0100738-source.json
Full Text
831-1.0100738-fulltext.txt
Citation
831-1.0100738.ris

Full Text

STRUCTURAL AND GENETIC MODULATORS OF VOLTAGE GATED POTASSIUM CHANNEL ACTIVATION KINETICS by Saman Rezazadeh-Roudsari B.Sc. (Hons), The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In The Faculty of Graduate Studies (Physiology) THE UNIVERSITY OF BRITISH C O L U M B I A August 2007 © Saman Rezazadeh-Roudsari, 2007 / Abstract Voltage-gated potassium (Kv) channels regulate membrane excitability and are therefore critical determinants of cellular function. However, the detailed mechanisms by which Kv channel activity is modulated are not well understood. This thesis investigates the modulation of activation of Kv l .2 and KCNQ1 channels. These studies reveal that K v l . 2 can activate via two different pathways that produce two distinct gating phenotypes/modes. In the 'slow' gating mode, the activation V1/2 was shifted by +30 mV and activation kinetics were at least 20-fold slower than those of channels gating through the 'fast' mode. This offers an explanation for the wide variations in the reported activation kinetics of K v l . 2 in the literature. Introduction of a positive charge at or around threonine 252 (T252) in the S2-S3 cytoplasmic linker of Kv l .2 trapped channels in the 'fast' activation mode, suggesting that this region may act as the molecular switch in Kv l . 2 . Consistent with this, the S2-S3 linker was shown to mediate the gating-modifying effect of a mutation (T46V) in the cytoplasmic T l domain of Kv l . 2 . Excision of patches containing K v l . 2 also trapped channels in the 'fast' gating mode, indicating cytoplasmic regulators may also modify the gating mode via the S2-S3 linker. We have ruled out cytoplasmic regulation by PIP2,' polyamines and phoshporylation. Interestingly, one kinase inhibitor, KN-93, a commonly used calcium/calmodulin-dependent protein kinase II inhibitor, was found to be a direct extracellular blocker of many different Kv channels including Kv l .2 . Finally, a novel missense mutation at the intracellular end of the S3 helix in a mutant KCNQ1 channel (V205M), detected in an aboriginal community with a high prevalence of long QT syndrome and sudden death, was shown to cause a depolarizing shift in the voltage dependence of activation and a slowing of activation kinetics. This resulted in reduced repolarization reserve during the cardiac action potential and a likely increased susceptibility to the ii initiation of arrhythmias. The close positioning of this mutation to the S2-S3 linker provides a putative structural working model for the gating switch in K v l . 2 that involves changes in the hydrophobic packing of the S3 helix and its influence on S4 voltage-sensor movement. iii Table of Contents Abstract i i Table of Contents iv List of Figures ix List of Abbreviations xi Acknowledgements xi i i Dedication xiv Co-Authorship statement xv Chapter 1: Introduction 1 1.1 Overview 2 1.2 Topology of K v channels 2 1.3 Conformational changes in the voltage-sensing domain in response to membrane depolarization 5 1.3.1 From sliding to the helical screw model 6 1.3.2 The paddle model 8 1.3.3 The paddle model contradicts experimental data from mammalian channels 9 1.3.4 Structure of the voltage-sensing domain in a native state 10 1.4 Conformational changes associated with channel opening 12 1.4.1 The activation gate 12 1.4.2 Electromechanical coupling between the voltage-sensing domain and the activation gate 14 1.5 Modulators of K v channel activation 17 1.5.1 Structural determinants of Kv channel activation kinetics 17 1.5.1.1 The role of cytoplasmic domains in regulation of gating 18 1.5.1.2 Transmembrane linkers and modulation of activation kinetics 19 1.5.2 Dynamic modification of Kv channel activation kinetics 22 1.5.2.1 Auxiliary subunits 23 1.5.2.2 Phosphorylation 24 1.5.2.3 Direct cyclic nucleotide binding 25 1.5.2.4 Lipids and ion channels 26 iv 1.5.2.5 SUMOylation 28 1.5.2.6 Ionic environment 28 1.6 Genetic modulators of channel activation 30 1.6.1 Kv channels and LQTS 33 1.6.1.1 IKr dysfunction (LQT2 and LQT6) 33 1.6.1.2 IKs dysfunction (LQT1 and LQT5) 35 1.6.2 Na+ channel mutations and LQT3 37 1.7 Scope of the thesis 38 1.8 References 42 Chapter 2: An activation gating switch in K v l . 2 is localized to a threonine residue in the S2-S3 Linker 61 2.1 Introduction 62 2.2 Materials and Methods 66 2.2.1 Cell preparation and transfection 66 2.2.2 Solutions 66 2.2.3 Electrophysiological procedures 67 2.2.4 Molecular biology and channel expression 67 2.2.5 Kvl.5/Kvl.2 chimeras 68 2.2.6 Data analysis and modeling 68 2.3 Results 71 2.3.1 Heterogeneous activation properties of Kvl.2 expressed in mammalian cell lines 71 2.3.2 Prepulse potentiation of Kvl.2 activation 74 2.3.3 Voltage-dependent recovery of 'slow' activation 77 2.3.4 Molecular determinants of prepulse potentiation 80 2.3.5 A critical threonine in the S2-S3 linker of Kvl.2 85 2.3.6 Cytoplasmic constituents regulate Kvl.2 channel gating 89 2.4 Discussion 92 2.4.1 'Cell-to-cell' versus 'pulse-to-pulse' heterogeneity of Kvl. 2 gating 92 2.4.2 A model scheme for fast' and 'slow' activation 93 2.4.3 Structural determinants of prepulse potentiation 98 2.5 Acknowlegements 102 2.6 References 103 Chapter 3: KN-93, a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels 109 3.1 Introduction 110 3.2 Materials and Methods 112 3.2.1 Materials : 112 3.2.2 Cell culture and transfection 112 3.2.3 Molecular biology and channel mutations 113 3.2.4 Electrophysiology solutions 113 3.2.5 Electrophysiological procedures 114 3.2.6 Data analysis 114 3.3 Results 116 3.3.1 KN-93 inhibits a wide range of Kv channels 116 3.3.2 KN-93 directly blocks the Kvl.5 channel 118 3.3.3 KN-93 is an extracellular open-channel blocker of Kvl.5 channels 122 3.3.4 KN-93 delays recovery from inactivation 125 3.4 Discussion 130 3.4.1 KN-93 Inhibits a wide variety of voltage-gated potassium channels 130 3.4.2 KN-93 stabilizes the inactivated state 130 3.5 Acknowledgements 133 3.6 References 134 Chapter 4: S2-S3 linker is associated with coupling of the T l domain to K v l . 2 channel gating 137 4.1 Introduction 138 4.2 Materials and Methods 141 4.2.1 Cell preparation and transfection 141 4.2.2 Solutions 141 4.2.3 Molecular biology and channel mutations 142 4.2.4 Kvl.5/Kvl.2 chimeras 142 4.2.5 RNA preparation and oocyte injection 142 4.2.6 Electrophysiological procedures 143 vi 4.2.7 Data analysis 143 4.3 Results 145 4.3.1 The T46Vmutation accelerates Kvl.2 deactivation without altering activation kinetics .' 145 4.3.2 Kvl.5 is insensitive to the T46V equivalent mutation 149 4.3.3 Effect of T46V mutation depends on regions outside the Tl domain 151 4.3.4 The Tl domain may exert its effect through coupling with the S2-S3 linker 153 4.4 Discussion 156 4.4.1 T46V has no effect on Kvl.2 activation properties 156 4.4.2 The role of the N-terminus in regulation of channel gating 156 4.4.3 Disparate effects ofTl domain mutations in Kvl.2 and Kvl.5 157 4.4.4 The S2-S3 linker connects the modification of the Tl domain to the channel gating 158 4.5 Acknowledgments 159 4.6 References 160 Chapter 5: Destabilization of the open state of I K s potassium channels by a KCNQ1 V205M missense mutation causes an inherited form of LQTS in a Canadian aboriginal community 164 5.1 Introduction 165 5.2 Materials and Methods 168 5.2.1 Molecular Biology 168 5.2.2 Cell Preparation and Transfection 168 5.2.3 Solutions 169 5.2.4 Electrophysiological procedures 169 5.2.5 Data Analysis 170 5.3 Results 171 5.3.1 V205M IKS channels traffic to the membrane 171 5.3.2 The V205M mutation alters activation and deactivation properties ofl^s channels 171 5.2.3 Behaviour of the V205M mutant channels during simulated cardiac action potentials 178 5.4 Discussion 182 vii 5.4.1 The biophysical defect in V205M 182 5.4.2 Summation oflas at high heart rates 183 5.4.3 Structural considerations 184 5.5 Acknowledgements 186 5.6 References 187 Chapter 6: Discussion 191 6.1 Novel heterogeneous gating of Kvl.2 192 6.2 Physiological importance of Kvl.2 modal gating 193 6.3 The S2-S3 linker plays an important role in regulation of Kvl.2 activation properties 195 6.4 Coupling of the T l domain to channel gating 196 6.5 Cytoplasmic regulators of channel gating 198 6.6 The role of hydrophobic residues in the S3 helix 200 6.8 References 203 viii List of Figures Figure 1.1. K v channel structure 4 Figure 1.2. Structural rearrangements associated with channel opening 16 Figure 1.3. Cardiac electrical activity 32 Figure 2.1. Bimodal gating of Kv l .2 expressed in mammalian cell systems 73 Figure 2.2. Twin pulses convert 'slow' to 'fast' activation in K v l . 2 76 Figure 2.3. Holding potential has little effect on slow activation kinetics of K v l .2 and voltage-dependent recovery of slow activation in K v l . 2 79 Figure 2.4. Chimeric study of Kvl .5 and K v l . 2 domains that regulate the activation rate. 82 Figure 2.5. Transfer of the S2 helix and S2-S3 linker from K v l . 2 to Kvl .5 confers modal activation gating kinetics 84 Figure 2.6. Point mutational study of the Kv l .2 S2 and S2-S3 linker 86 Figure 2.7. The activation effects of charged and uncharged substitutions of T252 in K v l . 2 , and of residues in close proximity 88 Figure 2.8. K v l . 2 channel gating is modified by interaction with a cytosolic component. 91 Figure 2.9. Model of'fast' and 'slow' activation in K v l . 2 95 Figure 2.10. Simulated and experimental observation of dual activation pathways of K v l . 2 channel opening r. 97 Figure 3.1. KN-93 inhibits Kv channels from a number of different subfamilies 117 Figure 3.2. KN-93 inhibition of Kvl .5 is independent of CaMK II activity 119 Figure 3.3. Voltage-dependence of KN-93 effect 121 Figure 3.4. Extracellular binding site of KN-93 124 Figure 3.5. Effect of KN93 on the rate of recovery from inactivation 128 Figure 3.6. Mutants that accelerate or slow inactivation alter KN-93 block of Kv l .5 . . . 129 Figure 4.1. The T46V mutations result in acceleration of K v l . 2 channel deactivation. 147 Figure 4.2. T46V mutation does not alter activation kinetics when expressed mXenopus oocytes 148 Figure 4.3. Kv l .5 channels are insensitive to the T46V equivalent mutation, T133V... 150 Figure 4.4. Acceleration of deactivation due to the T l domain mutation is linked to other regions of the channel 153 Figure 4.5. The S2-S3 linker is involved in modulation of gating by the T l domain.... 155 Figure 5.1. V205M mutation results in a depolarizing shift in the voltage dependence of IKS activation 173 Figure 5.2. V205M mutation accelerates the rate of K v L Q T l deactivation 175 Figure 5.3. Effect of V205M mutation on heteromultimeric Wt and mutant channels. 177 Figure 5.4. V205M eliminates K v L Q T l ionic current during the cardiac action potential. 179 Figure 5.5. V205M reduces repolarization reserve at high pulsing rates 181 List of Abbreviations Amino Acid 3 letter code 1 letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu E Glutamine Gin Q Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V T - time constant gmax- maximum conductance A g + - silver ion CaMKII- calcium/calmodulin-dependent protein kinase JJ Cav- voltage gated calcium (channel) C N B D - cyclic nucleotide-binding domain C N G - cyclic nucleotide-gated (channel) E A G - ether a-go-go (channel) E C G - electrocardiogram Fab- antigen binding fragment FRET- fluorescence resonance energy transfer g-V- conductance-voltage relationship I- current Itk- mouse fibroblast cell line lacking thymidine kinase H C N - hyperpolarization-activated cyclic-nucleotide-gated (channel) H E K - human embryonic kidney cells hERG- human ether a-go-go related gene (channel) HP- holding membrane potential IC50- concentration required to achieve half maximal block k- slope factor Kir- inwardly-rectifying potassium (channel) KN-93- (2-[N-(2-hydroxyethyl)]-N- (4-methoxybenzenesulfonyl)] amino -N chlorocinnamyl)-N-methylbenzylamine) Kv- voltage-gated potassium (channel) LQTS- long QT syndrome MTS- methanethiosulfonate mV- millivolt Nav- voltage gated sodium (channel) n- Hi l l coefficient P0- probability of opening pA- picoampere P K A - protein kinase A SIDS- sudden infant death syndrome T l - tetramerization domain TdP- torsades de pointes V1/2- half-activation voltage V G C C - voltage-gated calcium channel Wt- wild type Acknowledgements First and foremost I would like to thank my supervisor, Dr. David Fedida, who introduced me to research and has been very supportive during the entire duration of my graduate studies. He has given me the freedom to explore new areas of research but has always been there to guide me through. I would also like to thank Thomas Claydon. His enthusiasm for science has always been inspiring and his involvement in the KN-93 project made for an exciting and fun project. I thank Harley Kurata for starting the K v l . 2 project and also for being a great role model. I have been fortunate to have the privilege of learning molecular biology techniques from David Steele. Zhuran Wang has been a great source of knowledge for troubleshooting. Jodene Eldstom has always been there to listen, share and help me with my molecular biology problems and was also kind enough to proofread this thesis. My graduate supervisory committee, Dr. Steven Kehl, Dr. Eric Accili and Dr. Edgar Young has been extremely supportive and I thank them for that. I would also like to thanks Fifi Choi for all the assistance in the lab. I would like to thank my family, especially Arezoo, for their support throughout my university education. xiii Dedication my parents xiv Co-Authorship statement Chapter 2: An activation gating switch in Kvl.2 is localized to a threonine residue in the S2-S3 Linker Saman Rezazadeh was responsible for performing all the experiments, all the mutatgenesis and all the analysis presented in this chapter, except for the modeling of the bimodal activation property of K v l . 2 (Figures 2.9 and 2.10), which was performed kindly by Steven Kehl. Harley T. Kurata helped with the design of experiments and constructed the chimeric channels. Thomas W. Claydon helped with the manuscript preparation. Steve Kehl and David Fedida assisted in editing the manuscript. A version of this chapter is submitted to the Biophysical Journal. Saman Rezazadeh, Harley T. Kurata, Thomas W. Claydon, Steven J. Kehl, and David Fedida Chapter 3: KN-93, a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels Saman Rezazadeh was responsible for performing and analysis of the effect of KN-93 on various channels. Thomas Claydon contributed to design of experiments as well as performing the experiments (50% contribution). Saman Rezazadeh prepared the manuscript. Thomas Claydon and David Fedida assisted in editing the manuscript. A version of this chapter has been published. Saman Rezazadeh, Thomas W. Claydon and David Fedida. (2006). KN-93, a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. Journal of Pharmacology and Experimental Therapeutics 317, 292-299 Chapter 4: S2-S3 linker is associated with coupling of the T l domain to Kvl.2 channel gating Saman Rezazadeh was responsible for designing and performing all the experiments presented in this chapter. Harley Kurata constructed the chimeric channels and was also responsible for the site-directed mutagenesis. The manuscript was prepared by Saman Rezazadeh and edited by David Fedida. Chapter 5: Destabilization of the open state of IKS potassium channels by a KCNQ1 V205M missense mutation causes an inherited form of LQTS in a Canadian aboriginal community Saman Rezazadeh was responsible for the design of all the presented experiments, site-directed mutagenesis and performing all the experiments. Rosemarie Rupps, Shu Sanatani, and Laura Arbour carried out all the community-based experiments. Brett Casey performed the genomic screening of the patients. The manuscript was prepared by Saman Rezazadeh and edited by Jodene Eldstrom,.Glen Tibbits, Eric Accili and David Fedida. X V A version of this chapter wil l be submitted to Circulation. Saman Rezazadeh, Jodene Eldstrom, Rosemarie Rupps, Shu Sanatani, Glen Tibbits, Brett Casey, Eric Accil i , David Fedida and Laura Arbour. xvi Chapter 1: Introduction 1.1 Overview Ion channels are integral membrane proteins that span the lipid bilayer to form a pore through which selected ions can pass at near diffusion rate. These channels are ultimately responsible for generating and orchestrating the electrical signals in the brain, heart, muscles and other excitable cells. Potassium (K +) channels are the most diverse family of ion channels that share a common property of selectivity for K + over other ions (Gutman et ai., 2003). A group of K + channels that have been widely studied since their initial description by Hodgkin and Huxley are the voltage-gated potassium (Kv) channels (Hodgkin and Huxley, 1952). They are activated by changes in the transmembrane potential and are involved in repolarizing the membrane voltage from an excited state back to the resting level, and in reducing membrane excitability (Hille, 2001). Therefore, the gating properties of Kv channels directly influence cellular functions. Compromising the function of Kv channels can lead to many disorders such as epilepsy, episodic ataxia, long QT syndrome and sudden cardiac death (Ashcroft, 2000). Therefore, a detailed understanding of the gating properties of Kv channels and their physiological significance is important, and this forms the foundation of this thesis. The purpose of this chapter is to review our current structure-function understanding of Kv channels in terms of voltage-sensing and channel activation and to discuss different mechanisms through which properties of K v channels can be modified. 1.2 Topology of Kv channels Kv channels are multi-subunit complexes formed by multimerization of four a-subunits and may or may not contain additional accessory subunits. Each a-subunit is composed of six transmembrane (S1-S6) helices (Figure 1.1 A). Over the last decade, crystal 9 structures of several bacterial and one mammalian K + channel have been determined at atomic resolution (Doyle et al., 1998; Jiang et al, 2002a; Kuo et al, 2003; Jiang et al., 2003a; Long et al., 2005a). Although evolutionary well separated, all of these structures assume a tetrameric arrangement around a central pore through which K + ions can flow across the membrane. This structural organization is shown in Figure L I B , which depicts the structure of the Kv l .2 channel with each subunit highlighted in a different color (Long et al, 2005a). In addition to the tetrameric configuration, Kv channels are thought to assume a compartmentalized organization at the membrane. This was first revealed by electron microscopy images of the full-length Drosophila Shaker channel, the archetypical Kv channel (Sokolova et al., 2001). These images revealed tetrameric assembly of a well defined cytoplasmic density connected to the transmembrane density by thin 2 nm long linkers (Sokolova et al., 2001). The transmembrane density is attributed to the six transmembrane S1-S6 helices forming the core of the channel, while the bulk of the cytoplasmic electron density can be accounted for by the highly conserved N-terminal tetramerization (Tl) domain and possibly the C-terminus (Sokolova et al., 2003), although the structure of the C-terminus remains unknown. This overall organization of the channel is referred to as the "hanging gondola" model (Figure 1.1C), and was recently confirmed by the crystal structure of the mammalian K v l . 2 channel, which shows a distinct separation between the pore forming transmembrane domains and the cytoplasmic domains of the channel, that are connected by a-helical T l - S l linkers (Long et al., 2005a). Furthermore, the crystal structure shows that the linkers between the transmembrane and cytoplasmic domains form four lateral opening "windows" and that there is a central opening within the T l 3 tetrameric complex that aligns with the transmembrane pore domain. Interestingly, ions or blockers likely pass through the lateral windows to gain access to the channel pore rather than the central pore (Kobertz et al, 2000; Sokolova et al, 2001; Long et al, 2005a). Figure 1.1. Kv channel structure. (A) Schematic of the topology of a single K v channel ct-subunit. Each subunit has six transmembrane helices (S1-S6). S4 has a series of basic residues (indicated by (+) signs) positioned at regular intervals. S5/P-loop/S6 from each a-subunit collectively form the pore domain. The T l domain catalyzes tetramerization and prevents tetramerization of subunits from different K v subfamilies (B) Tetrameric crystal structure of K v l . 2 with each subunit colored differently. K + is shown as a black sphere in the central pore (from Long et al, 2005) (C) A 3D structure of Shaker channel generated using electron microscopy. The white lines represent the approximate position of the membrane. Note the separation between the transmembrane and cytoplasmic compartments (from Sokolova et al, 2001) Although the importance of the modular design of Kv channels is not clear, specific functions have been attributed to each module. The transmembrane helices of the channel comprise all of the structural elements required for voltage-dependent gating (the S1-S4 helices) and a K+-selective pore (formed by the S5, P-loop and S6 helices), and can function 4 as independent entities (Kurata et al., 2001). The cytoplasmic domains play regulatory roles in mediating channel assembly and subfamily specificity (Deutsch, 2003). They also allow for interaction with K v channel auxiliary proteins and other cytoplasmic regulatory components (Gulbis et al., 2000; Sokolova et al., 2003; Long et al., 2005a). In addition, the cytoplasmic domains may interact with the transmembrane domain directly to regulate • channel function (Minor et al., 2000; Cushman et al., 2000) by a currently unknown mechanism. 1 . 3 Conformational changes in the voltage-sensing domain in response to membrane depolarization In their seminal papers in 1952, Hodgkin and Huxley predicted that voltage-gated ion channels contain gating particles that are charged and respond to changes in the membrane potential, thus governing channel activation and deactivation. This, in essence, predicted the presence of small gating currents that are produced by movement of gating charges across the membrane, which were recorded in 1973 by Armstrong and Bezanilla (Armstrong and Bezanilla, 1973). The molecular determinants of these gating charges remained elusive until advances in the field of molecular biology allowed for cloning of voltage-gated ion channels. Cloning of Na + , C a 2 + and K + voltage-gated channels (Noda et al., 1984; Tempel et al., 1987; Papazian et al., 1987; Tanabe et al., 1987) revealed that the S4 helices were composed of positively charged basic amino acids positioned at every third residue. This cluster of positively charged amino acids has made the S4 helix the primary voltage sensor. In addition to the basic residues of the S4 segment, most voltage-dependent channels contain conserved negatively charged (acidic) amino acids in the S2 and S3 helices. Mutating these acidic residues alters activation gating, and it has been suggested that these residues participate in channel gating through electrostatic interactions with the basic residues in the 5 S4 helix (Seoh et al, 1996; Papazian and Bezanilla, 1997; Tiwari-Woodruff et al, 2000). Therefore, the SI to S4 transmembrane helices are collectively referred to as the "voltage-sensing domain". Upon depolarization, each voltage-sensing domain of the tetramer independently undergoes voltage dependent conformational changes, transferring several positively charged amino acid side chains in S4 between solvent accessible locations on opposite sides of the membrane (Yang and Horn, 1995; Larsson et al., 1996; Cha and Bezanilla, 1997; Lu et al., 2002; Lee et al., 2003). Once all the subunits are in this state, a conformation results that is permissive for pore opening, a concerted transition that involves only 10-15% of the total gating charge displacement (Smith-Maxwell et al., 1998a; Smith-Maxwell et al., 1998b; Pathak et al., 2005). Despite tremendous progress in the study of voltage-gated channels, the exact conformational changes associated with voltage-sensing have remained unclear and are subject to debate. Different models have been put forth (sliding, helical screw or paddle model) with a common requirement for transfer of three to four elementary charges per subunit across the membrane field to account for the steep voltage dependence of gating of most Kv channels (Schoppa et al., 1992; Seoh et al, 1996). These models are considered in detail in the following section. 1.3.1 From sliding to the helical screw model State-dependent accessibility of introduced cysteine residues within the voltage-sensing domain for covalent modification by reagents such as methanethiosulfonate (MTS) has provided a powerful tool to probe the conformational changes associated with channel activation. This technique was first applied to a voltage-gated skeletal muscle N a + channel, h S K M l (Yang et al., 1995). The accessibility of the basic residues in the S4 helix of domain IV of h S K M l appears to be extremely state-dependent. The outermost basic residue in the 6 S4 (R1448) is available for modification by MTS reagents only at depolarized membrane potentials. On the other hand, the second and third basic residues (R1451 and R1454), appear to be accessible from the extracellular side upon depolarization and from the intracellular side upon hyperpolarization; indicating that these residues fully traverse the membrane during channel gating (Yang et al, 1996). This led to the proposal of the sliding helix model where the S4 moves outward upon membrane depolarization. Accessibility experiments performed on the S4 helix of the Shaker channel suggest that the sliding-model can be extended to Kv channels. In Shaker, the first basic residue (R362) is about ten times more accessible for modification by external MTS reagents in the activated state of the channel; the second basic residue (R365) is only accessible to external MTS reagents in the activated state and is inaccessible to internal MTS reagents in the resting state; the third basic residue (R368) is only accessible to internal MTS reagents in the resting state and is inaccessible to either internal or external MTS reagents in the activated state. Furthermore, residue S376 at the intracellular end of the S4 helix is accessible for internal modification by MTS reagents only in the closed state, whereas modification of the G381 is state-independent and is accessible to internal MTS reagents at all times (Larsson et al., 1996). Collectivity, these data agree with the sliding-model and suggest that, at rest, S4 spans the membrane in five residues (363-367), while S4 movement due to membrane depolarization causes insertion of a minimum of nine S4 residues in the membrane. This cumulatively contributes the translocation of three elementary charges per subunit across the membrane (Mannuzzu et al, 1996; Larsson et al, 1996), thus satisfying the observed requirement for the transfer of ~12 elementary charges (Schoppa et al., 1992). 7 Application of resonance energy transfer between two fluorophores, one acting as a donor and the other acting as an acceptor, to Kv channels has enabled estimation of distances between residues in different subunits during channel gating (Cha et al., 1999; Glauner et al., 1999). This has allowed for estimation of changes in the distances between identical residues in S3-S4 linkers and S4 helices of different subunits during activation. In Shaker channels, during activation, a number of residues on one face of the S3-S4 linker and S4 helix move apart, while residues on the opposite face move closer to each other. Also, the distance from the central axis increases from residue 350 to 356, in both resting and activated channels, suggesting that the S3-S4 linker is tilted. If S4 is aligned with the S3-S4 linker, S4 may experience a tilting motion during activation. These findings have led to the proposal of a model where the S4 helix undergoes a 180° twist and possible tilt during activation (helical screw model; Cha et al., 1999; Glauner et al., 1999). 1.3.2 The paddle model Atomic resolution of the K v A P channel structure, a thermophilic bacterial Kv channel with properties similar to eukaryotic K v channels, allowed for direct visualization of the voltage-sensing domain (Jiang et al, 2003a; Jiang et al, 2003b). The pore-forming domain appeared to be surrounded by the SI and S2 helices, followed by S5, and finally S3 and S4 helices occupying the channel's outer perimeter. In addition, the S3 helix appeared to exist as two separate helices (S3a and S3b), and surprisingly the S3b helix was found to be closely packed with S4 to form a hairpin structure. The most unexpected finding from the crystal structure of KvAP was the position of the S4 helix, which was near the intracellular membrane surface, perpendicular to the pore axis. Jiang et al. (2003b) proposed that the S3b-S4 hairpin structure forms a "paddle" that moves across the membrane at the protein-lipid interface and in so doing satisfies the prerequisite of at least 12 elementary charges 8 crossing the electric field (Schoppa et al, 1992). Accessibility of biotin tethered to site-directed cysteine mutants in the "paddle" to avidin illustrated that the "paddle" moves a large distance through the membrane (-20 A) in contrast to the helical screw model that predicted less than 13.5 A movement (Cha et al, 1999; Glauner et al, 1999). 1.3.3 The paddle model contradicts experimental data from mammalian channels Given its significant departure from the conventional sliding or helical screw models, the paddle model has been subject to intense scrutiny. Hanatoxin, which binds to the extracellular end of the S3 helix and the S3-S4 linker of Kv2.1 channels (Swartz and MacKinnon, 1997a; Swartz and MacKinnon, 1997b), binds tightly to the voltage sensor even at hyperpolarized potentials and stabilizes the closed, state of the channel (Lee et al, 2003). This suggests that, contrary to the paddle model, the voltage sensor is exposed to the extracellular environment at rest. Consistent with this, the extracellular ends of SI, S2 and S3 helices of Shaker are available for modification by external reagents at rest (Gandhi et al., 2003). The availability of S2 for labeling is a departure from the K v A P crystal structure, since the S2 helix was described as being buried within the membrane (Jiang et al, 2003a). Additionally, mutation of the two acidic residues in the S3 segment to non-charged residues, or addition of a positive charge to this segment, does not alter the total gating charge movement implying that, unlike the suggestion from the paddle model, S3 does not undergo significant movement during gating in the Shaker channel (Gonzalez et al, 2005). Furthermore, mutation of the first basic residue in the S4 helix of Shaker to a histidine (R362H) results in the formation of a gating pore that conducts protons at hyperpolarized membrane potentials (Starace and Bezanilla, 2004). This indicates that site 362 is simultaneously accessible from both sides of the membrane at hyperpolarized potentials. The paddle model cannot accommodate this finding because the basic residues such as 362 are 9 inaccessible to internal Fab fragments (Jiang et al, 2003b), suggesting that these never face the intracellular solution, and therefore cannot offer a passage-way for protons. Similarly, Cuello et al. (2004) examined the structural dynamics of K v A P reconstituted in a lipid bilayer using site-directed spin labeling and electron paramagnetic resonance spectroscopy. They showed that the S1-S2, S2-S3 and S3-S4 linkers are accessible to the aqueous environment. This topological arrangement is in clear disagreement with the crystal structure of KvAP, which places the SI and S2 domains and their linker within the membrane (Cuello et al, 2004). It is possible that the voltage-sensing domain of KvAP was crystallized in a non-native conformation due to the Fab antibody fragment used to stabilize the channel (Tombola et al, 2005; Long et al., 2005a). This may explain the discrepancies between the biophysical data and the crystal structure of KvAP, as outlined above. 1.3.4 Structure of the voltage-sensing domain in a native state Recently, the structure of the mammalian Kv l .2 channel was resolved in its native state by using the interaction between the P subunit and the T l domain as a stabilizing anchor (Long et al., 2005a). This structure likely represents an activated and open conformation of the channel (Swartz, 2004; Long et al., 2005a). Unlike the KvAP crystal structure, the S3 helix of K v l . 2 appears as a single helix with a bend and not as two separate S3a and S3b helices. In addition, the S4 helix of K v l . 2 is noticeably perpendicular to the membrane, two findings that are different from the paddle model. Although the crystal structure of K v l . 2 provides only a static view of the voltage-sensing domain in the activated state, it provides a platform for generating in silico predictions of the structural dynamics of the sensor during gating from the closed to the open state (Yarov-Yarovoy et al., 2006). These models suggest that during activation, the voltage-10 sensing domain experiences conformational changes with translation and rotation of S4, as proposed in the original slide helix or helical screw models, coupled with movement of SI to 53 helices around S4 (Yarov-Yarovoy et al, 2006). Rotation of S4 relative to S3, coupled with the rolling of S2 around S4, allows for sequential interactions between basic residues in 54 and acidic residues in the S2 and S3 helices as shown previously (Papazian et al, 1995; Tiwari-Woodruff et al, 1997; Tiwari-Woodruff et al, 2000). Using the K v l . 2 crystal structure, Tombola et al. (2007) have provided new evidence for the helical screw model in Shaker. In the Shaker channel, mutation of the first arginine residue of the S4 helix to a smaller uncharged residue makes the voltage-sensing domain permeable to ions, giving rise to the so-called "omega current" (Starace et al, 2004). Tombola et al, (2007) mapped the results from mutation perturbation analysis in the voltage-sensing domain in resting and activated channels of the omega pathway onto the K v l . 2 crystal structure. Their data show that the pattern of mutations that influence the omega current is consistent with a rotational conformational change coupled with a displacement, in the voltage-sensing domain during channel activation. Interestingly, modeling of the K v A P channel in the closed state results in a similar structure to that predicted for Kv l .2 (Yarov-Yarovoy et al, 2006), although the open-state conformations of the voltage sensors of these channels are significantly different (Long et al, 2005a; Yarov-Yarovoy et al, 2006). The differences between the two channels may be due to intrinsic differences in the mechanisms of gating between the two channels. There is only about 20% sequence homology in the S4 helices of K v l . 2 and KvAP, leaving much scope for differences in amino acid sequence to result in altered structures. Therefore, the helical screw model that was proposed for Shaker channels (Cha et al, 1999; Glauner et al, 1999) 11 and modeled in the crystal structure of K v l . 2 (Tombola et al, 2007) may not apply universally to all Kv channels as the amino acid composition of the S4 helix varies significantly amongst the subfamilies. For instance, the sequence homology between the S4 helices of K v l . 2 and Kv2.1 channels is less than 50%. Therefore, the general applicability of the helical screw model to other channels cannot be taken for granted at this time and more experiments are required to determine if this model accurately describes voltage-sensing in channels other than K v l .2 and Shaker. 1.4 Conformational changes associated with channel opening 1.4.1 The activation gate Comparison of the crystal structure of KcsA (Doyle et al., 1998), which was 2_|_ crystallized in the closed state, with that of the MthK channel (Jiang et al, 2002b), a Ca sensitive bacterial K + channel, which was crystallized in the open state, reveals the striking conformational changes that occur during channel opening. The KcsA and MthK structures exhibit significant differences in the arrangement of the transmembrane M2 (equivalent to Kv channel S6) helices, while the structures of the selectivity filter region are comparable (Figure 1.2). In the KcsA channel, the M2 helices of all four subunits form a constriction near the cytoplasmic side of the channel, referred to as the inner helix "bundle crossing". This bundle crossing is clearly absent from the crystal structure of MthK channels, where the M2 helices are widely separated and the central cavity is connected to the cytoplasm. Based on the crystal structure of these two non-voltage-dependent bacterial channels, KcsA and MthK, a general model for the motion of the S6 segments during gating was proposed (Jiang et al, 2002a). According to this model, under conditions that promote channel opening, the M2 helices (equivalent to S6 helices in K v channels) undergo conformational changes 12 dependent on the flexibility of a highly conserved glycine residue referred to as the "glycine hinge", labeled red in Figure 1.2, leaving a wide open cytoplasmic entrance to the pore. The S6 helices of most K v channels contain a conserved amino acid sequence motif, proline-X-proline (PXP, where X represents a hydrophobic residue), seven residues downstream from the conserved "glycine hinge". The PXP motif induces a pronounced kink in the S6 helix as suggested from the K v l . 2 crystal structure (Long et al, 2005b). Accessibility studies in Shaker revealed the presence of an activation gate defined by the PXP motif of the S6 helix (Liu et al, 1997). In the closed state, pore-lining residues above and including residue 477 are inaccessible to C d 2 + or MTS reagents and even A g + , which is comparable in size to K + , while residues below residue 478 remain accessible in both closed and open states (Del Camino et al, 2000; Del Camino and Yellen, 2001). This suggests the presence of an intracellular gate that regulates the access of K + ions to the inner vestibule. In agreement with this, mutations of the PXP motif in the Shaker channel have revealed that alterations of residue P473 (the innermost proline of the PXP motif) results in a non-conducting channel by locking the channel closed, while mutation of P475 (the second proline of the PXP motif) results in a constitutively open channel, by locking the channel in the open state (Hackos et al, 2002). Furthermore, mutation of V478 and F481 residues to bulky amino acids (tryptophan) results in non-conducting and weakly conducting channels, respectively, suggesting that these residues form a "hydrophobic seal" that prevents entrance of ions into the internal cavity in the closed state of the channel (Hackos et al, 2002; Kitaguchi et al, 2004). Collectively, the "glycine hinge" and the PXP motif may bestow the Shaker S6 helix with the required flexibility to allow opening, as mutation of the conserved "glycine hinge" (G466) results in a nonfunctional channel that has a dominant negative effect 13 when heteromultimerized with wild type (Wt) channels (Cordes et al., 2002; Ding et al., 2005). Interestingly, in Kv l .5 channels, a proline scan of the lower S6 helix revealed that although the PXP motif is required for channel function, there is no absolute requirement for the position of the motif (Labro et al, 2003). As well, the PXP motif is absent in some K v channels such as human ether a-go-go related gene (hERG; Wartlike and Ganetsky, 1994) and the conserved "glycine hinge" is postulated to form the activation gate in these channels, although this has not been shown experimentally. 1.4.2 Electromechanical coupling between the voltage-sensing domain and the activation gate The conformational changes in the voltage sensor due to membrane depolarization must be translated to opening of the activation gate for the channel to open and conduct ions. A mechanism to explain the electromechanical coupling between the S4-S5 linker and the S6 helix was recently proposed based on the crystal structure of K v l . 2 (Long et al., 2005a). In the crystal structure, the S4-S5 helix runs parallel to the membrane plane inside the cell and crosses over the top of the C-terminal end of the S6 helix, which is kinked at the PXP motif, and makes several amino acid contacts in the open state of the channel (Long et al., 2005b). Inward movement of the voltage sensor with membrane hyperpolarization is proposed to compress the S4-S5 linkers against the S6 helices, resulting in closure of the activation gate (Long et al., 2005a) (Figure 1.2C). In agreement with this, chimeras of Shaker and KcsA channels containing the pore forming domain of KcsA inserted into the background of the voltage-sensing domain of Shaker (S1-S4 and S4-S5 linker) traffic normally to the membrane, but do no gate in response to membrane depolarization (Caprini et al, 2001). However, i f the C-terminal end of the Shaker S6 helix, which includes the PXP motif, is included in the chimera, the resulting channels open in a voltage dependent manner. This 14 suggests an interaction between the S4-S5 linker and the C-terminal end of the S6 helix during channel activation (Lu et al, 2002). Consistent with this, disruption of this putative interaction by a mutation (aspartate 540 to lysine) in the S4-S5 linker of the hERG channel causes channels to open at negative potentials, similar to the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channel (Tristani-Firouzi et al, 2002). Similarly, mutation of tyrosine 331 (Y331) in the S4-S5 linker of the HCN2 channel renders this channel constitutively open (Chen et al, 2001). Of interest, cyclic nucleotide gated (CNG) channels are not voltage-gated but have a S4 helix that functions as a voltage sensor i f placed in a permissive structural environment, such as the ether-a-go-go (EAG) K + channel background (Tang and Papazian, 1997). It has been proposed that the S4 helix of C N G channels is immobilized in the open state (Tang et al, 1997), but the role of S4-S5 linker and a possible disruption in S4-S5 linker coupling to channel opening has not yet been examined. Formation of a hydrophobic seal on the intracellular side of the pore (Hackos et al, 2002; Kitaguchi et al, 2004) in the closed conformation may not be a universal phenomenon. For example, in C N G channels, although the S5 and S6 helices are likely to form a bundle crossing and conformational rearrangements of S6 likely take place during channel activation by cyclic nucleotides (Johnson and Zagotta, 2001), cysteine residues substituted within the pore remain accessible to small ions such as A g + in the closed state. This suggests that there is an opening, even in the closed state, sufficient to permit the passage of small cations in these channels (Flynn and Zagotta, 2001). These findings argue against the existence of a tight hydrophobic seal on the cytosolic side of C N G channels and have led to speculation that conformational changes in the selectivity filter during opening and closing may act as the activation gate (Flynn et al, 2001). Similarly, in K v channels 15 the flow of ionic current can be impeded by conformational changes in the outer pore region and selectivity filter by a process known as slow inactivation (Starkus et al., 1997; Kiss and Korn, 1998; Kiss et al, 1999), although this is separate from the activation gate in K v channels. A B C Figure 1.2. Structural rearrangements associated with channel opening. In the KcsA structure (A) the M2 helices form a bundle crossing thus impeding the flow of ions. In the MthK structure (B) the M2 helices are displaced outward around the glycine hinge, shown in red, thus allowing the flow of K+ ions down their electrochemical gradient. (C) The putative electromechanical coupling between the S4-S5 linker and the C-terminal end of S6 helix of K v l . 2 . (O=open; C=closed; from Long et ah, 2005b). Although recent structural work has provided many novel insights into conformational changes underlying Kv channel gating, insights into mechanisms that alter K v channels gating properties to accommodate cellular requirements have been less forthcoming. In the following sections, a number of these mechanisms will be discussed. 16 1.5 Modulators of Kv channel activation The activation of K v channels is highly variable, with activation time constants ranging from less than 1 ms to more than 1 s at +80 mV. This diversity can be attributed, in part, to the structural determinants of channel gating i.e. the intrinsic structural features that are unique to each channel type and are dictated by the primary amino acid sequence, but also to extrinsic factors, such as interaction with auxiliary subunits or modification by phosphorylation or phospholipids. The extrinsic factors enable rapid and reversible modulation of K v channel gating to accommodate the continuous demand for adjustment of membrane excitability based on cellular requirements. 1.5.1 Structural determinants of Kv channel activation kinetics The biophysical characteristics of Kv channel activation vary dramatically, even though the overall topology of all Kv channels is the same i.e. S1-S4 transmembrane helices form the voltage-sensing domain and S5-S6 helices form the pore domain. For example, although the S4 helix of the hERG channel has the same number of basic residues as the Shaker channel, the number of gating charges transferred across the membrane upon activation in hERG channels is ~8 elementary charges (Zhang et al., 2004), which is 4-5 elementary charges fewer than in Shaker channels (Schoppa et al., 1992), and voltage sensor movement is ~50-fold slower in hERG channels compared to the Shaker channels (Smith and Yellen, 2002; Piper et al., 2003). The cause of the slow S4 movement in hERG is unclear, but may be due to different electrostatic interactions between the basic residues in the S4 and acidic residues in the S2 and S3 helices (Zhang et al., 2004) or different assembly of the voltage sensor relative to the channel (Subbiah et al, 2005). Alternatively, differences in the 17 cytoplasmic domains and the linkers connecting the transmembrane helices may underlie these contrasting kinetics. 1.5.1.1 The role of cytoplasmic domains in regulation of gating The cytoplasmic domains of Kv channels (N- and C-termini) play important roles in the regulation of channel gating without directly participating in voltage-sensing. The transmembrane segments of Kv channels are more highly conserved than the cytoplasmic isl-and C-termini, which show far more divergence, even amongst closely related channels. For example, comparison of primary sequence of K v l . 2 and Kvl .5 channels reveals that the N -termini of K v l . 2 and Kvl .5 are highly homologous within the T l domain, but exhibit very significant sequence divergence preceding the T l domain. The T l domain in Kv l .5 is preceded by 120 amino acids, while the T l domain in K v l . 2 is preceded by only 33 residues and no significant homology exists between the two. Truncation of the N - or C-terminus of Kv channels has allowed for investigation of the role of the cytoplasmic domain in the regulation of gating properties. Truncation of the N-terminus of Kv2.1 channels results in a significant depolarizing shift in the voltage dependence of activation coupled with slowing of activation kinetics, while truncation of the C-terminus has no apparent effect on the activation properties. Interestingly, truncation of both N - and C-termini restores normal Kv2.1 channel gating (VanDongen et al, 1990). Unlike Kv2.1 channels, however, truncation of the N-terminus of Kv4.2 has no apparent effect on the activation properties (Bahring et al, 2001). Truncation of the Kvl .5 N -terminus, on the other hand, results in a hyperpolarizing shift in the voltage dependence of activation and endows the channel with a unique closed-state inactivation property (Kurata et al, 2001; Kurata et al, 2002). Interestingly, deletion of the N-terminus of hERG results in no apparent change in the activation properties of the channel, but results in a dramatic 18 acceleration of deactivation kinetics (Spector et al, 1996; Schonherr and Heinemann, 1996; Wang et al., 1998). Point mutations within the N-terminus of K v l . l and K v l . 2 channels, particularly in the T l domain, have also been shown to cause changes in activation kinetics and shift the voltage dependence of activation without disrupting the structure (Minor et al., 2000; Cushman et al., 2000). These data collectively indicate that although the N - and C-termini are not necessary for channel gating, they can have profound modulatory effects on Kv channel gating properties that are unique to each channel type. The mechanisms by which the cytoplasmic domains modulate channel gating are not yet clear. However, we do know that the N - and C-termini are highly mobile during channel gating. The N - and C-termini of Kv2.1 physically interact (Ju et al, 2003) and fluorescence resonance energy transfer (FRET) combined with the voltage clamp technique indicates that there are voltage dependent rearrangements of the N - and C-termini in this channel (Kobrinsky et al, 2006). The C-terminus may affect gating via its direct connection to the pore-lining S6 helix and the interaction between the N - and C-termini may allow the N -terminus to exert its modulatory role on activation properties in a similar manner. It is also possible that the cytoplasmic domains influence the gating properties through direct coupling with the transmembrane core of the channel, perhaps by modifying the conformation of the transmembrane helices relative to each other or by altering voltage sensor movement, thus modifying activation kinetics. To date, however, there is no clear evidence for this hypothesized coupling between the cytoplasmic domains and the channel core. 1.5.1.2 Transmembrane linkers and modulation of activation kinetics In addition to the N - and C-termini, the connectors between the transmembrane helices are divergent in composition amongst Kv channels and have been implicated in the 19 regulation of channel gating. The extracellular linker connecting SI and S2 transmembrane helices has been shown to modulate the activation kinetics of Kv channels. The differences in the amino acid composition between the S1-S2 linker of HCN2 and HCN4 channels are thought to be responsible for the differences in the activation kinetics between these two channels (Stieber et al, 2003). Also, the extracellular S1-S2 linker of a number of Kv channels is glycosylated, which affects the activation properties of the channel via surface charge screening effects as a result of the incorporated sialic acids (Thornhill et al., 1996; Watanabe et al, 2003; Watanabe et al, 2007). In addition, the length of the S1-S2 linker varies dramatically amongst Kv channels. The linker is composed of 21 amino acids in Kv2.1 channels while 50 amino acids form the linker in Kvl .5 channels. This may have significant implications for the orientation of the transmembrane helices relative to each other and thus the conformational changes that take place during channel gating, although evidence for this is limited. Mutations within the S2-S3 linker of KCNQ1 and hERG channels result in dramatic alterations of the gating properties of these channels (Nakajima et al., 1998; Yamaguchi et ah, 2003). Similarly, mutation of the highly conserved glycine at residue 204 in the S2-S3 linker of C N G channels introduces voltage dependence to the activation of an otherwise voltage insensitive channel (Crary et al., 2000). It is possible that the S2-S3 linker engages in specific interactions with other parts of the channel, and is, therefore, directly involved in the normal allosteric transition(s) that occur during channel gating. This is supported by the predicted close proximity of the S2-S3 linker to the base of the S4 segment. Alternatively, the linker may simply act as a hinge that maintains the necessary distance between the S2 and S3 transmembrane segments to allow gating conformational changes. The structure of 20 the hinge may determine its flexibility and its ability to transduce or accommodate the dynamic movement of the transmembrane segments. Yet, another possibility is that structural changes in the S2-S3 linker may disrupt coordinated movements of the S2 and S3 transmembrane segments, thereby altering channel gating properties. Indeed, it is well established that the S2 and S3 transmembrane helices participate in channel gating via a charge interaction with the S4 segment (Papazian et al, 1995; Seoh et al, 1996; Zhang et al, 2004). The role of the S3-S4 linker in modulation of Kv channel gating has also been extensively studied. Interestingly, although point mutations in the S3-S4 linker of Shaker have only minor effects on the gating kinetics of the channel, substitution of the S3-S4 linker of the Shaker channel with that of the Shab channel results in slowing of the activation kinetics to resemble those of the donor channel (Mathur et al, 1997). This effect may be related to the difference in the length of the linker between these two channels given that the S3-S4 linker of the Shaker channel is composed of 31 amino acids while the linker of the Shab channel is only a 6 amino acid segment. Consistent with this, Shaker channels with S3-S4 linkers shortened to 5 or 0 amino acid residues exhibit dramatically slowed activation kinetics (Gonzalez et al, 2000), an effect that can be attributed to the slower S4 movement revealed by voltage clamp fluorimetry (Sorensen et al, 2000). This result suggests that decreasing the length of the S3-S4 linker restricts the movement of the voltage sensor, thus providing evidence for an important role of the S3-S4 linker in regulation of Kv channels activation properties. Furthermore, binding of hanatoxin, a protein toxin from spider venom, to the S3-S4 linker of Kv2.1 shifts the voltage dependence of activation in the depolarizing direction, again by impeding movement of the S4 voltage sensor (Lee et al, 2003). 21 The S4-S5 linker, which flanks the intracellular end of the S4 voltage sensor, is also thought to play an important role in electromechanical coupling and forms a crucial component of the activation gate. The activation properties of the Shaker channel are very sensitive to mutations in this linker. The hydrophobic substitution mutation (L370V, also known as the V2 mutation) in Shaker dramatically shifts the voltage dependence of activation in the depolarizing direction and decreases the apparent voltage sensitivity of channel opening (McCormack et al, 1991). Similarly, point mutations within the S4-S5 linkers of hERG and H C N channels result in dramatic alterations of activation properties (Sanguinetti and Xu, 1999; Prole and Yellen, 2006). Additionally, the activation gating properties of chimeric channels made from Kv2.1 and Kv3.1 channels are determined by the S4-S5 linker (Shieh et al, 1997). Collectively, these data provide strong evidence for the role of the S4-S5 linker in modulation of activation kinetics. 1.5.2 Dynamic modification of Kv channel activation kinetics Ion channels are ultimately responsible for the regulation of membrane excitability and as such, their biophysical properties must be constantly modified to reflect cellular requirements. Changes in Kv channel activation properties can lead to modification of action potential duration and modulation of repetitive neuronal action potential firing or pacemaker activity. For example, a hyperpolarizing shift in the voltage dependence of activation of H C N channels results in a reduced heart rate (DiFrancesco and Tortora, 1991), while a hyperpolarizing shift in the voltage dependence of activation of Kv2.1 channels allows for rapid neuronal action potential firing (Park et al, 2006). Therefore, it is important to understand the mechanisms by which Kv channels communicate with intracellular and extracellular modifying signals, which can rapidly and reversibly modify membrane excitability. 22 1.5.2.1 Auxiliary subunits Recombinant expression of K v channels in heterologous systems results in channels that do not always recapitulate all of the properties of native channels. For the most part, interaction of auxiliary subunits with Kv channels is thought to be responsible for this discrepancy. For example, the KCNQ1 gene codes for a Kv channel that activates rapidly and exhibits a small degree of inactivation (Pusch et al, 1998), but the presence of the auxiliary KCNE1 subunit results in channels with dramatically altered kinetics. Activation and deactivation kinetics are slow and the voltage dependence of activation is shifted to more depolarized potentials. Only when KCNQ1 is coexpressed with KCNE1 do channel kinetics recapitulate the phenotype of the slowly activating delayed rectifier current, hs, in the heart (Barhanin et al, 1996; Sanguinetti et al, 1996b). In addition, heterologous expression of the hERG channel, the main constituent of the rapid delayed rectifier current, produces currents that differ from those recorded from ventricular myocytes, although this cannot be reconciled by co-expression of its putative auxiliary subunits, KCNE2 (Abbott et al, 1999; Weerapura et al, 2002). It was recently suggested that interaction of two alternative hERG transcripts may contribute to hr in native tissue, which raises the interesting prospect of modulation of ion channel gating through alternative splicing (Jones et al, 2004). Interaction of KvP auxiliary subunits with K v channels affects the biophysical properties in addition to modifying cell surface expression, although the physiological importance of KvP subunits in native cells is not well understood (Nerbonne and Kass, 2005). Coexpression with KvP2 auxiliary subunits slightly shifts the voltage dependence of activation of Kvl .5 to more hyperpolarizing voltages and accelerates activation without affecting deactivation (Heinemann et al, 1996). Similarly, hKvpi.2 and Kv l .5 interaction 23 results in an apparent reduction in the current amplitude of K v l . 5 , which is thought to be a result of a hyperpolarizing shift in the activation gating due to rapid open channel block by the ball domain of Kvpi .2 (DeBiasi et al, 1997; Accili et al, 1997). The expression level of P-subunits may be dependent upon cellular requirements to allow for modulation of Kv channel surface expression and gating properties, however, there is no experimental evidence available for this. Crystallographic studies of Kvp subunit have revealed a structural homology with aldo-keto reductase, a class of redox enzymes that reduce aldehyde or ketone functional groups to primary or secondary alcohols using N A D P H as a cofactor (Gulbis et al., 1999; Gulbis et al., 2000; Long et al., 2005a). Recently, it was shown that the KvP2 subunit is indeed a functional aldo-keto reductase and exposure to substrate reduces the rate of inactivation of Kvl.4+KvP2 channel complexes, thus reducing membrane excitability (Weng et al., 2006). This possibly endows Kv channels or at least the Kv l .4 channel with a sensor module to detect the metabolic state of the cell, thus allowing modification of gating properties based on cellular requirements. It will be imperative to examine whether the gating properties of other Kv channel and Kvp subunit complexes are altered by the catalytic activity of Kvp subunits. 1.5.2.2 Phosphorylation The biophysical properties of Kv channels can be modified in a reversible and dynamic fashion through phosphorylation and dephosphorylation, processes that link channels residing on the cell surface to intracellular signaling cascades (Levitan, 1994). This link endows the channels with the ability to rapidly respond to cellular requirements. The effect of phosphorylation on Kv channel biophysical properties is attributed to the electrostatic 24 interactions between the voltage-sensing domain and the negatively-charged phosphate groups (Perozo and Bezanilla, 1990; Perozo et al, 1991). However, phosphorylation does not modify the biophysical properties of all channels in the same manner. Phosphorylation of the C-terminus of Kv2.1 channel reduces open probability by shifting the voltage dependence of activation to more depolarized potentials and this results in enhanced neuronal excitability (Park et al, 2006). On the other hand, phosphorylation of the cardiac KCNQ1-KCNE1 channel complex (IKs) in response to P-adrenergic stimulation results in increased channel open probability by inducing a hyperpolarizing shift of the voltage dependence of activation and by accelerating activation and slowing deactivation kinetics (Terrenoire et al., 2005). Furthermore, protein kinase A (PKA)-dependent phosphorylation of the hERG channel following P-adrenergic stimulation results in a reduction of current amplitude in heterologous systems, which is predicted to prolong action potential duration (Cui et al., 2000). However, this latter effect does not translate to native tissue because P-adrenergic stimulation has no detectable effect on hERG current amplitude recorded from guinea pig ventricular myocytes (Sanguinetti et al, 1991). This is thought to be due to a counteracting effect of binding of 14-3-3s protein to phosphorylated channels, which opposes the action of phosphorylation by accelerating activation and shifting the voltage dependence of activation in the hyperpolarizing direction (Kagan et al, 2002). 1.5.2.3 Direct cyclic nucleotide binding Elevation of cyclic nucleotides (cyclic adenosine monophosphate (cAMP) or guanosine monophosphate (cGMP) due to extracellular stimuli results in activation of kinases that can phosphorylate and modify properties of ion channels. Additionally, cyclic nucleotides can regulate properties of ion channels independently of kinases by binding 25 directly to the channel. Binding of cyclic nucleotides to the cyclic nucleotide-binding domain (CNBD) of C N G and H C N channels favors channel opening (Craven and Zagotta, 2006). In the heart, modification of H C N channel activation properties by c A M P contributes to heart rate regulation (DiFrancesco et al., 1991). Neurotransmitters, such as norepinephrine, or pharmacological agents, such as /3-adrenergic agonists, can elevate c A M P levels which increase heart rate through the binding of cAMP to the C-terminal C N B D of H C N channels. This causes a shift in the voltage dependence of activation to more depolarized potentials and increases both the rate of channel opening and the maximal current level (Craven et al., 2006). A putative C N B D is also present in the C-terminus of hERG channels, but its physiological relevance is not clear. Whereas direct binding of c A M P levels results in a hyperpolarizing shift in the voltage dependence of activation of hERG, thus increasing channel availability, activation of P K A by elevation of c A M P results in inhibition of hERG current by causing a depolarizing shift in the voltage dependence of activation (Cui et al., 2000). Furthermore, as mentioned earlier, phosphorylation of hERG recruits 14-3-3s, which counteracts the effect of phosphorylation (Kagan et al., 2002). Therefore, the final effect of /3-adrenergic stimulation on hERG is likely to reflect the balance of these putative regulatory mechanisms. This form of complex response may also be the case for other channels. 1.5.2.4 Lipids arid ion channels Membrane lipids and their metabolites have recently been shown to regulate K v channel properties. Phosphatidylinositol-4,5-bisphosphate (PIP2) is a membrane phospholipid enriched in the plasma membrane that is involved in many cellular functions (Czech, 2000). It is well known that PEP2 can interact directly with channels and modify their gating 26 properties, and the effect of PIP2 on the pancreatic ATP-sensitive K channel has been investigated in great detail. Positively charged residues in the cytoplasmic region of the channel near the membrane-fluid interface have been implicated in interactions with negatively-charged headgroups of anionic phospholipids in the inner leaflet of the plasma membrane, which result in increasing the open probability of the channel (Shyng and Nichols, 1998; Hilgemann et al, 2001; Enkvetchakul et al, 2005). Recently, phospholipids and their metabolites have also been implicated in the regulation of voltage-gated channels. Run-down of current in excised patches from cells expressing voltage-gated calcium (VGCC, Cav) (Wu et al, 2002), hERG (Bian et al, 2001), KCNQ1-KCNE1 (Loussouarn et al, 2003) or Kv2.1 (Hilgemann et al., 2001) channels could be largely prevented by application of internal PIP2. The open state of the KCNQ1-KCNE1 channel complex is stabilized by PIP2 possibly through an interaction with positively charged residues in the S4-S5 linker and the C-terminus (Park et al., 2005), leading to an increased current amplitude, slowed deactivation and a shift in the voltage dependence of activation (Loussouarn et al, 2003). PIP2 also inhibits N-type inactivation in Kv l .4 channels and in K v l . l channels co-expressed with a Kvp 1.1 subunit, effectively converting rapidly inactivating A-type currents into slowly inactivating delayed rectifiers (Oliver et al, 2004). Finally, arachidonic acid, a polyunsaturated fatty acid metabolite of phospholipids, has also been shown to modify Kv channel gating. Arachidonic acid directly activates a large conductance (160 pS) K + channel in cardiac atrial muscle (Kim and Clapham, 1989), endows delayed rectifier K + channels with rapid voltage-dependent inactivation (Oliver et al, 2004; Jacobson et al, 2007), or accelerates the inactivation rate of rapidly inactivating Kv4.2 in neurons (Keros and McBain, 1997). The mechanism of acceleration of inactivation of open channels by arachidonic acid 27 appears to depend upon interaction of the fatty acid with the channel protein and allosteric conformational alteration of the selectivity filter (Oliver et al, 2004). 1.5.2.5 SUMOylation Recently, SUMOylation of Kv channels has been suggested to modulate channel biophysical properties. SUMOylation is the post-translational modification of cytoplasmic lysine residues by covalent attachment of a small ubiquitin-related modifier protein (SUMO). Although SUMOylation is generally a nuclear phenomenon, K + channels residing on the membrane have been shown to be a substrate for SUMOylation. Interaction of SUMO with twin-pore domain channels silences channel function (Rajan et al, 2005), while SUMOylation of the N-terminus of Kvl .5 results in modification of inactivation kinetics without altering activation properties (Benson et al, 2007). Our current knowledge of SUMO modification of K + channels is limited to these two channels and clearly more research is required to understand the mechanism of action and physiological function of SUMO modulation. The implications are pertinent to the work in this thesis since SUMO can be removed from target proteins by an enzymatic reaction, making SUMOylation a reversible and dynamic process, thus allowing transient modification of channel properties. 1.5.2.6 Ionic environment Ion channels also respond to changes in the extracellular and intracellular ionic composition. Modification of the gating properties of ion channels and thus membrane excitability by divalent cations is well known. Divalent cations result in nonspecific shifts in the voltage dependence of activation kinetics of K + and N a + channels, an effect that is largely attributed to the surface charge screening effect of the ions (Hille, 2001). For example, elevation of the extracellular C a 2 + concentration results in a depolarizing shift in the voltage dependence of activation of both Nav and Kv channels of the squid giant axon 28 (Frankenhaeuser and Hodgkin, 1957; Hille et al, 1975). A similar effect is also reported for external M g 2 + ions (Hahin and Campbell, 1983). However, the effect of some divalent cations, such as Zn , cannot be explained solely by the charge screening effect since the 2+ effect is state-dependent (Gilly and Armstrong, 1982; Zhang et al., 2001b). External Zn results in a marked slowing of activation kinetics without altering deactivation kinetics in addition to inducing a depolarizing shift in the voltage dependence of activation due to the charge screening effect. This suggests the presence of a Z n 2 + binding site that is accessible during the resting state, but which disappears in the open state (Gilly et al., 1982). In addition to divalent cations, monovalent cations also alter the gating properties of ion channels. Modification of the extracellular K + concentration has dramatic effects on Kv channel properties. Elevation of external K + potentiates outward current through several Kv channels such as Kv l .4 (Pardo et al, 1992), Kv2.1 (Wood and Korn, 2000) and some Shaker mutants (Lopez-Barneo et al, 1993) despite a decrease in K + driving force. This is thought to be a result of increased channel availability, possibly due to the rescue of channels from inactivation (Lopez-Barneo et al, 1993; Wood et al, 2000). Additionally, reduction of the extracellular pH (acidosis) results in reduction of Kv channel current amplitude by shifting the voltage dependence of activation to more depolarized potentials and enhancing channel inactivation (Claydon et al, 2007). Our knowledge of the mechanisms involved in the modulation of Kv channel gating properties is not complete and investigation into additional forms of modification offers a potentially rich area of regulation of ion channels. Also, the ability to modify a specific subset of ion channels could have potential clinical implications. 29 1.6 Genetic modulators of channel activation Through clinical investigations of many inherited or acquired disorders, we know that defects in ion channels or ion channel regulatory subunits can result in diverse disease states. The term "channelopathies" has been introduced to define this class of diseases (Ashcroft, 2000). Extensive research into the ways in which ion channel dysfunction is manifested in disease states has led to a better understanding of the role ion channels play in regulation of different physiological functions such as the maintenance of normal heart rhythm or efficient insulin release. The list of channelopathies is extensive and this section will only focus on familial mutations in a number of different ion channels that have been linked with ventricular arrhythmias and sudden cardiac arrest by delaying ventricular relaxation. More than 45,000 Canadians die every year from sudden cardiac arrest, and in most cases, ventricular arrhythmias are assumed to be the underlying cause (Davis and Tang, 2004). Long QT interval syndrome (LQTS), which is a rare congenital disorder with an estimated incidence of 1 in 5,000, has been linked to sudden death due to cardiac arrest (Sanguinetti and Tristani-Firouzi, 2006) as well as sudden infant death syndrome (SIDS) (Schwartz et al, 1998). Clinically, LQTS is characterized by a prolongation of the QT interval of the electrocardiogram (ECG) (Figure 1.3B). Delayed repolarization (prolongation of the QT interval) increases the risk of a serious multifocal ventricular tachyarrhythmia, torsades de pointes (TdP), a unique cardiac arrhythmia characterized by an E C G trace that resembles a sinusoidal wave. TdP can revert back to normal sinus rhythm but can also degenerate into lethal ventricular fibrillation. A typical ventricular action potential is shown in Figure 1.3A with the contributing ionic currents marked. Substantial progress has been made toward elucidating the molecular 30 basis of LQTS and to date, mutations in eight different genetic loci have been associated with congenital LQTS: seven encode for cardiac ion channels and their accessory subunits (KCNQ1 or K v L Q T l (LQT1), KCNH2 or hERG (LQT2), SCN5A or Navl.5 (LQT3), KCNE1 (LQT5), KCNE2 (LQT6), KCNJ2 or Kir2.1 (LQT7), C A C N A 1 C or Cavl.2 (LQT8)) (Curran et al, 1995; Wang et al, 1995a; Wang et al, 1995b; Romey et al, 1997; Abbott et al, 1999; Plaster et al, 2001; Splawski et al, 2004); and one encoding for cardiac ankyrin (ANK2; LQT4), a scaffolding protein that anchors the channels at the membrane (Mohler et al, 2003). However, LQT1, LQT2 and LQT3 are the most common, comprising about 98% of all LQTS cases (Napolitano et al, 2005). Therefore, LQTS associated with mutations in hERG, KCNQ1 and Navl.5 channels along with their corresponding accessory subunits (KCNE1 and KCNE2) will be discussed in more detail in the following sections. 31 Figure 1.3. Cardiac electrical activity. (A) A typical human ventricular action potential with phases and selected contributing currents shown. (B) Normal E C G and one showing prolongation of the QT interval during one cardiac cycle are shown. The P wave represents atrial relaxation, QRS complex indicates ventricular contraction, while the T wave represents ventricular relaxation. (C) E C G showing conversion of normal sinus rhythm to torsades de pointes, which can lead to ventricular fibrillation (from Keating and Sanguinetti, 2001). 32 1.6.1 Kv channels and LQTS Two components of the delayed rectifier K + current, /jcr (rapid) and (slow), play a dominant role in ventricular action potential repolarization and thus govern the action potential duration (Sanguinetti and Jurkiewicz, 1990). KCNQ1 encodes for a K v channel subunit that coassembles with the accessory subunit KCNE1 to form the slowly activating delayed rectifier K + current, lKs (Barhanin et al., 1996; Sanguinetti et al., 1996b), while hERG encodes for the major subunit of the rapidly activating delayed rectifier K + current, IKV (Sanguinetti etal., 1995; Trudeau et al., 1995). 1.6.1.1 dysfunction (LQT2 and LQT6) iKr has been identified in several mammalian species including guinea pigs (Sanguinetti et al., 1990), dogs (Gintant, 1996), rabbits (Salata et al, 1996) and humans (Li et al, 1996). Pharmacological agents that block IKR (e.g. E-4031, dofetilide, cisapride) markedly increase the ventricular action potential duration in all species tested (Sanguinetti et al, 1990; Jost et al, 2005), underlining the importance of this current in the regulation of action potential repolarization and the maintenance of normal cardiac rhythms. Congenital mutations in hERG that result in suppression of IKK are associated with LQT2. The degree of channel impairment is variable, depending on the specific mutation involved (Sanguinetti et al, 1996a). Mutations can result in loss of function by: 1) impeding efficient trafficking to the plasma membrane, which is evident as a lack or reduced channel current density when expressed in heterologous expression systems (non-trafficking mutants) (Furutani et al., 1999; Ficker et al., 2003); 2) rendering the channel nonfunctional (nonconducting) (Zhou et al, 1998); or 3) by biophysical alteration, such as slowed activation, accelerated deactivation or enhanced inactivation, that limit the contribution of to ventricular action potential repolarization (Nakajima et al, 1998; Yang et al, 2002). Because most LQTS patients are 33 heterozygous for ion channel mutations, the relevant phenotype is determined when the mutant channel is coexpressed with Wt channels, and the effect of mutations seems to vary depending on the mutation (Sanguinetti et al, 1996a). For example, expression of Wt channel subunits is greatly impeded by coexpression with a non-trafficking mutant containing the A561V mutation in the S5 transmembrane segment (dominant-negative effect; Ficker et al, 2000). Deletion of nine amino acids in the S3 transmembrane helix.(AI500-F508) renders homomeric mutant channels unable to traffic to the membrane, while heteromultimeric assembly has no apparent effect on the expression of Wt subunits (rescue effect; Sanguinetti et al, 1996a). This suggests a variable degree of 7*> dysfunction in LQT2 patients, resulting in a variations in clinical severity. It has been suggested that co-assembly of the regulatory auxiliary subunit KCNE2 (also known as, M i R P l ; MinK-Related Peptide 1) with hERG a-subunits is required in order to recapitulate the biophysical and drug block properties of native (Abbott et al., 1999). Mutations in KCNE2 are associated with LQT6 and it is thought that this effect is exerted through modification of the biophysical properties of since mutant KCNE2 subunits result in reduced by slowing hERG channel activation and accelerating deactivation (Abbott et al, 1999; Isbrandt et al, 2002; Lu et al, 2003). However, the stoichiometry of the KCNE2 interaction with hERG is not clear (Deutsch, 2002) and the effect of coexpression of Wt and mutant KCNE2 subunits remains to be examined. Furthermore, two separate reports suggest that the presence of KNCE2 does not recapitulate /*> properties better than hERG alone (Weerapura et al, 2002; Lu et al, 2003) and KCNE2 has been shown to interact with KCNQ1 (Tinel et al, 2000), Kv4.2 (Zhang et al, 2001a) and H C N (Yu et al, 2001) channels. Although the physiological relevance of these observations is not 34 entirely clear, it remains to be seen i f interaction of mutant KCNE2 subunits with channels other than hERG can be an alternative mechanism for prolongation of the QT interval. 1.6.1.2 IKS dysfunction (LQT1 and LQT5) KCNQ1 encodes a Kv channel, which coassembles with KCNE1 (also known as MinK) to form the slowly activating K+-delayed rectifier current IKS (Barhanin et ah, 1996; Sanguinetti et ah, 1996b). As with hr, hs has been identified in several mammalian species (Sanguinetti et ah, 1990; L i et ah, 1996; Salata et ah, 1996; Gintant, 1996) and mutations in KCNQ1 and KCNE1 are associated with LQT1 and LQT5, respectively. LQT1 and LQT5 are attributed to a decrease or loss of hs by prevention of efficient KCNQ1 or KCNE1 protein folding and trafficking to the cell membrane (Shalaby et ah, 1997; Bianchi et ah, 2000; Huang et ah, 2001; Dahimene et ah, 2006), or by modification of the gating properties of hs channels. Altered gating, such as a depolarizing shift in the voltage dependence of activation, accelerated deactivation and slowing of activation result in reduced IKS contribution during an action potential (Chouabe et ah, 1997; Chouabe et ah, 2000; Huang et ah, 2001; Boulet et ah, 2006). Recently, LQTS mutations in the S4-S5 linker and the C-terminus of KCNQ1 were also reported to render hs less sensitive to modulation by PIP2, implicating signaling cascades in the generation of LQTS (Park et ah, 2005). As with mutations in hr, there is a spectrum of functional effects that is caused by different mutations in KCNQ1 and KCNE1 , which depend on the level of dominance of the mutated subunit in the heteromultimeric hs channels. For example, the E261D mutation in KCNQ1 imposes a strong dominant negative effect in the presence of Wt channel subunits (Huang et ah, 2001). In contrast, T59P/L60P mutations in KCNE1 abolish IKS in the absence of Wt KCNE1 subunits but in the presence of Wt KCNE1 subunits, current density and the biophysical properties of the KCNQ1+KCNE1 complex are normal (Huang et ah, 2001). 35 Patients with LQT1 and perhaps LQT5 experience ventricular arrhythmia during emotional stress and sympathetic stimulation. This is thought to be due to the major role that IKS plays in shortening the action potential duration and the QT interval only during rapid heart rates (Hund and Rudy, 2004), for two main reasons. Firstly, although the activation kinetics of IKS are very slow relative to the duration of a single action potential, the slow deactivation kinetics are thought to allow accumulation of IKS in the open state during repetitive high frequency activity (Romey et al., 1997). Such accumulation allows for greater outward K + efflux and this is referred to as a "repolarizing reserve" that allows abbreviation of the cardiac action potential, and therefore systole, at high heart rates (Stengl et al, 2006). Secondly, P-adrenergic stimulation results in phosphorylation of KCNQ1 channels and increases IKS by causing a hyperpolarizing shift in the voltage dependence of activation, accelerating activation and slowing deactivation kinetics. This contributes to shortening of the cardiac action potential in order to accommodate a rapid heart rate (Terrenoire et al., 2005). This is in agreement with the high risk for ventricular arrhythmia that is observed during periods of elevated sympathetic activity, such as exercise and stress in LQT1 patients (Keating and Sanguinetti, 2001; Schwartz et al., 2001; Moss, 2003). Additionally, while pharmacological block of IKS in the absence of P-adrenergic stimulation has only a small effect on the action potential duration recorded from isolated canine (Varro et al., 2000), rabbit (Lengyel et al, 2001) and human (Jost et al, 2005) ventricular myocytes, regardless of the stimulation frequency, IKS block under sympathetic stimulation results in prolongation of action potential duration (Volders et al, 2003; Stengl et al, 2003). Furthermore, the effect of IKS block on action potential duration in the presence of P-adrenergic stimulation is exacerbated following pharmacological block of suggesting that 36 IKS provides a repolarizing reserve to maintain normal action potential duration when other outward repolarizing currents are attenuated (Varro et al., 2000; Jost et al., 2005). This is a very important concept clinically since the effect of drugs that block hERG may be amplified in patients with IKS mutations (LQT1 and LQT5; Abbott et al., 1999). 1.6.2 Na+ channel mutations and LQT3 LQT3 is associated with mutations in the SCN5A gene that encodes the cardiac N a + channel, Navl.5. Although it only accounts for about 10% of cases of LQTS (Napolitano et al, 2005), LQT3 is the most lethal of the common LQTS variants (Schwartz et al, 2001). Normally, cardiac N a + channels open briefly to allow influx of Na + ions, which depolarizes the membrane and thus initiates the cardiac action potential. Following channel opening, the IFM motif in the cytoplasmic linker connecting domains III and IV rapidly occludes the open pore causing fast inactivation which impedes the flow of N a + (Patton et al., 1992; West et al., 1992). Inactivated channels remain nonconducting for the remainder of the action potential duration. However, gain-of-function mutations of the SCN5A gene that interfere with channel inactivation allow repetitive brief channel openings that occur throughout the action potential, producing a small but persistent inward depolarizing current which lengthens the action potential duration and thus causes LQTS and the associated increased risk of arrhythmias (Bennett et al, 1995; Wang et al, 1995a; Dumaine et al, 1996). Several mutations in this gene have been identified, including substitutions (N1325S in the S4-S5 linker of domain III and R1644H in the S4 of domain IV) and a 9 base-pair deletion involving three amino acids, AKPQ, in the domain III and IV linker (Wang et al., 1995a). The different mutations produce variable degrees of altered channel function. The AKPQ mutation causes both brief reopenings and longer-lasting reopenings, whereas the 37 substitutions result in only brief reopenings. As a consequence, the alteration in channel function is more severe with the AKPQ mutation (Dumaine et al, 1996). 1.7 Scope of the thesis The main aim of the studies detailed in this thesis is to better understand the regulation and modulation of Kv channel activation properties. The first set of experiments revolve around the K v l . 2 channel because, unlike any other Kv channel, the reported biophysical activation properties of Kv l .2 are highly variable, with reported half-activation potentials ranging from -43 mV to more than +20 mV (Grissmer et al., 1994; Steidl and Yool, 1999; Minor et al, 2000; Koopmann et al, 2001; Scholle et al, 2004). When we expressed K v l . 2 in cultured mammalian cells, we made the unprecedented observation that K v l . 2 can assume two different activation gating modes, with some cells expressing channels that exhibited very slow activation kinetics with half-activation potentials greater than +15 mV, and other cells expressing channels that showed rapid activation kinetics associated with half-activation potentials less than -15 mV. We hypothesized that there should be a molecular determinant within the Kv l .2 protein that dictates this unusual functional heterogeneity. Through a chimeric strategy with the Kvl .5 channel that normally shows non-variable gating, we determined that the S2-S3 linker of K v l . 2 is crucial for dictating the activation properties of Kv l . 2 . We extended this discovery to define a single amino acid (threonine 252) within the S2-S3 linker that acts as a switch between the two K v l . 2 gating modes. Mutation of the threonine at position 46 in the T l domain of K v l . 2 to valine (T46V) has been reported to result in a dramatic slowing of activation kinetics when expressed in Xenopus oocytes without altering the overall 3-dimensional structure of the T l domain 38 (Minor et al., 2000). We questioned whether this mutation exerted any effect on the activation properties of Kv l .2 channels expressed in mammalian cells in the light of the observed heterogeneous 'slow' and 'fast' gating modes. In these experiments, we determined that, when expressed in mammalian cells, the T46V mutant channels were trapped in the 'slow' gating mode, but that the deactivation kinetics were accelerated. While the equivalent mutation in the Kvl .5 channel (T133V) had no effect on channel kinetics, deactivation kinetics of a chimeric channel that consisted of the N-terminus of Kvl .5 attached to the transmembrane core of Kv l .2 were accelerated by the T133V mutation. Since the threonine to valine mutation in the T l domain only affected K v l . 2 gating properties, we hypothesized that the mechanism of coupling between the T l domain and the transmembrane core is different in Kv l .2 and Kvl .5 channels. Through a chimeric strategy between these two channels, we determined a putative link between the T l domain and the cytoplasmic S2-S3 linker of Kv l .2 . This is consistent with our previous finding that the S2-S3 linker acts as a gating switch in K v l .2 channels and offers the first report of a mechanism through which the cytoplasmic T l domain may couple to the transmembrane core of the channel. In addition to modification of the S2-S3 linker by the T l domain, we provide evidence that cytoplasmic constituents may interact with the S2-S3 linker in Kv l .2 and modify its gating characteristics. We demonstrated that excision of inside-out patches resulted in a permanent switch from the 'slow' gating mode in the cell-attached condition to the 'fast' mode upon excision of the patch. In addition, the 'fast' gating mode correlated well with high current density, suggesting that the 'slow' mode was stabilized by an endogenous cytoplasmic constituent that became overwhelmed with high channel expression levels. 39 K v l . 2 channels with threonine 252 in the S2-S3 linker mutated to arginine (T252R) could not be modified by the cytoplasmic environment and this led us to postulate the involvement of channel phosphorylation. However, neither non-specific broad-spectrum kinase inhibitors nor specific inhibitors altered the gating properties of Kv l .2 channels. Interestingly, during these experiments we discovered that KN-93, a commonly used calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of many different Kv channels that inhibits current by stabilizing inactivated channel states (Rezazadeh et al, 2006). We have also ruled out modification of K v l . 2 activation gating by PIP2 and polyamines. Further studies are required to identify the cytoplasmic gating-modifying component. The second set of experiments involve genetic modulation of Kv channel properties. Genetic screening of affected individuals from a Northern British Columbia aboriginal community with a disproportionately high incidence of LQTS revealed a novel missense mutation (V205M) at the intracellular end of the S3 transmembrane helix of KCNQ1, the pore-forming domain of the IKS channel complex in the heart. We have characterized the effects of the V205M mutation on IKS properties and have shown that it results in a depolarizing shift in the voltage dependence of activation and accelerated deactivation. Through action potential voltage clamp protocols, we have shown that V205M induces a dominant negative effect when co-expressed with Wt channels. The changes in channel kinetics produced by the V205M mutation are expected to decrease IKS current and reduce the repolarization reserve during the cardiac action potential, with a likely increased susceptibility to the initiation of arrhythmias, especially during periods of high sympathetic drive. These observations also provide a putative working model for the K v l . 2 activation 40 gating switch that involves S2-S3 linker mediated changes in the hydrophobic packing of the S3 helix and consequent alteration of S4 movement during activation. 41 1.8 References Abbott,G.W., F.Sesti, I.Splawski, M.E.Buck, W.H.Lehmann, K.W.Timothy, M.T.Keating, and S.A.N.Goldstein. 1999. MiRPT forms I K r potassium channels with hERG and is associated with cardiac arrhythmia. Cell 97:175-187. Accili ,E.A., J.Kiehn, Q.Yang, Z.G.Wang, A.M.Brown, and B.A.Wible. 1997. Separable Kv0 subunit domains alter expression and gating of potassium channels. J Biol Chem 272:25824-25831. Armstrong,C.M. and F.Bezanilla. 1973. Currents related to movement of the gating particles of the sodium channels. Nature 242:459-461. AshcroftF.M. 2000. Ion channels and disease. In Channelopathies. Academic Press. San Diego, California. Bahring,R., J.Dannenberg, H.C.Peters, T.Leicher, O.Pongs, and D.Isbrandt. 2001. Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating. JBiol Chem 276:23888-23894. Barhanin,J., F.Lesage, E.Guillemare, M.Fink, M.Lazdunski, and G.Romey. 1996. K V LQT1 and IsK (minK) proteins associate to form the 7KS cardiac potassium current. Nature 384:78-80. Bennett,P.B., K.Yazawa, N.Makita, and A.L.George, Jr. 1995. Molecular mechanism for an inherited cardiac arrhythmia. Nature 376:683-685. Benson,M.D., Q.J.Li, K.Kieckhafer, D.Dudek, M.R.Whorton, R.K.Sunahara, J.A.Iniguez-Lluhi, and J.R.Martens. 2007. SUMO modification regulates inactivation of the voltage-gated potassium channel K v l . 5 . Proc Natl Acad Sci USA 104:1805-1810. Bian,J., J.Cui, and T.V.McDonald. 2001. hERG K + channel activity is regulated by changes in phosphatidyl inositol 4,5-bisphosphate. Circ Res 89:1168-1176. Bianchi,L., S.G.Priori, C.Napolitano, K.A.Surewicz, A.T.Dennis, M.Memmi, P.J.Schwartz, and A.M.Brown. 2000. Mechanisms of 7K.S suppression in LQT1 mutants. Am J Physiol Heart Circ Physiol 279:H3003-H3011. 42 BouletJ.R., A.L.Raes, N.Ottschytsch, and D.J.Snyders. 2006. Functional effects of a KCNQ1 mutation associated with the long QT syndrome. Cardiovasc Res 70:466-474. Caprini,M., S.Ferroni, R.Planells-Cases, J.Rueda, C.Rapisarda, A.Ferrer-Montiel, and M.Montal. 2001. Structural compatibility between the putative voltage sensor of voltage-gated K + channels and the prokaryotic KcsA channel. J Biol Chem 276:21070-21076. Cha,A. and F.Bezanilla. 1997. Characterizing voltage-dependent conformational changes in the Shaker K + channel with fluorescence. Neuron 19:1127-1140. Cha,A., G.E.Snyder, P.R.Selvin, and F.Bezanilla. 1999. Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809-813. Chen,J., J.S.Mitcheson, M.Tristani-Firouzi, M.Lin , and M.C.Sanguinetti. 2001. The S4-S5 linker couples voltage sensing and activation of pacemaker channels. Proc Natl Acad Sci USA 10.1073:1-6. Chouabe,C, N.Neyroud, P.Guicheney, M.Lazdunski, G.Romey, and J.Barhanin. 1997. Properties of K v L Q T l K + channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias. EMBO J 16:5472-5479. Chouabe,C, N.Neyroud, P.Richard, I.Denjoy, B.Hainque, G.Romey, M.D.Drici, P.Guicheney, and J.Barhanin. 2000. Novel mutations in K v L Q T l that affect Iks activation through interactions with Isk. Cardiovasc Res 45:971-980. Claydon,T.W., M.Vaid, S.Rezazadeh, D.C.Kwan, S.J.Kehl, and D.Fedida. 2007. A Direct Demonstration of Closed-State Inactivation of K + Channels at Low pH.JGen Physiol 129:437-455. Cordes,F.S., J.N.Bright, and M.S.Sansom. 2002. Proline-induced distortions of transmembrane helices. J Moi Biol 323:951-960. Crary,J.I., D.M.Dean, F.Maroof, and A.L.Zimmerman. 2000. Mutation of a single residue in the S2-S3 loop of C N G channels alters the gating properties and sensitivity to inhibitors. J Gen Physiol 116:769-779. 43 Craven,K.B. and W.N.Zagotta. 2006. C N G and H C N channels: two peas, one pod. Annu Rev Physiol 68:375-401. Cuello,L.G., D.M.Cortes, and E.Perozo. 2004. Molecular architecture of the KvAP voltage-dependent K + channel in a lipid bilayer. Science 306:491-495. Cui,J., Y.Melman, E.Palma, G.I.Fishman, and T.V.McDonald. 2000. Cyclic A M P regulates the hERG K + channel by dual pathways. Curr Biol 10:671-674. Curran,M.E., I.Splawski, K.W.Timothy, G.M.Vincent, E.D.Green, and M.T.Keating. 1995. A molecular basis for cardiac arrhythmia: hERG mutations cause long QT syndrome. Cell 80:795-803. Cushman,S.J., M.H.Nanao, A.W.Jahng, D.DeRubeis, S.Choe, and P J.Pfaffinger. 2000. Voltage dependent activation of potassium channels is coupled to T l domain structure. Nature Structural Biology 7:403-407. Czech,M.P. 2000. PIP2 and PIP3: complex roles at the cell surface. Cell 100:603-606. Dahimene,S., S.Alcolea, P.Naud, P.Jourdon, D.Escande, R.Brasseur, A.Thomas, I.Baro, and J.Merot. 2006. The N-terminal juxtamembranous domain of KCNQ1 is critical for channel surface expression: implications in the Romano-Ward LQT1 syndrome. Circ ^99:1076-1083. Davis,D.R. and A.S.Tang. 2004. Implantable cardioverter defibrillators: therapy against Canada's leading killer. CMAJ 171:1037-1038. DeBiasi,M., Z.Wang, E.Accili , B.Wible, and D.Fedida. 1997. Open channel block of human heart hKvl .5 by the P-subunit hKvpl .2. Am J Physiol Heart Circ Physiol 272:H2932-H2941. Del Camino,D., M.Holmgren, Y.Liu , and G.Yellen. 2000. Blocker protection in the pore of a voltage-gated K + channel and its structural implications. Nature 403:321-325. Del Camino,D. and G.Yellen. 2001. Tight steric closure at the intracellular activation gate of a voltage-gated K + channel. Neuron 32:649-656. Deutsch,C. 2002. Potassium channel ontogeny. Annu Rev Physiol 64:19-46. 44 Deutsch,C. 2003. The birth of a channel. Neuron 40:265-276. DiFrancesco,D. and P.Tortora. 1991. Direct activation of cardiac pacemaker channels by intracellular cyclic A M P . Nature 351:145-147. Ding,S., L.Ingleby, C.A.Ahern, and R.Horn. 2005. Investigating the putative glycine hinge in Shaker potassium channel. J Gen Physiol 126:213-226. Doyle,D.A., J.M.Cabral, R.A.Pfuetzner, A.L.Kuo, J.M.Gulbis, S.L.Cohen, B.T.Chait, and R.MacKinnon. 1998. The structure of the potassium channel: Molecular basis of K + conduction and selectivity. Science 280:69-77'. Dumaine,R., Q.Wang, M.T.Keating, H.A.Hartmann, P.J.Schwartz, A.M.Brown, and G. E.Kirsch. 1996. Multiple mechanisms of N a + channel-linked long-QT syndrome. Circ Res 78:916-924. Enkvetchakul,D., I.Jeliazkova, and C.G.Nichols. 2005. Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem 280:35785-35788. Ficker,E., A.T.Dennis, C.A.Obejero-Paz, P.Castaldo, M.Taglialatela, and A.M.Brown. 2000. Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome. J Moi Cell Cardiol 32:2327-2337. Ficker,E., A.T.Dennis, L.Wang, and A.M.Brown. 2003. Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel hERG. Circ Res 92:e87-100. Flynn,G.E. and W.N.Zagotta. 2001. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron 30:689-698. Frankenhaeuser,B. and A.L.Hodgkin. 1957. The action of calcium on the electrical properties of squid axons. J Physiol (Lond) 137:218-244. Furutani,M., M.C.Trudeau, N.Hagiwara, A.Seki, Q.M.Gong, A.F.Zhou, S.Imamura, H . Nagashima, H.Kasanuki, A.Takao, K.Momma, C.T.January, G.A.Robertson, and R.Matsuoka. 1999. Novel mechanism associated with an inherited cardiac arrhythmia - Defective protein trafficking by the mutant hERG (G601S) potassium channel. Circulation 99:2290-2294. 45 Gandhi,C.S., E.Clark, E.Loots, A.Pralle, and E.Y.Isacoff. 2003. The orientation and molecular movement of a K + channel voltage-sensing domain. Neuron 40:515-525. Gilly,W.F. and C.M.Armstrong. 1982. Slowing of sodium channel opening kinetics in squid axon by extracellular zinc. J Gen Physiol 79:935-964. Gintant,G.A. 1996. Two components of delayed rectifier current in canine atrium and ventricle. Does IKs play a role in the reverse rate dependence of class III agents? Circ Res 78:26-37. Glauner,K.S., L.M.Mannuzzu, C.S.Gandhi, and E.Y.Isacoff. 1999. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813-817. Gonzalez,C, FJ.Morera, E.Rosenmann, O.Alvarez, and R.Latorre. 2005. S3b amino acid residues do not shuttle across the bilayer in voltage-dependent Shaker K + channels. Proc Natl Acad Sci USA 102:5020-5025. Gonzalez,C, E.Rosenman, F.Bezanilla, O.Alvarez, and R.Latorre. 2000. Modulation of the Shaker K + channel gating kinetics by the S3-S4 linker. J Gen Physiol 115:193-207. Grissmer,S., A.N.Nguyen, J.Aiyar, D.C.Hanson, R.J.Mather, G.A.Gutman, M.J.Karmilowicz, D.D.Auperin, and K.G.Chandy. 1994. Pharmacological characterization of five cloned voltage- gated K + channels, types K v l . l , 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Molec Pharmacol 45:1227-1234. Gulbis,J.M., S.Mann, and R.MacKinnon. 1999. Structure of a voltage-dependent K + channel p subunit. Cell 97:943-952. GulbisJ.M., M.Zhou, S.Mann, and R.MacKinnon. 2000. Structure of the cytoplasmic P subunit - T l assembly of voltage-dependent K + channels. Science 289:123-127. Gutman,G.A., K.G.Chandy, J.P.Adelman, J.Aiyar, D.A.Bayliss, D.E.Clapham, M.Covarriubias, G.V.Desir, K.Furuichi, B.Ganetzky, M.L.Garcia, S.Grissmer, L.Y.Jan, A.Karschin, D.Kim, S.Kuperschmidt, Y.Kurachi, M.Lazdunski, F.Lesage, H.A.Lester, D.McKinnon, C.G.Nichols, I.O'Kelly, J.Robbins, G.A.Robertson, B.Rudy, M.Sanguinetti, S.Seino, W.Stuehmer, M.M.Tamkun, C.A.Vandenberg, A.Wei, H.Wulff, and R.S.Wymore. 2003. International Union of Pharmacology. X L I . Compendium of voltage-gated ion channels: potassium channels. Pharmacol Rev 55:583-586. 46 Hackos,D.H., T.H.Chang, and K.J.Swartz. 2002. Scanning the intracellular S6 activation gate in the Shaker K + channel. J Gen Physiol 119:521-531. Hahin,R. and D.T.Campbell. 1983. Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations. J Gen Physiol 82:785-805. Heinemann,S.H., J.Rettig, H.R.Graack, and O.Pongs. 1996. Functional characterization of K v channel P-subunits from rat brain. J Physiol (Camb) 493:625-633. Hilgemann,D.W., S.Feng, and C.Nasuhoglu. 2001. The complex and intriguing lives of PIP2 with ion channels and transporters. Sci STKE 2001 :RE19. Hille,B. 2001. Ion channels of excitable membranes. 4 ed. Sinauer Associates Inc., Sunderland, Massachusetts. Hille,B., A.M.Woodhull, and B.I.Shapiro. 1975. Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH. Philos Trans R Soc Lond -5 5/o/5c/270:301-318. Hodgkin,A.L. and A.F.Huxley. 1952. The components of membrane conductance in the giant axon of Loligo. J Physiol (Lond) 116:473-496. Huang,L., M.Bitner-Glindzicz, L.Tranebjaerg, and A.Tinker. 2001. A spectrum of functional effects for disease causing mutations in the Jervell and Lange-Nielsen syndrome. Cardiovasc Res 51:670-680. Hund,T.J. and Y.Rudy. 2004. Rate dependence and regulation of action potential and calcium transient in a canine cardiac ventricular cell model. Circulation 110:3168-3174. Isbrandt,D., P.Friederich, A.Solth, W.Haverkamp, A.Ebneth, M.Borggrefe, H.Funke, K.Sauter, G.Breithardt, O.Pongs, and E.Schulze-Bahr. 2002. Identification and functional characterization of a novel KCNE2 (MiRPl) mutation that alters hERG channel kinetics. JMol Med 80:524-532. Jacobson,D.A., C.R.Weber, S.Bao, J.Turk, and L.H.Philipson. 2007. Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate. J Biol Chem 282:7442-7449. 47 Jiang,Y., A.Lee, J.Chen, M.Cadene, B.T.Chait, and R.MacKinnon. 2002a. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417:515-522. Jiang,Y., A.Lee, J.Chen, M.Cadene, B.T.Chait, and R.MacKinnon. 2002b. The open pore conformation of potassium channels. Nature 417:523-526. Jiang,Y., A.Lee, J.Chen, V.Ruta, M.Cadene, B.T.Chait, and R.MacKinnon. 2003a. X-ray structure of a voltage-dependent K + channel. Nature 423:33-41. Jiang,Y., V.Ruta, J.Chen, A.Lee, and R.MacKinnon. 2003b. The principle of gating charge movement in a voltage-dependent K + channel. Nature 423:42-48. Johnson,J.P. and W.N.Zagotta. 2001. Rotational movement during cyclic nucleotide-gated channel opening. Nature 412:917-921. Jones,E.M., E.C.Roti Roti, J.Wang, S.A.Delfosse, and G.A.Robertson. 2004. Cardiac IKr channels minimally comprise hERG la and lb subunits. J Biol Chem 279:44690-44694. Jost,N., L.Virag, M.Bitay, J.Takacs, C.Lengyel, P.Biliczki, Z.Nagy, G.Bogats, D.A.Lathrop, J.G.Papp, and A.Varro. 2005. Restricting excessive cardiac action potential and QT prolongation: a vital role for IKs in human ventricular muscle. Circulation 112:1392-1399. Ju,M., L.Stevens, E.Leadbitter, and D.Wray. 2003. The Roles of N - and C-terminal determinants in the activation of the Kv2.1 potassium channel. J Biol Chem 278:12769-12778. Kagan,A., Y.F.Melman, A.Krumerman, and T.V.McDonald. 2002. 14-3-3 amplifies and prolongs adrenergic stimulation of hERG K + channel activity. EMBO J21 : 1889-1898. Keating,M.T. and M.C.Sanguinetti. 2001. Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104:569-580. Keros,S. and C.J.McBain. 1997. Arachidonic acid inhibits transient potassium currents and broadens action potentials during electrographic seizures in hippocampal pyramidal and inhibitory interneurons. J Neurosci 17:3476-3487. 48 Kim,D. and D.E.Clapham. 1989. Potassium channels in cardiac cells activated by arachidonic acid and phospholipids. Science 244:1174-1176. Kiss,L. and S.J.Korn. 1998. Modulation of C-type inactivation by K + at the potassium channel selectivity filter. Biophys J 74:1840-1849. Kiss,L., J.LoTurco, and S.J.Korn. 1999. Contribution of the selectivity filter to inactivation in potassium channels. Biophys J 76:253-263. Kitaguchi,T., M.Sukhareva, and K.J.Swartz. 2004. Stabilizing the closed S6 gate in the Shaker Kv channel through modification of a hydrophobic seal. J Gen Physiol 124:319-332. Kobertz,W.R., C.Williams, and C.Miller. 2000. Hanging gondola structure of the T l domain in a voltage-gated K + channel. Biochemistry 39:10347-10352. Kobrinsky,E., L.Stevens, Y.Kazmi, D.Wray, and N.M.Soldatov. 2006. Molecular rearrangements of the Kv2.1 potassium channel termini associated with voltage gating. JBiol Chem 281:19233-19240. Koopmann,R., A.Scholle, J.Ludwig, T.Leicher, T.Zimmer, O.Pongs, and K.Benndorf. 2001. Role of the S2 and S3 segment in determining the activation kinetics in Kv2.1 channels. J Membrane Biol 182:49-59. Kuo,A., J.M.Gulbis, J.F.Antcliff, T.Rahman, E.D.Lowe, J.Zimmer, J.Cuthbertson, F.M.Ashcroft, T.Ezaki, and D.A.Doyle. 2003. Crystal structure of the potassium channel Ki rBac l . l in the closed state. Science 300:1922-1926. Kurata,H.T., G.S.Soon, J.R.Eldstrom, G.W.K.Lu, D.F.Steele, and D.Fedida. 2002. Amino-terminal determinants of U-type inactivation of voltage-gated K + channels. J Biol Chem 277:29045-29053. Kurata,H.T., G.S.Soon, and D.Fedida. 2001. Altered state dependence of C-type inactivation in the long and short forms of human K v l . 5 . J Gen Physiol 118:315-332. Labro,A.J., A.L.Raes, I.Bellens, N.Ottschytsch, and D.J.Snyders. 2003. Gating of Shaker-type channels requires the flexibility of S6 caused by prolines. J Biol Chem 278:50724-50731. 49 Larsson,H.P., O.S.Baker, D.S.Dhillon, and E.Y.Isacoff. 1996. Transmembrane movement of the Shaker K + channel S4. Neuron 16:387-397. Lee,H.C, J.M.Wang, and K.J.Swartz. 2003. Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels. Neuron 40:527-536. Lengyel,C, N.Iost, L.Virag, A.Varro, D.A.Lathrop, and J.G.Papp. 2001. Pharmacological block of the slow component of the outward delayed rectifier current (I(Ks)) fails to lengthen rabbit ventricular muscle QT(c) and action potential duration. Br J Pharmacol 132:101-110. Levitan,I.B. 1994. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56:193-212. Li,G.R., J.L.Feng, L.X.Yue, M.Carrier, and S.Nattel. 1996. Evidence for two components of delayed rectifier K + current in human ventricular myocytes. Circ Res 78:689-696. Liu,Y., M.Holmgren, M.E.Jurman, and G.Yellen. 1997. Gated access to the pore of a voltage-dependent K + channel. Neuron 19:175-184. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005a. Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309:897-903. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005b. Voltage sensor of K v l . 2 : structural basis of electromechanical coupling. Science 309:903-908. Lopez-Barneo,J., T.Hoshi, S.H.Heinemann, and R.W.Aldrich. 1993. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Recept Channels 1:61-71. Loussouarn,G., K.H.Park, C.Bellocq, I.Baro, F.Charpentier, and D.Escande. 2003. Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K + channels. EMBO722:5412-5421. Lu,Y., M.P.Mahaut-Smith, C.L.Huang, and J.I.Vandenberg. 2003. Mutant M i R P l subunits modulate hERG K + channel gating: a mechanism for pro-arrhythmia in long QT syndrome type 6. J Physiol 551:253-262. 50 Lu,Z., A.M.Klem, and Y.Ramu. 2002. Coupling between voltage sensors and activation gate in voltage-gated K + channels. J Gen Physiol 120:663-676. Mannuzzu,L.M., M.M.Moronne, and E.Y.Isacoff. 1996. Direct physical measurement of conformational rearrangement underlying potassium channel gating. Science 271:213-216. Mathur,R., J.Zheng, Y.Y.Yan, and F.J.Sigworth. 1997. Role of the S3-S4 linker in Shaker potassium channel activation. J Gen Physiol 109:191-199. McCormack,K., M.A.Tanouye, L.E.Iverson, J.-W.Lin, M.Ramaswami, T.McCormack, J.T.Campanelli, M.K.Mathew, and B.Rudy. 1991. A role for hydrophobic residues in the voltage-dependent gating of Shaker K + channels. Proc Natl Acad Sci USA 88:2931-2935. Minor,D.L.Jr., Y.F.Lin, B.C.Mobley, A.Avelar, Y.N.Jan, L.Y.Jan, and J.M.Berger. 2000. The polar T l interface is linked to conformational changes that open the voltage-gated potassium channel. Cell 102:657-670. Mohler,P.J., J.-J.Schott, A.O.Gramolini, K.W.Dilly, S.Guatimosim, W.H.duBell, L.S.Song, K.Haurogne, F.Kyndt, M . E . A l i , T.B.Rogers, W.J.Lederer, D.Escande, H.LeMarec, and V.Bennett. 2003. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421:634-639. Moss,A.J. 2003. Atrial fibrillation. Ann Noninvasive Electrocardiol 8:1-2. Nakajima,T., T.Furukawa, T.Tanaka, Y.Katayama, R.Nagai, Y.Nakamura, and M.Hiraoka. 1998. Novel mechanism of hERG current suppression in LQT2: shift in voltage dependence of hERG inactivation. Circ Res 83:415-422. Napolitano,C, S.G.Priori, P.J.Schwartz, R.Bloise, E.Ronchetti, J.Nastoli, G.Bottelli, M.Cerrone, and S.Leonardi. 2005. Genetic testing in the long QT syndrome: development and validation of an efficient approach to genotyping in clinical practice. JAMA 294:2975-2980. NerbonneJ.M. and R.S.Kass. 2005. Molecular physiology of cardiac repolarization. Physiol Rev 85:1205-1253. 51 Noda,M., S.Shimizu, T.Tanabe, T.Takai, T.Kayano, T.Ideda, H.Takahashi, H.Nakayama, Y.Kanaoka, N.Minamino, and et al. 1984. Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312:121-127. 01iver,D., C.C.Lien, M.Soom, T.Baukrowitz, P.Jonas, and B.Fakler. 2004. Functional conversion between A-type and delayed rectifier K + channels by membrane lipids. Science 304:265-270. Papazian,D.M. and F.Bezanilla. 1997. How does a channel sense voltage? NIPS 12:203-210. Papazian,D.M., T.L.Schwarz, B.L.Tempel, Y.N.Jan, and L.Y.Jan. 1987. Cloning of genomic and complementary D N A from Shaker, a putative potassium channel gene from Drosophila. Science 237:749-753. Papazian,D.M., X.M.Shao, S.-A.Seoh, A.F.Mock, Y.Huang, and D.H.Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K + channel. Neuron 14:1293-1301. Pardo,L.A., S.H.Heinemann, H.Terlau, U.Ludewig, C.Lorra, O.Pongs, and W.Stuhmer. 1992. Extracellular K + specifically modulates a rat brain K + channel. Proc Natl Acad Sci USA 89:2466-2470. Park,K.H., J.Piron, S.Dahimene, J.Merot, I.Baro, D.Escande, and G.Loussouarn. 2005. Impaired KCNQ1-KCNE1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome. Circ Res 96:730-739. Park,K.S., D.P.Mohapatra, H.Misonou, and J.S.Trimmer. 2006. Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science 313:976-979. Pathak,M., L.Kurtz, F.Tombola, and E.Isacoff. 2005. The Cooperative Voltage Sensor Motion that Gates a Potassium Channel. J Gen Physiol 125:57-69. Patton,D.E., J.W.West, W.A.Catterall, and A.L.Goldin. 1992. Amino acid residues required for fast Na+-channel inactivation: charge neutralizations and deletions in the III-IV linker. Proc Natl Acad Sci USA 89:10905-10909. Perozo,E. and F.Bezanilla. 1990. Phosphorylation affects voltage gating of the delayed rectifier K + channel by electrostatic interactions. Neuron 5:685-690. 52 Perozo,E., C.A.Vandenberg, D.S.Jong, and F.Bezanilla. 1991. Single channel studies of the phosphorylation of K + channels in the squid giant axon. I. Steady-state conditions. J Gen Physiol 98:1-17'. Piper,D.R., A.Varghese, M.C.Sanguinetti, and M.Tristani-Firouzi. 2003. Gating currents associated with intramembrane charge displacement in hERG potassium channels. Proc Natl Acad Sci USA 100:10534-10539. Plaster,N.M., R.Tawil, M.Tristani-Firouzi, S.Canun, S.Bendahhou, A.Tsunoda, M.R.Donaldson, S.T.Iannaccone, E.Brunt, R.Barohn, J.Clark, F.Deymeer, A.L.George, Jr., F.A.Fish, A.Hahn, A.Nitu, C.Ozdemir, P.Serdaroglu, S.H.Subramony, G.Wolfe, Y.H.Fu, and L.J.Ptacek. 2001. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell 105:511-519. Prole,D.L. and G.Yellen. 2006. Reversal of H C N channel voltage dependence via bridging of the S4-S5 linker and Post-S6. J Gen Physiol 128:273-282. Pusch,M., R.Magrassi, B.Wollnik, and F.Conti. 1998. Activation and inactivation of homomeric K v L Q T l potassium channels. Biophys J15:1'85-792. Rajan,S., L.D.Plant, M.L.Rabin, M.H.Butler, and S.A.Goldstein. 2005. Sumoylation silences the plasma membrane leak K + channel K2P1. Cell 121:37-47. Rezazadeh,S., T.W.Claydon, and D.Fedida. 2006. KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinn amyl)-N-methylbenzylamine), a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. J Pharmacol Exp Ther 317:292-299. Romey,G., B.Attali, C.Chouabe, I.Abitbol, E.Guillemare, J.Barhanin, and M.Lazdunski. 1997. Molecular mechanism and functional significance of the MinK control of the K v L Q T l channel activity. JBiol Chem 272:16713-16716. Salata,J.J., N.K.Jurkiewicz, B.Jow, K.Folander, P.J.Guinosso, Jr., B.Raynor, R.Swanson, and B.Fermini. 1996. 7K Of rabbit ventricle is composed of two currents: Evidence for /KS. Am J Physiol Heart Circ Physiol 271 :H2477-H2489. Sanguinetti,M.C., M.E.Curran, P.S.Spector, and M.T.Keating. 1996a. Spectrum of hERG K + -channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci USA 93:2208-2212. 53 Sanguinetti,M.C, M.E.Curran, A.Zou, J.Shen, P.S.Spector, D.L.Atkinson, and M.T.Keating. 1996b. Coassembly of K V LQT1 and minK (IsK) proteins to form cardiac 7KS potassium channel. Nature 384:80-83. Sanguinetti,M.C;, C.Jiang, M.E.Curran, and M.T.Keating. 1995. A mechanistic link between an inherited and an acquired cardiac arrhythmia: hERG encodes the Iicr potassium channel. Cell 81:299-307. Sanguinetti,M.C. and N.K.Jurkiewicz. 1990. Two components of delayed rectifier K + current. J Gen Physiol 96:195-215. Sanguinetti,M.C, N.K.Jurkiewicz, A.Scott, and P.K.Siegl. 1991. Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action. Circ Res 68:77-84. Sanguinetti,M.C. and M.Tristani-Firouzi. 2006. hERG potassium channels and cardiac arrhythmia. Nature 440:463-469. Sanguinetti,M.C. and Q.P.Xu. 1999. Mutations of the S4-S5 linker alter activation properties of hERG potassium channels expressed mXenopus oocytes. J Physiol (Camb) 514:667-675. Scholle,A., S.Dugarmaa, T.Zimmer, M.Leonhardt, R.Koopmann, B.Engeland, O.Pongs, and K.Benndorf. 2004. Rate-limiting reactions determining different activation kinetics of Kv l .2 andKv2.1 channels. JMembr Biol 198:103-112. Sch6nherr,R. and S.H.Heinemann. 1996. Molecular determinants for activation and inactivation of hERG, a human inward rectifier potassium channel. J Physiol (Camb) 493:635-642. Schoppa,N.E., K.McCormack, M.A.Tanouye, and F.J.Sigworth. 1992. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255:1712-1715. Schwartz,P.J., S.G.Priori, C.Spazzolini, A.J.Moss, G.M.Vincent, C.Napolitano, I.Denjoy, P.Guicheney, G.Breithardt, M.T.Keating, J.A.Towbin, A.H.Beggs, P.Brink, A.A.Wilde, L.Toivonen, W.Zareba, J.L.Robinson, K.W.Timothy, V.Corfield, D.Wattanasirichaigoon, C.Corbett, W.Haverkamp, E.Schulze-Bahr, M.H.Lehmann, K.Schwartz, P.Coumel, and R.Bloise. 2001. Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers for life-threatening arrhythmias. Circulation 103:89-95. 54 Schwartz,P.J., M.Stramba-Badiale, A.Segantini, P.Austoni, G.Bosi, R.Giorgetti, F.Grancini, E.D.Marni, F.Perticone, D.Rosti, and P.Salice. 1998. Prolongation of the QT interval and the sudden infant death syndrome. N EnglJ Med 338:1709-1714. Seoh,S.A., D.Sigg, D.M.Papazian, and F.Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K + channel. Neuron 16:1159-1167. Shalaby,F.Y., P.C.Levesque, W.P.Yang, W.A.Little, M.L.Conder, T.Jenkins-West, and M.A.Blanar. 1997. Dominant-negative K v L Q T l mutations underlie the LQT1 form of long QT syndrome. Circulation 96:1733-1736. Shieh,C.C, K.G.Klemic, and G.E.Kirsch. 1997. Role of transmembrane segment S5 on gating of voltage-dependent K + channels. J Gen Physiol 109:767-778. Shyng,S.L. and C.G.Nichols. 1998. Membrane phospholipid control of nucleotide sensitivity of K A T P channels. Science 282:1138-1141. Smith,P.L. and G.Yellen. 2002. Fast and slow voltage sensor movements in hERG potassium channels. J Gen Physiol 119:275-293. Smith-Maxwell,C.J., J.L.Ledwell, and R.W. Aldrich. 1998a. Role of the S4 in cooperativity of voltage-dependent potassium channel activation. J Gen Physiol 111 :399-420. Smith-Maxwell,C.J., J.L.Ledwell, and R.W.Aldrich. 1998b. Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation. J Gen Physiol 111:421-439. Sokolova,0., A.Accardi, D.Gutierrez, A.Lau, M.Rigney, and N.Grigorieff. 2003. Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvbeta2-subunit. Proc Natl Acad Sci USA 100:12607-12612. Sokolova,0., L.Kolmakova-Partensky, and N.Grigorieff. 2001. Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution. Structure (Camb) 9:215-220. Sorensen,J.B., A.Cha, R.Latorre, E.Rosenman, and F.Bezanilla. 2000. Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4. J Gen Physiol 115:209-221. 55 Spector,P.S., M.E.Curran, A.R.Zou, and M.C.Sanguinetti. 1996. Fast inactivation causes rectification of the her channel. J Gen Physiol 107:611-619. Splawski,L, K.W.Timothy, L.M.Sharpe, N.Decher, P.Kumar, R.Bloise, C.Napolitano, P J.Schwartz, R.M.Joseph, K.Condouris, H.Tager-Flusberg, S.G.Priori, M.C.Sanguinetti, and M.T.Keating. 2004. Cavl.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119:19-31. Starace,D.M. and F.Bezanilla. 2004. A proton pore in a potassium channel voltage sensor reveals a focused electric field. Nature 427:548-553. Starkus,J.G., L.Kuschel, M.D.Rayner, and S.H.Heinemann. 1997. Ion conduction through C-type inactivated Shaker channels. J Gen Physiol 110:539-550. SteidlJ.V. and A.J.Yool. 1999. Differential sensitivity of voltage-gated potassium channels Kvl .5 and K v l . 2 to acidic pH and molecular identification of pH sensor. Molec Pharmacol 55:812-820. Stengl,M., C.Ramakers, D.W.Donker, A.Nabar, A.V.Rybin, R.L.Spatjens, N.T.van der, W.K.Wodzig, K.R.Sipido, G.Antoons, A.F.Moorman, M.A.Vos, and P.G.Volders. 2006. Temporal patterns of electrical remodeling in canine ventricular hypertrophy: focus on IKs downregulation and blunted beta-adrenergic activation. Cardiovasc Res 72:90-100. Stengl,M., P.G.Volders, M.B.Thomsen, R.L.Spatjens, K.R.Sipido, and M.A.Vos. 2003. Accumulation of slowly activating delayed rectifier potassium current (IKs) in canine ventricular myocytes. J Physiol 551:777-786. Stieber,J., A.Thomer, B.Much, A.Schneider, M.Biel , and F.Hofmann. 2003. Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4. J Biol Chem 278:33672-33680. Subbiah,R.N., M.Kondo, T.J.Campbell, and J.I.Vandenberg. 2005. Tryptophan scanning mutagenesis of the hERG K + channel: the S4 domain is loosely packed and likely to be lipid exposed. J Physiol 569:367-379. Swartz,K.J. 2004. Opening the gate in potassium channels. Nat Struct Moi Biol 11:499-501. 56 Swartz,K.J. and R.MacKinnon. 1997a. Hanatoxin modifies the gating of a voltage-dependent K + channel through multiple binding sites. Neuron 18:665-673. Swartz,K.J. and R.MacKinnon. 1997b. Mapping the receptor site for hanatoxin, a gating modifier of voltage-dependent K + channels. Neuron 18:675-682. Tanabe,T., H.Takeshima, A.Mikami, V.Flockerzi, H.Takahashi, K.Kangawa, M.Kojima, H.Matsuo, T.Hirose, and S.Numa. 1987. Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328:313-318. Tang,C.Y. and D.M.Papazian. 1997. Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-a-go-go K + channel. J Gen Physiol 109:301-311. Tempel,B.L., D.M.Papazian, T.L.Schwarz, Y.N.Jan, and L.Y.Jan. 1987. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science 237:770-775. Terrenoire,C, C.E.Clancy, J.W.Cormier, K.J.Sampson, and R.S.Kass. 2005. Autonomic control of cardiac action potentials: role of potassium channel kinetics in response to sympathetic stimulation. Circ Res 96:e25-e34. Thornhill,W.B., M.B.Wu, X.Q.Jiang, X .Y .Wu, P.T.Morgan, and J.F.Margiotta. 1996. Expression of K v l . l delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function. J Biol Chem 271:19093-19098. Tinel,N., S.Diochot, M.Borsotto, M.Lazdunski, and J.Barhanin. 2000. KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel. EMBO J 19:6326-6330. Tiwari-WoodruffS.K., M.C.A.Lin , C.T.Schulteis, and D.M.Papazian. 2000. Voltage-dependent structural interactions in the Shaker K + channel. J Gen Physiol 115:123-138. Tiwari-Woodruff,S.K., C.T.Schulteis, A.F.Mock, and D.M.Papazian. 1997. Electrostatic interactions between transmembrane segments mediate folding of Shaker K + channel subunits. Biophys772:1489-1500. 57 Tombola,F., M.M.Pathak, P.Gorostiza, and E.Y.Isacoff. 2007. The twisted ion-permeation pathway of a resting voltage-sensing domain. Nature 445:546-549. Tombola,F., M.M.Pathak, and E.Y.Isacoff. 2005. Voltage-sensing arginines in a potassium channel permeate and occlude cation-selective pores. Neuron 45:379-388. Tristani-Firouzi,M., J.Chen, and M.C.Sanguinetti. 2002. Interactions between S4-S5 linker and S6 transmembrane domain modulate gating of hERG K + channels. J Biol Chem 277:18994-19000. Trudeau,M.C, J.W.Warmke, B.Ganetzky, and G.A.Robertson. 1995. hERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269:92-95. VanDongen,A.M.J., G.Freeh, J.A.Drewe, R.H.Joho, and A.M.Brown. 1990. Alteration and restoration of K + channel function by deletions at the N - and C-termini. Neuron 5:433.443. Varro,A., B.Balati, N.Iost, J.Takacs, L.Virag, D.A.Lathrop, L.Csaba, L.Talosi, and J.G.Papp. 2000. The role of the delayed rectifier component IKs in dog ventricular muscle and Purkinje fibre repolarization. J Physiol 523 Pt 1:67-81. Volders,P.G., M.Stengl, J.M.van Opstal, U.Gerlach, R.L.Spatjens, J.D.Beekman, K.R.Sipido, and M.A.Vos. 2003. Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation. Circulation 107:2753-2760. Wang,J.L., M.C.Trudeau, A.M.Zappia, and G.A.Robertson. 1998. Regulation of deactivation by an amino terminal domain in human ether-a-go-go-related gene potassium channels. J Gen Physiol 112:637-647. Wang,Q., J.Shen, Z .L i , K.Timothy, G.M.Vincent, S.G.Priori, P.J.Schwartz, and M.T.Keating. 1995a. Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Mol Genet 4:1603-1607. Wang,Q., J.Shen, I.Splawski, D.Atkinson, Z . L i , J.L.Robinson, AJ.Moss, J.A.Towbin, and M.T.Keating. 1995b. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 80:805-811. 58 Warmke,J.W. and B.Ganetsky. 1994. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U SA 91:3438-3442. Watanabe,L, H.G.Wang, J.J.Sutachan, J.Zhu, E.Recio-Pinto, and W.B.Thornhill. 2003. Glycosylation affects rat K v l . l potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism. J Physiol 550:51-66. WatanabeJ., J.Zhu, J.J.Sutachan, A.Gottschalk, E.Recio-Pinto, and W.B.Thornhill. 2007. The glycosylation state of K v l . 2 potassium channels affects trafficking, gating, and simulated action potentials. Brain Res 1144:1-18. Weerapura,M., S.Nattel, D.Chartier, R.Caballero, and T.E.Hebert. 2002. A comparison of currents carried by hERG, with and without coexpression of M i R P l , and the native rapid delayed rectifier current. Is M i R P l the missing link? J Physiol (Camb) 540:15-27. Weng,J., Y.Cao, N.Moss, and M.Zhou. 2006. Modulation of voltage-dependent Shaker family potassium channels by an aldo-keto reductase. J Biol Chem 281:15194-15200. WestJ.W., D.E.Patton, T.Scheuer, Y.Wang, A.L.Goldin, and W.A.Catterall. 1992. A cluster of hydrophobic amino acid residues required for fast Na+-channel inactivation. Proc Natl Acad Sci USA 89:10910-10914. Wood,M.J. and S.J.Korn. 2000. Two mechanisms of K+-dependent potentiation in Kv2.1 potassium channels. Biophys J79:2535-2546. Wu,L., C.S.Bauer, X.G.Zhen, C.Xie, and J.Yang. 2002. Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419:947-952. Yamaguchi,M., M.Shimizu, H.Ino, H.Terai, K.Hayashi, H.Mabuchi, N.Hoshi, and H.Higashida. 2003. Clinical and electrophysiological characterization of a novel mutation (F193L) in the KCNQ1 gene associated with long QT syndrome. Clin Sci (Lond) 104:377-382. Yang,N., A.L.George, Jr., and R.Horn. 1996. Molecular basis of charge movement in voltage-gated sodium channels. Neuron 16:113-122. Yang,N. and R.Horn. 1995. Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15:213-218. 59 Yang,P., H.Kanki, B.Drolet, T.Yang, J.Wei, P.C.Viswanathan, S.H.Hohnloser, W.Shimizu, P.J.Schwartz, M.Stanton, K.T.Murray, K.Norris, A.L.George, Jr., and D.M.Roden. 2002. Allelic variants in long-QT disease genes in patients with drug-associated torsades depointes. Circulation 105:1943-1948. Yarov-Yarovoy,V., D.Baker, and W.A.Catterall. 2006. Voltage sensor conformations in the open and closed states in ROSETTA structural models of K + channels. Proc Natl Acad Sci USA 103:7292-7297. Yu,H., J.Wu, I.Potapova, R.T.Wymore, B.Holmes, J.Zuckerman, Z.Pan, H.Wang, W.Shi, R.B.Robinson, M.R.E1 Maghrabi, W.Benjamin, J.Dixon, D.McKinnon, I.S.Cohen, and R.Wymore. 2001. MinK-related peptide 1: A beta subunit for the H C N ion channel subunit family enhances expression and speeds activation. Circ Res 88:E84-E87. Zhang,M., M.Jiang, and G.N.Tseng. 2001a. MinK-related peptide 1 associates with Kv4.2 and modulates its gating function - Potential role as (3 subunit of cardiac transient outward channel? Circ Res 88:1012-1019. Zhang,M., J.Liu, and G.N.Tseng. 2004. Gating charges in the activation and inactivation processes of the hERG channel. J Gen Physiol 124:703-718. Zhang,S.T., S.J.Kehl, and D.Fedida. 2001b. Modulation of Kv l .5 potassium channel gating by extracellular zinc. Biophys J S\:\25-\36. Zhou,Z., Q.Gong, M.I.Epstein, and C.T.January. 1998. hERG channel dysfunction in human long QT syndrome. Intracellular transport and functional defects. J Biol Chem 273:21061-21066. 60 Chapter 2: An activation gating switch in Kvl.2 is localized to a threonine residue in the S2-S3 Linker1 1 A version of this chapter is accepted for publication in the Biophysical Journal. Saman Rezazadeh, Harley T. Kurata, Thomas W. Claydon, Steven J. Kehl, and David Fedida 61 2.1 Introduction Much of the machinery associated with voltage-gated potassium (Kv) channel activation is localized to a voltage-sensing domain that comprises the transmembrane helices S1-S4, and includes a series of regularly spaced basic (Arg, Lys) residues in the S4 helix, together with acidic (Asp, Glu) residues in S2 and S3 helices (Papazian et al., 1991; Liman et al., 1991; Papazian et al., 1995; Larsson et al., 1996; Seoh et al, 1996; Aggarwal and MacKinnon, 1996; Bezanilla, 2000). Considerable effort has been devoted to understanding the structural rearrangements during voltage-gating, together with the conformational changes of the pore-forming domain that gate the permeation pathway (Gandhi et al., 2000; Elinder et al, 2001; Del Camino and Yellen, 2001). The detailed movements remain controversial (Cha et al., 1999; Glauner et al, 1999; Ahern and Horn, 2004; Long et al, 2005b), but it is well understood that membrane depolarization exerts forces on charged residues within the voltage-sensing domain, resulting in conformational changes that are coupled to the opening of a specialized ion-conducting pore formed by the S5 and S6 transmembrane helices (Bezanilla, 2000; Yellen, 2002; Long et al, 2005b). Much of the dynamic information that defines our understanding of K v channel activation comes from the Drosophila Shaker channel, and so specific mechanisms that regulate the individual activation properties of mammalian Kv channels remain incompletely understood. Although interactions between charged residues within the voltage-sensing domain (i.e. between basic residues in S4 and acidic residues in S2 and S3) are critical for normal activation gating (Papazian et al, 1995; Seoh et al, 1996), a number of studies demonstrate that other regions of the channel, especially the cytoplasmic N - and C- termini, can significantly modulate the time course and voltage dependence of activation. Deletion of 62 either the N - or C-terminus of many channels including K v l . l , K v l . 2 , K v l . 5 , Kv2.1, and hERG, can substantially shift the voltage dependence of activation (VanDongen et al, 1990; Wang et al, 1998; Chiara et al, 1999; Minor et al, 2000; Cushman et al, 2000; Kurata et al, 2001; Aydar and Palmer, 2001; Kurata et al, 2002; Ju et al, 2003). In addition, the MTSET modification rate of thiol groups in the T l - T l inter-subunit interface of Kv4 channels is state dependent, which suggests significant conformational changes take place outside the voltage-sensing domain and the pore domain during channel gating (Wang and Covarrubias, 2006). Detailed studies of individual transmembrane domains of Kv channels have also identified regions other than those involved in interactions between the basic and acidic residues in S2, S3 and S4 that affect activation gating. For example, substitution of three non-charged amino acids from the Shaw channel S4 into Shaker channels (the ILT mutation) results in a dissociation of S4 movement from the concerted step in channel opening (Smith-Maxwell et al, 1998). As well, exchange of the cytoplasmic half of S5 between Kv2.1 and Kv3.1 confers activation and deactivation properties similar to those of the donor channel, without altering the voltage-dependence of gating charge movements (Shieh et al, 1997). Finally, a swap of S2 or S3 of Kv2.1 with that of K v l . 2 profoundly alters the activation time course (Koopmann et al., 2001). In addition to these intrinsic structural influences on activation gating, the activation properties of Kv channels can also be modulated through protein-protein interactions, or post-translational modifications including phosphorylation and glycosylation. For example, it is well documented that the interaction of P subunits with K v channels results in modification of their activation and inactivation kinetics (Uebele et al, 1996; Heinemann et 63 al, 1996; DeBiasi et al., 1997; Accili et al., 1997). In addition, co-expression of the (3 subunit with B K c a channels increases the sensitivity of activation to voltage and calcium without affecting single-channel conductance or ionic selectivity (McManus et al., 1995). Phosphorylation of squid giant axon K + channels changes their activation kinetics, which is attributed to electrostatic interactions between the voltage-sensing domain and negatively charged phosphate groups (Perozo and Bezanilla, 1990; Perozo et al, 1991). Similarly, dephosphorylation of Kv2.1 and Kv3.1 channels has been shown to result in a hyperpolarizing shift in the Via of activation (Macica and Kaczmarek, 2001; Misonou et al., 2004; Mohapatra and Trimmer, 2006a). Finally, glycosylation of the S1-S2 linker of K v l . l and K v l . 2 channels results in a depolarizing shift in the Vm of activation, an effect that is thought to be partly due to the charge screening effect of sialic acids (Thornhill et al., 1996; Ponce et al, 1997; Watanabe et al, 2003; Watanabe et al, 2007). Kv l .2 is a Kv channel that shows quite variable activation kinetics. Most reports describe fast activation (x<10 ms at +40 mV) with a Via of activation ranging from -15 mV to -43 mV (Steidl and Yool, 1999; Minor et al., 2000; Koopmann et al, 2001; Scholle et al, 2004). However, Grissmer et al. (1994) briefly mentioned that K v l . 2 channels have a Via of activation of +27 mV and much slower activation gating (x -25 ms at +40 mV), which becomes faster upon repetitive pulsing. Here, we have extended these observations to show that K v l . 2 channels heterologously expressed in a number of different mammalian cell types can have two quite very distinct activation phenotypes. 'Slow' activation gating of Kv l .2 occurs during pulses applied after a rest interval and is associated with a depolarized Via of activation. Using a twin pulse protocol, we show that activation gating can be switched into a 'fast' mode, and that this is associated with a large hyperpolarizing shift in the voltage-64 dependence of activation gating. We demonstrate that this switch of gating is mediated at or around a single threonine residue, T252, in the S2-S3 linker that is uniquely present in K v l . 2 among the Kv channels, and that it involves an interaction with cytoplasmic gating-modifying constituents. Furthermore, transfer of the S2-S3 linker from K v l . 2 to Kv l .5 channels introduced the heterogeneous activation properties of K v l . 2 channels in the otherwise consistently fast gating Kvl .5 channels. These data identify a novel mode of gating in K v l . 2 channels that is regulated by the interaction of a cytosolic component either at, or associated with, T252 in the S2-S3 linker. This work has been presented in preliminary form as an abstract (Rezazadeh et al., 2007). 65 2.2 Materials and Methods 2.2.1 Cell preparation and transfection A l l experiments were carried out on transiently transfected mouse Uk- cells, H E K 293 cells, or Chinese Hamster Ovary (CHO) cells grown in Minimal Essential Medium (MEM) with 10% fetal bovine serum, at 37° C in an air/5% CO2 incubator. One day before transfection, cells were plated onto sterile glass coverslips in 35 mm Petri dishes at 20-30% confluence. On the day of transfection, cells were washed once with M E M containing 10% fetal bovine serum. In order to identify the transfected cells efficiently, channel D N A was co-transfected with a vector encoding green fluorescent protein (pGFP). Channel D N A was incubated with pGFP (1 pg of pGFP, 2 pg of channel DNA) and 3 uL of LipofectAMINE 2000 (Gibco-BRL) made up to 100 pL with serum-free OPTI-MEM (Gibco-BRL), then added to the dishes containing cells in 900 pL of M E M with 10% fetal bovine serum. Cells were allowed to grow overnight before recording. 2.2.2 Solutions Patch pipettes contained solution 1 (in mM): KC1, 135; EGTA, 5; HEPES, 10; and was adjusted to pH 7.2 with K O H . The bath solution contained solution 2 (in mM): NaCl, 135; KC1, 5; HEPES, 10; sodium acetate, 2.8; M g C l 2 , 1; CaCl 2 , 1; and was adjusted to pH 7.4 with NaOH. High external K + experiments were carried out using a bath containing solution 3 (in mM): KC1, 135; HEPES, 10; M g C l 2 , 1; glucose, 10 (adjusted to pH 7.4 with KOH). For cell attached and excised inside-out patch experiments, pipettes contained solution 2, while the bath contained solution 3. A l l chemicals were from Sigma Aldrich Chemical Co. (Mississauga, Ont.). Ser/Thr kinase inhibitors (bisindolylmaleimide, H-89, P K G inhibitor, KN-93, ML-7 and staurosporine) were purchased from Calbiochem (San Diego, CA) 66 2.2.3 Electrophysiological procedures Coverslips containing cells were removed from the incubator before experiments and placed in a superfusion chamber (volume 250 pi) containing the control bath solution at ambient temperature (22-23 °C), and perfused with bathing solution throughout the experiments. Whole-cell, excised and perforated patch current recording and data analysis were done using an Axopatch 200B clamp amplifier and pClamp 9 software (Axon Instruments, Foster City, CA) . Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments; FL, USA). Electrodes had resistances of 1-3 M Q when filled with pipette filling solution. Capacity compensation and 80% series resistance compensation were used in all whole-cell recordings. No leak subtraction was used when recording currents, and zero current levels are denoted by the dashed lines in the current panels. Data were sampled at 10 kHz and filtered at 2 kHz. For perforated patch experiments, 100 pg/ml of nystatin was added to the pipette solution (solution 1). After the gigaohm seal between tip and cell membrane had formed, negative pressure was released to await gradual opening of nystatin-induced pores. Recordings were made only after the access resistance was less than 20 M Q . Membrane potentials have not been corrected for small liquid junction potentials between bath and pipette solutions. 2.2.4 Molecular biology and channel expression The mammalian expression vector pcDNA3 was used for expression of all channel constructs used in this study. Kv l .2 channel was a kind gift from Dr. D. Minor (UCSF). A l l primers used were synthesized by Sigma Genosys (Oakville, Ontario, Canada). A l l constructs were sequenced to check for errors, and to ensure the correct reading frame (NAPS Unit, University of British Columbia, Vancouver, Canada). 67 2.2.5 Kvl.5/Kvl.2 chimeras Chimeras were constructed by PCR amplification of the desired segment of K v l . 2 , introducing restriction sites to allow subcloning into the Kvl .5 cDNA. With the exception of the Kv l .2 /Kv l .5C chimera, an EcoRIrestriction site was introduced at the C-terminal end of the fragment, and either a BspEI (for Kvl .5N/Kvl .2) , Pmll (for Kvl .5SlS2L/Kvl .2 ) , CM (for K v l .5S2S3L/Kvl .2), or StuI (for K v l .5S4S5L/Kvl .2) were introduced at the N-terminal end of the fragment. For construction of the Kv l . 2 /Kv l . 5C chimera, the Kv l .5 C-terminus was amplified by PCR, introducing 5' and 3' Hpal restriction sites, and subcloned into the K v l . 2 cDNA as a Hpal-Hpalfragment. Point mutations in K v l . 2 and Kv l .5 were generated using the Stratagene Quikchange kit (Stratagene, La Jolla, CA). 2.2.6 Data analysis and modeling g-Vcurves throughout the text were derived using the normalized chord conductance, which was calculated by dividing the maximum current elicited during a depolarizing step by the driving force derived from the calculated K + equilibrium potential. g-V curves were fitted with a single Boltzmann function of the form: y=\l(\ + exp[Vi/2 -V]/£) where y is the conductance normalized with respect to the maximal conductance, V\/2 is the half-activation potential, V is the test voltage and k is the slope factor. Data throughout the text and figures are shown as means ± S .E .M. Statistical significance was determined throughout using Student's t test with P values of less than 0.05 taken to be significant. Macroscopic K v l . 2 currents were simulated using a scientific graphing package (IGOR 5, Wavemetrics, Oregon) in which state occupancies as a function of time and voltage were derived from the spectral expansion of the _>-matrix (Colquhoun and Hawkes, 1995) generated from the state diagram in Figure 2.9A. For the calculation of currents, the open 68 channel I- V relationship was assumed to be ohmic. The rate constant for transition x at a given voltage (kx(V)) was calculated from the equation: kx(V) = kx(0 mV) exp(zxFV/RT) where F, R, and T have their usual meanings, ^(0 mV) is the rate constant at 0 mV, and zx is the equivalent charge moved between state x and the transition state. In the upper row the parameters were: k/j(0 mV) = 420 s"1, kb/(0 mV) = 373 s"1, zyj= 0.25, Z * A / = -0.8, kcoj = 8000 s"1 and k0cj — 100 s '. This approach and the values used to simulate fast activation are similar to those used for ShakerlR currents (Smith-Maxwell et al., 1998), and suggested to be appropriate for K v l . 2 (Scholle et ah, 2004). To replicate 'slow' activation, a parallel activation pathway was added (lower row) with the same kinetic behavior as the upper fast pathway except that the concerted transitions (Cin—»C>5 and Cio<— O 5 ) were slowed. Rate constants (kfs, ksf) for vertical transitions between the fast and 'slow' activation pathways were assumed, again for simplicity, to be voltage-independent and were assigned values to approximate the time-dependence for the relaxation from the fast activation pathway to the 'slow' activation pathway in a two-pulse voltage protocol (see below). The horizontal kinetic parameters were the same as the corresponding values in the upper row, except: 1) kffS(0 mV) was 140 s"1; 2) kCOtS was 40 s"1; and, 3) koc,s was 1 s"1. The variables g and d were required to conserve microscopic reversibility. With k/s = 0.022 and ks/= 0.0088, as was the case for all traces except for the fast-activating currents in Figure 2.9B, then g = (kcojkOCiSksf /kocjkco.skfsf'5 = 0.89442; and, d = (kfskb/kfjktJ0J which simplifies to d = (kfjkjjf'5 since the rate constants ktj(V) and kt,s(V) are identical and, because k/j and k/>s have the same voltage dependence, d has a fixed value of 0.57735. To reduce the number of fitting parameters it was assumed that the concerted opening ( C 4 —> O 5 , C10—> O 5 ) or concerted closing ( C 4 <— O 5 , 69 Cio <— O 5 ) transitions were voltage independent, but making these transitions voltage-dependent is not expected to substantially change the outcome. 70 2.3 Results 2.3.1 Heterogeneous activation properties of Kvl.2 expressed in mammalian cell lines Initial characterization of K v l . 2 using whole-cell patch clamp recordings from mouse Itk- cells revealed a considerable variation in both the time course and Vm of channel activation. Chord conductance was measured from currents during 400 ms activating voltage steps and individual conductance-voltage relationships from 70 cells are plotted in Figure 2.1 A to illustrate that activation relationships fell broadly into two groups, with isochronal Vj/2 estimates ranging between —35mV and ~+35 mV. It appears that K v l .2 shows two very distinct activation curve voltage-dependencies, with one population of cells showing isochronal activation F//.'s less than -10 mV, and a second population of cells with isochronal activation Virfs more positive than +10 mV. We also observed considerable differences in the speed of channel activation (Figure 2.1B-D). A proportion (27%) of cells expressing Kv l .2 exhibited a rapid activation time course, much of which could be fit with a single exponential ( r= 4.5 ± 1.7 ms at +35 mV), reminiscent of Shaker and related mammalian K v l channels (Figure IB). However, a larger proportion of cells exhibited much slower kinetics of activation (48%; x = 90 ± 6 ms at +35 mV, Figure 2.1C), or biphasic activation kinetics with fast and slow components in varying proportions (25%) (Figure 2.ID). As a first analysis we grouped individual 400 ms activation curves (Figure 2.1A) based on the activation kinetics into 'fast' cells (black lines), 'slow' cells (red lines) and 'mixed' cells (dashed black lines). Mean activation curves for cells falling into each group (Figure 2.IE) revealed that cells exhibiting 'fast' kinetics also exhibited current activation at negative voltages (V1/2 = -18.8 + 2.3 mV, n=\9). Cells that exhibited 'slow', or mixed activation kinetics had V^s of 16.6 ± 1.1 mV (n=33) and 14.5 ± 71 1.6 mV («=18), respectively (P>0.05). To illustrate the dramatic difference between the 'slow' and 'fast' gating phenotypes that we observed for Kv l . 2 , currents at +55 (from a slow cell) and +15 mV (from a fast cell), where P 0 was maximal, have been normalized to peak current and overlaid (Figure 2.IF). Critically, the differences in activation kinetics do not appear to be simply due to the different V/^s in cells exhibiting these various phenotypes. Single exponential fits to the activation time course to estimate activation kinetics demonstrated that even with strong depolarizations, the activation kinetics in 'slow' cells remained considerably slower than those observed in 'fast' cells even at the most positive potentials studied (Figure 2.1G). Overall, the proportion of cells exhibiting the various phenotypes was 27% (fast) and 73% (slow or mixed), respectively in Itk- cells and a similar ratio was observed for K v l . 2 channels expressed in other mammalian cell lines including CHO and H E K 293 cells (Figure 2.1H). Since Itk- cells showed little or no endogenous currents and because constructs expressed well in these cells, this cell line was used for the remaining experiments in the study. Previous experience with recordings from other K v channels in mammalian cell lines has accustomed us to high reproducibility of activation kinetics amongst individual cells. We ruled out the possibility of contamination of the D N A used for transfections by repeatedly selecting and sequencing clones from individual E. coli transformants. In addition, we noticed that although this dramatic variability has not been reported in any individual study of K v l . 2 , there are considerable discrepancies of reported activation V/a's, ranging from -43 mV (Steidl et al, 1999), -24.6 mV (Minor et al, 2000), and -17.9 mV (Koopmann et al, 2001), up to +27 mV (Grissmer et al, 1994) and +22 mV (Hulme et al, 1999). Figure 2.1. Bimodal gating of Kvl .2 expressed in mammalian cell systems. (A) Isochronal g-V relationships from seventy Itk- cells transiently expressing K v l . 2 . Normalized maximum chord conductance during 400 ms pulses was determined for each membrane potential and the data from individual cells were fitted with a Boltzmann function. (B-D) Examples of current-voltage data in (A). Currents were elicited by depolarizing steps from -65 to +55 mV in 20 mV increments. The gating kinetics of K v l . 2 showed three clear phenotypes: a group that expressed very fast activation kinetics (B); a group that exhibited dramatically slower opening (C), both of which were fitted with single exponential functions (insets illustrate the fitted currents recorded at +15 mV); and an intermediate phenotype (D) with a mixture of fast and 'slow' activating channels, whose activation time course was fitted with a double exponential function. (E) The mean isochronal activation Vm was -18.8 ± 2.3 mV (w=19, £=9.1 ± 0.5 mV) for fast activating channels (•), 16.6 ± 1.1 mV (A) for slowly activating channels (n=33, £=10.1 ± 0.3 mV), and 14.5 ± 1.6 mV («=18, £=12.5 ± 1.5 mV) for mixed channels (•). (F) Examples of normalized currents at +15 and +55 mV (P 0 -1.0) for 'fast' and 'slow' activating phenotypes. (G) Monoexponential activation time constants for 'fast' (•), and 'slow' (•) activating channels. (H) Modal gating was observed for K v l . 2 in Itk-, CHO and H E K 293 cells. 73 2.3.2 Prepulse potentiation of Kvl.2 activation One very early report examining the pharmacology of K v l . 2 had suggested a use-dependent activation process, with acceleration of activation kinetics and enhancement of Kv l .2 currents during trains of repetitive depolarizations (Grissmer et al., 1994). We investigated this phenomenon further with a twin-pulse protocol (Figure 2.2A). Cells expressing Kv l .2 were pulsed to voltages between -50 and +40 mV (PI, 1.5 s), repolarized (200 ms) to -80 mV, and finally pulsed to +20 mV (P2, 1.5 s). In cells exhibiting the 'slow' activation phenotype, the kinetics of activation in the P2 pulse depended dramatically on the voltage during the PI pulse (Figure 2.2A). If the voltage during the PI pulse did not lead to significant opening of channels (e.g. below -20 mV), the activation kinetics during P2 were very slow. In contrast, when the PI voltage led to significant channel opening (e.g. +40 mV), activation kinetics during P2 became very fast. This 'prepulse potentiation' resulted in significantly accelerated activation kinetics and greater current amplitude at the end of the P2 pulse (red trace, Figure 2.2B) than when the channels activated slowly (black trace, Figure 2.2B). K v l . 2 activation kinetics in cells exhibiting a 'fast' activation phenotype (Figure 2.IB) did not show prepulse potentiation (data not shown). Channel activation before and after prepulse conditioning had different voltage dependencies (Figure 2.2C, D). Cells exhibiting the 'slow' gating phenotype were pulsed to voltages between -65 mV and +35 mV for 2 s, and then repolarized to -40 mV, to allow calculation of an activation curve for slowly activating channels (Figure 2.2D, -prepulse). The Via of activation of slowly activating channels calculated from 2 s depolarizing steps in Figure 2.2D is somewhat hyperpolarized compared with the relation in Figure 2. IE, obtained from 400 ms pulses, and this is the result of allowing channel activation to reach a steady-74 state. In the same cells, the effect of prepulse potentiation was determined by including a 1 s prepulse to +40 mV before the activation protocol (Figure 2.2C). The prepulse accelerated channel activation, and resulted in a ~30 mV hyperpolarizing shift in the Via of channel activation (Figure 2.2D, data +prepulse). Such a prepulse-dependent gating shift in K v channels is unexpected, and these data raise the possibility that activation of 'slow' channels during the prepulse permits subsequent activation via a separate pathway that is more permissive to channel opening, presumably through the same pathway that the 'fast' activation gating proceeds. Consistent with this, application of a prepulse to 'fast' channels did not result in potentiation of ionic currents and resulted in no apparent shift in the Via of channel activation (data not shown). 75 Figure 2.2. Twin pulses convert 'slow' to 'fast' activation in Kvl.2. (A) Twin-pulse voltage protocol is shown above, currents below recorded between -50 mV and +40 mV in 10 mV increments for 1.5 s, repolarized to -80 mV for 200 ms, and then depolarized to +20 mV for 1.5 s. Labels on currents refer to the PI pulse potential. (B) Example of currents at +20 mV with (red trace) and without a prepulse to +40 mV (black trace). (C, D) Normalized g-V relations before and after a +40 mV prepulse. Original, continuous data shown in (C), for pulses between -65 and +35 mV in 10 mV steps obtained using the voltage protocol above. After the first set of voltage steps, the cell was repolarized to -80 mV for 4 s, and then given a l s prepulse to +40 mV before a 50 ms repolarization to -120 mV and finally the second set of voltage steps. (D) Maximum normalized conductance from three cells (gray symbols) obtained for the first (filled symbols) and the second set of steps (open symbols) plotted against membrane potential and fitted with a Boltzmann function. A switch in channel activation gating from 'slow' to fast between the first and second set of clamp pulses shifted the mean activation Via from 1.8 ± 1.5 mV to -25.7 ± 2.0 mV («=3). Mean relationships are plotted as black lines and symbols. 76 2.3.3 Voltage-dependent recovery of 'slow' activation It is possible that the acceleration of activation kinetics following a prepulse is simply the consequence of very slow deactivation after the first pulse, or very slow backwards transitions through one or more closed states upon repolarization. To test whether a very slow initial step in channel closure was responsible for the acceleration in activation kinetics, tail currents in the presence of 135 m M extracellular K + were examined (Figure 2.3A). Large inward tail currents were observed during repolarization to -80 mV that decayed rapidly and completely within the P1-P2 interval duration. The tails could be fit with single exponential functions and had a mean time constant of decay of 12.9 ± 3.2 ms at -80 mV (n=5). This suggested that failure of channel closure was not responsible for the rapid activation of current during the second step of a two-pulse protocol (Figure 2.2B, 2.3A). In addition, the slow activation kinetics of Kv l .2 were relatively insensitive to changes in holding potential, as it was observed that activation kinetics changed very little as the holding potential was altered between -40 mV and -120 mV (Figure 2.3B). This suggests that slow activation preferentially arose from a rate-limiting transition or transitions late in the activation pathway reached at potentials more positive than -40 mV. It was noted in Figure 2.2B, that when the interval between PI and P2 was brief, the P2 activation was very rapid. In Figure 2.3C-F, this experiment was repeated at different interpulse potentials, and as the P1-P2 interval was prolonged at each interpulse potential, the time-dependence of recovery of the slow component of channel activation could be seen. Clearly, the recovery of the slow component of channel activation was fastest when channels were held at voltages near the threshold of channel opening (eg. -40 mV) during the P1-P2 interval (Figure 2.3C), and recovery was much slower at extremely hyperpolarized voltages 77 (eg. -120 mV, Figure 2.3E). Fits to the time courses of recovery gave mean time constants of 1.45 s, 2.4 s and 2.75 s at interpulse potentials of -40 mV, -80 mV and -120 mV, respectively. This observation is the opposite of what would be expected if prepulse potentiation was the result of incomplete deactivation of channels. Collectively, these data demonstrate that the prepulse potentiation observed in K v l .2 was not the result of incomplete closure during the interpulse interval, and that it could instead arise from entry of channels into an alternative activation pathway more permissive to channel opening. 78 1 1 1 r 0.0 0.5 1.0 1.5 2.0 500 ms T i m e (S) Figure 2.3. Holding potential has little effect on slow activation kinetics of Kvl.2 and voltage-dependent recovery of slow activation in Kvl.2. (A) The rate of deactivation at -80 mV was assessed in symmetrical 135 m M K + . The cell was pulsed to +10 mV to reveal slow activation and then repolarized to -80 mV. Complete deactivation of the tail current was seen before the second pulse to +10 mV revealed rapid current activation. The inset illustrates the fitted tail current at -80 mV. (B) The cell was held at either -120 mV or -40 mV for 1 min prior to application of a depolarizing pulse to +30 mV. Each trace was fitted to a first-order exponential function and activation time constants were determined to be 92 ms and 72 ms for the -40 mV and -120 mV potentials, respectively. (C-E) Recovery of slow activation kinetics as a function of the interpulse holding potential, -40 mV (C), -80 mV (D), or -120 mV (E). Protocol is shown for -40 mV experiment. Activation of current traces was fit with a double exponential function and the normalized amplitude of the slow component (Ai/Amax) was plotted against recovery time (F). Fits to the time course gave mean time constants of recovery of 1.45 s, 2.4 s and 2.75 s at interpulse potentials of -40 mV, -80 mV and-120 mV, respectively (n =4). 79 2.3.4 Molecular determinants of prepulse potentiation Data presented thus far have demonstrated that the activation kinetics of K v l . 2 can show two distinct phenotypes. One population of cells exhibited 'fast' behavior, characterized by rapid activation kinetics insensitive to prepulse potentiation, while a second group of cells exhibited 'slow' behavior, characterized by extremely slow activation kinetics that could be accelerated by conditioning of the channels with a prepulse. To our knowledge, this heterogeneity of activation kinetics, if not unprecedented, is rare among the known Kv channels. Our previous studies of Kvl .5 and other K v l channels showed no hint of such regulation of channel activation, with extremely consistent kinetics and steady-state behavior observed between cells (Fedida et al., 1993). Therefore, we adopted a chimeric strategy, and progressively substituted the K v l . 2 sequence with corresponding residues from K v l . 5 , beginning from the N-terminus (Figure 2.4A). The purpose of this chimeric study was to isolate a segment of Kvl .5 that could abolish the unique 'slow' activation gating phenotype characterized in Kv l . 2 , and hence abolish the cell-to-cell heterogeneity of K v l . 2 activation gating. To this end, the activation kinetics of the Kvl .2 /Kvl .5 chimeras were characterized, with a particular focus on the proportion of cells exhibiting the 'slow' versus 'fast' activation phenotype. Substitution of the Kvl .5 sequence comprising the cytoplasmic N-terminus, or up to the beginning of the S2 transmembrane segment, did not abolish 'slow' activation in K v l . 2 . In the Kv l . 5N/ Kv l . 2 chimera, and the Kv l .5S lS2L/Kv l .2 chimera, the frequency of the 'slow' gating phenotype was reduced relative to Wt K v l . 2 channels, but was nevertheless clearly observed in one-third of cells (Figure 2.4B, C). Interestingly, further substitution of the Kvl .5 sequence comprising the S2 transmembrane segment and the cytoplasmic S2-S3 80 linker (Kvl.5S2S3L/Kvl.2 chimera, Figure 2.4B and 2.4C) completely abolished the 'slow' gating behavior in the 23 cells examined. Consistent with this observation, chimeras with further substitution of Kv l .5 sequence up to the cytoplasmic side of the S5 transmembrane helix (Kvl.5S4S5L/Kvl.2, Figure 2.4) exhibited none of the slowly activating currents that were observed in Wt Kv l .2 . A chimera comprising the C-terminus of K v l . 2 substituted with Kvl .5 sequence clearly did not abolish the 'slow' activation phenotype, but rather increased its frequency (this mutation expressed very poorly, hence the low sample size). 81 Figure 2.4. Chimeric study of Kvl.5 and Kvl.2 domains that regulate the activation rate. (A) Schematic diagram of chimeras constructed. K v l . 2 domains are shown in black boxes joined by continuous lines, while Kvl .5 domains are shown in hollow boxes joined by perforated lines. (B) The activation of each construct was assessed using the twin-pulse protocol (Figure 2.2) with either a -50 mV or +40 mV step, followed by a +40 mV test potential in the second step. The interpulse potential was -100 mV for 200 ms. Where constructs showed both 'fast'- and 'slow'-activating phenotypes, an example of each is shown. (C) The frequency of 'slow' and 'fast' activation for each construct is shown as the proportion of black and gray in the bar graph, respectively. The number of cells studied for each construct is shown above. 82 The chimeric studies, and particularly the difference between the Kv l . 5S lS2L /Kv l . 2 and Kvl.5S2S3L/Kvl.2 chimeras, suggested an important role for the S2 transmembrane helix and/or the S2-S3 linker in the regulation of Kv l .2 gating. To confirm this, we transferred this region from Kvl .2 into Kvl .5 and assessed whether this was sufficient to convert the consistently fast gating of Kv l .5 channels into a more heterogeneous phenotype (Figure 2.5). It is clear that following transfer of the S2 transmembrane helix and S2-S3 linker from K v l . 2 to K v l . 5 , activation properties could be divided into two groups. One group of cells exhibited 'fast' activation kinetics with a hyperpolarized Vm of activation (Figure 2.5A, B), whilst the other group exhibited 'slow' activation kinetics that could be accelerated by prepulse potentiation and displayed a depolarized Vm of activation. Together, these data demonstrate the importance of the S2 transmembrane helix and S2-S3 linker in the regulation of the activation properties of Kv l . 2 . 83 N S1 S2 S3 S4 S5 S6 C -60 -40 -20 0 20 40 60 Membrane Potential (mV) Figure 2.5. Transfer of the S2 helix and S2-S3 linker from Kvl.2 to Kvl.5 confers modal activation gating kinetics. (A) Above, the schematic diagram of the chimeric channel with the S2 helix and S2-S3 linker of K v l . 2 substituted into K v l . 5 . Representative ionic currents recorded during the twin-pulse protocol with either a -50 mV or a +40 mV step, followed by a +40 mV test potential in the second step. The interpulse potential was -100 mV for 500 ms. Transfer of the S2 helix and S2-S3 linker from K v l . 2 channels conferred variable gating kinetics to the normally invariant rapidly activating K v l . 5 channels. 'Slow' gating kinetics that demonstrated prepulse potentiation were observed in 50 % of cells. Typical examples of 'fast' and 'slow' gating in this channel are shown. (B) Isochronal g-V relationships from chimeric channels confirm the bimodal gating. The activation relationships fell into two groups with Vm values ranging from -19.5 mV in the 'fast' gating cells to +0.5 mV in the 'slow' gating cells. 84 2.3.5 A critical threonine in the S2-S3 linker of Kvl.2 Sequence alignment of the S2 and S2-S3 linker revealed six amino acid differences between K v l . 2 and Kv l .5 (Figure 2.6A, residues marked with *). Due to the chimera design, only five of these amino acids (Kvl.2 residues 230, 234, 237, 251, and 252) differed between the Kv l .5S lS2L/Kv l .2 and Kvl.5S2S3L/Kvl.2 chimeras (Figure 2.6A). These were individually replaced in Kv l .2 with the corresponding Kv l .5 residue and the activation kinetics of each point mutant characterized (Figure 2.6B), again paying particular attention to the relative frequency of 'fast' versus 'slow' activation gating (Figure 2.6C). Interestingly, in 4 of 5 point mutants, the 'slow' gating phenotype was predominant, although its frequency was somewhat reduced in the S234T mutant (Figure 2.6C). The T252R mutation, however, which is predicted to lie within the cytoplasmic S2-S3 linker, completely abolished the 'slow' gating phenotype in all 33 cells, which were tested over the course of many days (Figure 2.6C). To ensure that these data were not compromised by day-to-day variability of cell behavior, experiments with Wt Kv l .2 channels were generally performed in parallel, as shown by the Wt data bars in Figure 2.6C (11 of 12 showed slow gating). Apart from these functional data, sequence alignment with other K v l channels also suggests a unique and important role for T252 in Kv l . 2 . Among all other K v l channels, and nearly universally among Kv channels, the equivalent residue is basic, either arginine (R) or lysine (K) (Figure 2.6A). 85 222 254 * * * * ** KV1.2 F F I V E T L C I I W F S F E F L V R F F A C P S K A G F F T N I Kv1.5 F F I V E T T C V I W F T F E L L V R F F A C P S K A G F S R N I KV1.1 F F I V E T L C I I W F S F E L W R F F A C P S K T D F F K N 1 KV1.3 F F V V E T L C I I W F S F E L L V R F F A C P S K A T F S R N I Kv1.4 F F I V E T V C I V W F S F E F W R C F A C P S Q A L F F K N I Shaker F F L I E T L C I I W F T F E L T V R F L A C P N K L N F C R D V Kv2.1 LAHVEAVCIAWFTMEYLLRFLSSPKKVJKFFKGP Kv3.1 L T Y I E G V C W W F T F E F L M R V I F C F N K V E F I K N S Kv4.1 F F C M D T A C V L I F T G E Y L L R L FAAPSRCRFLRS V S2 S2-S3 Linker B I230V S234T F237L < c F251S T252R 400 ms Figure 2.6. Point mutational study of the Kvl.2 S2 and S2-S3 linker. (A) Alignment of K v l . 2 and Kv l .5 S2 helices and S2-S3 linker with Shaker and other Shaker-related channels, Kv2.1, Kv3.1 and Kv4.1. Only six residues (marked above) are different between K v l . 2 and K v l . 5 . A l l of these residues in Kv l .2 were mutated individually to the equivalent residues in K v l . 5 , with the exception of L228T which was covered in the K v l . 5 S l S 2 L / K v l . 2 chimera (Figure 2.4). (B) The activation properties of the constructs were studied using the twin-pulse protocol. Either a -50 mV (black trace) or +40 mV (red trace) step was followed by a +40 mV test potential in the second step. The interpulse potential was -100 mV for 200 ms. (C) The frequency of 'slow' and 'fast' activation for each point mutation is shown as the proportion of black and gray in the bar graph, respectively. The recordings for Wt K v l . 2 and all the mutants were done on the same day with the exception of T252R, which was performed over a number of days. The number of cells studied for each construct is shown above. 86 To further examine the functional importance of T252 in K v l . 2 , the effects of amino acid substitutions with various chemical properties were made at this site and the adjacent residues G249 to 1254 (Figure 2.7). As mentioned above, substitution with the basic amino acid arginine completely abolished the 'slow' gating phenotype observed in Wt K v l . 2 channels (Figure 2.6C). Similarly, substitution with lysine or cysteine completely abolished the 'slow' gating behavior of K v l . 2 (Figure 2.7A). With either substitution (T252C or T252R) ionic currents recorded over a wide range of potentials activated rapidly (Figure 2.7B, 2.7C) and at negative potentials (Figure 2.7E). The twin-pulse protocol applied to these mutants revealed no prepulse potentiation (Figure 2.7B,C, insets). Activation curves were reproducible between cells, and did not display the heterogeneity recorded from Wt K v l . 2 (Figure 2.1A, Figure 2.7E). The mean Vm of activation was -14 ± 1 mV («=12) in T252C and -11.2 ± 2.5 (n=6) in T252R and neither was significantly different from the Vm of 'fast' activating Wt Kv l .2 . The T252D mutant (Figure 2.7D) exhibited 'slow' gating behavior in 13 of 14 cells (93%) with a mean Vm of activation of +9.8 ± 2.3 mV. This 'slow' gating percentage was similar to that observed with Wt K v l . 2 (90%), recorded in parallel experiments (Figure 2.7A). Similar Wt-like heterogeneous gating was observed with T252A, T252E, T252M, and T252N (Figure 2.7A). On the other hand, substitution of arginine at position 250 or 251 for phenylalanine (F250R and F251R), was able to promote an exclusively 'fast' activation gating in Kv l . 2 , as it had for T252R and T252K. This effect appeared highly localized, as substitution with an arginine at 249 (G249R) or 253 (N253R) did not induce exclusively 'fast' activation gating. These results point to a local effect of charged substitutions around T252 as a potent regulatory mechanism for the time and voltage dependence of activation gating in Kv l . 2 . 87 1213 14 6 13 4 5 5 5 5 6 6 7 5 8 200 ms 200 ms T252R T252D 200 ms 1.0 0.8 | 0.6 0.2 0.0 / • T252C A T252R • T252D -80 -60 -40 -20 0 20 40 60 Potential (mV) Figure 2.7. The activation effects of charged and uncharged substitutions of T252 in Kvl.2, and of residues in close proximity. (A) The activation properties of mutant constructs indicated on the abscissa were assessed using the twin-pulse protocol. Either a -50 mV or +40 mV step was followed by a +40 mV test potential in the second step. The interpulse potential was -100 mV. N.E. = not expressed. The frequency of 'slow' and 'fast' activation for each point mutation is shown as the proportion of black and gray, respectively, in the bar graph. The number of cells studied for each construct is shown above. Wt K v l .2 cells studied concurrently are shown in the leftmost bar, for each data set. ( B - D ) Experimental examples of current-voltage data from constructs in (A), T252C, T252R, and T252D. Currents were elicited by depolarizing steps from -65 to +55 mV in 20 mV increments. Insets illustrate the mutant response to the twin-pulse protocol described above for (A). (E) The activation Vm was -14 ± 1 mV («=12, £=1 1.1 ± 0.6) for T252C (•), -11.2 ± 2.5 («=6, £=14 ± 1.3) for T252R (A), and +9.8 ± 2.3 mV (n=4, £=12.8 ± 0.8) for T252D (•). The broken curves illustrate the position of the mean isochronal conductance-voltage relations for 'fast' and 'slow' activating Wt channels from Figure 2.IE. 88 2.3.6 Cytoplasmic constituents regulate Kvl.2 channel gating In a number of cells expressing Wt K v l . 2 we observed a permanent switch from 'slow' to 'fast' activation gating kinetics over time (Figure 2.8A), suggesting that dialysis of the cellular constituents caused the switch. In support of this, prevention of dialysis, by using the perforated-patch clamp configuration (Figure 2.8B), preserved the 'slow' gating mode in all cells tested even during experiments that were 30 min in duration. Furthermore, excision of inside-out patches from the cytosolic environment (Figure 2.8C) permanently switched channels from the 'slow' to the 'fast' gating mode upon formation of the excised patch. Moreover, in whole-cell experiments we observed a clear relationship between the gating mode and cell surface expression (Figure 2.8D). The data in Figure 2.8D suggest that when there are large numbers of channels, the modifying cytosolic component is so overwhelmed that the majority of channels exhibit the 'fast' gating mode. Taken together, these data suggest that K v l . 2 activation gating is regulated by a cytoplasmic gating-modifying component. Many cytoplasmic mediators of K + channel function have been identified (Shyng and Nichols, 1998; Oliver et al., 2004; Mohapatra and Trimmer, 2006b). In order to identify the cytosolic factor modifying Kv l .2 gating, we investigated the effects of key candidates on the ability of Kv l .2 channels to operate in the two modes of activation. Selective inhibition of a number of Ser/Thr kinases (PKC, P K A , P K G , CaMKII and M L C K ) by incubation or dialysis of cells expressing K v l . 2 channels with selective blockers (bisindolylmaleimide, H-89, P K G inhibitor, KN-93 and ML-7, respectively) or a broad range inhibitor (staurosporine) resulted in no measurable change in the frequency of the two activation gating modes (data not shown). Furthermore, cleavage of all the bound phosphate groups by dialysis of cells, 89 through the patch pipette, with a very high concentration of alkaline phosphatase (100 U/ml; New England BioLabs, Ipswich, M A ) resulted in no change in channel activation properties (data not shown). Other than phosphorylation, K + channel function is also known to be modulated by phospholipids, such as PIP2, and cytoplasmic polyamines. However, addition of 10 p.g/ml PIP2 or 1 m M spermidine to the intracellular face of excised inside-out patches of membranes containing Wt K v l . 2 channels had no effect on the channel gating mode (data not shown). These observations appear to rule out a role for phosphorylation, PIP2 and polyamines in switching between K v l . 2 gating modes; however, further study is required to identify the interacting component that is responsible for the activation gating switch in Kv l .2 channels. 90 100 ms 0 2 4 10 Time (min) Fast Slow Figure 2.8. K v l . 2 channel gating is modified by interaction with a cytosolic component. (A, B) Representative ionic currents (left) recorded in the whole-cell (A) and perforated patch (B) configuration. Traces represent the current obtained immediately after breaking into the cell (black trace; time = 0) and that recorded from the same cell 90 s (in A) or 30 min (in B) later (gray trace). We observed a marked time-dependence of gating mode modification in 3 of 10 cells in the whole-cell configuration (right), but prevention of the dialysis of cytoplasmic constituents in the perforated patch configuration preserved the 'slow' gating mode (right). (C) Representative ionic currents (left) recorded from a cell-attached patch (black trace) and following excision of the inside-out patch (gray trace). We observed an immediate switch from the 'slow' to the 'fast' gating mode in eight of out eight patches upon excision from the cell cytoplasm. In all cases ionic currents were recorded during depolarizing voltage steps to +40 mV at 30 s intervals from a holding potential of -80 mV. (D) Mean K v l . 2 current densities observed in cells favoring 'slow' and 'fast' gating modes. In cells expressing large numbers of K v l . 2 channels, the 'fast' activation gating mode is favored (PO.0001, Student's t-test). The n values for the 'fast' and 'slow' groups were 23 and 31, respectively. 91 2.4 Discussion 2.4.1 'Cell-to-cell' versus 'pulse-to-pulse' heterogeneity of Kvl.2 gating This study has characterized a significant heterogeneity in the activation properties of heterologously expressed K v l . 2 channels. Our experiments demonstrate that K v l . 2 channels can exhibit at least two different gating phenotypes or modes in mammalian cells, which we have referred to as 'fast' or 'slow'. Heterogeneity of the K v l . 2 current activation time course and voltage dependence between cells arises from differences in the proportion of channels occupying these two modes. K v l . 2 exhibits purely 'fast' gating in a fraction of cells, purely 'slow' gating in another fraction, and a mixture of the 'fast' and 'slow' modes in the remainder of cells (Figure 2.1). The 'fast' gating Vm at —20 mV is reminiscent of reports of expression of K v l . 2 in oocytes (Koopmann et al. 2001; -17.9 ± 0.5 mV) and in the same preparation as that we have used here, mouse fibroblasts (Scholle et al., 2004). Within individual cells exhibiting the 'slow' gating phenotype, prepulse potentiation resulted in a predictable and reproducible acceleration of channel activation in a subsequent test pulse (Figure 2.2), as first noticed by Grissmer et al. (1994). Importantly, we could readily shift channels from the 'slow' gating mode towards the 'fast' gating mode, using depolarizing pulses above the threshold of current activation, and we were able to return channels from the 'fast' gating mode and place them into the 'slow' gating mode by holding cells for rest periods of 2s or more at potentials of -40 mV and more negative (Figure 2.3). Several lines of evidence suggest that the acceleration of activation during twin-pulse experiments is not due to incomplete deactivation during the interpulse interval. Ionic current deactivation appeared complete with time constants of less than 20 ms at negative potentials (Figure 2.3A). Additionally, changing the holding potential over a wide range of 92 voltages did not change the activation rate of channels exhibiting the 'slow' phenotype, which suggests that 'slow' and 'fast' activation steps do not coexist in a single common activation pathway. As well, recovery of the 'slow' gating phenotype occurred most slowly at negative holding potentials, when channel deactivation would be expected to occur most rapidly (Figure 2.3). Furthermore, conditioning channels with a depolarizing prepulse appeared to alter the voltage-dependence of activation, resulting in a ~30 mV hyperpolarizing shift of the activation Via (Figure 2.2D). Collectively, these data suggest that prepulse potentiation of K v l . 2 reflects the existence of two pathways or modes of activation for this channel, with depolarization (sufficient for channel activation) favoring a shift of channels from the 'slow' gating mode into the 'fast' gating mode. 2.4.2 A model scheme for fast' and 'slow' activation A potential scheme describing the experimental data is shown in Figure 2.9 (and see Methods). In the state diagram (Figure 2.9A), the upper row is similar to that modeled for ShakerlR when slow inactivation is excluded (Smith-Maxwell et ah, 1998). Briefly, a voltage-dependent conformational change in each of the four independent but identical subunits leads to a fully activated, not-open state (C4), which can then undergo a concerted transition to the open state (O5). As expected (Smith-Maxwell et al, 1998), slowing the rate of the concerted transition (kcos) in the lower gating tier so that it became the rate-limiting step and strongly biasing the vertical transitions to the 'slow' activation pathway (k/s» ksj), produced currents with a predominantly slow activation time course that was well-fitted by a single exponential (not shown). However, constrained by the measured activation time constant-V relationship (Figure 2.1G), it was not possible, solely by slowing the concerted transitions (Cio-^Os and Cio<— O 5 ) , to reproduce the depolarizing shift of the g- V relationship 93 (Figure 2.IE). For that reason, in the final working version of the model to produce data in Figures 9B-D, an additional modification was made to the 'slow' activation pathway to decrease the value for the microscopic activation rate constant ^.,(0 mV). This modification, in turn, required the incorporation of the variable dt, where x = 1 - 4, in the vertical transitions to conserve microscopic reversibility. Replication of currents showing only fast activation kinetics from a holding potential of -80 mV (Figure 2.1C) was achieved by altering the values for ks/ and k/s. The fast currents illustrated in Figure 2.9B were obtained by increasing hsjto 5 s"1, which corresponds to a decrease of activation energy of 3.8 kcal/mol at 20° C. The output of the model with the twin-pulse protocol is shown in Figure 2.9E. Both in the simulated and the experimental traces (Figure 2.2A), larger depolarizing prepulses increased the fast-activating current component. In the model this behavior arose because channels that reached O 5 along the 'slow' pathway during the pre-pulse deactivated primarily along the upper gating tier, reaching Co and Ci by the end the interpulse interval. Consequently, the larger the proportion of channels that reached O 5 during the prepulse, the larger the relative proportion of channels that activated via the 'fast' pathway during the test pulse. Both the time dependence of the relaxation from the 'fast' to the 'slow' activation pathway in the two-pulse protocol (Figure 2 . 3 D , T « 4.5 s at -80 mV in the model), as well as a progressive enhancement of the peak current and fast-activating component with repeated 100 ms pulses to 10 mV at 2 Hz (seen experimentally, but not shown), were reproduced quite well by this gating scheme. 94 0.1s Figure 2.9. Model of 'fast' and 'slow' activation in Kvl.2. (A) Model gating scheme for K v l . 2 . Two interconnected, parallel pathways representing 'fast' (upper) and 'slow' (lower) activation are connected to a single open state (O5). Model kinetics are described in Materials and Methods. (B) To simulate the family of 'fast'-activating currents in the left panel, ks/was increased to 5 s"1. Slowly activating currents are shown in the right panel. (C) Normalized g-V curves, derived from simulated currents and fitted to a Boltzmann function, gave V1/2 of activation and k values, respectively, of -19.4 mV and +8.9 mV for the 'fast'-activating currents (•) and +20.7 mV and +8.4 mV for 'slow'-activating currents (•). This approximates the nearly parallel, roughly 35 mV rightward shift of the g-V curve observed experimentally (Figure 2.IE). (D) For comparison to Figure 2.1G, the r-V relationship for the two families of currents was obtained as described in the Materials and Methods (i.e., exponential fit to the rising phase of the 'slow' current; exponential fit from the half-amplitude time to the steady-state for 'fast', sigmoidal currents). (E) With a two-pulse voltage protocol, increasing the amplitude of the depolarizing pre-pulse from -10 to +50 mV increased the 'fast'-activating component of the test current evoked at 20 mV (compare with Figure 2.2A). The test current decay seen following a +50 mV pre-pulse is due to a 'slow' relaxation of channels from C4 into the 'slow' activation pathway (C7 -Cio). 95 An interesting outcome of the twin-pulse voltage protocol is that, following a strong depolarizing pre-pulse, the fast activating test current at +20 mV showed a small, slow decay (Figure 2.9E). When a similar pattern was observed in experimental records (Figure 2.2A) it was provisionally attributed to coupling between activation and slow inactivation. However, the model, which explicitly excludes inactivation, indicated that a similar decay could arise from a slow transition of channels from C 4 into C10 and subsequent redistribution into less activated states ( C 7 - C 9 ) . A prediction of the model is that this decay becomes less prominent at test voltages above +20 mV, because both activation pathways are more strongly biased toward the open state (Figure 2.1 OA). Indeed, this is what is observed with this voltage protocol in experimental currents (Figure 2.1 OB) where inactivation is not an obvious confounding factor. 96 Figure 2.10. Simulated and experimental observation of dual activation pathways of Kvl.2 channel opening. (A) Traces were generated using the model of K v l . 2 gating (Figure 2 . 9 A ) . A 600 ms pulse to +40 mV, was followed by repolarization to -80 mV for 500 ms and then steps to potentials between —45 mV and +35 mV in 10 mV increments for 600 ms. The model predicts that following the +40 mV conditioning pulse, depolarization to potentials up to +25 mV (i.e. intermediate potentials) should give rise to current traces that activate rapidly and show a slow decay due to transitions from the late 'fast' closed state (C4) to the 'slow' closed states (C7-C10), while depolarizing pulses to potentials greater than +25 mV should give rise to a fast activating current that is followed by a slow rising current, which is attributed to the opening of the channels that have made the transition from the late 'fast' closed states to the 'slow' gating state. (B) Experimental currents elicited by the voltage protocol shown above, closely replicate the traces generated by the model. While prepulse potentiation can transiently move channels into the 'fast' gating mode, there is clearly a time-dependent recovery of 'slow' gating (Figure 2.3), with the balance of 'fast' versus 'slow' gating eventually returning to the level observed upon break-in. This 'basal' balance of 'fast' versus 'slow' channels is generally quite stable over durations required to complete the experiments reported here, although in several cells we were able to maintain a whole-cell recording for 30-40 minutes, and observed a gradual 97 disappearance of the 'slow' gating phenotype (Figure 2.8A). This was prevented by performing experiments in the perforated patch clamp configuration (Figure 2.8B) and dramatically accelerated upon excision of an inside-out patch (Figure 2.8C). The 'slow' versus 'fast' balance is therefore likely to be influenced by a cytoplasmic gating modifying component that is uncontrolled in our experiments. On the basis of our model we suggest that modification of the channel (phosphorylation, hydrogen bonding or interaction with other proteins) can alter the values for transition rates between the 'slow' and 'fast' activation pathways and in doing so change the relative contributions of the two activation pathways to overall channel activation. Possible molecular mechanisms for this include variable expression of auxiliary subunits/binding partners, or variable activation of one or more signaling pathways, or other reversible post-translational modification that could promote the 'slow' gating mode of K v l . 2 . 2.4.3 Structural determinants of prepulse potentiation Chimeric constructs of K v l . 2 and Kvl .5 allowed identification of the structural elements responsible for the 'slow' gating behavior in K v l . 2 and its acceleration during twin pulses. Substitution of the S2-S3 linker of K v l . 2 with the sequence from the Kv l .5 channel completely abolished the 'slow' gating phenotype, and we further delimited this effect to a single threonine (T252) in the K v l . 2 S2-S3 linker (Figs. 2.4-2.6). This segment of the protein is thought to be cytoplasmic (Long et al., 2005a), although its spatial relationship with other regions of the channel (either the cytoplasmic T l domain, or the transmembrane helices) remains uncertain. The S234T mutation in the K v l . 2 S2 segment was able to modify the relative frequency of cells exhibiting 'fast' versus 'slow' gating phenotypes (Figure 2.6C), however only mutations at T252 or close by at F251 and F250 were able to 98 abolish 'slow' gating of K v l . 2 (Figure 2.1 A). A possible explanation is that T252 and/or its immediate environment is directly involved in interactions that regulate the 'slow' gating mode in K v l . 2 , while mutations in other residues in the S2 segment can slightly alter the positioning/conformational arrangement of T252. Interestingly, substitution of the K v l . 2 S2 and S2-S3 linker into Kv l .5 imparted a 'slow' gating phenotype in some cells (Figure 2.5). However, the mutation Kvl .5 R356T (equivalent to Kv l .2 residue T252) did not confer 'slow' gating. This suggests that other residues in the K v l . 2 S2 segment and S2-S3 linker may also be involved in regulating the 'slow' gating behavior of this channel. These data demonstrate that the S2 segment and S2-S3 linker are involved in the regulation of activation gating in Kv l .2 channels. Several studies that have demonstrated a critical role for the S2 segment in stabilizing positively charged residues in the S4 'voltage-sensor' may be relevant to our findings (Papazian et al., 1995; Tiwari-Woodruff et al., 1997). It has also been suggested that residues in the S2 transmembrane helix undergo rapid conformational changes early in the Shaker activation pathway (Cha and Bezanilla, 1997). Whether this voltage-sensing role of S2 is involved in regulating the balance of channels occupying 'fast' versus 'slow' gating modes is unknown. However, it is worth mentioning again that the 'slow' gating behavior (with essentially mono-exponential activation kinetics, i.e. having little or no sigmoid character) of Kv l .2 appears to be governed by a rate-limiting transition very late in the activation pathway. Our most straightforward experimental support for this assertion is that slow activation kinetics in K v l . 2 persist even with very positive holding potentials just below the threshold for channel activation (Figure 2.3). Thus, energetic effects on voltage-sensor movement at negative voltages do not provide an obvious 99 explanation for the regulation of 'slow' K v l . 2 gating by amino acids in the S2 segment or S2-S3 linker. Alignments within and across Kv channel subfamilies demonstrate that the homologous position to K v l . 2 T252 is almost universally occupied by a positively charged basic residue (eg. lysine or arginine). Introduction of lysine and arginine (T252R and T252K; Figure 2.7) restored 'fast' activation gating properties that are evident in other K v channels. The presence of threonine at 252 in Kv l .2 raises the interesting possibility that phosphorylation of this residue may play a role in regulation of 'slow' gating. However, our attempt to mimic a phosphorylated state by the introduction of a fixed negative charge (T252D; Figs. 6 and 7) did not alter the heterogeneity of activation gating, given that the proportion of channels exhibiting 'slow' activation kinetics was similar to Wt (Figure 2.7). Additionally, the sequence in the region of T252 does not form a recognition sequence for any known protein kinase, and attempts with a broad spectrum kinase inhibitor (e.g. staurosporine) or mixtures of specific Ser/Thr kinase inhibitors were unable to abolish the 'slow' gating phenotype of Kv l .2 (data not shown). Another possible mechanism for regulation is interaction with membrane phospholipids. In the Kir channel family, positively charged residues that lie near the membrane-fluid interface have been implicated in interactions with negatively charged headgroups of anionic phospholipids (eg. PIP2) in the inner leaflet of the plasma membrane, and these can dramatically alter channel gating by various ligands (Enkvetchakul et al., 2005). However, application of PIP2 to K v l . 2 inside-out patches did not alter activation gating properties (data not shown). Further studies are required to identify the cytoplasmic component that is involved with regulation of K v l . 2 activation gating. 100 Despite the lack of a role for phosphorylation and PIP2 in the regulation of K v l . 2 activation properties, the observation that dialysis of the cell or separation of channels from the cytoplasmic constituents by forming excised patches causes a permanent switch of the activation gating from 'slow' to 'fast' (Figure 2.8) suggests the involvement of a cytosolic component capable of modifying activation. In the present study, we have been unable to identify the nature of this component, although we have ruled out phosphorylation by PKC, P K A , P K G , CaMKII and M L C K , and interaction of PIP2 and polyamines (data not shown). The current data do, however, support the conclusion that activation gating in K v l . 2 is regulated by interaction of a cytosolic gating modifier at or associated with T252 in the S2-S3 linker. 101 2.5 Acknowlegements We would like to thank Ms. Ka Kee Chiu for preparation of cells. This work was supported by grants from the Heart and Stroke Foundations of British Columbia and Yukon and the CIHR to DF and SJK. S.R. was supported by a University of British Columbia Graduate Fellowship and a Heart and Stroke Foundation of British Columbia and Yukon scholarship. H.T.K was funded by the Canadian Institutes of Health Research and Michael Smith Foundation for Health Research. T.W.C was supported by a postdoctoral research fellowship funded by a Focus on Stroke strategic initiative from The Canadian Stroke Network, the Heart and Stroke Foundation, the CIHR Institute of Circulatory and Respiratory Health and the CIHR/Rx&D Program along with AstraZeneca Canada. 102 2.6 References Accili ,E.A., J.Kiehn, Q.Yang, Z.G.Wang, A.M.Brown, and B.A.Wible. 1997. Separable Kvb subunit domains alter expression and gating of potassium channels. J Biol Chem 272:25824-25831. Aggarwal,S.K. and R.MacKinnon. 1996. Contribution of the S4 segment to gating charge in the Shaker K + channel. Neuron 16:1169-1177. Ahern,C.A. and R.Horn. 2004. Stirring up controversy with a voltage sensor paddle. Trends Neurosci 27:303-307. A.ydar,E. and C.Palmer. 2001. Functional characterization of the C-terminus of the human ether-d-go-go related gene K + channel (hERG). J Physiol (Camb) 534:1-14. Bezanilla,F. 2000. The voltage sensor in voltage-dependent ion channels. Physiological Reviews 80:555-592. Cha,A. and F.Bezanilla. 1997. Characterizing voltage-dependent conformational changes in the Shaker K + channel with fluorescence. Neuron 19:1127-1140. Cha,A., G.E.Snyder, P.R.Selvin, and F.Bezanilla. 1999. Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809-813. Chiara,M.D., F.Monje, A.Castellano, and J.Lopez-Barneo. 1999. A small domain in the N terminus of the regulatory a-subunit Kv2.3 modulates Kv2.1 potassium channel gating. J Neurosci 19:6865-6873. Colquhoun,D. and A.G.Hawkes. 1995. A Q-matrix cookbook. In Single Channel Recording. B.Sakmann and E.Neher, editors. Plenum, New York. 589-633. Cushman,S.J., M.H.Nanao, A.W.Jahng, D.DeRubeis, S.Choe, and P.J.Pfaffinger. 2000. Voltage dependent activation of potassium channels is coupled to T l domain structure. Nature Structural Biology 7:403-407. 103 DeBiasi,M., Z.Wang, E.Accili , B.Wible, and D.Fedida. 1997. Open channel block of human heart h K v l .5 by the b-subunit hKvbl .2. Am J Physiol Heart Circ Physiol 272:H2932-H2941. Del Camino,D. and G.Yellen. 2001. Tight steric closure at the intracellular activation gate of a voltage-gated K + channel. Neuron 32:649-656. Elinder,F., R.Mannikko, and H.P.Larsson. 2001. S4 charges move close to residues in the pore domain during activation in a K channel. J Gen Physiol 118:1-10. Enkvetchakul,D., I.Jeliazkova, and C.G.Nichols. 2005. Direct modulation of Kir channel gating by membrane phosphatidylinositol 4,5-bisphosphate. J Biol Chem 280:35785-35788. Fedida,D., B.Wible, Z.Wang, B.Fermini, F.Faust, S.Nattel, and A.M.Brown. 1993. Identity of a novel delayed rectifier current from human heart with a cloned K + channel current. Circ Res 73:210-216. Gandhi,C.S., E.Loots, and E.Y.Isacoff. 2000. Reconstructing voltage sensor-pore interaction from a fluorescence scan of a voltage-gated K + channel. Neuron 27:585-595. Glauner,K.S., L.M.Mannuzzu, C.S.Gandhi, and E.Y.Isacoff. 1999. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813-817. Grissmer,S., A.N.Nguyen, J.Aiyar, D.C.Hanson, R.J.Mather, G.A.Gutman, M.J.Karmilowicz, D.D.Auperin, and K.G.Chandy. 1994. Pharmacological characterization of five cloned voltage- gated K + channels, types K v l . l , 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Molec Pharmacol 45:1227-1234. Heinemann,S.H., J.Rettig, H.R.Graack, and O.Pongs. 1996. Functional characterization of K v channel b-subunits from rat brain. J Physiol (Camb) 493:625-633. Hulme,J.T., E.A.Coppock, A.Felipe, J.R.Martens, and M.M.Tamkun. 1999. Oxygen sensitivity of cloned voltage-gated K + channels expressed in the pulmonary vasculature. Circ Res 85:489-497. Ju,M., L.Stevens, E.Leadbitter, and D.Wray. 2003. The Roles of N - and C-terminal determinants in the activation of the Kv2.1 potassium channel. JBiol Chem 278:12769-12778. 104 Kooprnarin,R., A.Scholle, J.Ludwig, T.Leicher, T.Zimmer, O.Pongs, and K.Benndorf. 2001. Role of the S2 and S3 segment in determining the activation kinetics in Kv2.1 channels. J Membrane Biol 182:49-59. Kurata,H.T., G.S.Soon, J.R.Eldstrom, G.W.K.Lu, D.F.Steele, and D.Fedida. 2002. Amino-terminal determinants of U-type inactivation of voltage-gated K + channels. J Biol Chem 277:29045-29053. Kurata,H.T., G.S.Soon, and D.Fedida. 2001. Altered state dependence of C-type inactivation in the long and short forms of human K v l .5. J Gen Physiol 118:315-332. Larsson,H.P., O.S.Baker, D.S.Dhillon, and E.Y.Isacoff. 1996. Transmembrane movement of the Shaker K + channel S4. Neuron 16:387-397. Liman,E.R., P.Hess, F. Weaver, and G.Koren. 1991. Voltage-sensing residues in the S4 region of a mammalian K + channel. Nature 353:752-756. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005a. Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309:897-903. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005b. Voltage sensor of K v l . 2 : structural basis of electromechanical coupling. Science 309:903-908. Macica,C.M. and L.K.Kaczmarek. 2001. Casein kinase 2 determines the voltage dependence of the Kv3.1 channel in auditory neurons and transfected cells. J Neurosci 21:1160-1168. McManus,0., L.M.H.Helms, L.Pallanck, B.Ganetsky, R.Swanson, and R.J.Leonard. 1995. Functional role of the b subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650. Minor,D.L.Jr., Y.F.Lin, B.C.Mobley, A.Avelar, Y.N.Jan, L.Y.Jan, and J.M.Berger. 2000. The polar T l interface is linked to conformational changes that open the voltage-gated potassium channel. Cell 102:657-670. Misonou,H., D.P.Mohapatra, E.W.Park, V.Leung, D.Zhen, K.Misonou, A.E.Anderson, and J.S.Trimmer. 2004. Regulation of ion channel localization and phosphorylation by neuronal activity. Nat Neurosci 7:711-718. 105 Mohapatra,D.P. and J.S.Trimmer. 2006b. The Kv2.1 C terminus can autonomously transfer Kv2.1-like phosphorylation-dependent localization, voltage-dependent gating, and muscarinic modulation to diverse K v channels. J Neurosci 26:685-695. Mohapatra,D.P. and J.S.Trimmer. 2006a. The Kv2.1 C terminus can autonomously transfer Kv2.1-like phosphorylation-dependent localization, voltage-dependent gating, and muscarinic modulation to diverse Kv channels. J Neurosci 26:685-695. 01iver,D., C.C.Lien, M.Soom, T.Baukrowitz, P.Jonas, and B.Fakler. 2004. Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids. Science 304:265-270. Papazian,D.M., X.M.Shao, S.A.Seoh, A.F.Mock, Y.Huang, and D.H.Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K + channel. Neuron 14:1293-1301. Papazian,D.M., L.C.Timpe, Y.N.Jan, and L.Y.Jan. 1991. Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. Nature 349:305-310. Perozo,E. and F.Bezanilla. 1990. Phosphorylation affects voltage gating of the delayed rectifier K + channel by electrostatic interactions. Neuron 5:685-690. Perozo,E., C.A.Vandenberg, D.S.Jong, and F.Bezanilla. 1991. Single channel studies of the phosphorylation of K + channels in the squid giant axon. I. Steady-state conditions. J Gen Physiol 98:1-11'. Ponce,A., E.V.S.De Miera, C.Kentros, H.Moreno, B.Thornhill, and B.Rudy. 1997. K + channel subunit isoforms with divergent carboxy-terminal sequences carry distinct membrane targeting signals. J Membrane Biol 159:149-159. Rezazadeh,S., H.T.Kurata, T.W.Claydon, S.J.Kehl, and D.Fedida. 2007. Modal gating of K v l . 2 channels mediated by the S2-S3 linker. Biophys J467A-467A Suppl. S. Scholle,A., S.Dugarmaa, T.Zimmer, M.Leonhardt, R.Koopmann, B.Engeland, O.Pongs, and K.Benndorf. 2004. Rate-limiting reactions determining different activation kinetics of K v l . 2 and Kv2.1 channels. JMembr Biol 198:103-112. Seoh,S.A., D.Sigg, D.M.Papazian, and F.Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K + channel. Neuron 16:1159-1167. 106 Shieh,C.C., K.G.Klemic, and G.E.Kirsch. 1997. Role of transmembrane segment S5 on gating of voltage-dependent K + channels. J Gen Physiol 109:767-778. Shyng,S.L. and C.G.Nichols. 1998. Membrane phospholipid control of nucleotide sensitivity of K A T P channels. Science 282:1138-1141. Smith-Maxwell,C.J., J.L.Ledwell, and R.W.Aldrich. 1998. Role of the S4 in cooperativity of voltage-dependent potassium channel activation. J Gen Physiol 111:399-420. SteidlJ.V. and A.J.Yool. 1999. Differential sensitivity of voltage-gated potassium channels Kv l .5 and K v l . 2 to acidic pH and molecular identification of pH sensor. Molec Pharmacol 55:812-820. Thornhill,W.B., M.B.Wu, X.Q.Jiang, X .Y .Wu, P.T.Morgan, and J.F.Margiotta. 1996. Expression of K v l . l delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function. J Biol Chem 271:19093-19098. Tiwari-WoodruffS.K., C.T.Schulteis, A.F.Mock, and D.M.Papazian. 1997. Electrostatic interactions between transmembrane segments mediate folding of Shaker K + channel subunits. Biophys 772:1489-1500. Uebele,V.N., S.K.England, A.Chaudhary, M.M.Tamkun, and D.J.Snyders. 1996. Functional differences in Kvl .5 currents expressed in mammalian cell lines are due to the presence of endogenous Kvb2.1 subunits. J Biol Chem 271:2406-2412. VanDongen,A.M.J., G.Frech, J.A.Drewe, R.H.Joho, and A.M.Brown. 1990. Alteration and restoration of K + channel function by deletions at the N - and C-termini. Neuron 5:433-443. Wang,G. and M.Covarrubias. 2006. Voltage-dependent gating rearrangements in the intracellular T l - T l interface of a K+ channel. J Gen Physiol 127:391-400. Wang,J.L., M.C.Trudeau, A.M.Zappia, and G.A.Robertson. 1998. Regulation of deactivation by an amino terminal domain in human ether-a-go-go-related gene potassium channels. J Gen Physiol 112:637-647. 107 WatanabeJ., H.G.Wang, J.J.Sutachan, J.Zhu, E.Recio-Pinto, and W.B.Thornhill. 2003. Glycosylation affects rat K v l . l potassium channel gating by a combined surface potential and cooperative subunit interaction mechanism. J Physiol 550:51-66. WatanabeJ., J.Zhu, J.J.Sutachan, A.Gottschalk, E.Recio-Pinto, and W.B.Thornhill. 2007. The glycosylation state of K v l . 2 potassium channels affects trafficking, gating, and simulated action potentials. Brain Res 1144:1-18. Yellen,G. 2002. The voltage-gated potassium channels and their relatives. Nature 419:35-42. 108 Chapter 3: KN-93, a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels2 2 A version of this chapter has been published. Rezazadeh, S., Claydon, T. W., & Fedida, D. (2006). KN-93 (2-|>l-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnarnyl)-N-methylbenzylarnine), a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. Journal of Pharmacology and Experimental Therapeutics 317, 292-299 109 3.1 Introduction Voltage-gated potassium (Kv) channels, which are activated by changes in the transmembrane potential, play an important role in the control of excitability. There are a number of structurally related sub-families of K v channels (Chandy 1991) that are expressed in a wide range of tissues, such as heart (e.g. K v l , Kv2, Kv4 and hERG), brain ( K v l , Kv2, Kv3 and Kv4), pancreas and smooth muscle (e.g. K v l ) (Shieh et al, 2000). It is well documented that K v channels are crucial targets for protein kinase activation and that phosphorylation can affect channel characteristics (Levitan, 1994). Calcium/calmodulin kinase II (CaMK II) is a multi-functional cytoplasmic calcium and calmodulin-dependent protein kinase that phosphorylates and alters the function of a variety of substrates. Given that members of a number of Kv channel subfamilies are expressed in the brain (Shieh et al, 2000) and the abundance of expression of C a M K II in the brain (Braun and Schulman, 1995), it comes as no surprise that CaMK II has been suggested to phosphorylate K v channels (Roeper et al, 1997). Roeper et al. (1997) showed that the rate of fast N-type inactivation of the Shaker related Kv l .4 channel is modulated by CaMKII phosphorylation of serine 123 in the N-terminus. Similarly, the rate of fast inactivation of Kv4.3 was recently shown to be slowed by CaMK II phosphorylation of serine 550 in the C-terminus (Sergeant et al, 2005). In addition to these effects on channel gating, C a M K II also increased surface expression of Kv4.2 and Drosophila ether a go-go (EAG) potassium channels (Wang et al, 2002; Varga et al, 2004; Sun et al, 2004). The regulatory effect of CaMK II is not limited to potassium channels, since CaMK II has been shown to alter the activation properties of the sodium channel, Nayl.5 (Young and Caldwell, 2005). 110 The effect of CaMK II on ion channel function has been widely studied through the use of specific inhibitors such as KN-93. KN-93 is a methoxybenzenesulfonamide compound, which exerts its effect by competing for the calmodulin (CaM) binding site of CaMK II with an IC50 of 370 nM (Sumi et al, 1991) and is an extensively used inhibitor of CaMK II. For example, inhibition of CaMK II with KN-93 incubation enhanced N-type inactivation of Kv l .4 and Kv4.3 (Roeper et al, 1997; Sergeant et al, 2005), an observation that is consistent with CaMK II induced slowing of inactivation. However, Ledoux et al. (1999) have shown that caution must be taken in the interpretation of such experiments since KN-93 affected Kv currents in rabbit portal vein smooth muscles independently of CaMK II action. In the present study, we have extended these initial observations to show a direct effect of KN-93 on a wide range of cloned Kv channels heterologously expressed in mammalian cells is independent of CaMK II. KN-93 is a potent extracellular open channel blocker of all Kv channels tested and exerts its effect by enhancing C-type inactivation. 3.2 Materials and Methods 3.2.1 Materials Cell culture supplies were purchased from Invitrogen (Burlington, ON). KN-92 (2-pSf-(4-Methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, Phosphate), KN-93 (2-[N-(2-hydroxyethyl)]-N- (4-methoxybenzenesulfonyl)] amino -N -(4 -chlorocinnamyl)-N-methylbenzylamine) and the CaMK II inhibitory peptide fragment 281-301 (CIP) were obtained from Calbiochem (San Diego, CA). 5 m M stock solutions of K N compounds were made in 100% DMSO (Sigma, Mississauga, ON). The maximum final working concentration of DMSO was 0.06%, which had no effect on K v channels tested (data not shown). CIP was reconstituted in distilled water as a 1 mM stock solution. A l l other chemical reagents used to make solutions were purchased from Sigma. 3.2.2 Cell culture and transfection Experiments were performed on H E K 293 cells either stably (human ether a-go-go related gene (hERG), K v l . 5 , Kv l .5 R487V, Kv l .4 A 2-147 and Kv4.2) or transiently (Kvl .2, Kv2.1, Kv3.2 and all other Kvl .5 point mutations) expressing cloned channels. H E K 293 cells stably expressing channels were grown in minimum essential medium (MEM), 10 % fetal bovine serum (FBS), 160 unils ml"1 penicillin-streptomycin and 1 mg ml"1 gentamicin. Transient transfections were performed with H E K 293 cells plated at 20-30 % confluency on sterile coverslips in 25 mm Petri dishes one day prior to transfection. two pg of ion channel D N A was incubated with 1 pg of eGFP D N A (to enable detection of transfected cells) and 3 pi of lipofectAMINE 2000 (Invitrogen) in 100 pi of Opti-MEM and added to the cells after changing the media with 900 pi of M E M with 10 % FBS. A l l cells were maintained at 37 °C in an atmosphere of 5 % C02 / a i r . 112 3.2.3 Molecular biology and channel mutations The mammalian expression system pcDNA3.1 (Invitrogen) was used for expression of all constructs in this study. A l l point mutations in Kvl .5 were performed using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene, San Diego, CA) using primers constructed by Sigma-Genosys (Oakville, ON). A l l constructs were sequenced at the University of British Columbia core facilities (Vancouver, BC) to ensure the fidelity of the PCR reactions. 3.2.4 Electrophysiology solutions For recording potassium current, the pipette solution contained (mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na + 2 ATP, 4; GTP, 0.1 (adjusted to pH 7.2 with KOH). The bath solution contained (mM): KC1, 5; NaCl, 135; M g C l 2 , 1; sodium acetate, 2.8; HEPES, 10 (adjusted to pH 7.4 with NaOH). High extracellular K + experiments were performed using bath solution containing (mM): KC1, 135; HEPES, 10; M g C l 2 , 1; Dextrose, 10 (adjusted to pH 7.4 with KOH). For recording currents through Kvl .5 R487V, the pipette solution contained (mM): NaCl, 130; Na + 2 ATP, 4; M g C l 2 , 1; HEPES, 5; EGTA, 10; GTP, 0.1 (pH adjusted to 7.2 with NaOH). The bath solution contained (mM): NaCl, 5; N M D G , 130; HEPES, 10; Dextrose, 10; M g C l 2 , 1 (pH adjusted to 7.4 with HC1). K N compounds were diluted in bath solution at the appropriate concentration immediately prior to each experiment and kept in the dark, in order to avoid photo-inactivation of the drug. For experiments with CIP and internal KN-93, the compounds were diluted in pipette solution at the appropriate concentration. 113 3.2.5 Electrophysiological procedures Glass coverslips to which cells had adhered were removed from the incubator immediately prior to experiments and placed in a recording chamber mounted on the stage of an inverted phase contrast microscope at room temperature. The bath solution was constantly flowing. Patch electrodes fashioned from thin-walled borosilicate glass (World Precision Instruments, FL, USA) had a resistance of 1.5-2.5 M Q when filled with the pipette solution. Whole-cell current recording and data analysis were performed using an Axopatch 200B amplifier, DigiData 1322A digitizer and pClamp8 software (Axon instruments, Foster City, CA). Capacity compensation and 80% serial resistance compensation were used in all whole-cell recordings. Data were sampled at 10 kHz and filtered at 2 kHz. No leak subtraction was used and dashed lines on current records represent the zero current level. 3.2.6 Data analysis Potency of each drug was determined by fitting the concentration-response relationships with the Hi l l equation (1) using GraphPad Prism 3.02 (San Diego, CA): y=V(\+(IC50/[KN-93])h) (1) where y is the fraction of current remaining at a given membrane potential, ICso is the concentration required to achieve half maximal block, [KN-93] is the concentration of K N -93 in the bath solution, and h is the Hil l coefficient. Chord conductance (G) at a given potential was calculated by dividing the current at the end of a 300 ms pulse by the driving force calculated from the Nernst equation. g-V curves were fitted with a single Boltzmann function: y = l / ( l + e x p [ ( F / / 2 - V ) / £ ] ) (2) where y is the conductance normalized with respect to the maximal conductance, Vm is the half-activation potential, V is the test voltage and k is the slope factor. 114 The pore structure of Kvl .5 was modeled using the known crystal structure of the related Kv l .2 channel (accession: 2A79) as a template using Swiss-Model's "First Approach Mode" (http://swissmodel.expasy.org/SM FIRST.html) and "Swiss-Pdbviewer". Data throughout the text and figures are shown as means ± S.E.M. Statistical significance was determined throughout using Student's t test with P values of less than 0.05 taken to be significant. 115 3.3 Results 3.3.1 KN-93 inhibits a wide range of Kv channels Given the initial observations of Ledoux et al. (1999) in rabbit portal vein smooth muscle, where the types of Kv channels directly affected by KN-93 were not identified, we examined the effect of KN-93 on a range of K v channel representatives from a number of different subfamilies. Figure 3.1A shows typical current traces recorded from Kv l .2 , K v l . 5 , Kv l .4 A2-147, Kv2.1, Kv3.2, Kv4.2 and hERG channels during perfusion of control solution or solution containing 1 p M KN-93. Kvl .4 A2-147 was used to remove fast inactivation from the channels and reveal any effect of KN-93. Currents were recorded during voltage pulses to +60 mV for 5 s every 40 s with the exception of Kv4.2 currents, which were recorded during 200 ms pulses every 10 s and hERG currents, which were recorded during 4 s pulses to -50 mV following a 4 s pulse to +20 mV. In all channels tested, KN-93 enhanced current decay so that the current amplitude at the end of the pulse was significantly reduced (Figure 3. IB, filled bars). In some channels, KN-93 also inhibited the peak current (e.g. K v l . 2 , Kv2.1 in Figure 3.1 A), but this was probably due to the slower activation kinetics of these channels (as shown by the scaled traces) rather than any additional effect of KN-93 on these channels. To demonstrate that the effect of KN-93 on Kv channels was independent of CaMK II, we repeated these experiments using KN-92, the inactive, but structurally very similar, form of KN-93. The open bars in Figure 3.IB show that, like KN-93, KN-92 significantly reduced the sustained current amplitudes at the end of the 5 s depolarizing pulses of all channels tested. These data show that KN-93 inhibits a wide range of K v channels in a manner that is independent of CaMK II. 116 Figure 3.1. KN-93 inhibits Kv channels from a number of different subfamilies. ( A ) Effect of 1 u M KN-93 on K v l . 2 , K v l . 5 , K v l . 4 A2-147, Kv2.1, Kv3.2, Kv4.2 and hERG currents. A l l currents were recorded during 5 s depolarizing pulses to +60 mV from a holding potential of -80 mV, in the presence and absence of 1 u M KN-93, with the exception of Kv4.2 (200 ms pulses to +60 mV) and hERG (4 s depolarization to +20 mV followed by a 4 s pulse to -50 mV to record tail currents). The pulse interval was 30 s for Kv4.2 and 40 s for all other channels in order to prevent cumulative inactivation. Gray lines depict current traces obtained in the presence of KN-93 scaled to the peak of the current in the absence of KN-93 to illustrate the effect of KN-93 on current decay. In case of K v l . 2 and Kv2.1, the slow 117 activation results in underestimation of normalized block. (B) Summary of current inhibition by 1 p M KN-93 (filled bars) and the inactive form of KN-93, KN-92 (open bars). Fractional sustained current refers to the current at the end of the depolarizing pulse in the presence of drug normalized to the control value, with the exception of hERG currents, where peak tail currents in the presence of drug were normalized to those in the absence of drug. Numbers above bars represent n values. Significantly different from control: *, P<0.05; **, P<0.01 (Paired t test). 3.3.2 KN-93 directly blocks the Kvl.5 channel Since Kvl .5 is thought to be the major constituent of the delayed rectifier current in rabbit portal vein smooth muscle cells (Overturf et al., 1994) and KN-93 has been shown to act as a direct blocker of Kv currents in these cells (Ledoux et al., 1999), we used Kvl .5 to examine the nature of the KN-93 inhibition of K v currents. Data in Figure 3.2A show typical traces recorded from Kvl .5 channels in the presence of a number of different concentrations of KN-93. The current amplitude at the end of the 5 s pulse at each concentration was used to construct the concentration-response curve shown in Figure 3.2B. A non-linear least-squares fit of the Hi l l equation to the concentration-response data yielded an IC50 value of 307 ± 12 nM and a Hi l l coefficient, h, of 1.3 ± 0.3 (Figure 3.2B). In order to rule out the involvement of CaMK II inhibition by KN-93 in the observed inhibition of Kv current, we used the CaMK II inhibitor peptide (CIP), a 29-amino-acid peptide corresponding to the CaM-binding domain of CaMK II. Dialysis of cells with 10 p M of CIP for 5 min had no effect on peak or sustained current amplitudes; after CIP treatment, average peak and sustained currents were 90 ± 3 % and 91 + 5 % of the control pretreated value (n=5, Paired t test, not significant). A typical example is shown in the diary plot of K v l . 5 sustained current in Figure 3.2C. Furthermore, inhibition of C a M K II with CIP did not prevent KN-93 from exerting its effect as demonstrated by the reduction 118 of sustained current upon application of KN-93 to a value similar to that of cells not dialyzed with CIP; KN-93 inhibited current by 88 ± 2 % in the presence of CIP compared with 87 ± 2 % in the absence of CIP (Figure 3.2C; «=5, t test, not significant). These data are consistent with the observation that the inactive inhibitor, KN-92, induced a similar inhibition of sustained current to KN-93 (Figure 3. IB). Figure 3.2. KN-93 inhibition of Kvl.5 is independent of CaMK II activity. (A) K v l . 5 currents recorded during 5 s pulses to +60 mV in the presence of increasing bath concentrations of KN-93. (B) Concentration-response curve for the effect of KN-93 on K v l . 5 , from data such as those in (A). Data were fitted to a Hi l l equation (see Methods). The IC50 and Hi l l coefficient (h) are shown («=4). (C) Diary of sustained current amplitude measured at the end of 5 s depolarizing pulses to +60 mV. Dialysis of cells with 10 p M C a M K II inhibitory peptide fragment 281-301 (CIP) is indicated by the broken line, and addition of KN-93 to the same cell by the first continuous line. Note that only KN-93 addition reduced sustained current amplitude. 119 In Figure 3.3 the effect of KN-93 on the voltage-dependence of Kv l .5 channel activation was examined. Figure 3.3A shows currents recorded during 300 ms pulses from -60 mV to +60 mV (in 10 mV increments) from a holding potential of -80 mV in the absence and presence of 1 p M KN-93. Conductance-voltage curves (Figure 3.3B) were generated by calculating the chord conductance from the current amplitude at the end of each pulse. The data were fitted to a single Boltzmann function (see Eqn. (2) in Materials and Methods). The deviation from the Boltzmann fit seen at depolarized potentials most likely represents an artifact that arises from the calculation of conductance from the current at the end of the pulse. It was not possible to calculate conductance from tail currents in these experiments because KN-93 reduced tail currents to such an extent that it was not possible to obtain accurate measurements. Figure 3.3B shows that KN-93 appeared to shift the voltage-dependence of channel opening to more hyperpolarizing potentials as reported for the sustained K + currents of rabbit portal vein smooth muscle cells (Ledoux et al, 1999), however, the data in Figure 3.3B did not reach statistical significance. The V1/2 of activation was -7.7 ± 1.0 mV during control conditions and -12.8 ± 3.0 mV during perfusion of 1 p M KN-93. KN-93 did result in a significant decrease in the slope factor, k, from 8.6 + 0.7 mV during control conditions to 4.8 ± 0.3 mV. Note that in Figure 3.3A on the first pulse following channel opening at -20 mV, there is no apparent block due to KN-93. From data such as those recorded in Figure 3.3A, we measured the degree of channel block in the presence of different concentrations of KN-93 over a range of potentials where the probability of channel opening was close to 1 (Vm>10 mV) to construct concentration-response curves at each potential (Figure 3.3C). The inset of Figure 3.3C shows the dependence of the IC50 on membrane potential and highlights that KN-93 is only a weakly voltage-dependent blocker of K v l .5. —1 1 1 1 1 1 1— 0.0 -I 1 — ' — 1 ' — ' — 1 -60 -40 -20 0 20 40 60 10 100 1000 Membrane Potential (mV) KN-93 Concentration (nM) Figure 3.3. Voltage-dependence of KN-93 effect. (A) Kv l .5 currents recorded in the absence (left) and presence (right) of 1 p M KN-93 during 300 ms pulses from -60 mV to +60 mV in 10 mV increments followed by a 200 ms pulse to -50 mV to measure tail currents. (B) Normalized conductance-voltage relationships in the absence and presence of 1 p M KN-93 determined based on the conductance at the end of each depolarizing pulse. Data were fitted to a single Boltzmann function. The Vm of activation was -7.7 ± 1 . 0 mV in control conditions and -12.8 ± 3.0 mV in the presence of 1 p M KN-93 («=4; Paired t test, /»0.05). The slope factor, k, was 8.6 + 0.7 mV and 4.8 ± 0.3 mV, respectively («=4; Paired t test, /K0.05). (C) Voltage-dependence of KN-93 inhibition determined from data such as those recorded in (A) in the presence of increasing concentrations of KN-93. The inset shows IC50 values plotted against the membrane potential. Points were connected by a line to guide the eye. 121 3.3.3 KN-93 is an extracellular open-channel blocker of Kvl.5 channels To examine the state dependence of accessibility of KN-93, a control current trace was recorded (Figure 3.4A, left) and then 1 uM KN-93 was added to the bath while channels were held in the closed state (-80 mV) (Figure 3.4A). The peak current obtained on the first opening of the channels following the 3 min incubation was not different from that of control; the average peak current on the first pulse following a 3 min rest was 96 + 2 % that of the control value (n=6, Paired t test, not significant). There was however, a pronounced effect on the peak current during the second depolarizing pulse, suggesting that KN-93 could not access its binding site in the closed state of the channel. We further examined the open state-dependent binding of KN-93 by inspecting the tail currents (Figure 3.4B). Currents were recorded during 200 ms pulses to allow KN-93 binding before a hyperpolarizing pulse to -80 mV to observe tail currents. These experiments were performed in symmetrical K + conditions to increase tail current amplitude and therefore aid analysis. Tail currents obtained from the same cell in control conditions and in the presence of 1 uM KN-93 are shown in Figure 3.4B. The tail currents were fitted to a double exponential function, which show that KN-93 significantly slowed the slow component of the tail current (mean time constant, T_, increased from 52 ± 8 to 77 ± 5 ms, n=5; Paired /-test, p<0.05) causing the tail currents to crossover, a phenomenon that suggests that KN-93 is an open channel blocker that must unbind before the channel can close. To identify which side of the membrane KN-93 exerts its effect, we dialyzed the cells with intracellular solution containing 1 p M KN-93 for 5 min after formation of the whole-cell configuration. Constant drug-free bath perfusion was maintained throughout. Cells were held at -80 mV and pulsed to +60 mV every 40 s for 5 min to document the effect of 122 intracellularly applied KN-93. Figure 3.4C and 3.4D show typical traces and a diary plot from such an experiment. It is clear that at the end of the 5 min period with KN-93 applied intracellularly, sustained currents were not different from those obtained immediately after formation of the whole-cell configuration (Figure 3.4C). To the same cell, we then applied KN-93 extracellularly by bath perfusion. Both Figure 3.4C and 3.4D document that addition of KN-93 to the extracellular solution caused a rapid reduction of sustained currents. These data suggest that KN-93 exerts its effect from the extracellular side and is ineffective as a blocker from the intracellular solution. Since nearly all known blockers of hERG involve drug binding at a site located in the central cavity of the pore (Mitcheson et ah, 2000), we were interested to know whether K N -93 was an extracellular blocker of hERG, as it is in K v l . 5 . Figure 3.4E and 3.4F show that hERG currents were not affected following a 5 min period with KN-93 applied intracellularly («=3, Paired t test, not significant). In contrast, external KN-93 dramatically reduced hERG currents. These data suggest that KN-93 does not act by binding at the "classical" internal drug binding site of the hERG channel. 123 B KN -93 3 min Control <y , 5 min Int. KN -93 Control 5 min Int. KN -93 Ext. KN -93 "crossover" 20 ms 1 LLM KN -93: r, = 6.9 + 0.8 ms; a, = 0.78 + 0.03 r, = 77 +5 ms*; a, = 0.16 + 0.02* Control: r, = 6.7 + 0.8 ms; a, = 0.91+ 0.01 ; a, = 0.07+ 0.01 Int. KN -93 10 c 8 a> 3 O •a 6 4 4 Ext. KN -93 • Wash 200 300 < 250 C L £ 200 6 150 '5 0. 100 50 400 Time (s) Int. KN -93 600 800 Ext. KN -93 Wash 100 200 300 Time (s) 400 500 Figure 3.4. Extracellular binding site of KN-93. (A) After a depolarizing pulse to +60 mV for 100 ms, the cell was held in the closed state (-80 mV) while the bath was perfused with 1 p M KN-93. This was followed by depolarizing pulses every 10 s. (B) Tail currents recorded from K v l . 5 channels with high extracellular K + (135 mM) at -80 mV after a 200 ms depolarizing pulse to +60 mV in the presence and absence of KN-93. Crossover of the two tracings is indicated by the arrow. Tail currents were fitted by a double exponential function. In the absence and presence of 1 p M KN-93, the mean fast time constant (z>) was 6.7 ± 0.8 and 6.9 ± 0.8 ms, respectively (n=5; Paired t test, p>0.05) and the mean slow time constant (T2) was 52 ± 8 and 77 ± 5 ms, respectively («=5; Paired t test, p<0.05). The fractional amplitudes a, and a2 were 0.91 + 0.01 and 0.78 ± 0.01 (a,) and 0.07 ± 0.01 and 0.16 ± 0.02 fa) in control and KN-93 respectively. (C, D) Intracellular dialysis of cells with 1 p M K N -124 93 for 5 min did not change Kvl .5 currents recorded during 5 s depolarizations to +60 mV. Individual currents are shown in (C) and the diary of sustained current amplitude in (D). (E, F) Intracellular dialysis of cells with 1 p M KN-93 for 5 min did not change hERG currents recorded during 4 s depolarizations to +20 mV followed by 4 s pulses to -50 mV. Individual currents are shown in (E) and the diary of peak tail current amplitude in (F). Note that the addition of 1 p M KN-93 to the bath solution (i.e. extracellularly) resulted in a rapid decline of both K v l .5 and hERG currents. 3.3.4 KN-93 delays recovery from inactivation Data in Figure 3.1 show that Kvl .5 channels activate rapidly upon depolarization and then undergo slow inactivation over the course of a number of seconds, which results in the decay of the current while depolarization is sustained. Slow inactivation is thought to be caused by a local conformational change in the outer pore (P-type inactivation) followed by an energetically and structurally more complete stabilization of the inactivated conformation (C-type inactivation; DeBiasi et al, 1993; Loots and Isacoff, 1998; Kurata and Fedida, 2005). We measured the rate of recovery of Kv l .5 channels from inactivation in the absence (Figure 3.5A) and presence of 1 p M KN-93 (Figure 3.5B) by applying a +60 mV conditioning pulse for 5 s followed by brief (10 ms) test pulses to +60 mV applied at increasing intervals. The peak current amplitude obtained during each test pulse was normalized to that obtained during the conditioning pulse and plotted against the interpulse interval (Figure 3.5C). The data points were fitted to a double exponential function, to represent recovery from P-type inactivation (Tfasl) and the more stable C-type inactivation (fsiow)- The data show that KN-93 slowed the fast phase of recovery from 255 ± 28 ms to 546 ± 61 ms (n=5; Paired t test, /?<0.05) and increased its contribution a/ast from 0.13 ± 0.04 to 0.32 ± 0.09 of the total inactivation (n=5; Paired Mest,/?<0.05). KN-93 had no effect on the slow component of recovery from inactivation (n=5; Paired /-test, p =N.S.). 125 These results showed that inactivation was deeper and recovery from inactivation was delayed in the presence of KN-93, suggesting that KN-93 might interact with residues that regulate P-type inactivation in Kv l .5 and stabilize channels in the inactivated state. Given our data (Figure 3.4) demonstrating extracellular binding of KN-93, we focused on extracellular residues known to be near the site of constriction responsible for inactivation and examined the effect of KN-93 on mutated Kv l .5 channels that showed reduced or accelerated slow inactivation. Data in Figure 6A show currents recorded during a 1 s depolarizing pulse to +60 mV from mutant channels in which the arginine at position 487 in the outer pore was replaced with a valine (R487V; this is equivalent to the T499V mutation in Shaker (Lopez-Barneo et al, 1993; Wang et al, 2000)). R487V mutant channels showed little inactivation during the pulse and the effect of 1 p M KN-93 was significantly reduced (Figure 3.6A). Figure 3.6B shows a model of the primary sequence of the Kv l .5 outer pore, based on that of the recently crystallized structure of the K v l . 2 channel (Long et al, 2005), and reveals that the outer vestibule of the K v l . 5 pore is formed by the extracellular loops between the fifth and sixth transmembrane helices (S5-P-S6) and R487 is positioned close to the external mouth of the pore. In contrast to the R487V mutation, mutation of threonine 462 to cysteine (T462C) in the outer pore of the channel (Figure 3.6B) resulted in an increased rate of inactivation (Figure 3.6C). T462C currents inactivated so rapidly that currents were recorded during much shorter pulses; during a 500 ms depolarizing pulse to +60 mV, inactivation was comparable to that in the wild-type channel following a 5 s depolarization (Figure 3.IB). Enhancement of inactivation by the T462C mutant was accompanied by increased block upon exposure to KN-93 (Figure 3.6C). Concentration-response curves generated using T462C current amplitudes at the end of the 500 ms depolarizing pulses in 126 Figure 3.6D show that the IC50 of block was significantly reduced to 69 ± 18 nM (n=4; PO.05, Paired Mest, compared with wild-type). To test the correlation between KN-93 potency and outer pore mediated inactivation, we mutated each residue within the pore lining region of the outer vestibule (residues highlighted in Figure 3.6B) and assessed the effect of KN-93. Of the eight mutations made, four were non-functional. However, H463C, S465C, S466C and P468C showed robust currents and these showed similar inactivation kinetics to the wild-type channel. The bar graph in Figure 3.6E shows the effect of all of the mutations tested on the fractional current, normalized to control, remaining at the end of the pulse following KN-93 block. In contrast to the R487V and T462C mutations, which alter inactivation, the pore mutations that did not alter inactivation (H463C, S465C, S466C and P468C) showed a level of block that was not significantly different from control. 127 A Control B 1 uM KN-93 0.0 i 1 1 1 1 — 0 10 20 30 40 Time (s) Figure 3.5. Effect of KN93 on the rate of recovery from inactivation. Currents recorded (from the same cell) during a 5 s conditioning pulse to +60 mV to allow slow inactivation followed by 10 ms test pulses to +60 mV at different intervals to measure the recovery of channels from inactivation in control conditions (A) and with 1 p M KN-93 (B). (C) Peak test pulse current normalized to the peak conditioning pulse current plotted again pulse interval. Data were fitted to a double exponential function and the time constants (rfasl and r x/ o w) and the amplitudes (afast and asiow) of each component are shown (n-5). * Significantly different (p<0.05) from control. 128 Control 10 nM 100 nM c JS0 .8 (A - 5 0 . 6 t _ 0.4 I 0.2 H z 0.0 / C M = 69 + 18 nM n =0.98 + 0.13 100 ms 1 10 100 1000 KN-93 Concentration (nM) Figure 3.6. Mutants that accelerate or slow inactivation alter KN-93 block of Kvl.5. (A) K v l . 5 R487V currents recorded during 1 s depolarizing pulses to +60 mV from the holding potential of-80 mV in the absence and presence of KN-93. (B) A model of the outer pore of K v l . 5 based on the known crystal structure of K v l . 2 (accession: 2A79). The outer pore regions of only two subunits are shown for clarity. The side chains that were mutated are highlighted to show their position within the outer pore region. Spheres indicate K + ion coordination sites within the pore. (C) T462C currents recorded during 500 ms pulses to +60 mV from the holding potential of -80 mV in the absence and presence of increasing concentrations of KN-93 as indicated. (D) Concentration-response curve for KN-93 block of T462C mutant channels (w=4). (E) Summary of the effect of KN-93 on each mutant tested. Bars show the fractional current remaining at the end of the pulse. * Significantly different (p<0.05) from wild-type K v l . 5 . n.f. stands for non-expressing channels (n = 3-6). 129 3.4 Discussion 3.4.1 KN-93 Inhibits a wide variety of voltage-gated potassium channels This study has demonstrated, for the first time, the direct interaction of KN-93, a specific CaMK II Inhibitor, with members of K v l , Kv2, Kv3, Kv4, and hERG voltage-gated potassium channel families. The effect of KN-93 on K v channels was shown to be independent of CaMK II as: 1) KN-92, an inactive form of KN-93, resulted in a similar inhibition of ionic currents (Figure 3.IB); 2) dialysis of cells with CIP resulted in no detectable change in Kv l .5 currents or gating kinetics (Figure 3.2C); and, 3) internal KN-93 did not alter channel gating (Figure 3.4C and 3.4D). A number of lines of evidence suggest that KN-93 has a direct action as an open channel blocker of the Kvl .5 channel. Firstly, the currents shown in Figure 3.3A and 3.3B recorded from the same cell in the absence and presence of KN-93 show that there was no block evident in the presence of the drug on the first pulse following channel opening (note the -20 mV traces). Secondly, incubation of cells held in the closed state with 1 p M KN-93 for 3 min resulted in no detectable effect on the peak current amplitude on the first depolarizing pulse (Figure 3.4A). These results indicate that KN-93 cannot access its binding site when the channel is in the closed state. Finally, superposition of the tail currents in the presence and absence of KN-93 shows crossover (Figure 3.4B), which suggests that channel closure is slowed by KN-93 unbinding from the open channel. 3.4.2 KN-93 stabilizes the inactivated state Here, we have shown that KN-93 induces a rapid decay of current, and a reduction of peak current that is dependent upon the ability of the channel to inactivate. Reduction of the rate of inactivation, as in the Kvl .5 R487V mutation, markedly attenuated the effect of K N -130 93 (Figure 3.6A). This suggests that KN-93 binding to open, and/or inactivated channels promotes the transition of channels to the inactivated state, and stabilizes them there. Consistent with this, recovery of channels from inactivation was slowed in the presence of KN-93 (Figure 3.5). Since the data in Figure 3.6 suggest that KN-93 binds with a lower affinity to channels that inactivate slowly (e.g. R487V) this suggests that the concentration-response curve of R487V channels is right-shifted and we therefore expect that higher concentrations of KN-93 would induce greater block of R487V. Interestingly, we observed enhanced inactivation of Kv l .4 even in the absence of the N-terminus and therefore N-type inactivation (Figure 3.1 A), suggesting that at least part of the decay of current observed in Kv l .4 by Roeper et al. (1997) is due to a direct effect of KN-93 on slow inactivation of the channel. Enhancement of inactivation by the mutation Kvl .5 T462C in the outer pore enhanced the potency of KN-93 action (Figure 3.6C and 3.6D). This increased block was unlikely to be due to allosteric effects on the outer pore structure because mutation of neighboring residues had no effect on either the rate of inactivation or the potency of KN-93 binding (Figure 3.6E). Given that KN-93 acts at the extracellular side of membrane and interacts with the open channel (Figure 3.4) and that the drug slows the recovery from the P-type inactivated state (Figure 3.5), which involves a reconfiguration of the outer pore, these results are consistent with the conclusion that, following open channel block, KN-93 promotes and stabilizes outer pore-dependent inactivation. As a precedent for such an action, it is well documented that drug binding to hERG channel requires channel opening (Trudeau et al, 1995; Zhou et al, 1998), and there is a developing body of evidence suggesting that the block for most drugs occur via the inactivated state of the channel (Ficker et al, 1998; Mitcheson et al, 2000). Molecular 131 determinants of high-affinity hERG block by a wide range of agents has been attributed to two aromatic residues in the S6 domain (Mitcheson et al., 2000). It has been suggested that through inactivation, these two aromatic residues undergo a rotation and become exposed for drug block (Chen et al, 2002). Since inactivation of Kv channels involves a physical conformational change in the outer pore (Liu et al., 1996) a similar mechanism may provide a favorable binding site for KN-93 in these channels. In conclusion, the CaMK II inhibitor KN-93 and its inactive form, KN-92, inhibit a wide range of K v channels. The present study shows that KN-93 is an external open channel blocker that shows little voltage-dependence and exerts its action by enhancement of inactivation. Since KN-93 is a potent blocker of many K v channels, it must be used with caution. However, since KN-93 had no effect on K v l . 5 when applied intracellularly, C a M K Il-independent effects of KN-93 on K v channels can probably be circumvented by its intracellular application, although this should be confirmed for each channel system in which it is used. 132 3.5 Acknowledgements We thank Ka-Kee Chiu for preparation of cells, and Cyrus Eduljee for the Kv l .5 T462C mutation. This work was supported by grants from the Heart and Stroke Foundations of British Columbia and Yukon and the CIHR to D.F. S.R. was supported by University of British Columbia Graduate Fellowship. T.W.C was supported by a Postdoctoral Research Fellowship funded by a Focus on Stroke strategic initiative from The Canadian Stroke Network, the Heart and Stroke Foundation, the CIHR Institute of Circulatory and Respiratory Health and the CIHR/Rx&D Program along with AstraZeneca Canada. 133 3.6 References Braun,A.P. and H.Schulman. 1995. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57:417-445. Chen,J., G.Seebohm, and M.C.Sanguinetti. 2002. Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of hERG and eag potassium channels. Proc Natl Acad Sci USA 99:12461-12466. DeBiasi,M., H.A.Hartmann, J.A.Drewe, M.Taglialatela, A.M.Brown, and G.E.Kirsch. 1993. Inactivation determined by a single site in K + pores. Pflugers Arch 422:354-363. Ficker,E., W.Jarolimek, J.Kiehn, A.Baumann, and A.M.Brown. 1998. Molecular determinants of dofetilide block of hERG K + channels. Circ Res 82:386-395. Kurata,H.T. and D.Fedida. 2005. A structural interpretation of voltage-gated potassium channel inactivation. Prog Biophys Molec Biol in press. LedouxJ., D.Chartier, and N.Leblanc. 1999. Inhibitors of calmodulin-dependent protein kinase are nonspecific blockers of voltage-dependent K + channels in vascular myocytes. J Pharmacol Exp Ther 290:1165-1174. Levitan,I.B. 1994. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56:193-212. Liu,Y., M.E.Jurman, and G.Yellen. 1996. Dynamic rearrangement of the outer mouth of a K channel during gating. Neuron 16:859-867. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005. Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309:897-903. Loots,E. and E.Y.Isacoff. 1998. Protein rearrangements underlying slow inactivation of the Shaker K + channel. J Gen Physiol 112:377-389. Lopez-Barneo,J., T.Hoshi, S.H.Heinemann, and R.W.Aldrich. 1993. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Recept Channels 1:61-71. 134 MitchesonJ.S., J.Chen, M.Lin , C.Culberson, and M.C.Sanguinetti. 2000. A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA 97:12329-12333. Overturf,K.E., S.N.Russell, A.Carl, F.Vogalis, P.J.Hart, J.R.Hume, K.M.Sanders, and B.Horowitz. 1994. Cloning and characterization of a K v1.5 delayed rectifier K + channel from vascular and visceral smooth muscles. Am J Physiol Cell Physiol 267:C1231-C1238. RoeperJ., C.Lorra, and O.Pongs. 1997. Frequency-dependent inactivation of mammalian A -type K + channel Kyi .4 regulated by Ca2+/calmodulin-dependent protein kinase. J Neurosci 17:3379-3391. v Sergeant,G.P., S.Ohya, J.A.Reihill, B.A.Perrino, G.C.Amberg, Y.Imaizumi, B.Horowitz, K.M.Sanders, and S.D.Koh. 2005. Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II. Am J Physiol Cell Physiol 288:C304-C313. Shieh,C.C, M.Coghlan, J.P.Sullivan, and M.Gopalakrishnan. 2000. Potassium channels: Molecular defects, diseases, and therapeutic opportunities. Pharmacol Rev 52:557-593. Sumi,M., K.Kiuchi, T.Ishikawa, A.Ishii, M.Hagiwara, T.Nagatsu, and H.Hidaka. 1991. The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Commun 181:968-975. Sun,X.X., J.J.Hodge, Y.Zhou, M.Nguyen, and L.C.Griffith. 2004. The eag potassium channel binds and locally activates calcium/calmodulin-dependent protein kinase II. J Biol Chem 279:10206-10214. Trudeau,M.C, J.W.Warmke, B.Ganetzky, and G.A.Robertson. 1995. hERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269:92-95. Varga,A.W., L.L.Yuan, A.E.Anderson, L.A.Schrader, G.Y.Wu, J.R.Gatchel, D.Johnston, and J.D.Sweatt. 2004. Calcium-calmodulin-dependent kinase II modulates Kv4.2 channel expression and upregulates neuronal A-type potassium currents. J Neurosci 24:3643-3654. Wang,Z., G.F.Wilson, and L.C.Griffith. 2002. Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel. JBiol Chem 277:24022-24029. 135 Wang,Z.R., J.C.Hesketh, and D.Fedida. 2000. A high-Na+ conduction state during recovery from inactivation in the K + channel K v l .5. Biophys J 79:2416-2433. Young,K.A. and J.H.Caldwell. 2005. Modulation of skeletal and cardiac voltage-gated sodium channels by calmodulin. J Physiol 565:349-370. Zhou,Z.F., Q.M.Gong, B.Ye, Z.Fan, J.C.Makielski, G.A.Robertson, and C.T.January. 1998. Properties of hERG channels stably expressed in H E K 293 cells studied at physiological temperature. Biophys J 74:230-241. 136 Chapter 4: S2-S3 linker is associated with coupling of the T l domain to Kvl.2 channel gating5 3 A version of this chapter will be submitted for publication. Saman Rezazadeh, Harley T. Kurata and David Fedida 137 4.1 Introduction Voltage-gated K + (Kv) channels activate and open in response to membrane depolarization, and play an important physiological role in the repolarization of action potentials in all excitable tissues. A large number of intracellular signals, which regulate K v channel activity, have been identified, and many of these involve interactions or phosphorylation of residues within the T l domain (Huang et al, 1993; Huang et al., 1994; Tsai et al., 1999). The T l domain is a cytosolic N-terminal domain present in K v channels, and is highly conserved within Kv channel subfamilies (Deutsch, 2002). Crystal structures determined for the T l domains of Shaker, Shaw and Shal family of K v channels demonstrate that the T l domains are arranged as a rotationally symmetrical tetramer, which is thought to lie in alignment with the channel pore, and despite significant primary sequence differences between different K v channel subfamilies, the structural scaffold of the T l domain is common to all Kv channels (Kreusch et al., 1998; Bixby et al, 1999; Gulbis et al., 2000; Minor et al., 2000; Long et al, 2005; Wang et al, 2007). Tetramerization of the T l domains is an early step in channel biosynthesis (Deutsch, 2002), and crystallographic, biochemical, and electrophysiological evidence suggest that the T l tetramer exists as a module distinct from the transmembrane channel core, adopting a "hanging gondola" structure away from the inner pore of intact K v channels (Baro et al., 1902; Gulbis et al, 2000; Kobertz et al, 2000; Sokolova et al., 2001; Sokolova et al, 2003; Long et al, 2005). The T l domain influences numerous fundamental channel functions, including interaction of channels with Kvfi subunits (Rettig et al, 1994; Sewing et al, 1996; Pongs et al, 1999; Gulbis et al, 2000), interaction with many intracellular signaling molecules (Huang et al, 1993; Huang et al, 1994; Jing et al, 1999; Tsai et al, 1999), and prevention of heteromultimerization between 138 different Kv channel subfamilies (Li et al, 1992; Shen et al., 1993; X u et al., 1995; Shen and Pfafiinger, 1995; Kreusch et al, 1998; Bixby et al, 1999; Kobertz et al, 2000). Furthermore, a wide variety of point mutations and deletion mutations within the T l domain have been demonstrated to substantially alter the voltage-dependence and kinetics of activation in Shaker-related channels, suggesting conformational coupling of the T l domain and the transmembrane segments of the channel (Kobertz and Miller, 1999; Minor et al., 2000; Cushman et al., 2000; Kurata et al, 2005). However, despite the clearly delineated role for the T l domain in cytosolic regulation of channel gating, the mechanism(s) of interaction between the T l domain and the gating elements of the channel remains unclear. The structure of the T l domain of Kv l .2 containing the T46V mutation has been previously solved by X-ray crystallography, and exhibits no major changes in the overall 3-dimensional structure. In spite of the very minor structural effects of the T46V mutation, the activation gating of this channel was reported to be significantly shifted to depolarized voltages when expressed in Xenopus oocytes, by stabilizing the closed state of the channel through an unknown mechanism (Minor et al, 2000). However, when we expressed the Kv l .2 T46V mutant channels in mammalian cells we made an unexpected observation. The T46V mutation resulted in a dramatic acceleration of deactivation without altering the activation kinetics compared to Wt channels. Furthermore, when we expressed K v l . 2 T46V in oocytes, we again failed to detect any effect on channel activation kinetics, but the acceleration of deactivation kinetics persisted. Interestingly, we have determined that the equivalent mutation to T46V in Kv l .5 (T133V) exerts no effect on channel gating properties. We have exploited this observation to probe the mechanism by which the T l domain is coupled to the gating machinery of Kv channels. We report that the T l domain may exert its modulatory effects on channel gating properties by interacting with the S2-S3 linker. 140 4.2 Materials and Methods 4.2.1 Cell preparation and transfection A l l experiments were carried out on transiently transfected mouse Itk- cells grown in Minimal Essential Medium (MEM) with 10% fetal bovine serum, at 37° C in an air/5% C 0 2 incubator. One day before transfection, cells were plated on sterile glass coverslips in 35 mm Petri dishes and grown to 20-30% confluence. On the day of transfection, cells were washed once with M E M with 10% fetal bovine serum. In order to identify the transfected cells efficiently, channel cDNA was co-transfected with a vector encoding green fluorescent protein (pGFP). Channel cDNA was incubated with pGFP (1 pg of pGFP, 2 ug of channel cDNA) and 3 uL of LipofectAMINE 2000 (Invitrogen) in 100 uL of serum-free OPTI-MEM (Gibco-BRL), then added to the dishes containing cells in 1 mL of M E M with 10% fetal bovine serum. Cells were allowed to grow overnight before recording. 4.2.2 Solutions For mammalian cells, patch pipettes contained (in mM): NaCl, 5; KC1, 135; Na 2 ATP, 4; GTP, 0.1; M g C l 2 , 1; EGTA, 5; HEPES, 10; and was adjusted to pH 7.2 with K O H . The bath solution contained (in mM): NaCl, 135; KC1, 5; HEPES, 10; sodium acetate, 2.8; M g C l 2 , 1; CaCl 2 , 1; and was adjusted to pH 7.4 with NaOH. For Xenopus oocytes, Barm's solution contained (in mM): NaCl, 88; KC1, 1; NaHC0 3 , 2.4; MgS0 4 , 0.82; Ca(N0 3 ) 2 , 0.33; CaCl 2 , 0.41 and HEPES, 0.41; titrated to pH 7.4 using NaOH. ND96 bath solution contained (in mM): NaCl, 96; KC1, 3; MgCb, 1; CaCl 2 , 0.3; HEPES, 5 mM, titrated to pH 7.5 using NaOH. A l l chemicals were from Sigma Aldrich Chemical Co. (Mississauga, Ont). 141 4.2.3 Molecular biology and channel mutations K v l . 2 was a kind gift from Dr. Dan Minor. The mammalian expression vector pcDNA3 was used for expression of all channel constructs used in this study. A l l primers used were synthesized by Sigma Genosys (Oakville, Ontario, Canada). A l l constructs were sequenced to check for sequence errors, and to ensure the correct reading frame (NAPS Unit, University of British Columbia, Vancouver, Canada). Site-directed mutagenesis of Kv l .5 and K v l . 2 were performed using the Stratagene QuikChange kit (La Jolla, CA, USA). 4.2.4 Kvl.5/Kvl.2 chimeras Chimeras were constructed by PCR amplification of the desired segment of Kv l . 2 , introducing restriction sites to allow subcloning into the Kvl .5 cDNA. A n EcoRIrestriction site was introduced at the C-terminal end of the fragment, and either a BspEI (for Kvl .5N/Kvl .2) , Pmll (for Kvl .5SlS2L/Kvl .2) , CM (for Kvl.5S2S3L/Kvl.2) , or StuI (for Kvl.5S4S5L/Kvl.2) were introduced at the N-terminal end of the fragment. 4.2.5 RNA preparation and oocyte injection A modified pBluescript SKII oocyte expression vector (pEXO) was used to express K v l . 2 channel. cRNA was synthesized from linear cDNA using the T7 mMessage mMachine T7 Ultra cRNA transcription kit (Ambion, Austin, TX). Xenopus oocytes were prepared and isolated as described previously (Claydon et al, 2007). Briefly, Xenopus laevis were terminally anaesthetized and stage V - V I oocytes were isolated and defolliculated using a combination of collagenase treatment (1 h in 1 mg/ml collagenase type 1; Sigma-Aldrich) and manual defolliculation. Oocytes were injected with 50 nl (5-10 ng) cRNA using a Drummond dispenser (Fisher Scientific, ON, Canada) and then incubated in Barth's medium at 19 °C. Currents were recorded 1-3 days after injection. 4.2.6 Electrophysiological procedures Coverslips containing cells were removed from the incubator before experiments and placed in a superfusion chamber (volume 250 pi) containing the control bath solution at ambient temperature (22-23 °C), and perfused with bathing solution throughout the experiments. Whole-cell current recording and data analysis were done using an Axopatch 200B amplifier and pClamp 8 software (Axon Instruments, Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments; FL, USA). Electrodes had resistances of 1-3 M Q when filled with pipette filling solution. Capacity compensation and 80% series resistance compensation were used in all whole-cell recordings. No leak subtraction was used when recording currents. Data were sampled at 10 kHz and filtered at 2 kHz. Throughout the text, data are presented as mean ± S.E.M. Voltage clamp of oocytes and acquisition of the current and voltage signals was achieved using the two-microelectrode voltage-clamp technique with a Warner Instruments OC-725C amplifier, Axon Digidata 1322, and pClamp9 software. Microelectrodes were filled with 3 M KC1 and had a resistance of 0.2-0.5 M Q . 4.2.7 Data analysis g-W curves throughout the text were derived using the normalized chord conductance, which was calculated by dividing the maximum current elicited during the depolarizing step by the driving force derived from the K + equilibrium potential. g-W curves were fitted with a single Boltzmann function: y=\/(l+exp[V1/2-V\/k) where y is the conductance normalized with respect to the maximal conductance, Vj/2 is the half-activation potential, V is the test voltage and k is the slope factor. Data throughout 143 the text and figures are shown as mean ± S.E.M. Statistical significance was determined throughout using Student's t test with P values of less than 0.05 taken to be significant. 144 4.3 Results 4.3.1 The T46Vmutation accelerates Kvl.2 deactivation without altering activation kinetics Data in Figures 4.1 A and 4.IB illustrate the ionic currents recorded from Uk- cells expressing Wt Kv l . 2 during depolarizing pulses to potentials between -65 mV and +55 mV in 20 mV increments followed by repolarizing pulses to -40 mV. In agreement with our previous report (Chapter 2), the activation kinetics of the Wt K v l . 2 channel are heterogeneous when expressed in mammalian cells and can assume two distinct activation gating modes, 'slow' (Figure 4.1A) and 'fast' (Figure 4.IB) a categorization based on the time course of channel activation. Single exponential fits to the activation time course to estimate activation kinetics clearly illustrate that at even very strong depolarizing pulses, the activation kinetics in 'slow' cells remained considerably slower than those observed in 'fast' cells (Figure 4.ID). Furthermore, the 'slow' gating mode is associated with a dramatic 35 mV depolarizing shift in the voltage dependence of activation relative to the 'fast' gating mode (Figure 4.IF). However, despite these marked differences between the activation properties of the 'slow' and the 'fast' gating modes, the deactivation properties of the channel are independent of the gating mode (Figure 4. IE). The time constant of deactivation (tdeact) was determined by fitting the tail current at -40 mV following a strong depolarizing pulse to a single exponential function, ideact values were 29.2 + 0.4 ms (n=5) and 28.0 ± 1 . 2 ms (n=6) for 'slow' and 'fast' gating modes, respectively. Figure 4.IE illustrates the normalized tail currents for the 'slow' and 'fast' gating modes. Minor et al. (2000) reported that the T46V mutation in the intersubunit interface of the T l domain of Kv l . 2 resulted in a dramatic depolarizing shift in the Via of activation, slowing of activation and acceleration of deactivation kinetics, possibly through 145 destabilization of the open state of the channel when expressed in Xenopus oocytes (Minor et al, 2000). In our hands, the T46V mutation resulted in a dramatic reduction of K v l . 2 channel surface expression in mammalian cells, and therefore ionic currents greater than the endogenous level (-100 pA at +60 mV) could only be recorded in 3 out of the 21 cells examined. Furthermore, the activation properties of the T46V mutation (Figure 4.1C) appeared indistinguishable from the 'slow' mode Wt K v l . 2 (Figure 4.1 A) both in terms of time constant of activation (Figure 4.ID) and Vm of activation (Figure 4.IF). However, in agreement with Minor et al (2000), K v l . 2 deactivation kinetics were accelerated in the presence of the T46V mutation. The time constant of deactivation of T46V mutant channels at -40 mV was 6.0 ± 0.9 ms (n=3; Figure 4.IE), which is ~5-fold smaller than the deactivation time constant of Wt K v l . 2 channels at -40 mV, as evident in the overlain normalized tail currents shown in Figure 4.IE. We hypothesized that this discrepancy between data in Figure 4.1 and those reported previously might be due to differences in the expression system used. Therefore, Wt and T46V Kvl .2 channels were expressed in oocytes and ionic currents were recorded during depolarizing pulses from -60 mV to +60 mV in 20 mV increments from the holding potential of-80 mV followed by repolarizing pulses to -60 mV (Figure 4.2A and 4.2B). Wt Kv l .2 only exhibited the 'fast' gating mode in oocytes (Figure 4.2A), but the T46V mutation resulted in no detectable alteration in the activation kinetics of the channel (Figure 4.2B). Furthermore, the voltage dependence of activation was not altered by the T46V mutation and the V!/2s of activation were -12.8 ± 1.6 («=3) and -11.9 ± 2.1 (w=8) for Wt and T46V channels, respectively (Figure 4.2C), despite attempts to record from oocytes prepared from different frogs on different days to allow for biological variability. However, the T46V 146 mutation resulted in a significant acceleration of channel deactivation {jdeaa values were 12.2 ± 0.9 ms («=3) and 7.15 ± 0.25 ms («=8) for Wt and T46V channels, respectively (PO.001), as evident from the superimposed normalized tail currents in Figure 4.2D, in agreement with data collected from mammalian cells (Figure 4.IE). B 100 ms 100 ms 100 ms T46V1 50 ms 1000 _ 100 S E ro E O a 10 1 o D • • • O Wt Slow • Wt Fast • T46V -40 -20 0 20 40 p Membrane Potential (mV) 1.0 0.8 0.6 0.4 0.2 0.0 • Wt Fast O Wt Slow • T46V -80 -60 -40 -20 0 20 40 60 80 100 Membrane Potential (mV) Figure 4.1. T46V mutations result in acceleration of Kvl.2 channel deactivation. Wt (A and B) and T46V (C) ionic currents were recorded during depolarizing steps from -65 to +55 mV in 20 mV steps. Representative traces from cells expressing K v l . 2 channels exhibiting the 'slow' (A) or 'fast' (B) gating modes are shown. (D) Monoexponential activation time constants for 'fast' and 'slow' activating Wt K v l . 2 and T46V mutant channels. (E) 147 Normalized tail currents at -40 mV following membrane depolarization to +55 mV. r</ac, values were 29.2 ± 0.9 ms («=4) for 'fast' channels, 27.8 ± 1.2 ms («=5) for 'slow' channels and 6.0 + 0.9 ms (n=3) for T46V channels. (F) Normalized maximum chord conductance during 400 ms pulses was determined for each membrane potential and the data from individual cells were fitted with a Boltzmann function. The mean isochronal activation Vm values for Wt K v l . 2 channels were -18.8 ± 2.3 mV («=19, £=9.1 ± 0.5 mV) for 'fast' activating channels and 16.6 ± 1.1 mV for slowly activating channels («=33, £=10.1 ± 0.3 mV). The Vm of activation for T46V mutation was 18.2 ± 1.1 0=3, £=9.5 ± 1.0 mV). Figure 4.2. The T46V mutation does not alter activation kinetics when expressed in Xenopus oocytes. Ionic currents from Xenopus oocytes expressing Wt (A) or T46V (B) were recorded during depolarizing pulses from -60 mV to +60 mV in 20 mV steps followed by repolarizing pulses to -60 mV. (C) Normalized maximum chord conductance during 400 ms pulses was determined for each membrane potential and the data from individual oocytes were fitted with a Boltzmann function. The mean activation Vm values were -12.8 ± 1 . 6 mV («=3, £=12.6 ± 3.0 mV) and -11.9 ± 2.1 mV 0=8, £=15.5 ± 1.4 mV) for Wt and T46V channels, respectively. (D) Normalized tail currents for Wt and T46V channels at -60 mV are shown, ideact values were 12.2 + 1.5 ms (n=3) and 7.1 ± 0.2 ms 0=8) for Wt and T46V channels, respectively. 148 4.3.2 Kvl.5 is insensitive to the T46Vequivalent mutation The T46V equivalent mutation (T133V) in Kvl .5 exerted no detectable effect on channel gating, despite more than 90% homology in the amino acid composition of the K v l .5 T l domain to that of K v l . 2 . Data in Figures 4.3A and 4.3B illustrate the ionic currents recorded from Wt and T133V Kvl .5 channels, respectively, during depolarizing pulses to potentials between -65 and +55 mV in 20 mV increments, followed by repolarizing pulses to -40 mV. It is evident that the presence of the T133V mutation resulted in no apparent change in the time course of channel activation (Figure 4.3C) or the voltage dependence of activation (Figure 4.3D). Furthermore, unlike the effect of the T46V mutation on Kv l . 2 , T133V resulted in no detectable change in the deactivation kinetics recorded at -40 mV (Figure 4.3E). 149 0 \ , , , , , , , i 1 1 . . --20 -10 0 10 20 30 40 50 -60 -40 -20 0 20 40 Membrane Potential (mV) Membrane Potential (mV) E 50 ms Figure 4.3. Kvl.5 channels are insensitive to the T46V equivalent mutation, T133V. Ionic currents from cells expressing Wt (A) and T133V mutant (B) K v l . 5 channels were recorded during depolarizing pulses to potentials between -65 mV and +55 mV in 20 mV increments. (C) Monoexponential time constants for the activation time course of Wt and T133V channels. (D) Normalized maximum chord conductance during 400 ms pulses fitted with a Boltzmann function. The mean activation Via values were -8.5 ± 2 . 1 mV (n=3, k=7.0 ± 0.8 mV) and -5.0 ± 2.1 mV («=7, £=8.1 ± 0.7 mV) for Wt and T133V channels, respectively. (E) Normalized tail currents at -40 mV recorded after a +55 mV depolarizing pulse, ideact values were 19.7 ± 0.9 (n=3) and 18.1 ± 0.6 ms (n=l) for Wt (black trace) and mutant (red trace) channels, respectively. 150 4.3.3 Effect of T46V mutation depends on regions outside the Tl domain The N-termini of Kv l .2 and Kvl .5 are highly homologous within the T l domain, but exhibit significant sequence divergence preceding the T l domain, as the T l domain in Kv l .5 is preceded by 120 amino acids, while the T l domain in K v l . 2 is preceded by only 33 residues. Therefore, differences in the sensitivity of K v l . 2 and Kv l .5 channels to an equivalent mutation in the T l domain might be caused by structural differences between the proximal N-termini of the two channels. Data in Figure 4.4 illustrate the effects of substituting the N-terminus of K v l .2 with that of K v l .5 (Kvl .5N/Kvl .2). The modal gating observed in Wt K v l . 2 (Figure 4.1) persisted in this chimeric channel with two well-separated g-V curves (Figure 4.4A) with activation Vyfs similar to those of Wt K v l . 2 channels (Figure 4.1 A and IB). However, attachment of the N-terminus of Kv l .5 to the Kv l .2 transmembrane domain resulted in a dramatic acceleration of channel deactivation compared to Wt K v l . 2 . Figure 3.4C illustrates the normalized tail currents recorded from Kvl .5N/Kv l .2 channels at -40 mV following a strong depolarization, with the Wt K v l . 2 tail current shown as reference. Interestingly, the time course of deactivation of the Kv l . 5 N/ Kv l . 2 channel was markedly faster than the deactivation kinetics of the Wt K v l . 2 channel (xdeact = 28.1 ± 0.9 ms (n=\\) and 16.7 ± 1.2 («=12) for Wt K v l . 2 and Kv l .5N/Kv l .2 , respectively, PO.0001) but was similar to the deactivation kinetics of Wt Kvl .5 (Tdeact = 19.7 ± 0.9 ms (n=6) and 16.7 ± 1.2 («=12) for Wt Kvl .5 and Kv l .5N/Kv l .2 , respectively, P>0.05). This suggests that the N-terminus plays a role in regulating the deactivation properties. When the T133V mutation was introduced into Kv l .5N/Kvl .2 chimeric channels (Kvl.5N-T133V/Kvl.2), the deactivation kinetics were accelerated from 16.7 ± 1.2 ms 151 («=12) for K v l . 5 N / K v l . 2 to 12.4 ± 0.8 ms (w=8) for Kvl .5N-T133V/Kvl .2 (P<0.05, Figure 4.4C) without altering the activation properties of the channel (Figure 4.4B). This is interesting as it suggests a unique interaction between the T l domain and the transmembrane core of the K v l . 2 channel. Furthermore, substitution of the Kv l .5 N-terminus with K v l . 2 N -terminus carrying the T46V mutation (Kvl .2N/Kvl .5) resulted in no detectable change in the gating properties of the channel (data not shown). Collectively, these data suggest that significant differences exist in the mechanism of coupling between the T l domain and channel gating in K v l . 2 versus K v l . 5 . Figure 4.4. Acceleration of deactivation due to the T l domain mutation is linked to other regions of the channel. Normalized maximum chord conductance during 400 ms pulses fitted with a Boltzmann function from cells expressing K v l . 5 N / K v l . 2 channels (Kvl .5 N-terminus attached to the transmembrane domain of Kvl .2) in the absence (A) and presence (B) of the T133V mutation. The T133V mutation did not alter the voltage dependence of either mode of K v l . 2 channel activation. Open symbols (o) and filled symbols (•) correspond to 'slow' and 'fast' cells, respectively. (C) The T133V mutation accelerated the rate of channel deactivation as evident from the normalized tail currents at -40 mV, Tdeact values were 16.7 ± 1.1 ms («=3) and 12.4 ± 0.8 ms («=8) for K v l . 5 N / K v l . 2 and Kvl .5N-T133V/Kvl .2 channels, respectively (PO.05). 152 4.3.4 The Tl domain may exert its effect through coupling with the S2-S3 linker A chimeric strategy was devised to investigate the mechanism of coupling between the T l domain and channel deactivation. Unlike the T46V mutation, the presence of the T133V mutation in Kv l .5N/Kv l .2 chimeric channels resulted in acceleration of deactivation without significantly compromising channel trafficking. Therefore, we used Kv l .5N/Kv l .2 as the background channel in our chimeric approach and asked which domains of Kv l .5 should be substituted in order to abolish the acceleration of deactivation caused by the T133V mutation. Data in Figure 4.5A show the schematic view of the chimeric channels constructed. Normalized tail currents recorded at -40 mV following membrane depolarization to +55 mV are shown in Figure 4.5B from the chimeric channels in the presence (red traces) or absence (black traces) of the T133V mutation. From Figure 4.5B it is evident that substitution of the Kvl .5 sequence up to the beginning of the S2 transmembrane segment (Kvl .5SlS2L/Kvl .2 ; Figure 4.5Ba) did not abolish the acceleration of deactivation due to the T133V mutation (tdeact values were 18.5 ± 0.5 ms («=4) and 14.3 ± 0.5 (n=\4) in the absence or presence of T133V, respectively (PO.001)). Interestingly, further substitution of the Kvl .5 sequence comprising the S2 transmembrane segment and the cytoplasmic S2-S3 linker (Kvl.5S2S3L/Kvl.2, Figure 4.5Bb) completely abolished the effect of the T133V mutation. Consistent with this observation, chimeras with further substitution of Kvl .5 sequence up to the cytoplasmic side of the S5 transmembrane helix (Kvl.5S4S5L/Kvl.2, Figure 4.5Bc) also appeared insensitive to the T133V mutation, suggesting a possible role for the cytoplasmic S2-S3 linker of K v l . 2 in the regulation of channel deactivation properties through interaction with the T l domain. In agreement, a chimeric channel with the K v l . 2 S2 helix and the S2-S3 linker substituted for that of Kv l .5 153 (Figure 4.5A) is sensitive to the T133V mutation as the time constant of deactivation was reduced from 37.8 ± 4.8 ms (n=4) to 21.8 ± 3.3 ms (n=6) in the presence of the T133V mutation (P<0.05). This underlines the importance of the S2-S3 linker in the interaction with the T l domain (Figure 4.5Bd). The deactivation time constants of the chimeric channels are summarized in Figure 4.5C. 154 Kv1.5 :---IZZ}aC3CD-CD-CD-CZH Kv1.5N/Kv1.2i-l l-l Kv 1. 5S1 S2UKv1.2 > • • LZZr-IZZH Kv1.5S2S3UKV1.2 i • • I I • [ _ • •__} | Kv1 5S4S5UKV1.2 H l-l H H H I-Kv1.5S1S2L/KV1.2S2S3L/KV1.5 i-l H l-l H l-l l-r B Kv1.5S1S2L/Kv1.2 Kv1.5S2S3L/Kv1.2 100 ms 100 ms Kv1.5S4S5UKv1.2 d . Kv1.5S1S2L7Kv1.2S2S3L/Kv1.5 100 ms 100 ms 50 40 E 30 I 20 10 Hum • > N > s1 y y > > Figure 4.5. The S2-S3 linker is involved in modulation of gating by the T l domain. (A) Schematic diagram of the chimeras constructed. K v l . 2 domains are shown in black boxes joined by continuous lines, while Kv l .5 domains are shown in hollow boxes joined by perforated lines. (B) The deactivation properties of the chimeric channels in the absence (black traces) and presence (red traces) of the T133V mutation. (C) Summary of the deactivation time constants determined by fitting the tail currents at -AO mV to a single exponential function. Black and gray bars represent the time constant of deactivation for constructs in the absence or presence of the T46V for Wt K v l . 2 and T133V for WtKvl .5 and chimeric channels, respectively. N.S. stands for not significant. (***) represents P<0.0001, (**) represents P<0.01 and (*) represents P<0.05. 155 4 . 4 Discussion 4.4.1 T46V has no effect on Kvl.2 activation properties We report here that the T46V mutation, unlike a previous report by Minor et al. (2000), resulted in no detectable change in the time course or voltage dependence of activation in K v l . 2 when expressed in mammalian cells (Figure 4.1) or Xenopus oocytes (Figure 4.2). We have previously shown that the Kv l .2 channel can assume two distinct gating modes when expressed in mammalian cells, 'slow' and 'fast' (Figure 4.1 A and 4.IB) (Chapter 2). In Itk- cells, the activation kinetics of T46V Kvl .2 channels were not different from those of 'slow' Wt Kv l .2 channels either in terms of the time course or the voltage dependence of activation. We believe that the observed exclusive 'slow' gating mode due to the T46V mutation arises because of the marked reduction of the surface expression as a result of the mutation (Figure 4.1). We have previously shown that the activation gating mode of K v l . 2 correlates with the channel surface expression, where low surface expression of K v l . 2 predominantly results in slowly activating currents and vice versa (Chapter 2). In our hands, the K v l . 2 channel activated exclusively through the 'fast' mode when expressed in oocytes (Figure 4.2A), and T46V failed to modify the activation properties (Figure 4.2B and 2C). Regardless of the discrepancy in the effect of the T46V on the activation kinetics of Kv l . 2 , the T46V mutation resulted in a dramatic acceleration of channel deactivation in both mammalian and oocyte expression systems, which suggests that this mutation can result in alteration of channel gating without imposing any detectable structural changes (Minor et al, 2000; Figures. 4.1 and 4.2). 4.4.2 The role of the N-terminus in regulation of channel gating Attachment of the N-terminus of the Kvl .5 to the transmembrane domain of K v l . 2 resulted in marked acceleration of K v l . 2 deactivation, which matched closely the kinetics of 156 Kvl .5 deactivation, without altering channel activation properties (Figure 4.4). In K v channels, the N-terminus has been implicated in playing a significant regulatory role in the deactivation properties. The N-terminus of human ether a-go-go (hERG) channels is thought to be involved in regulation of deactivation by a mechanism similar to N-type inactivation (Wang et al., 1998; Wang et al, 2000), where the N-terminus binds to the S4-S5 linker and prevents deactivation (Terlau et al, 1997). A number of mutations in the N-terminus of hERG have been reported to result in acceleration of deactivation (Chen et al., 1999), possibly by altering the structure of the N-terminus. Even though the degree of structural modification by these mutations is not clear in the hERG channel, in Aplysia K v l . l and Kv l . 2 , point mutations in the T l domain alter channel gating with minimal perturbation of the 3D structure of this domain (Minor et al., 2000; Cushman et al., 2000), suggesting that very small changes in the T l domain can be reliably linked to alteration of channel gating. Additionally, in Kv2.1 channels, deletion of a part of the N terminus (VanDongen et al., 1990), or application of methylmethanesulfonate which attaches a thiomethyl group to native 9+ N-terminal cysteines involved in Zn coordination (Pascual et al., 1997), as well as replacing part of the Kv2.1 N-terminus with corresponding regions of Kv l .5 (Kurata et al., 2002), all result in channels with slow activation, deactivation, and altered inactivation properties. Given the significant role of the N-terminus in regulating Kv channel gating, the possible structural differences between the N-termini of K v l . 2 and Kvl .5 may explain the acceleration of K v l . 2 deactivation by the Kvl .5 N-terminus. 4.4.3 Disparate effects ofTl domain mutations in Kvl.2 and Kvl.5 The T46V equivalent mutation in Kvl .5 (T133V) failed to induce any significant change in the gating properties of the channel (Figure 4.3), while substitution of the N -terminus of Kvl .5 with the T133V mutation in K v l . 2 still resulted in acceleration of 157 deactivation (Figure 4). An energetic link between the T l domain structure and gating in the transmembrane pore provides an opportunity to modulate the gating state of the Kv channel by directly coupling cellular regulatory signals that interact with the T l domain to channel gating states (Sewing et al, 1996; Y u et al., 1996; Accili et al., 1997). However, the differential response of K v l . 2 and Kvl .5 channels to an equivalent mutation suggests that modification of the structure of the T l domain does not necessarily translate into the same response in different channels, and thus allows for selective modulation of a specific family of channels. 4.4.4 The S2-S3 linker connects the modification of the Tl domain to the channel gating The possible involvement of the S2-S3 linker in coupling of the T l domain to channel gating is exciting as this is the first report offering a mechanism through which the T l domain and the channel core may interact. It is possible that the S2-S3 linker has a very distinct interaction with other parts of the channel, and is therefore directly involved in the normal allosteric transition(s) that occur during channel gating. Alternatively, the linker may simply be a hinge that maintains a particular distance between the S2 and S3 transmembrane helices, and therefore, the structure of such a hinge may determine its flexibility. This may be critical during the conformational changes that constitute channel gating, a process that most likely requires movement of the transmembrane segments in the membrane. The S2 and S3 transmembrane helices participate in channel gating via a charge interaction with the S4 segment (Papazian et al., 1995; Seoh et al, 1996; Zhang et al, 2004), therefore, structural changes in the S2-S3 linker may disrupt coordinated movements of the S2 and S3 transmembrane segments, thereby altering channel gating properties. As indicated by our data, it is plausible that the T l domain may exert its regulatory effect on the gating property of the channel by modifying the structure of the S2-S3 linker. 158 4.5 Acknowledgments We would like to thank Grace Lu for assisting with mutagenesis and Ka Kee Chiu for preparation of cells. This work was supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon and the CIHR to DF. S.R. was supported by a University of British Columbia Graduate Fellowship and a Heart and Stroke Foundation of British Columbia and Yukon scholarship. H.T.K. was funded by the CIHR and Michael Smith Foundation for Health Research. 159 4.6 References Accili ,E.A., J.Kiehn, Q.Yang, Z.G.Wang, A.M.Brown, and B.A.Wible. 1997. Separable Kvp subunit domains alter expression and gating of potassium channels. J Biol Chem 272:25824-25831. Baro,D.J., L.Quinones, C.C.Lanning, R.M.Harris-Warrick, and M.Ruiz. 2001. Alternate splicing of the Shal gene and the origin of I A diversity among neurons in a dynamic motor network. Neurosci 106:419-432. Bixby,K.A., M.H.Nanao, N.V.Shen, A.Kreusch, H.Bellamy, P J.Pfaffinger, and S.Choe. 1999. Zn 2 +-binding and molecular determinants of tetramerization in voltage-gated K + channels. Nature Structural Biology 6:38-43. Chen,J., A.R.Zou, I.Splawski, M.T.Keating, and M.C.Sanguinetti. 1999. Long QT syndrome-associated mutations in the Per-Arnt-Sim (PAS) domain of hERG potassium channels accelerate channel deactivation. JBiol Chem 274:10113-10118. Claydon,T.W., M.Vaid, S.Rezazadeh, D.C.Kwan, S.J.Kehl, and D.Fedida. 2007. A Direct Demonstration of Closed-State Inactivation of K + Channels at Low pH. J Gen Physiol 129:437-455. Cushman,S.J., M.H.Nanao, A.WJahng, D.DeRubeis, S.Choe, and P.J.Pfaffinger. 2000. Voltage dependent activation of potassium channels is coupled to T l domain structure. Nature Structural Biology 7:403-407. Deutsch,C. 2002. Potassium channel ontogeny. Annu Rev Physiol 64:19-46. GulbisJ.M., M.Zhou, S.Mann, and R.MacKinnon. 2000. Structure of the cytoplasmic (3 subunit - T l assembly of voltage-dependent K + channels. Science 289:123-127. Huang,X.-Y., A.D.Morelli, and E.G.Peralta. 1993. Tyrosine kinase-dependent suppression of a potassium channel by the G-protein-coupled ml muscarinic receptor. Cell 75:1145-1156. Huang,X.-Y., A.D.Morelli, and E.G.Peralta. 1994. Molecular basis of cardiac potassium channel stimulation by protein kinase A . Proc Natl Acad Sci USA 91:624-628. 160 Jing,J., D.Chikvashvili, D.Singer-Lahat, W.B.Thornhill, E.Reuveny, and I.Lotan. 1999. Fast inactivation of a brain K + channel composed of K v l . l and K v p i . l subunits modulated by G protein beta-gamma subunits. EMBO J 18:1245-1256. Kobertz,W.R. and C.Miller. 1999. K + channels lacking the 'tetramerization' domain: implications for pore structure. Nature Structural Biology 6:1122-1125. Kobertz,W.R., C.Williams, and C.Miller. 2000. Hanging gondola structure of the T l domain in a voltage-gated K + channel. Biochemistry 39:10347-10352. Kreusch,A., P.J.Pfaffinger, C.F.Stevens, and S.Choe. 1998. Crystal structure of the tetramerization domain of the Shaker potassium channel. Nature 392:945-948. Kurata,H.T., K.Doerksen, J.R.Eldstrom, S.Rezazedeh, and D.Fedida. 2005. Separation of P/C- and U-type inactivation pathways in Kvl .5 potassium channels. J Physiol. Kurata,H.T., G.S.Soon, J.R.Eldstrom, G.W.K.Lu, D.F.Steele, and D.Fedida. 2002. Amino-terminal determinants of U-type inactivation of voltage-gated K + channels. J Biol Chem 277:29045-29053. L i , M . , Y.N.Jan, and L.Y.Jan. 1992. Specification of subunit assembly by the hydrophilic amino- terminal domain of the Shaker potassium channel. Science 257:1225-1230. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005. Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309:897-903. Minor,D.L.Jr., Y.F.Lin, B.C.Mobley, A.Avelar, Y.N.Jan, L.Y.Jan, and J.M.Berger. 2000. The polar T l interface is linked to conformational changes that open the voltage-gated potassium channel. Cell 102:657-670. Papazian,D.M., X.M.Shao, S.-A.Seoh, A.F.Mock, Y.Huang, and D.H.Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K + channel. Neuron 14:1293-1301. Pascual,J.M., C.C.Shieh, G.E.Kirsch, and A.M.Brown. 1997. Contribution of the N H 2 terminus of Kv2.1 to channel activation. Am J Physiol 273:C1849-C1858. 161 Pongs,0., T.Leicher, M.Berger, J.Roeper, R.Bahring, D.Wray, K.P.Giese, A.J.Silva, and J.F. Storm. 1999. Functional and molecular aspects of voltage-gated K + channel P subunits. Ann NY Acad Sci 868:344-355. RettigJ., S.H.Heinemann, F.Wunder, C.Lorra, D.N.Parcej, J.O.Dolly, and O.Pongs. 1994. Inactivation properties of voltage-gated K + channels altered by presence of P-subunit. Nature 369:289-294. Rezazadeh,S., H.T.Kurata, T.W.Claydon, S.J.Kehl, and D.Fedida. 2007. An activation gating switch in K v l .2 is localized to a threonine residue in the S2-S3 linker. Biophys J. Seoh,S.A., D.Sigg, D.M.Papazian, and F.Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K + channel. Neuron 16:1159-1167. Sewing,S., J.Roeper, and O.Pongs. 1996. K v p i subunit binding specific for STza&er-related potassium channel a subunits. Neuron 16:455-463. Shen,N.V., X.Chen, M.M.Boyer, and P.J.Pfaffmger. 1993. Deletion analysis of K +channel assembly. Neuron 11:67-76. Shen,N.V. and P.J.Pfaffmger. 1995. Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K + channel subunit proteins. Neuron 14:625-633. Sokolova,0., A.Accardi, D.Gutierrez, A.Lau, M.Rigney, and N.Grigorieff. 2003. Conformational changes in the C terminus of Shaker K + channel bound to the rat Kvbeta2-subunit. Proc Natl Acad Sci USA 100:12607-12612. Sokolova,0., L.Kolmakova-Partensky, and N.Grigorieff. 2001. Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution. Structure (Camh) 9:215-220. Terlau,H., S.H.Heinemann, W.Stuhmer, O.Pongs, and J.Ludwig. 1997. Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment. J Physiol (Camh) 502:537-543. Tsai,W., A.D.Morielli, T.G.Cachero, and E.G.Peralta. 1999. Receptor protein tyrosine phosphatase alpha participates in the m l muscarinic acetylcholine receptor-dependent regulation of Kv 1.2 channel activity. EMBO 718:109-118. 162 VanDongen,A.M.J., G.Frech, J.A.Drewe, R.HJoho, and A.M.Brown. 1990. Alteration and restoration of K + channel function by deletions at the N - and C-termini. Neuron 5:433-443. Wang,H., Y.Yan, Q.Liu, Y.Huang, Y.Shen, L.Chen, Y.Chen, Q.Yang, Q.Hao, K.Wang, and J.Chai. 2007. Structural basis for modulation of Kv4 K + channels by auxiliary KChIP subunits. Nat Neurosci 10:32-39. Wang,J.L., C.D.Myers, and G.A.Robertson. 2000. Dynamic control of deactivation gating by a soluble amino-terminal domain in hERG K + channels. J Gen Physiol 115:749-758. Wang,J.L., M.C.Trudeau, A.M.Zappia, and G.A.Robertson. 1998. Regulation of deactivation by an amino terminal domain in human ether-a-go-go-related gene potassium channels. J Gen Physiol 112:637-647. Xu,J., W.F.Yu, Y.N.Jan, L.Y.Jan, and M . L i . 1995. Assembly of voltage-gated potassium channels - Conserved hydrophilic motifs determine subfamily-specific interactions between the a-subunits. JBiol Chem 270:24761-24768. Yu,W., J.Xu, and M . L i . 1996. N A B domain is essential for the subunit assembly of both a-a and o>P complexes of Shaker-like potassium channels. Neuron 16:441-453. Zhang,M., J.Liu, and G.N.Tseng. 2004. Gating charges in the activation and inactivation processes of the hERG channel. J Gen Physiol 124:703-718. 163 Chapter 5: Destabilization of the open state of IK s potassium channels by a KCNQ1 V205M missense mutation causes an inherited form of LQTS in a Canadian aboriginal community4 4 A version of this chapter will be submitted to Circulation. Saman Rezazadeh, Jodene Eldstrom, Rosemarie Rupps, Shu Sanatani, Glen Tibbits, Brett Casey, Eric Accili, David Fedida and Laura Arbour 164 5.1 Introduction Long QT syndrome (LQTS), which can be acquired or congenital, results from delayed repolarization of cardiac ventricular action potentials, and is characterized by a prolongation in the QT interval of the electrocardiogram (ECG). It can be associated with a serious multifocal ventricular tachyarrhythmia, torsades de pointes (TdP), which may lead to syncope and sudden death (Keating and Sanguinetti, 2001). To date, mutations in eight different genes have been associated with congenital LQTS: seven encode for cardiac ion channels or their accessory subunits (KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), KCNE2 (LQT6), KCNJ2 (LQT7), C A C N A 1 C (LQT8)(Wang et al, 1995b; Curran et al, 1995; Wang et al, 1995a; Romey et al, 1997; Abbott et al, 1999; Plaster et al, 2001; Splawski et al, 2004). The eighth gene encodes for cardiac ankyrin (ANK2; LQT4), a scaffolding protein that anchors the ion channels at the membrane (Mohler et al., 2003). Acquired LQTS can be caused by common medications but, unlike congenital LQTS, it predominantly results from an inhibition of function or expression of hERG (KCNH2) channels (Sanguinetti and Tristani-Firouzi, 2006). In addition, genetic predisposition may play an important role in drug-induced LQTS (Abbott et al, 1999). Congenital LQTS is a relatively rare condition affecting an estimated 1 in 5,000 to 10,000 people (Piippo et al, 2001), but we have recently determined that it is disproportionately prevalent in a Canadian aboriginal community of 10,000 people in Northern British Columbia, where at least 40 related people have demonstrated prolonged QT intervals. Based on family studies, 200 people are thought to be at risk (1 in 500 people; unpublished data). Screening of the KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 genes of two unrelated but severely affected patients in the Canadian native community, who had 165 suffered episodes of syncope and cardiac arrest after emotional stress, has revealed a novel missense mutation (valine substitution with methionine at position 205, V205M) in the S3 transmembrane region of the KCNQ1 channel, the a-subunit for the slow delayed rectifier potassium channel, IKS- Furthermore, out of the 124 members of the extended family of these two patients, the V205M mutation was detected in 21 (1 in 6 people). Tetramers of four KCNQ1 subunits co-assemble with KCNE1 accessory subunits to form the slowly activating and slowly deactivating IKS in the heart, which modulates the repolarization of cardiac action potentials (Sanguinetti et al, 1996; Barhanin et al, 1996; Tai and Goldstein, 1998), particularly during sympathetic activation and tachycardia (Romey et al, 1997; Yang et al, 1997). LQTS linked to mutations in the KCNQ1 gene is usually attributed to a decrease or suppression of IKS by the prevention of efficient KCNQ1 protein folding and trafficking to the cell membrane (Shalaby et al, 1997; Dahimene et al, 2006; Huang et al, 2001; Bianchi et al., 2000), and less often to modification of the response of IKS channels to changes in voltage such that their contribution to repolarization is diminished (Boulet et al, 2006; Chouabe et al, 2000; Chouabe etal, 1997). In this study we find that KCNQ1 V205M channels co-expressed with K C N E 1 , can give rise to outward currents that are comparable to wild-type (Wt) channels in terms of surface expression, but the electrophysiological characteristics of KCNQ1 V205M + KCNE1 expressed in mammalian cells are markedly different from those of Wt channels. The V205M mutation can suppress IKS by causing a dramatic depolarizing shift in the voltage dependence of activation, coupled with a 3-fold acceleration of channel deactivation. This impairs the physiological ability of IKS channels to respond to rapid heart rhythms by beat-to-beat sumrnation of current levels and leads to a reduction of the overall repolarization reserve in affected hearts (Romey et al, 1997). 167 5.2 Materials and Methods 5.2.1 Molecular Biology KCNQ1 and KCNE1 genes were purchased from Origene Technologies (Rockville, MD). The missense mutation V205M (substitution of methionine for valine at position 205) within the third transmembrane (S3) region of KCNQ1 channel was constructed using a two-step PCR reaction. PCR products from reaction one using 5'-gtaatacgactcactatagg-3' and 3'-catggaggccacgaccatgatgaggtcgatgatgg-5' primers and reaction two using 5'-catcatcgacctcatcatggtcgtggcctccatg-3' and 3'-attaggacaaggctggtggg-5' primers were used as templates for the second PCR reaction using 5'-gtaatacgactcactatagg-3' and 3'-gtgagatgtgggtgatgg-5' primers. A l l the primers used were synthesized by Integrated D N A Technologies (Coralville, IA). Pfu Turbo polymerase (Stratagene, La Jolla , CA) was used for all the PCR reactions. The PCR product was subcloned in the KCNQ1 gene using EcoRI and Bglll restriction enzymes and the final product was sequenced to check for errors, and to ensure the correct reading frame (NAPS Unit, University of British Columbia, Vancouver, Canada). Genetic tests on patients were carried out by Familion (New Haven, CT). 5.2.2 Cell Preparation and Transfection A l l experiments were carried out on transiently transfected mouse Itk- cells grown in Minimal Essential Medium (MEM) with 10% fetal bovine serum, at 37° C in an air/5% C 0 2 incubator. This mammalian cell line is an excellent model system in which to compare the functions of Wt and mutant forms of KCNQ1 channels as they allow Wt KCNQ1 subunits to fold and assemble in a normal fashion to form functional channels and possess no endogenous K + channel activity. One day before transfection, cells were plated onto sterile glass coverslips in 35 mm Petri dishes with 20-30% confluence. On the day of transfection, 168 cells were washed once with M E M with 10% fetal bovine serum. In order to identify the transfected cells efficiently, channel D N A was co-transfected with of vector encoding green fluorescent protein (pGFP). 1 pg of KCNQ1 channel D N A and 2.5 pg of KCNE1 accessory subunit was incubated with 1 pg of pGFP and 3 pL of LipofectAMINE 2000 (Gibco-BRL) in 100 pL of serum-free OPTI-MEM (Gibco-BRL), then added to the dishes containing cells in 900 pL of M E M with 10% fetal bovine serum. In order to express heteromeric channels, 0.5 pg of Wt and 0.5 pg of V205M KCNQ1 channel D N A was used. Cells were allowed to grow overnight before recording. 5.2.3 Solutions Patch pipettes contained (in mM): KC1, 130; EGTA, 5; M g C l 2 , 1; HEPES, 10; Na 2 ATP, 4; GTP, 0.1; and was adjusted to pH 7.2 with K O H . The bath solution contained (in mM): NaCl, 135; KC1, 5; HEPES, 10; sodium acetate, 2.8; M g C l 2 , 1; CaCl 2 , 1; and was adjusted to pH 7.4 with NaOH. 5.2.4 Electrophysiological procedures Coverslips containing cells were removed from the incubator before experiments and placed in a superfusion chamber (volume 250 pi) containing the control bath solution at 35 °C and perfused continuously with bathing solution heated to 35 °C by an in-line heater (Warner Instruments, Hamden, CT). Whole-cell current recording and data analysis were done using an Axopatch 200B clamp amplifier and pClamp 8 software (Axon Instruments, Foster City, CA). Patch electrodes were fabricated using thin-walled borosilicate glass (World Precision Instruments; FL). Electrodes had resistances of 1-2 M Q when filled with control filling solution. Capacity compensation and 80% series resistance compensation were used in all whole-cell recordings. No leak subtraction was used when recording 169 currents, and zero current levels are denoted by the dashed lines in the current panels. Data were sampled at 10 kHz and filtered at 2 kHz. Membrane potentials have not been corrected for small liquid junction potentials between bath and pipette solutions. An action potential voltage-clamp protocol was generated using LabHeart v4.9.5. 5.2.5 Data Analysis To determine the voltage dependence of activation g-V plots were constructed using the normalized tail currents at -50 mV and fitted to a single Boltzmann function: y=l/(l+Qxp[Vi/2-V]/k) where y is the conductance normalized with respect to the maximal conductance, Via is the half-activation potential, V is the test voltage and k is the slope factor. Throughout the text, data are presented as mean ± S .E .M. Significance was tested using Student's* t-test or one-way A N O V A as appropriate and a value of P<0.05 was considered significant. 170 5.3 Results 5.3.1 V205MIKs channels traffic to the membrane Wt and mutant KCNQ1 channels were co-expressed with the KCNE1 accessory subunit in Itk- cells to form IKS channels and patch-clamp experiments were performed to examine channel expression and kinetics. Both Wt and mutant channels exhibited robust outward currents with equivalent surface expression densities (827 ± 140 pA/pF (n=9) for Wt channels and 650 ± 104 pA/pF (»=10) for V205M channels at +80 mV, (p=0.33). These data suggest that the LQTS observed in patients carrying KCNQ1 V205M is unlikely to be due to defects in processing and trafficking of the KCNQ1 channels to the cell surface. 5.3.2 The V205M mutation alters activation and deactivation properties oflfcs channels The responses of wildtype and mutant IK s channels to voltage were characterized and compared by recording ionic currents at 35 °C from cells expressing Wt KCNQ1 + KCNE1 channels (Figure 5.1 A) or V205M KCNQ1 + KCNE1 channels (Figure 5.IB) during 2 s depolarizing pulses. The ionic currents recorded from cells expressing the mutant channels exhibited kinetics distinctly different from Wt channels. Firstly, current activation was shifted to more depolarized potentials in the presence of the V205M mutation as outward currents were first detected positive to -10 mV in the Wt channels (Figure 5.1 A , IC) while ionic currents became apparent at potentials more positive than +20 mV in the mutant channels (Figure 5.IB, IC). The voltage dependence of activation was determined by measuring the peak tail currents at -50 mV and plotting them as a function of the depolarizing pre-pulse (Figure 5.1C). The half-activation voltages iVui) for Wt and V205M channels were 10.4 ± 3.2 mV (n=7) and 44.1 ± 5.0 mV (n=6) with slope factor values of 8.5 ± 0.7 mV and 9.9 ± 1.1 mV, respectively. In the V205M channels, tail current amplitudes 171 did not saturate even for the +70 mV prepulse, which is indicative of incomplete activation. Therefore, the extent to which the V 1 / 2 is shifted in the positive direction for the mutant channels is likely underestimated by our protocol, and the difference between the abilities of the Wt and mutant channels to open is probably even larger than we observed. The V205M mutation also resulted in a dramatic slowing of the rate of channel opening in response to membrane depolarization, as shown in Figure 5.IB. A steady level of outward ionic current was not achieved during 2 s depolarizations, even at the most positive potentials studied. The time constant of activation was estimated by fitting the ionic currents during the test pulses in Figures 5.1 A and 5.IB to single exponential functions, and was voltage dependent for both Wt and V205M channels (Figure 5.ID), but remained faster for the Wt channels at all potentials tested. 172 Figure 5.1. V205M mutation results in a depolarizing shift in the voltage dependence of IKs activation. Wt KCNQ1 + KCNE1 (A) and V205M KCNQ1 + KCNE1 (B) ionic currents were recorded at 35 °C during 2 s depolarizing pulses between -50 mV and +70 mV in 10 mV increments followed by a 2 s repolarizing pulse to -50 mV. (C) g-V relationships were constructed by plotting the peak tail currents at -50 mV and the data were fit to single Boltzmann functions. Values for the Vm of activation were 10.4 ± 3 . 2 mV and 44.5 ± 5 . 0 mV with k values of 8.5 ± 0.7 mV and 9.9 ± 1.1 for Wt KCNQ1 + KCNE1 (n=7) and V205M KCNQ1 + KCNE1 («=6), respectively. (D) Time constants of activation (r f lC /) were determined by fitting the test currents in (A) and (B) to single exponential functions and plotting them as a function of membrane potential. In addition to modulating i j_ channel activation properties, the V205M mutation appeared to accelerate the rate of channel deactivation (Figure 5.1 A , 5.IB). The time constant of deactivation was determined by applying a range of hyperpolarizing steps to Wt and V205M channels (Figure 5.2A, 5.2B) and fitting the tail currents between -40 and -100 mV to single exponential functions (Figure 5.2C). It is clear that the V205M mutation 173 accelerated deactivation at all the potentials tested compared to Wt channels. Altogether, these data suggest that the V205M mutation reduces IKs channel availability by shirting the voltage dependence of activation to more depolarized potentials, in addition to slowing the rate of activation and accelerating the rate of channel deactivation. This depolarizing shift would reduce the contribution of the mutant channels to repolarization in the heart of affected individuals. 174 + 80 mV Wt KCNQ1 + KCNE1 -40 mV -100 mV 200 ms B V205M KCNQ1 + KCNE1 -100 -90 -80 -70 -60 -50 -40 Membrane Potential (mV) Figure 5.2. V205M mutation accelerates the rate of KvLQTl deactivation. Ionic currents were recorded from cells expressing WT KCNQ1 + KCNE1 (A) and V205M KCNQ1 + KCNE1 (B) during 2 s depolarizing pulses to +80 mV at 35 °C followed by repolarizing pulses to potentials between -40 mV and -100 mV in 10 mV increments. The ionic currents were normalized to the outward current at the end of the pulse to +80 mV. Deactivation time constants (Tdeact) were determined by fitting the tail currents to a single exponential function and were plotted as a function of membrane potential (C) (n=5 and 4 for Wt and V205M channels, respectively). 175 The effects of the V205M mutation were also evaluated in conditions aimed at simulating the heterozygous composition of native channels in patients, with one allele containing the Wt KCNQ1 gene and the other allele containing the V205M mutation. Cells were co-transfected with equal amounts of cDNA coding for Wt and V205M mutant KCNQ1 channels in conjunction with the KCNE1 accessory subunit and the activation (Figure 5.3A-C) and deactivation (Figure 5.3D, 5.3E) properties of these heteromultimeric channels were examined. Co-expression of Wt and mutant channels caused a significant depolarizing shift in the voltage dependence of activation (Vm = 23.3 ± 3.5 mV (n=\0) compared to 10.4 ± 3.2 mV for Wt alone, P<0.05) without significantly altering the slope factor of activation (k = 11.0 ± 1.2 mV, n=\0) (Figure 5.3B). In addition, the time constants of activation of heteromultimeric channels were slower than in Wt channels and closer to the values obtained for V205M alone (Figure 5.3C). Similarly, the deactivation kinetics of the heteromultimeric channels were intermediate between Wt and V205M homomultimeric channels (Figure 5.3E). Deactivation was significantly accelerated in heteromultimeric channels compared to homomultimeric Wt channels at all potentials tested and somewhat slower than the kinetics of homomultimeric V205M channels. These results indicate that in the model system that we have used, the presence of mutant V205M subunits is sufficient to significantly modulate the function of the overall channel population even when mixed with equal proportions of Wt subunits. 176 Membrane Potential (mV) Membrane Potential (mV) 1 1 1 1 1 1 1 -100 -90 -80 -70 -60 -50 -40 Membrane Potential (mV) Figure 5.3. Effect of V205M mutation on heteromultimeric Wt and mutant channels. Wt and V205M KCNQ1 channels (1:1) were coexpressed with the KCNE1 accessory subunit and ionic currents were recorded at 35 °C. (A) Voltage dependence of activation was determined during 2 s depolarizing pulses between -50 mV and +70 mV in 10 mV increments followed by a 2 s repolarizing pulse to -50 mV to record tail currents. (B) g-\ relationships were determined by fitting the peak tail current amplitudes at -50 mV, plotted as a function of test potential, to a Boltzmann function. Vm of activation was 23.3 ± 3.5 mV with k value of 11.0 ± 1.1 mV (w=10). (C) Time constant of activation (racl) was determined by fitting the test currents in (A) to single exponential functions and plotted as a function of membrane potential (A) (For ease of comparison, values for homomultimeric Wt (•) and V205M (0) channels are shown). (D) Deactivation properties of heteromultimeric channels were determined by applying 2 s depolarizing pulses to +80 mV at 35 °C followed by repolarizing pulses to potentials between -40 mV and -100 mV in 10 mV increments. The ionic currents were normalized to peak outward current. Deactivation time constants (tdeaa) were determined by fitting the tail currents to a single exponential function and were plotted as a function of membrane potential (A, n=9) (•, 0 symbols represent Wt and V205M channels, respectively). 177 5.2.3 Behaviour of the V205M mutant channels during simulated cardiac action potentials The above experiments show that mutant channel opening in response to voltage is modified but do not define how the contribution of IKS is compromised during the heart beat. To understand better how the contribution of IKS is compromised by the V205M mutation during a cardiac action potential, we recorded ionic currents elicited by Wt KCNQ1, V205M KCNQ1 or Wt:V205M KCNQ1 + KCNE1 channels during simulated ventricular action potentials (Figure 5.4). Upon stimulation of the membrane with the action potential voltage clamp protocol (Figure 5.4Aa) at 1 Hz, Wt IKS channels displayed a bell-shaped current waveform with relatively slow activation kinetics and a peak of ionic current during the repolarization phase of the action potential. The ionic currents recorded during the subsequent pulses were potentiated relative to the first pulse (Figure 5.4Ab) and the total potassium charge movement, estimated by integrating the recorded ionic currents during the action potential protocol, was increased by -14% during the second pulse compared to the first pulse). In sharp contrast, V205M channels displayed little detectable ionic current due to hs during the action potential (Figure 5.4Ac), and the total charge during the simulated action potential was only 15% of that for Wt channels. In cells expressing both constructs, small ionic currents (Figure 5.4Ad) were observed that exhibited a significant but limited potentiation (8% increase in total charge movement) upon repetitive depolarization at 1 Hz. In order to compare current amplitudes recorded during the action potential protocol between individual cells, peak repolarization outward currents were normalized to the instantaneous tail current amplitude from each cell at -50 mV following a 2 s depolarizing pulse to +80 mV (an indicator of the total number of channels that are present at the cell surface). Mean repolarization current is plotted in Figure 5.4B from different cells for the 178 three groups of channels. Current amplitude was significantly reduced in the V205M mutant channels, compared with Wt channels and, importantly, was not significantly increased by co-expression with Wt subunits (Wt:V205M heteromultimeric channels, Figure 5.4B). 100 ms 100 ms Figure 5.4. V205M eliminates K v L Q T l ionic current during the cardiac action potential. (A) Typical current traces recorded at 35 °C from cells expressing Wt KCNQ1 + KCNE1 (Ab), V205M KCNQ1 + KCNE1 (Ac) or Wt:V205M KCNQ1 + KCNE1 (Ad) channels during an applied ventricular action potential clamp (LabHeart v.4.9.5) (Aa) applied at 1 Hz. (B) Current amplitude elicited during the action potential protocol was compared among different channels by normalizing the peak outward current recording during the action potential protocol to the peak tail current amplitude at -50 mV using the protocol explained in Figure 5.3A-C. Significance between groups was tested using one-way A N O V A . *, represents (<0.015), **, represents (<0.01) and N.S. stands for not significant. 179 Physiologically, the activation and deactivation properties of IKS provides increased repolarizing outward current at high heart rates by allowing accumulation of open IKS channels. The shifted V1/2 of activation, slowed activation, and the accelerated deactivation in the V205M mutant should all reduce the number of open IKS channels during rapid action potential firing. To examine this, Wt and V205M currents were recorded while the action potential protocol was applied at 3 Hz (Figure 5.5A) and the total ionic charge movement was estimated by integrating the ionic currents (Figure 5.5B). In the Wt channels, the current amplitude at the beginning of the pulse was dramatically increased upon 3 Hz stimulation as was the overall current amplitude (Figure 5.5Ab), and outward charge movement increased markedly (66 ± 7%, n=l) from the first pulse to a plateau at the seventh pulse (Figure 5.5B). In contrast, 3 Hz application of the action potential protocol to cells expressing V205M I^S channels resulted in little discernable ionic current (Figure 5.5Ac) and the outward charge movement did not significantly increase with subsequent pulsing (Figure 5.5B, n=3). The ionic currents recorded from cells expressing heteromultimeric Wt:V205M IKS channels increased moderately (42 ± 4%, rc=8) during the 3 Hz action potential protocol (Figure 5.5Ad, 5.5B) but the increase was significantly less than in the homomultimeric Wt channels. The total charge movement was significantly reduced in Wt:V205M heteromultimeric channels compared to Wt channels even after 3 Hz stimulation (176.1 ± 3.4 pC for Wt (n=l) vs. 40.3 ± 8 pC for Wt:V205M channels («=8), pO.OOOl). 180 Figure 5.5. V205M reduces repolarization reserve at high pulsing rates (A) Current traces recorded during a ventricular action potential (Aa) applied at 3 Hz (Aa) from cells expressing Wt KCNQ1 + KCNE1 (Ab), V205M KCNQ1 + KCNE1 (Ac) or Wt:V205M KCNQ1 + KCNE1 (Ad) channels. (B) The increase in charge movement during repetitive action potential clamps at 3 Hz was quantitated by integrating the ionic currents in Ab-Ad and plotting the total charge as a function of trace number. Data are normalized to the total charge during the first applied action potential stimulus. 181 5.4 Discussion Genetic screening in a Northern British Columbia aboriginal community with a high prevalence of LQTS (1 in 500 people) revealed a novel missense mutation (V205M) in the S3 transmembrane region of the KCNQ1 channel. Coassembly of the KCNQ1 a-subunit with its accessory KCNE1 subunit recapitulates the properties of cardiac IKS (Yang et al, 1997; Barhanine/ al, 1996; Sanguinetti et al, 1996), which is one of the outward currents responsible for termination of the action potential plateau and initiation of final action potential repolarization. IKS is known to provide a "repolarizing reserve" when the action potential duration is prolonged due to reduction of other outward repolarizing currents, for example by remodeling of cell surface expression of ion channels during heart failure (Volders et al, 1999; Tomaselli et al, 1994), or block of hERG channels, for example by drugs. 5.4.1 The biophysical defect in V205M The majority of KCNQ1 mutations typically produce a loss of function by disrupting protein folding, assembly and/or trafficking to the membrane (Shalaby et al, 1997; Dahimene et al, 2006; Huang et al, 2001; Bianchi et al, 2000). In contrast, the V205M mutation resulted in functional IKS channels that apparently trafficked as efficiently as Wt channels to the membrane of Itk- cells, but exhibited markedly slowed activation (Figure 5.1) and accelerated deactivation (Figure 5.2) compared to Wt channels. The V205M mutation shifted the voltage dependence of activation more than 30 mV in the depolarizing direction without altering the slope factor. This represents a destabilization of the open state and/or a favoring of the closed states of the channel without an alteration of the apparent gating 182 valence of activation and/or a change in the cooperative interactions between subunits during the activation process (Schoppa et al, 1992; Perozo et al, 1994; Planells-Cases et al, 1995). The heteromultimeric Wt:V205M IKS channels showed intermediate kinetics between Wt and mutant V205M channels with significantly slower activation kinetics and faster deactivation properties than Wt channels (Figure 5.3). Ionic currents recorded during a cardiac action potential voltage clamp revealed that the shift in the voltage dependence of activation and the slowing of the activation kinetics are sufficient to virtually eliminate outward potassium currents through V205M IKS channels (Figure 5.4). More importantly, the total outward flux of potassium ions through Wt:V205M heteromultimeric IKS channels during an action potential was not significantly different from that of homomultimeric V205M channels (Figure 5.4), suggesting that the V205M mutation functionally has a dominant negative action on IKS- Similar results have been reported for the Q357R mutation in the S6 region (Boulet et al, 2006), R294H in S4 (Chouabe et al, 2000) and R555C in the C-terminus (Chouabe et al, 1997), but the effect of the V205M mutation on the kinetics of IKS channels reported here is more severe. 5.4.2 Summation of IKs at high heart rates Physiologically, IKS current amplitude increases during sympathetic stimulation (Yang et al, 1997) and indirectly during fast pacing. Block of IKS during sympathetic stimulation results in a lengthening of the ventricular action potential that can be translated into a prolonged QT interval (Jost et al, 2005) suggesting that hs is critically important for the shortening of the QT interval during sympathetic drive and tachycardia (Stengl et al, 2006; Jost et al, 2005). Any reduction of IKS due to inherited mutations in the KCNQ1 genes could 183 result in LQT1, with high risk for ventricular arrhythmia during periods of elevated sympathetic activity such as exercise and emotion (Schwartz et al, 2001; Moss, 2003; Keating and Sanguinetti, 2001). Ordinarily, the activation kinetics of Wt IKS channels are very slow relative to the duration of a single action potential, but the deactivation kinetics allow accumulation of IKS channels in the open state during repetitive activity. This provides a "repolarizing reserve" to allow abbreviation of the cardiac action potential, and therefore systole, at high heart rates (Jost et al, 2005; Roden, 1998; Stengl et al, 2006). Data in Figures 5.4 and 5.5 showed that the current amplitude increased slightly in Wt channels stimulated with an action potential waveform at 1 Hz, but at 3 Hz the ionic currents increased dramatically, and more importantly the IKS channel current was present throughout the action potential due to the summation effect. This increase was not observed in V205M channels and was greatly reduced in the Wt:V205M heteromultimeric channels. Together, these data provide evidence that the effect of the V205M mutation is to reduce the amount of IKS during the cardiac action potential, an effect that is likely to be more pronounced at high heart rates and during stress. This conclusion is consistent with the findings in the index case, an individual who experienced cardiac arrest during an episode of emotional stress while exercising. 5.4.3 Structural considerations There is strong evidence that the S2 and S3 transmembrane regions of Kv channels play an important role in modulating channel gating (Papazian et al, 1995, Planells-Cases et al, 1995, Seoh et al, 1996). These domains contain acidic residues in highly conserved positions, which interact specifically with basic residues in the S4 domain to regulate channel folding and gating (Papazian et al, 2002; Tiwari-Woodruff et al, 2000). Although the role 184 of non-charged hyrdrophobic residues in the S3 domain is not fully understood, mutation of hydrophobic residues in the S4 domain of Drosophila Shaker channels to the equivalent residues in Drosophila Shaw channels (ILT mutation) greatly alters the activation of channels by dissociating the voltage sensor movement from channel opening (Smith-Maxwell et al., 1998). Our data here suggest that, at least in IKS channels, the hydrophobic residues of the S3 domain also regulate channel activation. Interestingly, four other mutations (D202H, I204M, S209F and V215M) have been detected in the S3 region of KCNQ1 channel of patients with LQTS (Napolitano et al., 2005). However, none of these mutations has yet been characterized, so it would be not only of great interest to investigate the effect of these mutations on the gating properties of IKS channel, but imperative to do so in order to develop therapeutic strategies designed to ameliorate the functional deficits accompanying these specific mutations. 185 5.5 Acknowledgements We would like to thank Ms. Ka Kee Chiu for preparation of cells. This work was supported by grants from the Heart and Stroke Foundations of British Columbia and Yukon and the CIHR to DF and L A . S.R. was supported by a Heart and Stroke Foundation of British Columbia and Yukon pre-doctoral scholarship. 186 5.6 References Abbott,G.W., F.Sesti, I.Splawski, M.E.Buck, W.H.Lehmann, K.W.Timothy, M.T.Keating, and S.A.N.Goldstein. 1999. M i R P l forms 1^ potassium channels with H E R G and is associated with cardiac arrhythmia. Cell 97:175-187. BarhaninJ., F.Lesage, E.Guillemare, M.Fink, M.Lazdunski, and G.Romey. 1996. K V LQT1 and IsK (minK) proteins associate to form the 7Ks cardiac potassium current. Nature 384:78-80. Bianchi,L., S.G.Priori, C.Napolitano, K.A.Surewicz, A.T.Dennis, M.Memmi, P.J.Schwartz, and A.M.Brown. 2000. Mechanisms of 7Ks suppression in LQT1 mutants. Am J Physiol Heart Circ Physiol 279:H3003-H3011. Boulet,I.R., A.L.Raes, N.Ottschytsch, and D.J.Snyders. 2006. Functional effects of a KCNQ1 mutation associated with the long QT syndrome. Cardiovasc Res 70:466-474. Chouabe,C., N.Neyroud, P.Guicheney, M.Lazdunski, G.Romey, and J.Barhanin. 1997. Properties of K v L Q T l K + channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias. EMBO J 16:5472-5479. Chouabe,C., N.Neyroud, P.Richard, I.Denjoy, B.Hainque, G.Romey, M.D.Drici, P.Guicheney, and J.Barhanin. 2000. Novel mutations in K v L Q T l that affect Iks activation through interactions with Isk. Cardiovasc Res 45:971-980. Curran,M.E., I.Splawski, K.W.Timothy, G.M.Vincent, E.D.Green, and M.T.Keating. 1995. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80:795-803. Dahimene,S., S.Alcolea, P.Naud, P.Jourdon, D.Escande, R.Brasseur, A.Thomas, I.Baro, and J.Merot. 2006. The N-terminal juxtamembranous domain of KCNQ1 is critical for channel surface expression: implications in the Romano-Ward LQT1 syndrome. Circ Res 99:1076-1083. Huang,L., M.Bitner-Glindzicz, L.Tranebjaerg, and A.Tinker. 2001. A spectrum of functional effects for disease causing mutations in the Jervell and Lange-Nielsen syndrome. Cardiovasc Res 51:670-680. Jost,N., L.Virag, M.Bitay, J.Takacs, C.Lengyel, P.Biliczki, Z.Nagy, G.Bogats, D.A.Lathrop, J.G.Papp, and A.Varro. 2005. Restricting excessive cardiac action potential and QT 187 prolongation: a vital role for IKs in human ventricular muscle. Circulation 112:1392-1399. Keating,M.T. and M.C.Sanguinetti. 2001. Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104:569-580. Mohler,P.J., J.-J.Schott, A.O.Gramolini, K.W.Dilly, S.Guatimosim, W.H.duBell, L.S.Song, K.Haurogne, F.Kyndt, M . E . A l i , T.B.Rogers, W.J.Lederer, D.Escande, H.LeMarec, and V.Bennett. 2003. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature 421:634-639. Moss,A.J. 2003. Atrial fibrillation. Ann Noninvasive Electrocardiol 8:1-2. Napolitano,C, S.G.Priori, P.J.Schwartz, R.Bloise, E.Ronchetti, J.Nastoli, G.Bottelli, M.Cerrone, and S.Leonardi. 2005. Genetic testing in the long QT syndrome: development and validation of an efficient approach to genotyping in clinical practice. JAMA 294:2975-2980. Papazian,D.M., W.R.Silverman, M.C.Lin , S.K.Tiwari-Woodruff, and C.Y.Tang. 2002. Structural organization of the voltage sensor in voltage-dependent potassium channels. Novartis Found Symp 245:178-190. Perozo,E., L.Santacruz-Toloza, E.Stefani, F.Bezanilla, and D.M.Papazian. 1994. S4 mutations alter gating currents of Shaker K channels. Biophys J 66:345-354. Piippo,K., H.Swan, M.Pasternack, H.Chapman, K.Paavonen, M.Viitasalo, L.Toivonen, and K.Kontula. 2001. A founder mutation of the potassium channel KCNQ1 in long QT syndrome: implications for estimation of disease prevalence and molecular diagnostics. J Am Coll Cardiol 37:562-568. Planells-Cases,R., A.V.Ferrer-Montiel, C.D.Patten, and M.Montal. 1995. Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K + channel selectively modulates channel gating. Proc Natl Acad Sci U S A 92:9422-9426. Plaster,N.M., R.Tawil, M.Tristani-Firouzi, S.Canun, S.Bendahhou, A.Tsunoda, M.R.Donaldson, S.T.Iannaccone, E.Brunt, R.Barohn, J.Clark, F.Deymeer, A.L.George, Jr., F.A.Fish, A.Hahn, A.Nitu, C.Ozdemir, P.Serdaroglu, S.H.Subramony, G.Wolfe, Y.H.Fu, and L.J.Ptacek. 2001. Mutations in Kir2.1 cause 1 8 8 the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell 105:511-519. Roden,D.M. 1998. Taking the "idio" out of "idiosyncratic": predicting torsades de pointes. Pacing Clin Electrophysiol 21:1029-1034. Romey,G., B.Attali, C.Chouabe, I.Abitbol, E.Guillemare, J.Barhanin, and M.Lazdunski. 1997. Molecular mechanism and functional significance of the MinK control of the K v L Q T l channel activity. JBiol Chem 272:16713-16716. Sanguinetti,M.C, M.E.Curran, A.Zou, J.Shen, P.S.Spector, D.L.Atkinson, and M.T.Keating. 1996. Coassembly of K V LQT1 and minK (IsK) proteins to form cardiac 7K.S potassium channel. Nature 384:80-83. Sanguinetti,M.C. and M.Tristani-Firouzi. 2006. hERG potassium channels and cardiac arrhythmia. Nature 440:463-469. Schoppa,N.E., K.McCormack, M.A.Tanouye, and F.J.Sigworth. 1992. The size of gating charge in wild-type and mutant Shaker potassium channels. Science 255:1712-1715. Schwartz,P.J., S.G.Priori, C.Spazzolini, A.J.Moss, G.M.Vincent, C.Napolitano, I.Denjoy, P.Guicheney, G.Breithardt, M.T.Keating, J.A.Towbin, A.H.Beggs, P.Brink, A.A.Wilde, L.Toivonen, W.Zareba, J.L.Robinson, K.W.Timothy, V.Corfield, D.Wattanasirichaigoon, C.Corbett, W.Haverkamp, E.Schulze-Bahr, M.H.Lehmann, K.Schwartz, P.Coumel, and R.Bloise. 2001. Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers for life-threatening arrhythmias. Circulation 103:89-95. Shalaby,F.Y., P.C.Levesque, W.P.Yang, W.A.Little, M.L.Conder, T.Jenkins-West, and M.A.Blanar. 1997. Dominant-negative K v L Q T l mutations underlie the LQT1 form of long QT syndrome. Circulation 96:1733-1736. Smith-Maxwell,C.J., J.L.Ledwell, and R.W.Aldrich. 1998. Uncharged S4 residues and cooperativity in voltage-dependent potassium channel activation. J Gen Physiol 111:421-439. Splawski,L, K.W.Timothy, L.M.Sharpe, N.Decher, P.Kumar, R.Bloise, C.Napolitano, P.J.Schwartz, R.M.Joseph, K.Condouris, H.Tager-Flusberg, S.G.Priori, M.C.Sanguinetti, and M.T.Keating. 2004. Ca(V)l .2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. Cell 119:19-31. 189 Stengl,M., C.Ramakers, D.W.Donker, A.Nabar, A.V.Rybin, R.L.Spatjens, N.T.van der, W.K.Wodzig, K.R.Sipido, G.Antoons, A.F.Moorman, M.A.Vos, and P.G.Volders. 2006. Temporal patterns of electrical remodeling in canine ventricular hypertrophy: focus on IKs downregulation and blunted beta-adrenergic activation. Cardiovasc Res 72:90-100. Tai,K.K. and S.A.N.Goldstein. 1998. The conduction pore of a cardiac potassium channel. Nature 391:605-608. Tiwari-Woodruff,S.K., M.A.Lin , C.T.Schulteis, and D.M.Papazian. 2000. Voltage-dependent structural interactions in the Shaker K + channel. J Gen Physiol 115:123-138. Tomaselli,G.F., D.J.Beuckelmann, H.G.Calkins, R.D.Berger, P.D.Kessler, J.H.Lawrence, D.Kass, A.M.Feldman, and E.Marban. 1994. Sudden cardiac death in heart failure. The role of abnormal repolarization. Circulation 90:2534-2539. Volders,P.G., K.R.Sipido, M.A.Vos, R.L.Spatjens, J.D.Leunissen, E.Carmeliet, and H.J.Wellens. 1999. Downregulation of delayed rectifier K + currents in dogs with chronic complete atrioventricular block and acquired torsades de pointes. Circulation 100:2455-2461. Wang,Q., J.Shen, Z .L i , K.Timothy, G.M.Vincent, S.G.Priori, P.J.Schwartz, and M.T.Keating. 1995a. Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Moi Genet 4:1603-1607. Wang,Q., J.Shen, I.Splawski, D.Atkinson, Z .L i , J.L.Robinson, A.J.Moss, J.A.Towbin, and M.T.Keating. 1995b. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 80:805-811. Yang,W.P., P.C.Levesque, W.A.Little, M.L.Conder, F.Y.Shalaby, and M.A.Blanar. 1997. K v L Q T l , a voltage-gated potassium channel responsible for human cardiac arrhythmias. Proc Natl Acad Sci USA 94:4017-4021. 190 C h a p t e r 6: Discussion 6.1 Novel heterogeneous gating of Kvl.2 There are considerable discrepancies in the reported activation Vm's for K v l . 2 ranging from -43 mV up to +27 mV in the literature (Grissmer et al, 1994; Steidl and Yool, 1999; Hulme et al, 1999; Minor et al, 2000; Koopmann et al, 2001). Most reports describe fast activation (x<10 ms at +40 mV) with a Vm ranging from -15 mV to -43 mV (Steidl et al, 1999; Minor et al, 2000; Koopmann et al, 2001; Scholle et al, 2004), but there are also reports of K v l . 2 channels with Vm% of activation of +27 mV and much slower activation gating (T -25 ms at +40 mV) (Grissmer et al, 1994). This wide range of reported activation properties is unprecedented amongst Kv channels. The reported gating properties of other K v channels fall within a narrow range throughout the literature and any variability, which is relatively small compared to that seen for Wt Kv l . 2 , is often attributed to differences in the heterologous expression systems used (Uebele et al, 1996). This suggests the existence of a previously uncharacterized regulatory mechanism for K v l . 2 channel activation. Unlike any other report, we showed that K v l . 2 channels heterologously expressed in mammalian cell lines exhibit two distinct gating phenotypes or modes. The voltage dependence of activation of Kv l .2 channels appeared to depend on the gating mode. In the 'slow' gating mode, the activation Vm was 16.6 ± 1.1 mV and the time course of activation of channels was at least 20-fold slower than that of channels gating through the 'fast' mode, which had an activation Vm of -18.8 ± 2.3 mV. This showed, for the first time, two modes of K v l . 2 activation gating (Figure 2.1). Moreover, it was possible to switch channels from the 'slow' to the 'fast' mode by applying a prepulse (Figure 2.2). By using a computer simulation, it was shown that the observed heterogeneity of K v l . 2 activation time course and voltage dependence arises from differences in the proportion of channels occupying the 192 'slow' or the 'fast' gating modes (Figure 2.9) and that prepulses switch channels from activation along the 'slow' gating pathway to the 'fast' gating pathway. The heterogeneous gating behavior of K v l . 2 channels persisted, albeit to different ratios, in all the expression systems that were examined by us apart from Xenopus oocytes (Figures 2.1 and 4.2). This novel bimodal gating property of Kv l .2 channels offers an explanation for the inconsistencies observed in the reported time course and voltage dependence of activation in the literature. 6.2 Physiological importance of Kvl.2 modal gating K v l . 2 is abundantly distributed throughout the rodent brain, and recent work has illustrated the juxtaperinodal distribution of this channel (Rasband et ah, 1998; Rasband and Trimmer, 2001). Others have shown the presence of K v l . 2 in the cardiovascular system (Paulmichl et al., 1991; Barry et ah, 1995; Bertaso et ah, 2003). The existence of two gating modes, and the ability of channels to switch from the 'slow' to the 'fast' mode upon depolarization may have important implications for repolarization in these tissues. Unlike other delayed rectifier K v channels, where repetitive trains of depolarization results in the accumulation of channels in P/C-type inactivated states and the reduction of repolarizing current, the unique activation gating features of K v l . 2 channels may enable cumulative activation in vivo. Within trains of repetitive depolarizations, shunting of channels from the 'slow' to the 'fast' gating mode results in a prominent cumulative activation of K v l . 2 channels. In each successive depolarization, rather than cumulative inactivation, more channels switch to the 'fast' gating mode and form a gradually increasing pool of readily activated channels. Although we have no direct evidence as to the physiological role of this behavior, this unique accumulation of available channels raises many intriguing possibilities. In continuously activating cardiac tissue, the balance of'fast' and 'slow' K v l . 2 channels would likely be highly sensitive to heart rate, and could therefore potentially impact action potential duration. In firing bursts of neuronal action potentials, multiple gating modes of Kv l .2 could provide a possible mechanism to enhance the repolarizing K + conductance during bursts of action potentials, contributing to termination of the burst or altered patterns of firing. Clearly, further studies are required to understand the role of this activation-gating switch in intact tissues. Additionally, a number of reports suggest that K v l . 2 channels heteromultimerize with K v l . l , Kv l .4 , Kv l .5 or Kv l . 6 channels (Sheng et al, 1993; X u et al, 1996; Rhodes et al, 1997; Shamotienko et al, 1997; Kerr et al, 2001), in addition to forming homomultimeric channels in rat atrial myocytes (Van Wagoner et al, 1996; Bou-Abboud and Nerbonne, 1999; Nerbonne, 2000) and rat olfactory bulb neurons (Wang et al, 1994). This raises the exciting possibility that heteromultimerization of K v l . 2 subunits with other channel subunits may introduce modal gating into heteromultimeric channels and give rise to an array of different activation kinetics unlike those recorded in heterologous systems from homomultimeric channels. This will potentially increase the repertoire of Kv channels available for fine-tuning of membrane excitability and action potential duration. However, elimination of modal gating by substitution of basic residues for T252 in K v l . 2 raises the question of whether the modal gating persists in heteromultimeric channels since the equivalent residue in all other K v l channels is either arginine or lysine. More recent data suggests that heteromultimeric channels formed by co-expression of 'fast' gating K v l . 2 194 T252R mutant subunits with K v l . 2 wild-type subunits displayed predominantly wild-type behavior with variable gating, suggesting that the bi-modal gating phenotype, in these channels at least, may be a dominant feature. 6.3 The S2-S3 linker plays an important role in regulation of Kvl.2 activation properties Through a chimeric strategy we determined that the transfer of the S2-S3 linker from Kvl .5 into K v l . 2 abolished the heterogeneous gating observed in Wt K v l . 2 channels. Furthermore, transfer of the S2-S3 linker of K v l . 2 to Kv l .5 introduced heterogeneous gating in Kvl .5 channels that otherwise had non-variable activation properties (Figure 2.4 and 2.5). This suggests a crucial role for the S2-S3 linker of K v l . 2 in dictating the gating mode exhibited by these channels. Furthermore, we showed that mutation of the T252 amino acid in the linker of Kv l .2 to positively charged amino acids or a cysteine residue was sufficient to abolish the heterogeneous gating of K v l . 2 and 'trapping' the channels in the 'fast' gating mode (Figure 2.4 and 2.6). How does the S2-S3 linker and in particular the T252 residue modulate channel gating? The S2-S3 linker has previously been reported to regulate the gating properties of other Kv channels. Mutations within the S2-S3 linker of KCNQ1 and hERG channels result in dramatic alterations of the gating properties and have been associated with long QT syndrome (Nakajima et al., 1998; Yamaguchi et al., 2003). Similarly, mutations of the highly conserved glycine at residue 204 in the S2-S3 linker of C N G channels introduces voltage dependence to the activation of an otherwise voltage insensitive channel (Crary et al., 2000). Although these observations set a precedent for a role of the S2-S3 linker in determining activation properties in Kv channels, they provide little insight into the 195 mechanism of coupling between this region and the voltage sensor or the intracellular activation gate. It is possible that the S2-S3 linker has a very distinct interaction with another part of the channel, and is, therefore, directly involved in the normal allosteric transition(s) that occur during channel gating. Although the structure of the S2-S3 linker was not resolved in the recent crystal structure of Kv l . 2 , the predicted position of the S2-S3 linker in the crystal structure of K v l . 2 that positions the linker in a close proximity to the base of the S4 segment. Alternatively, the linker may simply act as a hinge that maintains the necessary distance between the S2 and S3 transmembrane segments to allow gating conformational changes. Additionally, it is well accepted that the S2 and S3 transmembrane helices participate in channel gating via a charge interaction with the S4 segment (Papazian et al, 1995; Seoh et al., 1996; Zhang et al., 2004) and therefore the structural changes in the S2-S3 linker may disrupt coordinated movements of the S2 and S3 transmembrane segments, thereby altering channel gating properties. Unfortunately, the structures of the S2-S3 linker of K v l . 2 was not resolved in the recent crystal structure of K v l . 2 (Long et al, 2005) suggesting that the S2-S3 linker of the channel may have an inherent flexibility, which is consistent with its important role in regulation of channel gating that we have described. 6.4 Coupling of the T l domain to channel gating The T l domain influences numerous fundamental channel functions, including interaction of channels with Kv(3 subunits (Rettig et al, 1994; Sewing et al, 1996; Pongs et al, 1999; Gulbis et al., 2000), interaction with many intracellular signaling molecules (Huang et al, 1993; Huang et al, 1994; Jing et al, 1999; Tsai et al, 1999), and prevention 196 of heteromultimerization between different Kv channel subfamilies (Li et al, 1992; Shen et al, 1993; X u et al, 1995; Shen and Pfaffinger, 1995; Kreusch et al, 1998; Bixby et al, 1999; Kobertz et al, 2000). Furthermore, it has been demonstrated that a wide variety of point mutations and deletion mutations within the T l domain substantially alter the voltage-dependence and kinetics of activation in Kv channels, suggesting conformational coupling of the T l domain and the transmembrane segments of the channel (Kobertz and Miller, 1999; Minor et al, 2000; Cushman et al, 2000; Kurata et al, 2005). Despite the clearly delineated role for the T l domain in cytosolic regulation of channel gating, the mechanism(s) of interaction between the T l domain and the gating elements of the channel remains unclear. Comparison of the crystal structures of the T l domain of Kv l .2 with the T46V mutation to the crystal structures of the Wt domain, indicates that this mutation results in minimal changes in the 3D structure of the channel (Minor et al, 2000). We showed that the T46V mutation in the T l domain of the K v l . 2 channel has no apparent effect on the activation properties of the channel, but induced a marked acceleration in deactivation kinetics (Figure 4.1). Furthermore, we demonstrated that the equivalent mutation in Kvl .5 (T133V) results in no detectable changes in channel properties. By taking advantage of the disparate sensitivity of K v l . 2 and Kvl .5 channels to this mutation, through a chimeric strategy, we determined that there could be a mechanistic link between the T l domain and the S2-S3 linker of K v l . 2 (Figure 4.5). This offers the first report of a mechanism through which the cytoplasmic T l domain may couple to the transmembrane core of the channel. This is an interesting concept as cytoplasmic molecules can potentially interact and modify the structure of the T l domain and at least in the K v l . 2 channel, this modification 197 can lead to alteration of channel gating through interaction with the S2-S3 linker. In an evolutionary sense, differential sensitivity of K v l . 2 and Kvl .5 to mutations in their T l domains indicates that the properties of Kv channels can be modified differently with the same modification of the T l domain, thus allowing for selective modification of only a specific family of channels. 6 . 5 Cytoplasmic regulators of channel gating Our data suggest that the switch between the 'slow' and 'fast' gating modes of Kv l .2 is the result of an interaction with a cytosolic component (Figure 1.7). In the whole-cell configuration, there was a permanent switch from the 'slow' to the 'fast' gating mode over time suggesting that dialysis of a cellular constituent caused the switch. In support of this, prevention of dialysis, by using the perforated-patch clamp configuration, preserved the slow gating mode. Furthermore, excision of inside-out membrane patches containing K v l . 2 channels from the cytosolic environment permanently switched channels from the 'slow' to the 'fast' gating mode. In addition, we observed a significant correlation in whole-cell experiments between the gating mode and cell surface expression, which demonstrated that the 'fast' gating mode is favored when current density is high. This suggests that when there is a high channel density, the modifying cytosolic component is so overwhelmed that the majority of channels exhibit the 'fast' gating mode. Taken together, these data demonstrate that interaction of a cytosolic gating-modifying component favors the slow gating mode of K v l . 2 channels and that prevention of the interaction favors the fast gating mode. Many cytoplasmic regulators of Kv channel function have been described (see section 1.5). Despite substantial effort, we were not able to identify the regulatory cytoplasmic constituent that causes the gating switch in K v l . 2 channels. However, our experiments rule 198 out regulation due to phosphorylation by P K C , P K A , P K G , CaMKII and M L C K , interaction with phospholipids such as P I P 2 , and interaction with polyamines. Initial data examining the effect of kinase inhibitors to probe the possible role of channel phosphorylation in regulation of activation properties illustrated dramatic modulation of the Kv l .2 channel gating as a result of treatment with KN-93, a CaMK II inhibitor; however, further studies showed instead that this drug is an extracellular blocker of Kv l .2 and other Kv channels (Figure 3.1). Does the putative cytoplasmic regulator have to bind within the S2-S3 linker to modify channel gating? Transmembrane linkers have previously been implicated to play a regulatory role in channel gating. For instance, the S1-S2 linker of the K v l . l channel is glycosylated and removal of the incorporated sialic acid group results in alteration of channel activation properties due to charge screening effects (Thornhill et al., 1996). Also, binding of hanatoxin, a protein toxin from spider venom, to the S3-S4 linker of Kv2.1 shifts the voltage dependence of activation in the depolarizing direction (Lee et al., 2003) and interaction of P I P 2 with the S4-S5 linker of KCNQ1 channels is thought to modulate channel gating (Park et al., 2005). Therefore, direct interaction of the cytosolic constituents with the cytoplasmic S2-S3 linker could modify the gating properties of the channel by changing the conformation of the linker or altering the alignment of the S2 and S3 helices relative to the S4 helix. This may alter the electrostatic interactions between the acidic residues in the S2 and S3 helices and the basic residues in S4 and thus alter channel gating (Papazian et al, 1995; Tiwari-Woodruff et al; 1997; Tiwari-Woodruff et al, 2000). Furthermore, it was shown in this thesis that the T l domain may interact with the S2-S3 linker of Kv l .2 and modify its gating (Figure 4.5). Therefore, it is possible that the 199 cytoplasmic constituents that regulate K v l . 2 channel activation may interact with the T l domain and exert their effect through the S2-S3 linker. It would be interesting to perform pull-down assays with the Wt K v l . 2 and T252R mutant Kv l .2 channels, which abolished the 'slow' gating mode, in an attempt to determine possible interacting proteins. However, the interaction between K v l . 2 and the putative interacting compounds may not be strong enough to allow for successful co-precipitation, since excision of inside-out patches permanently switches the gating phenotype from 'slow' to 'fast' (Figure 2.8). 6.6 The role of hydrophobic residues in the S3 helix Genetic screening in a Northern British Columbia aboriginal community with a high prevalence of LQTS (1 in 500 people) revealed a novel missense mutation (V205M) in the S3 transmembrane region of the KCNQ1 channel. We showed that this mutation resulted in a dramatic depolarizing shift in the voltage dependence of activation of the KCNQ1 + KCNE1 channel complex (IKS) and also increased the rate of deactivation. This is predicted to compromise the ability of IKS to contribute to action potential repolarization in these individuals. But how does the V205M mutation at the intracellular end of the S3 transmembrane helix alter channel activation? Can this mutation provide insight into the role of the S2-S3 linker in regulating K v l . 2 activation properties? In the Shaker channel, a tryptophan scan of the transmembrane SI, S2 and S3 helices revealed that one face of these helices packs against the membrane lipid, while the opposing face is packed against other transmembrane domains (Monks et al, 1999; Hong and Miller, 2000). Therefore, mutations on the face of the helix that is packed against other transmembrane domains are expected to have more pronounced effects on the kinetics of the channels. Although there is only 25% 200 homology in the primary sequence between the S3 segments of Shaker and KCNQ1 channels, alignment of these two segments gives a plausible explanation for the dramatic effect of V205M mutation on IKS gating properties. Residue V205 in KCNQ1 aligns with residue A319 in Shaker, which is thought to be packed against other transmembrane domains (Hong et al., 2000). Therefore, the V205M mutation may alter the alignment of the helices around the S3 segment and thus disturb the necessary interactions between the acidic residues in the S2 and S3 helices and the basic residues in the S4 helix (Papazian et al., 1995; Seoh et al., 1996; Tiwari-Woodruff et al., 1997). Interestingly, four other mutations (D202H, I204M, S209F and V215M) have been detected in the S3 region of KCNQ1 channels of patients with LQTS (Napolitano et al., 2005), and with the exception of residue 1204, the other residues are expected to occupy the same face of the helix as V205M and as such would be expected to have major effects on channel biophysical properties. Alteration of channel gating by the V205M point mutation highlights the importance of the positioning of the helices relative to each other, thus underlining the role of the S2-S3 linker in regulation of channel gating as it possibly plays an important role in aligning the S2 and S3 helices and their positioning relative to the rest of the channel, especially the S4 helix. 6.7 Summary Recent structural work has provided many novel insights into the underlying principles of the conformational changes associated with Kv channel activation. However, the details of the mechanisms by which Kv channel function is modulated and fine-tuned are less clear. In this thesis I have demonstrated that the cytoplasmic linker connecting the S2 and S3 transmembrane helices acts as a novel regulator of Kv l .2 channel activation kinetics. This linker could switch channel gating modes through an interaction with 201 cytosolic constituents. Furthermore, cytoplasmic T l domain modulation of channel gating may be mediated by the S2-S3 linker. These studies have identified the S2-S3 cytoplasmic linker as a regulatory site with the ability to convert cytoplasmic signals to modulation of Kv channel gating via hydrophobic packing and changing structural dynamics of the voltage sensing domain. Finally, I have shown that the integrity of the structure of the S3 transmembrane helix is also an important determinant of the activation properties of the KCNQ1 Kv channel. 202 6.8 References Barry,D.M., J.S.Trimmer, J.P.Merlie, and J.M.Nerbonne. 1995. Differential expression of voltage-gated K + channel subunits in adult rat heart: Relation to functional K + channels. Circ Res 77:361-369. Bertaso,F., C.C.Sharpe, B.M.Hendry, and A.F.James. 2003. Expression of voltage-gated K + channels in human atrium. Basic Res Cardiol 97:424-433. Bixby,K.A., M.H.Nanao, N.V.Shen, A.Kreusch, H.Bellamy, P.J.Pfaffinger, and S.Choe. 1999. Zn 2 +-binding and molecular determinants of tetramerization in voltage-gated K + channels. Nature Structural Biology 6:38-43. Bou-Abboud,E. and J.M.Nerbonne. 1999. Molecular correlates of the calcium-independent, depolarization-activated K + currents in rat atrial myocytes. J Physiol (Camb) 517:407-420. Crary,J.L, D.M.Dean, F.Maroof, and A.L.Zimmerman. 2000. Mutation of a single residue in the S2-S3 loop of C N G channels alters the gating properties and sensitivity to inhibitors. J Gen Physiol 116:769-779. Cushman,S.J., M.H.Nanao, A.W.Jahng, D.DeRubeis, S.Choe, and P.J.Pfaffinger. 2000. Voltage dependent activation of potassium channels is coupled to T l domain structure. Nature Structural Biology 7:403-407. Grissmer,S., A.N.Nguyen, J.Aiyar, D.C.Hanson, R.J.Mather, G.A.Gutman, M.J.Karmilowicz, D.D.Auperin, and K.G.Chandy. 1994. Pharmacological characterization of five cloned voltage- gated K + channels, types K v l . l , 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Molec Pharmacol 45:1227-1234. Gulbis,J.M., M.Zhou, S.Mann, and R.MacKinnon. 2000. Structure of the cytoplasmic (3 subunit - T l assembly of voltage-dependent K + channels. Science 289:123-127. Hong,K.H. and C.Miller. 2000. The lipid-protein interface of a Shaker K + channel. J Gen Physiol 115:51-58. 203 Huang,X.-Y., A.D.Morelli, and E.G.Peralta. 1993. Tyrosine kinase-dependent suppression of a potassium channel by the G-protein-coupled m l muscarinic receptor. Cell 75:1145-1156. Huang,X.-Y., A.D.Morelli, and E.G.Peralta. 1994. Molecular basis of cardiac potassium channel stimulation by protein kinase A. Proc Natl Acad Sci USA 91:624-628. Hulme,J.T., E.A.Coppock, A.Felipe, J.R.Martens, and M.M.Tamkun. 1999. Oxygen sensitivity of cloned voltage-gated K + channels expressed in the pulmonary vasculature. Circ Res 85:489-497. Jing,J., D.Chikvashvili, D.Singer-Lahat, W.B.Thornhill, E.Reuveny, and I.Lotan. 1999. Fast inactivation of a brain K + channel composed of K v 1.1 and K vp1.1 subunits modulated by G protein pgamma subunits. EMBOJ 18:1245-1256. Kerr,P.M., O.Clement-Chomienne, K.S.Thorneloe, T.T.Chen, K.Ishii, D.P.Sontag, M.P.Walsh, and W.C.Cole. 2001. Heteromultimeric Kvl .2 -Kvl .5 channels underlie 4-aminopyridine-sensitive delayed rectifier K + current of rabbit vascular myocytes. Circ Res 89:1038-1044. Kobertz,W.R. and C.Miller. 1999. K + channels lacking the 'tetramerization' domain: implications for pore structure. Nature Structural Biology 6:1122-1125. Kobertz,W.R., C.Williams, and C.Miller. 2000. Hanging gondola structure of the T l domain in a voltage-gated K + channel. Biochemistry 39:10347-10352. Koopmann,R., A.Scholle, J.Ludwig, T.Leicher, T.Zimmer, O.Pongs, and K.Benndorf. 2001. Role of the S2 and S3 segment in determining the activation kinetics in Kv2.1 channels. J Membrane Biol 182:49-59. Kreusch,A., P.J.Pfaffinger, C.F.Stevens, and S.Choe. 1998. Crystal structure of the tetramerization domain of the Shaker potassium channel. Nature 392:945-948. Kurata,H.T., K.Doerksen, J.R.Eldstrom, S.Rezazedeh, and D.Fedida. 2005. Separation of P/C- and U-type inactivation pathways in K v l .5 potassium channels. J Physiol. 204 Lee,H.C, J.M.Wang, and K.J.Swartz. 2003. Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels. Neuron 40:527-536. L i , M . , Y.N.Jan, and L.Y.Jan. 1992. Specification of subunit assembly by the hydrophilic amino- terminal domain of the Shaker potassium channel. Science 257:1225-1230. Long,S.B., E.B.Campbell, and R.MacKinnon. 2005. Crystal structure of a mammalian voltage-dependent Shaker family K + channel. Science 309:897-903. Minor,D.L.Jr., Y.F.Lin, B.C.Mobley, A.Avelar, Y.N.Jan, L.Y.Jan, and J.M.Berger. 2000. The polar T l interface is linked to conformational changes that open the voltage-gated potassium channel. Cell 102:657-670. Monks,S.A., D.J.Needleman, and C.Miller. 1999. Helical structure and packing orientation of the S2 segment in the Shaker K + channel. J Gen Physiol 113:415-423. Nakajima,T., T.Furukawa, T.Tanaka, Y.Katayama, R.Nagai, Y.Nakamura, and M.Hiraoka. 1998. Novel mechanism of hERG current suppression in LQT2: shift in voltage dependence of hERG inactivation. Circ Res 83:415-422. Napolitano,C, S.G.Priori, P.J.Schwartz, R.Bloise, E.Ronchetti, J.Nastoli, G.Bottelli, M.Cerrone, and S.Leonardi. 2005. Genetic testing in the long QT syndrome: development and validation of an efficient approach to genotyping in clinical practice. JAMA 294:2975-2980. Nerbonne,J.M. 2000. Molecular basis of functional voltage-gated K + channel diversity in the mammalian myocardium. J Physiol (Camh) 525:285-298. Papazian,D.M., X.M.Shao, S.-A.Seoh, A.F.Mock, Y.Huang, and D.H.Wainstock. 1995. Electrostatic interactions of S4 voltage sensor in Shaker K + channel. Neuron 14:1293-1301. Park,K.H., J.Piron, S.Dahimene, J.Merot, I.Baro, D.Escande, and G.Loussouarn. 2005. Impaired KCNQ1-KCNE1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome. Circ Res 96:730-739. 205 Paulmichl,M., P.Nasmith, R.Hellmiss, K.Reed, W.A.Boyle, J.M.Nerbonne, E.G.Peralta, and D.E.Clapham. 1991. Cloning and expression of a rat cardiac delayed rectifier potassium channel. Proc Natl Acad Sci USA 88:7892-7895. Pongs,0., T.Leicher, M.Berger, J.Roeper, R.Bahring, D.Wray, K.P.Giese, A.J.Silva, and J.F.Storm. 1999. Functional and molecular aspects of voltage-gated K + channel (3 subunits. Ann NY Acad Sci 868:344-355. Rasband,M.N. and J.S.Trimmer. 2001. Subunit composition and novel localization of K + channels in spinal cord. J Comp Neurol 429:166-176. Rasband,M.N., J.S.Trimmer, T.L.Schwarz, S.R.Levinson, M.H.Ellisman, M.Schachner, and P.Shrager. 1998. Potassium channel distribution, clustering, and function in remyelinating rat axons. J Neurosci 18:36-47. RettigJ., S.H.Heinemann, F.Wunder, C.Lorra, D.N.Parcej, J.O.Dolly, and O.Pongs. 1994. Inactivation properties of voltage-gated K + channels altered by presence of P-subunit. Nature 369:289-294. Rhodes,K.J., B.W.Strassle, M.M.Monaghan, Z.Bekele-Arcuri, M.F.Matos, and J.S.Trimmer. 1997. Association and colocalization of the K v p l and Kvp2 p-subunits with K v l ct-subunits in mammalian brain K + channel complexes. J Neurosci 17:8246-8258. Scholle,A., S.Dugarmaa, T.Zimmer, M.Leonhardt, R.Koopmann, B.Engeland, O.Pongs, and K.Benndorf. 2004. Rate-limiting reactions determining different activation kinetics of Kv l .2 andKv2.1 channels. JMembr Biol 198:103-112. Seoh,S.A., D.Sigg, D.M.Papazian, and F.Bezanilla. 1996. Voltage-sensing residues in the S2 and S4 segments of the Shaker K + channel. Neuron 16:1159-1167. Sewing,S., J.Roeper, and O.Pongs. 1996. K v p i subunit binding specific for Shaker-related potassium channel a subunits. Neuron 16:455-463. Shamotienko,O.G., D.N.Parcej, and J.O.Dolly. 1997. Subunit combinations defined for K + channel K v l subtypes in synaptic membranes from bovine brain. Biochemistry 36:8195-8201. 206 Shen,N.V., X.Chen, M.M.Boyer, and P.J.Pfaffmger. 1993. Deletion analysis of K +channel assembly. Neuron 11:67-76. Shen,N.V. and P.J.Pfaffmger. 1995. Molecular recognition and assembly sequences involved in the subfamily-specific assembly of voltage-gated K + channel subunit proteins. Neuron 14:625-633. Sheng,M., Y.J.Liao, Y.N.Jan, and L.Y.Jan. 1993. Presynaptic A-current based on heteromultimeric K + channels detected in vivo. Nature 365:72-75. SteidlJ.V. and A.J.Yool. 1999. Differential sensitivity of voltage-gated potassium channels Kvl .5 and K v l . 2 to acidic pH and molecular identification of pH sensor. Molec Pharmacol 55:812-820. Thornhill,W.B., M.B.Wu, X.Q.Jiang, X .Y.Wu, P.T.Morgan, and J.F.Margiotta. 1996. Expression of K v l . 1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function. J Biol Chem 271:19093-19098. Tiwari-WoodruffS.K., M.C.A.Lin , C.T.Schulteis, and D.M.Papazian. 2000. Voltage-dependent structural interactions in the Shaker K + channel. J Gen Physiol 115:123-138. Tiwari-WoodruffS.K., C.T.Schulteis, A.F.Mock, and D.M.Papazian. 1997. Electrostatic interactions between transmembrane segments mediate folding of Shaker K + channel subunits. Biophys J12:\A%9-\500. Tsai,W., A.D.Morielli, T.G.Cachero, and E.G.Peralta. 1999. Receptor protein tyrosine phosphatase alpha participates in the m l muscarinic acetylcholine receptor-dependent regulation of K v l .2 channel activity. EMBO J18:109-118. Uebele,V.N., S.K.England, A.Chaudhary, M.M.Tamkun, and D.J.Snyders. 1996. Functional differences in Kvl .5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv|32.1 subunits. J Biol Chem 271:2406-2412. Van Wagoner,D.R., M.Kirian, and M.Lamorgese. 1996. Phenylephrine suppresses outward K + currents in rat atrial myocytes. Am J Physiol Heart Circ Physiol 271 :H937-H946. 207 Wang,H., D.D:Kunkel, P.A.Schwartzkroin, and B.L.Tempel. 1994. Localization of Kvl .1 and K v l . 2 , two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain. J Neurosci 14:4588-4599. Xu,H.D., J.E.Dixon, D.M.Barry, J.S.Trimmer, J.P.Merlie, D.McKinnon, and J.M.Nerbonne. 1996. Developmental analysis reveals mismatches in the expression of K + channel a subunits and voltage-gated K + channel currents in rat ventricular myocytes. J Gen Physiol 108:405-419. Xu,J., W.F.Yu, Y.N.Jan, L .YJan, and M . L i . 1995. Assembly of voltage-gated potassium channels - Conserved hydrophilic motifs determine subfamily-specific interactions between the a-subunits. J Biol Chem 21'0:247'61 -247'68. Yamaguchi,M., M.Shimizu, H.Ino, H.Terai, K.Hayashi, H.Mabuchi, N.Hoshi, and H.Higashida. 2003. Clinical and electrophysiological characterization of a novel mutation (F193L) in the KCNQ1 gene associated with long QT syndrome. Clin Sci (Lond) 104:377-382. Zhang,M., J.Liu, and G.N.Tseng. 2004. Gating charges in the activation and inactivation processes of the hERG channel. J Gen Physiol 124:703-718. 208 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0100738/manifest

Comment

Related Items