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Moment-to-moment regulation of voltage-gated potassium channel function Baronas, Victoria 2018

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   MOMENT-TO-MOMENT REGULATION OF VOLTAGE-GATED POTASSIUM CHANNEL FUNCTION   by  VICTORIA BARONAS  B.Sc. (Hons), The University of British Columbia, 2013     A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in   THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Pharmacology)       THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)     July 2018  © Victoria Baronas, 2018ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Moment-to-moment regulation of a voltage-gated potassium channel  submitted by Victoria Baronas  in partial fulfillment of the requirements for the degree of Doctor of philosophy in Pharmacology  Examining Committee: Harley Kurata Co-supervisor Filip van Petegem Co-supervisor  Filip Van Petegem Supervisory Committee Member Ann Marie Craig University Examiner Brian MacVicar University Examiner  Additional Supervisory Committee Members: Lynn Raymond Supervisory Committee Member Shernaz Bamji Supervisory Committee Member Robert Molday Supervisory Committee Member iii  ABSTRACT  Kv1.2 channels are prominently expressed in neurons where they help to set the threshold of action potential firing. While we have a good understanding of the mechanism of voltage sensing and gating, we have comparatively little information on the compendium of regulatory molecules that can impact Kv1.2 expression and function. Kv1.2 channels are subject to a unique mechanism of regulation whereby a train of brief, repetitive depolarizations elicit increasing amounts of current, a phenotype we term ‘use-dependent activation’. In heterologous cells expressing Kv1.2 and primary hippocampal cultures from rats, there is remarkable diversity in this phenotype. While use-dependent activation is absent in all other Kv1 channels, it persists in heteromeric channels containing at least one Kv1.2 subunit. Exposing cells expressing Kv1.2 to reducing conditions causes a dramatic shift in use-dependent activation where there is very little or no current elicited by the first pulse, but over the course of the train there is a hundred-fold or more increase in current. Additionally, reducing conditions cause a depolarizing shift in the activation curve of Kv1.2 by +64 mV. Taken together, we postulate that use-dependence arises from an extrinsic, redox-sensitive inhibitory regulator that associates with Kv1.2 preferentially in the closed, reduced state.  We have identified a new regulator of Kv1.2 function, Slc7a5, an amino acid transporter. Co-expression of these two proteins decreases Kv1.2 expression and produces a hyperpolarizing shift of the activation and inactivation curves. Together these effects result in Kv1.2 channels being caught in an ‘inactivation trap’. These effects of Slc7a5 can be rescued by co-expressing a third protein, Slc3a2, which is known to heterodimerize with the Slc7a5 channel. Using BRET we iv  show that Slc7a5 and Kv1.2 can be within 10 nm of each other. Other Kv1 channels we have tested (Kv1.1 and Kv1.5) are insensitive to the activation shift produced by Slc7a5, however Kv1.1 channels are exquisitely sensitive to current inhibition.  Overall, the work in this thesis expands our knowledge of how Kv1.2 channels are regulated and opens the door to examining how these interactions contribute to normal neuronal function.      v  LAY SUMMARY  Brain cells communicate with each other through electrical signals. One family of proteins that are key in generating precise electrical signals are potassium channels. Through many structural and functional studies, the details of how these proteins function is well understood. What is not well known is how these proteins behave in a physiological system where they interact with many other proteins and molecules. To address this gap in knowledge, we have examined the interacting network of a specific potassium channel that is critical for normal electrical activity in the brain. We have characterized in detail a mechanism which allows this channel to respond to changes in neuronal activity on a moment-to-moment basis. We have also identified a novel interactor, a protein that transports amino acids. We demonstrate how a potassium channel can be strongly influenced by its environment, and open the door to examine the physiological ramifications.    vi  PREFACE  CHAPTER 3: USE-DEPENDENT ACTIVATION OF NEURONAL KV1.2 CHANNEL COMPLEXES Harley Kurata and I conceived the project. I performed all electrophysiology experiments except Figure 3.2 and 3.9 which were done by Yury Vilin and Robin Kim, and Figure 3.1 which was done by Brandon McGuinness and I. Runying made all cDNA constructs. Stefano Brigidi and Rachel Gomm isolated and plated rat hippocampal neurons, and I recorded from them. I analyzed all data. This chapter is adapted from: Baronas VA, McGuinness BR, Brigidi GS, Gomm Kolisko RN, Vilin YY, Kim RY, Lynn FC, Bamji SX, Yang R, Kurata HT (2015). Use-dependent activation of neuronal Kv1.2 channel complexes. J Neurosci. 35(8):3515-24. Baronas VA, Yang R, Vilin YY, Kurata HT. Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels. Channels (Austin). 10(2):158-66.  CHAPTER 4: EXTRACELLULAR REDOX SENSITIVITY OF KV1.2 POTASSIUM CHANNELS Harley Kurata and I conceived the project, Filip van Petegem conceived of experiment shown in Figure 4.8A. I performed all electrophysiological recordings and GRX1-roGFP measurements, and Runying Yang and I made all cDNA constructs. I analyzed all data. This chapter is adapted from: Baronas VA, Yang R, Kurata HT. Extracellular redox sensitivity of Kv1.2 potassium channels. Sci Rep. 7(1):9142.  CHAPTER 5: SLC7A5 REGULATION OF KV1.2 POTASSIUM CHANNELS BY AN INACTIVATION GATING TRAP MECHANISM Harley Kurata and I conceived the project. Runying Yang performed the crosslinking and co-immunoprecipitation assay, and the mass spectrometry was done at the UBC Proteomics Core. Runying Yang and I made all cDNA constructs. I performed all electrophysiology and BRET experiments. Harley Kurata and I analyzed the mass spectrometry data and I analyzed the electrophysiology and BRET data. This chapter is adapted from: Baronas VA, Yang R, Kurata HT. Slc7a5 regulation of Kv1.2 potassium channels by an inactivation trap mechanism. In submission.  CHAPTER 6: SPECIFICITY OF KV1.2 AND SLC7A5 INTERACTIONS Harley Kurata and I conceived the project. I performed all electrophysiology experiments and Runying Yang and I made all the cDNA constructs. Lucy Chin assisted with construction and recording from Kv1.2-Kv1.5 chimeric channels. I analyzed all data. vii  TABLE OF CONTENTS  ABSTRACT ............................................................................................................................ iii LAY SUMMARY ...................................................................................................................... v PREFACE  ............................................................................................................................. vi TABLE OF CONTENTS ........................................................................................................... vii LIST OF FIGURES ................................................................................................................... xi LIST OF ABBREVIATIONS ...................................................................................................... xiii ACKNOWLEDGEMENTS ......................................................................................................... xv CHAPTER 1: INTRODUCTION ............................................................................................. 1 POTASSIUM CHANNEL STRUCTURE AND GATING ...................................................................... 2 Cloning voltage-gated potassium channels............................................................................. 2 Overview of Kv channel structure and tetrameric assembly ................................................... 2 Pore domain and channel activation ....................................................................................... 4 Voltage-sensing domain and voltage-dependent activation .................................................. 6 Inactivation .............................................................................................................................. 8 Mechanism of voltage sensing and gating of Kv1.2 .............................................................. 10 PHYSIOLOGICAL FUNCTION OF KV1.2 ....................................................................................... 12 Localization and function ....................................................................................................... 12 Kv1.2 channels in disease ...................................................................................................... 15 Other Kv1 family members .................................................................................................... 19 GENERATION OF DIVERSITY IN Kv1.2 CHANNELS ..................................................................... 24 Kv1.2 channel heteromerization ............................................................................................ 24 Post-translational modifications ........................................................................................... 25 Interacting proteins ............................................................................................................... 27 AUXILIARY PROTEINS AND THEIR NON-CANONICAL FUNCTIONS............................................. 31 a. Sodium channel β subunit (Navβ) ...................................................................................... 31 b. Potassium channel interacting protein (KChIP) ................................................................. 32 c. Fragile X Mental Retardation Protein (FMRP) ................................................................... 34 d. Calmodulin (CaM) .............................................................................................................. 35 V. MOMENT-TO-MOMENT REGULATION OF ION CHANNELS .................................................. 36 N-type Voltage-gated calcium channels ................................................................................ 37 AMPA receptor and stargazin................................................................................................ 38 Use-dependent activation of Kv1.2 channels ........................................................................ 39 THESIS OVERVIEW ..................................................................................................................... 40 CHAPTER 2: METHODS .................................................................................................... 41 viii  Constructs and expression cell lines ...................................................................................... 41 Electrophysiology ................................................................................................................... 42 MTSET delivery ....................................................................................................................... 43 Hippocampal cell culture preparation and electrophysiology ............................................... 43 Western blot analysis ............................................................................................................ 44 Grx1-roGFP ............................................................................................................................ 44 Redox potential measurements ............................................................................................. 45 Data analysis ......................................................................................................................... 45 Co-immunoprecipitation ........................................................................................................ 46 Mass spectrometry ................................................................................................................ 47 Mass spectrometry data analysis and selecting candidate interacting proteins .................. 48 Bioluminescence resonance energy transfer ......................................................................... 48 Fluorescence-activated flow cytometry ................................................................................. 49 CHAPTER 3: USE-DEPENDENT ACTIVATION OF NEURONAL Kv1.2 CHANNEL COMPLEXES .. 50 INTRODUCTION ......................................................................................................................... 50 RESULTS ..................................................................................................................................... 52 Prepulse potentiation of Kv1.2 channels ............................................................................... 52 Prepulse potentiation generates use-dependent activation ................................................. 54 Use-dependent activation is transferred to heteromeric channels ....................................... 58 Functional demonstration of dominance of Kv1.2 use-dependence in dimeric channels ..... 60 Use-dependent activation in heteromeric channels containing one Kv1.2 subunit .............. 63 Use-dependent activation is evident in a primary culture of hippocampal neurons ............ 64 Model for Kv1.2 regulation .................................................................................................... 68 Duty-cycle dependence of Kv1.2 potentiation ....................................................................... 70 Structure-function analysis of the S2-S3 linker region........................................................... 72 Effects of S2-S3 linker mutations on surface expression ....................................................... 75 DISCUSSION ............................................................................................................................... 77 Kv1.2 regulation of excitability in the hippocampus ............................................................. 78 Ion channel responses to repetitive stimuli ........................................................................... 79 Integration of signaling mechanisms in heteromeric Kv channels ........................................ 81 Conclusion .............................................................................................................................. 82 CHAPTER 4: EXTRACELLULAR REDOX SENSITIVITY OF Kv1.2 POTASSIUM CHANNELS ........ 83 INTRODUCTION ......................................................................................................................... 83 RESULTS ..................................................................................................................................... 85 Redox conditions strongly regulate voltage-dependence of Kv1.2 ....................................... 85 DTT accelerates recovery of use-dependent gating .............................................................. 89 Kv1.2[T252R] weakens use-dependent activation and redox sensitivity .............................. 92 Kv1.2 recruits redox sensitivity to heteromeric channel complexes ...................................... 95 Extracellular redox environment controls Kv1.2 channel gating ........................................... 97 ix  Cysteine residues in Kv1.2 do not control redox sensitivity ................................................. 100 Calibrated redox potential measurements of Kv1.2 use-dependent gating ....................... 103 Rescue of normal use-dependence with redox in an epilepsy-causing mutant of Kv1.2 .... 104 DISCUSSION ............................................................................................................................. 106 Variability of use-dependent activation of Kv1.2 channels ................................................. 106 Role of Kv1.2 in diseases of electrical hyperexcitability ...................................................... 107 Redox regulation of use-dependent activation ................................................................... 108 Many ion channels are regulated by extracellular redox .................................................... 109 Conclusion ............................................................................................................................ 110 CHAPTER 5: SLC7A5 REGULATION OF Kv1.2 POTASSIUM CHANNELS BY AN INACTIVATION GATING TRAP MECHANISM ............................................................................................... 112 INTRODUCTION ....................................................................................................................... 112 RESULTS ................................................................................................................................... 114 Identification of novel Kv1.2-associated proteins ............................................................... 114 Suppression of Kv1.2 currents by Slc7a5 ............................................................................. 116 Slc7a5 induces a prominent hyperpolarizing shift of Kv1.2 activation ................................ 119 Slc7a5 promotes Kv1.2 inactivation .................................................................................... 121 Kv1.2 and Slc7a5 are in close physical proximity................................................................. 123 Variable responses of a tripartite Kv1.2:Slc7a5:Slc3a2 complex ......................................... 125 Disease-linked mutants of Slc7a5 attenuate the WT-dependent current reduction and activation shift ..................................................................................................................... 128 Disease-linked mutants of Kv1.2 are extremely susceptible to Slc7a5 ................................ 129 DISCUSSION ............................................................................................................................. 132 Identification of novel interactors of the Kv1.2 channel ..................................................... 132 ‘Inactivation trap’ is a novel mechanism of Kv1.2 regulation ............................................. 133 Reconciling heterogeneity of Kv1.2 disease mutants that lead to the same disease phenotype ............................................................................................................................ 134 Pleiotropic functions of the Slc7a5 transporter ................................................................... 135 CHAPTER 6: DISSECTING THE SPECIFICITY OF Kv1.2 AND SLC7A5 INTERACTIONS ............ 137 INTRODUCTION ....................................................................................................................... 137 RESULTS ................................................................................................................................... 139 Slc7a5 inhibits Kv1.1 but does not affect Kv1.5 ................................................................... 139 Slc7a5 sensitivity depends on heteromeric channel composition ....................................... 141 The S1 segment mediates channel interaction with Slc7a5 ................................................ 143 Ile164 is a key residue in mediating the interaction between Kv1.2 and Slc7a5 ................ 145 Mutations of Kv1.1[Val168] affect its sensitivity to Slc7a5 ................................................. 147 Kv1.5 Ala251 mutations cannot reconstitute Slc7a5 sensitivity .......................................... 149 Slc7 family screen for functional effects on Kv1.2 ............................................................... 149 x  Slc7a5-Slc7a6 chimeras isolate the key regions that determine the Slc7a5 effect on Kv1.2 ............................................................................................................................................. 150 Slc7a5 alters redox sensitivity of the Kv1.2 channel ............................................................ 151 DISCUSSION ............................................................................................................................. 154 Structural insights into the interaction between Kv1.1/Kv1.2 and Slc7a5 .......................... 154 Slc7a5 has partial gating effects on Kv1.2-containing heteromeric channels .................... 155 Use-dependent activation competes with Slc7a5 on Kv1.2 ................................................. 155 Conclusion ............................................................................................................................ 157 CHAPTER 7: DISCUSSION ............................................................................................... 158 REGULATORY FACTOR MEDIATING USE-DEPENDENT ACTIVATION ....................................... 159 Evidence for an extrinsic regulatory protein ........................................................................ 159 Evidence for an inhibitory regulatory protein ...................................................................... 160 Evidence for a transmembrane, redox sensitive regulatory protein ................................... 163 What is left to learn about use-dependent activation? ....................................................... 165 SLC7A5 AND KV1.2 INTERACTION AND PHYSIOLOGICAL CONSEQUENCES ............................ 168 Expanding the role of Slc7a5 ............................................................................................... 168 Significance of the Slc7a5-Kv1.2 interaction ....................................................................... 170 What is left to learn about the Slc7a5-Kv1.2 interaction? .................................................. 170 SUMMARY AND CONCLUSION ................................................................................................ 171 BIBLIOGRAPHY .................................................................................................................. 173    xi  LIST OF FIGURES  Figure 1.1. Kv1.2 channel structural elements. .............................................................................. 4 Figure 1.2. Disease-causing mutations of the Kv1.2 channel. ...................................................... 18 Figure 1.3. Kv1.2 channel modulating interactions and modifications. ....................................... 26 Figure 3.1. Prepulse potentiation of Kv1.2 channels. ................................................................... 53 Figure 3.2. Use-dependent activation is observed in Kv1.2 but not other Kv1 channel subtypes........................................................................................................................................................ 56 Figure 3.3. Use-dependent activation persists in Kv1.2-containing heteromeric channel complexes with other Kv1 channel subtypes. .............................................................................. 59 Figure 3.4. Functional contribution of Kv1.2 and Kv1.5 to heteromeric channel complexes that exhibit use-dependent activation. ................................................................................................ 62 Figure 3.5. Use-dependent activation persists in channels containing a single Kv1.2 subunit. ... 64 Figure 3.6. Isolation of tityustoxin sensitive currents in dissociated hippocampal neurons. ...... 65 Figure 3.7. Variability of gating properties of tityustoxin sensitive channels in dissociated hippocampal neurons. .................................................................................................................. 67 Figure 3.8. Hypothetical model for interpretation of use-dependent activation of Kv1.2 channels. ....................................................................................................................................... 70 Figure 3.9. Frequency and pulse duration effects on use-dependent activation. ....................... 71 Figure 3.10. Effects of S2-S3 linker mutations on use-dependent activation. ............................. 73 Figure 3.11. Substitution of Thr at the 252 equivalent position of Kv1.2 into use-dependent activation insensitive channels. .................................................................................................... 74 Figure 3.12. Cell surface expression and current density for a panel of different Kv1.2 S2-S3 linker mutants. .............................................................................................................................. 76 Figure 3.13. Comparison of peak current to use-dependent activation in Kv1.2, Kv1.2[T252S] and Kv1.2[T252V]. ......................................................................................................................... 77 Figure 4.1. Reducing conditions promote use-dependent activation. ......................................... 87 Figure 4.2. Oxidizing agents do not affect use-dependent activation. ......................................... 89 Figure 4.3. Time dependent recovery of inhibited gating mode. ................................................. 91 Figure 4.4. Quantification of recovery of inhibited gating mode in ambient redox and reducing conditions. ..................................................................................................................................... 92 Figure 4.5. Weakened use-dependence and redox sensitivity in Kv1.2[T252R] channels. .......... 94 Figure 4.6. Quantification of recovery of inhibited gating mode in Kv1.2[T252R] channels. ...... 95 Figure 4.7. Redox-sensitive use-dependence is transferable in heteromeric channels containing Kv1.2 subunits. .............................................................................................................................. 96 Figure 4.8. Extracellular redox environment modulates Kv1.2 use-dependent activation. ......... 98 Figure 4.9. Dose-dependent shift in use-dependence with membrane impermeant reducing agents. ......................................................................................................................................... 100 Figure 4.10. Systematic mutagenesis of transmembrane cysteine residues in Kv1.2. .............. 102 Figure 4.11. Calibrated effects of redox potential on Kv1.2 use-dependent activation. ........... 104 xii  Figure 4.12. Kv1.2[R297Q] hyerpolarizes the activation curve and slows deactivation kinetics...................................................................................................................................................... 105 Figure 4.13. Use-dependent activation of Kv1.2[R297Q] in ambient and reducing extracellular conditions. ................................................................................................................................... 106 Figure 5.1. Mass spectrometry and screening potential Kv1.2 interacting proteins. ................ 116 Figure 5.2. Effects of Slc7a5 on Kv1.2 expression and current density. ..................................... 118 Figure 5.3. Hyperpolarization disinhibits Kv1.2 co-expressed with Slc7a5. ............................... 119 Figure 5.4. Slc7a5 co-expression hyper-polarizes the Kv1.2 activation curve. ........................... 120 Figure 5.5. Other Slc7 and Kv1 subtypes do not alter gating. .................................................... 121 Figure 5.6. Slc7a5 influences inactivation of Kv1.2..................................................................... 122 Figure 5.7. Slc7a5 shifts steady state inactivation of Kv1.2[V381T]. .......................................... 123 Figure 5.8. Measurement of the proximity of Slc7a5 and Kv1.2 with bioluminescence resonance energy transfer (BRET). ............................................................................................................... 125 Figure 5.9. Using split YFP constructs to measure competition for Slc7a5 between Kv1.2 and Slc3a2. ......................................................................................................................................... 127 Figure 5.10. Disease-linked Slc7a5 mutations have attenuated effects on Kv1.2...................... 129 Figure 5.11. Kv1.2 disease-linked mutations are strongly suppressed by Slc7a5. ..................... 131 Figure 5.12. Model demonstrating the effect of Slc7a5 on Kv1.2. ............................................. 135 Figure 6.1. Kv1.1 channels are highly susceptible to Slc7a5 regulation. .................................... 140 Figure 6.2. Kv1.5 channels are insensitive to Slc7a5 regulation. ................................................ 141 Figure 6.3. Heteromeric channels containing Kv1.2 can restore some Slc7a5 sensitivity. ........ 142 Figure 6.4. Testing chimeric Kv1.2/Kv1.5 channels to introduce Slc7a5 sensitivity into Kv1.5. . 144 Figure 6.5. Testing Kv1.2-Kv1.5 chimeric channels for Slc7a5 effects. ....................................... 145 Figure 6.6. Testing S1 mutations of Kv1.2 to isolate a key residue in mediating Slc7a5 sensitivity...................................................................................................................................................... 146 Figure 6.7. Mutation of Kv1.2 Ile164Ala abolishes Slc7a5 sensitivity in a dose-dependent manner. ....................................................................................................................................... 147 Figure 6.8. Kv1.1 Val168Ala diminishes Slc7a5 sensitivity. ......................................................... 148 Figure 6.9. Kv1.5 Val168 mutation cannot introduce Slc7a5 sensitivity. ................................... 149 Figure 6.10. Screening Slc7 family for effects on Kv1.2 activation and current. ........................ 150 Figure 6.11. Slc7a5-7a6 chimeric channels isolate the TM1 and TM1-2 linker as the effectors of Kv1.2 gating shift. ........................................................................................................................ 151 Figure 6.12. Slc7a5 attenuates the redox sensitivity of Kv1.2 channels. ................................... 153 Figure 6.13. Slc3a2 and Slc3a2+Slc7a5 leave redox sensitivity of Kv1.2 unaffected. ................. 154 Figure 6.14. Slc7a5 and use-dependence regulator likely have overlapping binding sites. ....... 156 Figure 7.1. Comparison of use-dependent potentiation of AMPA receptors and disinhibition of voltage-gated potassium channels with Kv1.2. .......................................................................... 162 Figure 7.2. Comparison of time-dependent changes in use-dependent activation. .................. 165 Figure 7.3. mRNA expression patterns of Slc7a5, Kv1.2, and Kv1.1 in the central nervous system in mice. ........................................................................................................................................ 169  xiii  LIST OF ABBREVIATIONS  AIS – axon initial segment AMPAR - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AP – action potential BRET – bioluminescence resonance energy transfer CaM – calmodulin  Cav – voltage-gated calcium channel CySS – cystine  DTT – dithiothreitol  EC50 – concentration to reach half maximal response FMRP – fragile X mental retardation protein GSH – reduced glutathione GSSG – oxidized glutathione HEK – human embryonic kidney IKA – A-type potassium current IKD – D-type potassium current  IKto – transient outward potassium current IKUR – ultrarapid potassium current k – slope factor KChIP – potassium channel interacting protein Kv – voltage-gated potassium channel α subunit Kvβ – voltage-gated potassium channel β subunit LM – mouse Itk fibroblast cells MNTB – medial nucleus of the trapezoid body ms - milliseconds MTSET – 2-(Trimethylammonium)ethyl methanethiosulfonate mV – millivolt  N – Hill coefficient nA - nanoamps NADP - Nicotinamide adenine dinucleotide phosphate Nav – voltage-gated sodium channel α subunit Navβ – voltage-gated sodium channel β subunit pA – picoamps  PA – phosphatidic acid PIP2 – Phosphatidylinositol 4,5-bisphosphate  Po – open probability RMP – resting membrane potential s – seconds  Sig-1R – sigma 1 receptor TARP – transmembrane AMPA receptor regulatory protein V1/2 – half activation voltage WT – wild type  xiv  Amino acid name Three letter abbreviation One letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamine Gln Q Glutamate Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tyrosine Tyr Y Tryptophan Trp W Valine Val V     xv  ACKNOWLEDGEMENTS The work presented in this thesis is one of my proudest achievements and would have been impossible if not for all of the individuals that have shown me support and encouragement along the way. First and foremost, I would like to thank my supervisor, Harley Kurata for his guidance, scientific (and life) advice and his tireless dedication to the project at every step along the way. I would also like to thank my co-supervisor Filip van Petegem for his patience and helpful suggestions, and my supervisory committee, Lynn Raymond, Shernaz Bamji and Robert Molday for their input and discussion. A special thank you to Runying Yang for her tireless help in the lab. And of course my sincerest gratitude to the UBC MD/PhD program and pharmacology departments at the University of British Columbia and University of Alberta, especially Jane Lee, Wynne Leung, and Jennifer Beattie for their help in easing the transition from Vancouver to Edmonton and back. To all of the people that have made this a fun ride (RK, CW, AW, WL, LM, MW, ET) from all the nights out, all the eating and especially all the commiserating, you all have made this a memorable journey and you all rock! My eternal gratitude to my family for sticking through the highs and lows of life with me. Last and most of all, thank you to my other half, CM.  1  CHAPTER 1: INTRODUCTION   Voltage gated potassium (Kv) channels are integral to electrical signaling in excitable cells, where they regulate action potential threshold, shape and frequency. Through work done in many laboratories since the seminal work of Hodgkin and Huxley (Hodgkin and Huxley, 1952, 1952), we now understand in great detail the structure and mechanisms of voltage sensing and gating of many different types of potassium channels. In contrast, comparatively little is known about the full compendium of potential interacting partners of the many different types of channels, and how these interactions contribute to normal physiological function. The focus of this thesis is to outline two regulatory mechanisms which have a profound impact on Kv1.2 channel function, much more so than other known regulators. The objective of this introduction is to provide an overview of what is known about the structure and gating of the prototypical potassium channel, Shaker, and its mammalian homologues in the Kv1 family. The known interacting partners of Kv1.2 and their effects on channel function are highlighted to demonstrate the substantial impact regulatory molecules can have on ion channels. I also describe a variety of known regulators of ion channel function, to showcase that these factors often play key roles in cells, beyond their function in ion channel regulation.     2  POTASSIUM CHANNEL STRUCTURE AND GATING Cloning voltage-gated potassium channels The Shaker channel was the first potassium channel to be cloned from Drosophila via chromosome walking (Kamb et al., 1987; Papazian et al., 1987; Tempel et al., 1987). This discovery soon multiplied as new and distinct potassium channel genes were isolated from Drosophila, some of which are splice variants of the Shaker gene, including ShA, ShB, ShC and ShD (Schwarz et al., 1988; Timpe et al., 1988) whereas others are encoded by independent genes (Shab, Shaw, Shal) (Butler et al., 1989). In just a few short years in the late 80s and early 90s the bridge between Drosophila and mammalian potassium channels was bridged as many laboratories cloned potassium channel homologues in rats, mice, and humans (Chandy et al., 1990; Christie et al., 1989; Stühmer et al., 1989; Swanson et al., 1990; Tempel et al., 1988). The Shaker homologues in mammals correspond to the Kv1 family of ion channels, the Shab family to Kv2, Shaw to Kv3, and Shal to Kv4. Mammalian channels in these subfamilies are encoded by individual genes, rather than being splice variants of one gene (Chandy et al., 1990). At present, 12 families of potassium channels comprising ~70 distinct human genes have been discovered, revealing one piece of the puzzle of how electrical diversity between different cell types is generated (Gutman et al., 2005; Miller, 2000). Overview of Kv channel structure and tetrameric assembly Before the advent of crystallography of membrane proteins, hydropathy plots and sequence alignment suggested a six transmembrane domain topology (S1-S6) for Kv channels, with intracellular N and C termini (Pongs et al., 1988; Schwarz et al., 1988). Remarkably, the degree of sequence conservation between select mammalian and Drosophila potassium channels is 68% 3  based on DNA sequence, and 82% based on amino acid sequence, even though the common ancestor of rat and Drosophila dates back more than 600 million years. The implication of this observation is that there must have been substantial pressure for these channels to survive intact through millions of years of evolution in order to serve a fundamental purpose in the life of these distinct organisms (Baumann et al., 1988). In most cases, the highly conserved portions are the hydrophobic, transmembrane domains whereas the linkers between S1-S2 and S3-S4, and the N and C termini are much more variable (Stühmer et al., 1989).  Four potassium channel subunits assemble symmetrically around a central ion conducting pore to form a functional channel (Long et al., 2005a; MacKinnon, 1991). These four subunits are arranged with the S5 and S6 of each subunit lining a central, ion-conducting pore, and the S1-S4 voltage-sensing domain at the periphery (Doyle et al., 1998). The subunit composition of a channel is strictly regulated, limited to subunits that are in the same Kv channel subfamily. For many Kv channel subfamilies, specificity of subunit assembly depends on a region in the amino terminus referred to as the T1 domain (Fig. 1.1). This region is composed of 114 amino acids (83-196 in Shaker), and is highly conserved between Shaker homologues (Li et al., 1992; Sheng et al., 1995). Absence of this region allows Kv channels from different families to assemble promiscuously – for instance, elimination of this region in Kv1.4 and Kv2.1 led to formation of heteromultimers between these two channels, something that cannot otherwise occur (Lee et al., 1994).  4    Pore domain and channel activation Selectivity Filter: The pore domain of potassium channels is a specialized structure that supports diffusion-limited passage of potassium ions (ten million ions per second) while maintaining high selectivity for potassium over sodium ions, which are 10 000 times less permeant (Doyle et al., 1998). Potassium channels achieve this remarkable selectivity with a signature sequence, TXXTXGY/FG (Shaker TMTTVGYG), within the S5-S6 linker (Heginbotham et al., 1994; Yellen et al., 1991) (Fig. 1.1). Deletion or mutation of residues within this region results in either non-selective or non-Figure 1.1. Kv1.2 channel structural elements.  Central structure shows two subunits, with the voltage-sensing domain containing transmembrane segments S1-S4 and the N-terminal T1 domain in light grey, and the pore domain containing S5-S6 in dark grey. For clarity, the subunits from which the voltage sensing domains are shown are perpendicular to the subunits from which the pore domain is shown. The selectivity filter is highlighted in top left box, with pore lining residues shown in yellow, and residues that impact C-type inactivation in orange. Coordination sites for potassium ions are numbered. ‘PVP’ gating motif in S6 highlighted in bottom left box, with the PVP residues shown in cyan. The voltage-sensing domain S2-S4 segments and S4-S5 linker are shown in the right box, with the S4 voltage sensing residues coloured in red (Arg and Lys) and in S2 coloured green (Glu). Structure from PDB files 3LUT (Chen et al., 2010) and 2A79 (Long et al., 2005). 5  functional channels (Heginbotham et al., 1994). This linker forms a re-entrant structure where the first 22 residues after the S5 segment enter the pore as an alpha helix, and the remaining 11 residues exit the pore and connect to the top of the S6 segment. The latter half of this re-entrant loop contains the amino acids TVGY. The side chains of these amino acids are facing away from the pore, forming hydrophobic contacts with residues in the S6 segment to staple open the pore. The backbone carbonyl oxygens of these amino acids face the pore, where two adjacent rings of carbonyls coordinate one potassium ion in the filter, forming four potassium coordination sites: between the carbonyl oxygens of G445+Y445 (1), V444+G445 (2) and T443+V444 (3) and the hydroxyl and the carbonyl oxygen of T443 (4). The four-fold symmetry of the channel allows eight carbonyl oxygens at each site to coordinate one potassium ion, such that it can lose its hydration shell in the vestibule and become ‘pseudo-hydrated’ by the carbonyl oxygens  (Zhou et al., 2001b). Two potassium ions occupy the filter at the same time, at the 1 and 3 positions, followed by the 2 and 4 positions, before the terminal potassium is knocked off into the extracellular space and a new potassium binds the 1 position (Morais-Cabral et al., 2001). The repulsive force of the second potassium ion in the filter together with the binding force of the carbonyl oxygens combine to make potassium channels highly selective but also high throughput (Morais-Cabral et al., 2001). Beneath the selectivity filter lies a hydrophobic vestibule which allows diffusion of hydrated potassium ions, and at either end of the pore, acidic residues may attract potassium ions, creating a local increase in potassium concentration to facilitate throughput (Doyle et al., 1998), though this effect of the acidic residues remains controversial (Chatelain et al., 2005).  Gate: 6  The S5 and S6 helices form an ‘inverted teepee’ structure, with a wider diameter at the extracellular end to accommodate the re-entrant loop and the selectivity filter, and a narrower diameter at the intracellular end. At the bottom of this teepee is the ion channel gate which controls access of ions to the vestibule (Camino et al., 2000; Holmgren et al., 1997; Liu et al., 1997). This gate is formed by a sharp bend in the S6 helix created by a PVP motif (Camino et al., 2000; Lu et al., 2002). This kink in the helix behaves like a hinge, which does not permit access to the pore at hyperpolarized voltages when the hinge is bent, but unbends upon voltage sensor activation to allow access to the pore (S.P. Sansom and Weinstein, 2000; Webster et al., 2004). Voltage-sensing domain and voltage-dependent activation The voltage sensor is composed of the first four transmembrane segments, S1-S4 (Fig. 1.1). Every third residue within the S4 segment contains a positive charge, which was speculated early on to be the domain responsible for allowing an ion channel to change conformation in response to voltage (Noda et al., 1984). Movement of the voltage sensor in response to a change in membrane potential is a function of the amount of charge (z) and the distance it moves through the electric field (δ) within the membrane. Initial estimates of zδ were quantified using the limiting slope of the log[Po] vs voltage curve, which was an estimate of the charge moved per channel, most of which occurs in the initial stages of activation (Almers, 1978). Using this technique in Shaker and Kv1.1 channels typically predicted total charge displacement of ~6-7 e per channel (Liman and Hess, 1991; Logothetis et al., 1992; Papazian et al., 1991), although this approach was used to predict a more accurate ~16 e per channel (Zagotta et al., 1994). Neutralization of the first or second arginine in Kv1.1 channels (Arg 362 or Arg365) resulted in a smaller zδ, and therefore channels that were less sensitive to voltage, confirming that the 7  charged residues in S4 make up a major component of the region of the channel that responds to voltage (Liman and Hess, 1991; Logothetis et al., 1992). Gating currents, which are small currents generated by charged amino acids in the channel, mainly from Arg and Lys in S4 (Armstrong and Bezanilla, 1973), were also used to estimate the amount of charge moved per channel. Measuring the area under the curve of gating currents gives the total charge displaced by all channels in the membrane. When divided by the total number of channels, this measurement yielded a higher and more accurate measure of ~12-13 e per channel (Aggarwal and MacKinnon, 1996; Schoppa et al., 1992). Measurement of arginine neutralizations now revealed that each arginine contributed ~4 e to the total charge displacement. Interestingly, addition of the contribution of each arginine was ~18 e, more than the measured charge displacement per channel, suggesting that the arginine charges can contribute to the total charge displacement both by their positive charge as well as a secondary effect of changing the charge distribution of the residues around them (Aggarwal and MacKinnon, 1996; Tao et al., 2010). It is not solely the S4 positive charges that underlie voltage sensitivity of Kv channels. A residue within the S2 segment, E293 (Shaker) also contributes to charge displacement (Seoh et al., 1996). Confirmation that the S2 and S4 segments are indeed involved in responding to changes in voltage came with the advent of voltage clamp fluorometry (Mannuzzu et al., 1996). Measurement of the quenching/dequenching of a fluorescent probe attached to the S4 or S2 segments revealed that movements in these segments precede ionic currents arising from channel opening, suggesting that they are tracking voltage sensor movement (Cha and Bezanilla, 1997).  8  A long-standing question in the field is how the voltage sensor moves in response to changes in voltage. One argument is that there is a focused electric field within the membrane (Ahern and Horn, 2005; Tao et al., 2010) through which only a small tilting and helical screw movement of the S4 can generate the observed charge displacement (Cha et al., 1999; Chanda et al., 2005; Glauner et al., 1999; Larsson et al., 1996). Another argument is that a large displacement in the voltage sensor ‘paddle’ comprised of the second portion of the S3 segment, S3b, and S4 helices moves across the length of the membrane 15-20 Å to generate the observed charge displacement (Aggarwal and MacKinnon, 1996; Banerjee and MacKinnon, 2008; Long et al., 2005b; Ruta et al., 2005). Regardless of the mechanism, it is clear that the voltage sensor structure allows for movement of charged amino acids within the lipid bilayer which then transfers this movement through the S4-5 linker to the S6 segment, leading to ‘unbending’ of the S6 and opening of the channel pore to allow ion conduction. Inactivation N-type inactivation – N-type inactivation is a rapid form of inactivation that is coupled to channel opening and can be seen during a depolarizing pulse as a decrease in current after channels have opened (Zagotta et al., 1989). This type of inactivation is usually mediated by an amino-terminal sequence consisting of a chain of hydrophobic residues followed by hydrophilic, positively charged residues. The amino portion can be digested by application of intracellular trypsin, which results in removal of N-type inactivation (Hoshi et al., 1990). N-type inactivation may also be conferred on non-inactivating channels by an auxiliary Kvβ subunit which has an amino terminus similar to N-type inactivating channels (Rettig et al., 1994). The amino terminus enters the cavity 9  and blocks potassium ions, and therefore N-type inactivation can only occur once channels activate (Isacoff et al., 1991; Zhou et al., 2001a).  C-type inactivation – C-type inactivation is a distinct form of inactivation characterized by a rearrangement at the outer mouth of the pore that prevents potassium conduction (Cuello et al., 2010). Cysteine accessibility experiments showed inactivation alters pore conformation as Shaker amino acids M448C, T449C and P450C, located just before the S6 segment, all become modified by methanethiosulfonate reagents at a much greater rate when channels are inactivated than when they are closed or open (Liu et al., 1996). C-type inactivation is usually voltage-independent and coupled to opening (Hoshi et al., 1991). The rate of C-type inactivation varies widely between channels, and one determinant of inactivation is the identity of the residues near the outer mouth of the pore, though this is not the only factor, especially in Kv1 channels (Kurata and Fedida, 2006). In Shaker, Ala463 produces slow C-type inactivation whereas Ala463Val accelerates inactivation (Hoshi et al., 1991) (Fig. 1.1, see equivalent residue Kv1.2[A395]). C-type inactivation is highly sensitive to the residue at position 449 in Shaker (Fig. 1.1, see equivalent residue in Kv1.2, V381). A Val, Tyr or His at this position virtually prevents inactivation, whereas a Glu, Lys, Ala and Thr all promote inactivation (López-Barneo et al., 1993). This type of inactivation occurs in a concerted manner where all subunits simultaneously enter into the inactivated state (Ogielska et al., 1995; Panyi et al., 1995). C-type inactivation is dependent on ion occupancy of the selectivity filter. If the external potassium concentration is increased, inactivation is delayed, whereas if the external solution lacks permeant ions, C-type inactivation is greatly enhanced (López-Barneo et al., 1993). Occupancy of the S2 filter site in the absence of other ions in the filter accelerates inactivation, whereas occupancy at the other sites and 10  elimination of the S2 site will greatly delay inactivation (Matulef et al., 2013). In contrast, Pau et al. generated a constitutively inactivated Kv1.2-2.1 paddle chimera channel through a single mutation, V406W in the S6 domain, and crystallographic studies demonstrated that occupancy of the S1 filter site was perturbed (Pau et al., 2017). Therefore, the specific mechanism of C-type inactivation remains controversial, but it is clear that it involves rearrangement of the outer pore (Hoshi and Armstrong, 2013). Recovery from C-type inactivation requires channels to be closed before the pore returns to its conductive conformation (Zagotta et al., 1989). C- and N-type inactivation are not independent processes. N-type inactivation can enhance the rate of C-type inactivation. Blocking the inner mouth of the pore with the N-terminus inhibits potassium flux and therefore increases the likelihood of a vacant selectivity filter. In addition, N-terminus binding to the inner mouth will prop the activation gate open, even after membrane repolarization, increasing the time that the gate is open and channels are able to undergo C-type inactivation (Hoshi et al., 1991). Mechanism of voltage sensing and gating of Kv1.2 Kv1.2 channels are canonical potassium channels with six transmembrane domains and a selectivity filter with the signature sequence SMTTVGYG that selects for potassium. Transition from the closed to the open conformation occurs after all voltage sensors have activated, and a final, weakly voltage-dependent concerted transition to the open state occurs (Smith-Maxwell et al., 1998). Kv1.2 channels show a change in activation kinetics depending on the holding potential. As the holding potential becomes more negative, there is an increase in the sigmoidicity of channel opening and at more depolarized holding potentials, the activation kinetics become exponential (Ishida et al., 2015). This is a consequence of channels having to 11  traverse multiple closed states before opening. However, at more depolarized holding voltages, channels have gone through these ‘deeper’ closed states, and they can now simply activate in response to a depolarization (Hoshi and Armstrong, 2015). Once all voltage sensors are in their active conformations, Kv1.2 channels undergo a final concerted gate opening step (Ishida et al., 2015). This distinct final step was elucidated by mutagenesis of three neutral residues within the S4 domain. In Shaker channels, these amino acids were mutated to amino acids present in Shaw channels (Kv3) with drastic effects on the final gating transition (Ledwell and Aldrich, 1999; Smith-Maxwell et al., 1998). Mutation of V369I, I372L, and S376T in Shaker channels (V301I, I304L, S308T in Kv1.2) leads to a right shift of the activation curve and a significant shallowing of the slope, without affecting movement of the voltage sensors, as measured through gating currents (Smith-Maxwell et al., 1998). These three mutations depolarize the voltage requirement for the final, concerted opening transition so that the weak voltage dependence of this step, which accounts for ~13% of the total charge moved, becomes the rate-limiting step. As a consequence, the activation curve becomes shallower and shifts to very depolarized voltages (Ledwell and Aldrich, 1999).  Kv1.2 channels do not undergo N-type inactivation, but it can be conferred by coassembly with Kvβ1 (Heinemann et al., 1996; Rettig et al., 1994). In addition, C-type inactivation occurs at a very slow rate as Kv1.2 channels contain a Val at position 381, equivalent to the 449 position in Shaker, a residue which is known to drastically slow down the rate of inactivation (López-Barneo et al., 1993). 12  PHYSIOLOGICAL FUNCTION OF KV1.2 Localization and function Central nervous system  Kv1.2 channels are distributed throughout the central and peripheral nervous system where they have unique patterns of subcellular distribution depending on the neuronal type. They are rarely expressed as homomers. Rather, they usually form heteromeric channels with Kv1.1, Kv1.4 or Kv1.6 and may also associate with the Kvβ1 and Kvβ2 auxiliary subunits (Arroyo et al., 1999; Chung et al., 2001; Monaghan et al., 2001; Rhodes et al., 1995, 1997; Sheng et al., 1993, 1994; Wang et al., 1993a, 1994). This formation of heteromeric channels and assembly with additional subunits creates additional mechanisms for diversity between neurons and fine-tuned control of individual electrical properties. In general, Kv1 channels conduct low levels of current at subthreshold voltages, through which they regulate electrical excitability by setting the voltage at which action potentials (AP) fire and the number of APs fired per current stimulus (Bean, 2007; Bekkers and Delaney, 2001; Brew and Forsythe, 1995; Golding et al., 1999; Guan et al., 2007; Halliwell et al., 1986; Ishikawa et al., 2003; Veh et al., 1995). Myelinated axons: Kv1.2 channels are colocalized with Kv1.1 channels in the juxtaparanodes of myelinated axons throughout the brain (Rasband et al., 1998; Sheng et al., 1994; Vabnick et al., 1999; Wang et al., 1993a, 1994). This localization is mediated by Caspr2, of the neurexin superfamily, and TAG-1, a neuronal cell adhesion molecule (Poliak et al., 2003). The post-synaptic density protein, PSD95, while present and part of the complex, appears to be unnecessary for localization (Rasband et al., 2002).  13  Hippocampus: Kv1.2 channels are found in many regions of the hippocampus. Localization studies using subunit-specific antibodies show that they are present in axons and axonal terminals in the perforant path II (axons from the entorhinal cortex to dentate gyrus (DG) and CA1) (Monaghan et al., 2001; Rhodes et al., 1995, 1997; Sheng et al., 1993), in mossy fibers (axons from DG to CA3) (Sheng et al., 1993) and in Schaffer collaterals (axons from CA3 to CA1) (Monaghan et al., 2001; Veh et al., 1995). Kv1.2 is also present at the axon initial segment (Lorincz and Nusser, 2008) and in dendrites of pyramidal cells in CA3 and, to a lesser extent, in CA1 (Sheng et al., 1994). Functional studies where dendrotoxin (DTX) application is used to isolate Kv1.1, Kv1.2 and Kv1.6-specific currents leads to an increase in action potential firing, demonstrating the importance of these potassium channels in regulating excitability within the hippocampus (Halliwell et al., 1986; Wu and Barish, 1992). Furthermore, this effect of dendrotoxin on Kv1-containing neurons propagates into other neurons, resulting in aberrant network firing (Cudmore et al., 2010).  Auditory system: One part of the auditory pathway is composed of the medial nucleus of the trapezoid (MNTB), where neurons from the contralateral cochlear nucleus synapse. This synapse, the calyx of Held, is one of the largest synapses in the brain and consequently has been studied in detail (Ishikawa et al., 2003; Nakamura and Takahashi, 2007). Kv1.2-containing channels are located presynaptically where they regulate action potential firing and therefore synaptic vesicle release timing (Ishikawa et al., 2003). Kv1.2 channels heteromerize with Kv1.1 channels postsynaptically in MNTB neurons, where they regulate the threshold for AP firing and therefore the temporal fidelity of AP firing, ensuring that one presynaptic action potential will only cause one post-synaptic AP to fire (Brew and Forsythe, 1995; Dodson et al., 2002).   14  Cerebellum: Kv1.2 channels are coexpressed with Kv1.1 channels very prominently at nerve terminals of basket cells within the cerebellum. Basket cells are inhibitory interneurons that regulate the output of inhibitory Purkinje cells from the cerebellum to the deep cerebellar nuclei, the chief output from the cerebellum (Rhodes et al., 1995; Sheng et al., 1994; Veh et al., 1995; Wang et al., 1993a). Loss of function mutations within either Kv1.1 or Kv1.2 can lead to ataxic disorders where basket cell input is increased, thereby altering Purkinje cell inhibitory output and promoting an ataxic phenotype (Herson et al., 2003; Southan and Robertson, 1998). Kv1.2 is also present in Purkinje cells where it composes 15% of the total potassium current (McKay et al., 2005; Veh et al., 1995). If this current is blocked, Purkinje cells become hyperexcitable, increasing the inhibitory output which results in a decreased frequency of APs in deep cerebellar nuclei (McKay et al., 2005).  Cortex: Kv1.2 is expressed in cortical pyramidal neurons, primarily in layer V in the apical dendrites (Bekkers and Delaney, 2001; Sheng et al., 1994) and in axons (Guan et al., 2007; Rhodes et al., 1995), whereas others have argued that Kv1.2 is only weakly expressed or absent (Albert and Nerbonne, 1995). Kv1.2 is additionally expressed at the axon initial segment (AIS) within layer V pyramidal neurons (Lorincz and Nusser, 2008) where it is targeted and anchored by PSD-93 (Ogawa et al., 2008). Application of DTX  to pyramidal neurons increases excitability, where a smaller current injection is required to elicit an AP, demonstrating the importance of Kv1.2 in cortical neurons (Guan et al., 2007). Kv1.2 is additionally found in the neuropil of the cortex (Sheng et al., 1993, 1994; Wang et al., 1994).  Peripheral nervous system 15  Kv1 channels, especially Kv1.2, Kv1.1 and Kv1.4, are located in afferent neurons, where they have been documented on large diameter, mechanosensitive and proprioceptive dorsal root ganglion (DRG) neurons (Rasband et al., 2001), in addition to smaller diameter, nociceptive, Aδ and C fibers (Fan et al., 2014; Ishikawa et al., 1999; Kim et al., 2002). The physiological significance of Kv1.2 channels at juxtaparanodes is clear, though the mechanism is not well understood (Rasband et al., 2002). During development, Kv1.1/Kv1.2 heteromeric channel localization plays an important role in stabilizing the nodal resting potential in mice (Vabnick et al., 1999). Furthermore, impairing juxtaparanodal localization of Kv1.1/Kv1.2 channels through Caspr2 deletion in mice leads to pain hypersensitivity (Dawes et al., 2018; Poliak et al., 2003).  In humans, deletion mutations of Caspr2 lead to epilepsy, autistic features and intellectual disability (Rodenas-Cuadrado et al., 2016), bearing a striking similarity to Kv1.2 channelopathies. Cardiovascular system Kv1.2 is expressed in low levels in cardiac atria (Bertaso et al., 2002), ventricles (Barry et al., 1995) as well as in the right and left coronary arteries (Gautier et al., 2007), however the prominent Kv1 subunit in the heart is Kv1.5. Kv1.2 makes a minor contribution to the ultrarapid potassium current, IKur, in the heart (Nattel et al., 1999). In addition, Kv1.2 forms heteromeric channels with Kv1.5 in vascular smooth muscle cells (Kerr et al., 2001; Xu et al., 1999; Yuan et al., 1998) where they contribute to the RMP (Yuan, 1995). This channel complex is important in modulating the hypoxic response in vascular cells (Conforti et al., 2000).  Kv1.2 channels in disease Mouse models of Kv1.1 and Kv1.2 knockouts 16  Kv1.2 knockout mice exhibit the most dramatic phenotype of all Kv1 knockouts reported to date. All homozygous Kv1.2 null mice die shortly after birth, with an average lifespan of 17 days. These mice exhibit spontaneous seizures which consist of an episode of wild running and bouncing (RBS) followed by a period of tonic extension (TE), and then a 5-20 min post-ictal period where they are susceptible to spontaneous myoclonic jerks and tremors. These seizures are termed RBS/TE seizures, and mice died due to respiratory arrest during the TE phase (Brew et al., 2007). Other than seizures, the mice are the same compared to their Kv1.2 +/+ and +/- littermates, exhibiting normal motor function. RBS/TE seizures are characteristic of brainstem seizures, which also usually lead to respiratory arrest and death (Wenzel et al., 2007). They bear a remarkable resemblance to seizures in audiogenic seizure Wistar rats and audiogenic kindled rodents in that they all result in brainstem seizures (Wenzel et al., 2007). Brew et al. reported that RBS/TE seizures were often triggered by bumping the cage, and speculated that it might be due to the noise (Brew et al., 2007). The authors directly tested Kv1.2-/- MNTB neurons, a known locus of Kv1.1/1.2 channels, and found that these neurons are hypoexcitable (Brew et al., 2003, 2007). They require a larger amount of current injection to see one AP, and fire less APs per current injection than Kv1.2 +/+ mice (Brew et al., 2007). This is an unexpected outcome of knocking out a potassium channel, as generally hyperexcitability is expected. For instance, MNTB neurons isolated from Kv1.1 -/- mice are hyperexcitable, firing more action potentials in response to increasing current injections, and require less current injection to fire one AP (Brew et al., 2003).   Genetic diseases resulting from Kv1.2 channelopathies 17  Mutations within the KCNA2 gene causing disease are very rare. Initially, a mouse model with a I402T substitution, a mildly perturbative mutation, was the only instance of a disease-causing mutation in a mammal, leading to cerebellar ataxia (Xie et al., 2010). The first report of human patients heterozygous for KCNA2 mutations came in 2015, where five different mutations were described in six patients (Syrbe et al., 2015). Two gain of function mutations were found (R297Q and L298F), two loss of function (I263T and P405L), and a mutant which had no discernible functional effect (R147L) (Fig. 1.2). Interestingly, all patients presented with a similar clinical picture of infantile-onset seizures, intellectual disability and sometimes ataxia. The four patients with loss of function mutations overall had a less severe phenotype than patients with gain of function mutations. A few case reports since then have detailed two other patients with Kv1.2[R297Q] with a similarly severe phenotype as previously reported (Corbett et al., 2016; Pena and Coimbra, 2015) and one with a deletion from 255-257 which was predicted to impact the structural integrity of the voltage sensor (Corbett et al., 2016). Another patient, also with early-onset epileptic encephalopathy and developmental delay, was discovered to have a Kv1.2[T374A] mutation, which makes up the first threonine in the ‘signature sequence’ of the Kv1.2 channel (Hundallah et al., 2016). More recently, a study looking at a larger number of patients expanded these findings (Masnada et al., 2017; Syrbe et al., 2015) (Fig. 1.2). They also added a new category, ‘gain and loss of function’, which describes mutations that have mixed effects on activation, inactivation and current density. For instance, T374A hyperpolarizes the activation curve and reduces current density. Interestingly, patients with ‘gain and loss of function’ mutations had the most severe phenotype, all having seizures at an earlier onset than the other patients, movement disorders including ataxia and tremor, and severe developmental delay, half 18  of whom were non-verbal. All together, these studies demonstrate that the effects of mutations on intrinsic Kv1.2 channel function in isolation is not a good predictor of disease – all of them cause the same constellation of phenotypes of epilepsy, developmental delay and movement disorders – while having drastically different effects on channel function. Therefore, it is likely that these mutations impact some other non-canonical channel function, perhaps mediated by auxiliary subunits and/or other interacting molecules.  Acquired diseases Anti-Kv1.2 antibodies: Morvan syndrome is a diverse disease, with most patients reporting insomnia, neuromyotonia (muscle twitching, cramps, fasciculations, myokymia) and a variety of other symptoms including hallucinations and seizures (Abou-Zeid et al., 2012). It was originally thought to result from antibodies targeted to Kv1.1, Kv1.2 and Kv1.6 channels (Kleopa et al., 2006). It is now recognized that this syndrome does not only arise from Kv1 antibodies, but also from antibodies targeted against Kv1-associated proteins Caspr2 and leucine-rich glioma-1 (Irani et al., 2010). Both of these auxiliary proteins are involved in localization of Kv1.1/1.2  at the  Figure 1.2. Disease-causing mutations of the Kv1.2 channel. Kv1.2 mutations found in patients with epilepsy, intellectual disability and movement disorders. Mutations range from gain of function (GoF, red), loss of function (LoF, blue), mixed gain and loss of function effects on different channel properties (G/LoF, green) and no/unknown effect (unk, black x). Voltage sensor segments coloured light grey, pore segments dark grey. Figure adapted from (Masnada et al., 2017).    19  juxtaparanode (Poliak et al., 2003) and at the axon initial segment (Seagar et al., 2017). Therefore, targeting of Kv1 channels or their auxiliary partners through autoimmune antibodies leads to an acquired disease involving aberrant potassium conduction. Decreased Kv1.2 expression: The role of Kv1.2 in nociceptive fibers is pronounced. There are various models of neuropathic pain in mice which show decreased Kv1.1, Kv1.4 and Kv1.2 expression, including chronic constriction injury (Kim et al., 2001, 2002) and peripheral nerve injury and axotomy (Fan et al., 2014; Ishikawa et al., 1999; Yang et al., 2004). Strikingly, replacing Kv1.2 via a viral vector post peripheral nerve injury blunts neuropathic pain (Fan et al., 2014). Decreased Kv1.2 expression following peripheral nerve injury seems to be one of the causal factors in neuropathic pain in patients with type 2 diabetes mellitus (T2DM) (Zenker et al., 2012). In T2DM patients with neuropathic pain, Kv1.2 suppression via long noncoding RNA is one of the causes of decreased Kv1.2 expression and resultant hyperexcitability (Zhao et al., 2013). Another mechanism to explain decreased expression of Kv1.2 is DNA methyltransferase DNMT3a activation post nerve injury, and resultant methylation of the KCNA2 gene, leading to reduced Kv1.2 expression (Zhao et al., 2017). These studies in various models of neuropathic pain demonstrate the importance of Kv1.2 in normal conduction in nociceptive fibers. Other Kv1 family members Kv1.1  Kv1.1 channels are abundantly expressed in the CNS and PNS. In the PNS, Kv1.1 channels are co-expressed with Kv1.2 at the juxtaparanode in peripheral afferent neurons (Kim et al., 2002; Yang et al., 2004). In the CNS, they are expressed at the juxtaparanodes of most myelinated neurons, 20  in cerebellar basket cells with Kv1.2 channels, as well as in many neurons in the hippocampus with either Kv1.2 or Kv1.4 (Monaghan et al., 2001; Rhodes et al., 1995, 1997; Veh et al., 1995; Wang et al., 1993a). Mutations in the Kv1.1 channel are known to cause episodic ataxia type 1 (EA1) (Browne et al., 1994; Litt et al., 1994; Scheffer et al., 1998; Zuberi et al., 1999). Most EA1 causing mutations in the Kv1.1 channel lead to a loss of function of Kv1.1 through different mechanisms including decreased membrane expression and accelerated C-type inactivation (Adelman et al., 1995). This loss of function is carried to heteromeric Kv1.1/1.2 channels, which leads to increased action potential firing of basket cells, perturbing Purkinje cell output, leading to an ataxic phenotype (Herson et al., 2003; Rajakulendran et al., 2007). Similarly, application of dendrotoxin to block Kv1.1/1.2 channels produces the same ataxic phenotype in mice (Southan and Robertson, 1998).  Kv1.1 knockout mice are susceptible to seizures, with half of the litter dying 3-5 weeks after birth from generalized seizures (Smart et al., 1998). Seizures exhibited by these mice are characteristic of limbic seizures (episodic eyeblink, twitching whiskers, hyperstartle response, etc.), and resemble how seizure development occurs in humans from childhood where seizures start early, into adulthood where there are recurrent spontaneous seizures. For this reason, Kv1.1 -/- have been suggested to be good models for developmental seizures (Rho et al., 1999). Interestingly, in these mice, there is no evidence of ataxia or motor impairment, other than cold swim-induced tremors (Smart et al., 1998; Zhou et al., 1998, 1999). Kv1.3  21  Kv1.3 channels exhibit significant C-type inactivation. They are expressed in effector T cells of the immune system, where they are upregulated in response to T cell activation (Wulff et al., 2003). Therefore, in T cell-mediated autoimmune diseases like multiple sclerosis and rheumatoid arthritis, Kv1.3 is a potential therapeutic target (Beeton et al., 2006; Wulff et al., 2003). Kv1.3 -/- mice are resistant to high-fat diet induced obesity, suggesting that these channels also play a metabolic function (Tucker et al., 2012).  Kv1.4 Kv1.4 channels are distinct among Kv1 channels in that they possess an intrinsic N-type inactivation mechanism. They are expressed in the hippocampus in glutamatergic perforant path neurons, mossy fiber axons and Schaffer collateral axons, where they are strongly localized at unmyelinated neurons and at axonal terminals (Cooper et al., 1998; Sheng et al., 1992, 1993). In the hippocampus, they likely play a role in regulating neurotransmitter release and action potential firing (Cooper et al., 1998). In cortical pyramidal neurons, Kv1.4 channels make up the A-type, rapidly inactivating potassium current (IKA) current along with Kv4.2 and Kv4.3 channels (Norris and Nerbonne, 2010). Additionally, Kv1.4 channels contribute to IKA in suprachiasmatic neurons, along with Kv4.2 and Kv4.3 (Alvado and Allen, 2008; Bouskila and Dudek, 1995) where they help to set the circadian rhythm within these neurons (Granados-Fuentes et al., 2012, 2015). In turn, suprachiasmatic neurons govern circadian rhythms in mammals (Dibner et al., 2010). Kv1.4 channels are expressed to a lesser extent in cardiac tissue (Barry et al., 1995; Bertaso et al., 2002) where they make up part of the transient outward current (IKto) (Oudit et al., 2001). Kv1.4 and 1.6 subunits are present in the pancreas where, along with Kv2.1, they regulate membrane 22  repolarization and termination of insulin secretion after depolarization via Cav channels (MacDonald et al., 2001). Kv1.5 Kv1.5 is predominantly expressed in the heart. Within cardiac tissues, Kv1.5 is most abundant in atria (Bertaso et al., 2002; Mays et al., 1995), whereas expression in ventricular tissue is controversial (Barry et al., 1995; Dixon and McKinnon, 1994; Li et al., 1996). Functionally, Kv1.5 plays a predominant role in the atria (Marczenke et al., 2017). In cardiac tissues, Kv1.5 is responsible for generating IKUR (Fedida et al., 1993; Wang et al., 1993b). In human pluripotent stem cells induced into cardiac tissue, Kv1.5 -/- atrial cells had a decreased beating frequency and longer QT interval, as a consequence of delayed cardiac repolarization (Marczenke et al., 2017). Underscoring the importance of Kv1.5 channels in atria, mutations in this channel have been implicated in atrial arrhythmias in humans (Christophersen et al., 2013; Colman et al., 2017; Olson et al., 2006; Yang et al., 2009).  Kv1.5 channels form heteromeric complexes with Kv1.2 in smooth muscle cells in the vasculature, where they are involved in mediating hypoxic pulmonary vasoconstriction (HPV), which ensures blood is shunted to oxygen-rich alveoli (Archer et al., 2001; Kerr et al., 2001; Xu et al., 1999). In pulmonary arterial cells, Kv1.2/Kv1.5 channels are inhibited by oxygen, allowing blood vessels connected to alveoli rich in oxygen to dilate (Archer et al., 1998; Hulme et al., 1999). Excessive hypoxia can lead to pulmonary hypertension, and decreased expression of Kv1.2 and Kv1.5 channels (Wang et al., 1997, 2005). Replacing Kv1.5 channels via an adenovirus restores normal 23  HPV (Pozeg et al., 2003). Additionally, Kv1.5 is also present in airway smooth muscle cells where it contributes to smooth muscle tone (Adda et al., 1996). Kv1.6 Kv1.6 is found in the central nervous system primarily in the midbrain and brainstem (Grupe et al., 1990). Kv1.6 is coexpressed with Kv1.1, Kv1.2 and Kv1.4 in the central nervous system, especially in the hippocampus (Rhodes et al., 1996; Veh et al., 1995). It is the main component of the delayed rectifier current in astrocytes, where it is responsible for maintaining a low extracellular potassium concentration (Smart et al., 1997). In addition, it is expressed in sensory neurons with Kv1.1 and Kv1.2 where it regulates excitability (Glazebrook et al., 2002). Kv1.7 Kv1.7 is the only KCNA/Kv1 channel gene to contain an intron (Kalman et al., 1998). Overall expression in the body is not high, but Kv1.7 channels have been localized to skeletal muscle, heart and kidney (Kashuba et al., 2001). It may play a role in the Ikur in the heart (Bardien-Kruger et al., 2002). Kv1.8 Kv1.8 channels are expressed in renal blood vessels as well as the heart where they are hypothesized to play a role in the action potential (Lang et al., 2000).  24  GENERATION OF DIVERSITY IN Kv1.2 CHANNELS Some ways in which diversity of Kv1.2 function can be generated are known, however it is likely that this description is incomplete. Many of the described mechanisms relate to changing Kv1.2 expression at the cell membrane, however some describe relatively small changes in activation or inactivation. In this thesis, I present two different mechanisms of regulation that have much more pronounced effects on Kv1.2 gating, and it is likely that this is only a small subset of all regulators of Kv1.2 function. In this section I outline the known regulators of Kv1.2, and in the next section I summarize four regulators of other ion channels that have profound and diverse effects to illustrate that we have a lot left to learn specifically about Kv1.2, but also many other ion channels. Kv1.2 channel heteromerization Kv channels are the largest and most diverse ion channel family. There are ~70 potassium channels that have been cloned, and many of these have a variety of splice variants (Gutman et al., 2005). Soon after Kv channels were cloned, the scope of the diversity that could be generated was beginning to come to light. Co-injection of different subunits within the same subfamily yielded currents with intermediate phenotypes (Christie et al., 1990; Isacoff et al., 1990; Po et al., 1993; Ruppersberg et al., 1990). These observations suggested that heteromerization may be an important source of diversity in cells. This was proven resoundingly with most localization and co-immunoprecipitation studies demonstrating that Kv1 channels rarely assemble as homomers, but commonly assemble as heteromers (Rhodes et al., 1997; Sheng et al., 1993, 1994; Veh et al., 1995; Wang et al., 1993a). Heteromerization generates unique channels that amalgamate the 25  biophysical and regulatory properties of each different subunit. It is no surprise that most channels in neurons are composed of distinct subunits. Post-translational modifications Phosphorylation  Phosphorylation determines the stability of Kv1.2 at the cell membrane by regulating its association with the cytoskeleton in various ways (Hattan et al., 2002). Cortactin acts as a bridge, where it binds to Kv1.2 and to actin to anchor the channel at the cell surface (Williams et al., 2007). The association of Kv1.2 with cortactin is dependent on Tyr phosphorylation at the N- and C-termini (Fig. 1.3, green residues), where phosphorylation causes cortactin dissociation and suppression of Kv1.2 current via endocytosis (Nesti et al., 2004; Yang et al., 2007). One protein that can mediate phosphorylation is protein tyrosine kinase 2, whose activity is promoted by increases in intracellular calcium (Lev et al., 1995). Phosphorylation is also promoted by a small GTPase, RhoA (Cachero et al., 1998), which leads to activation of tyrosine kinases and consequent tyrosine phosphorylation of many proteins within the cell, including Kv1.2 (Rankin et al., 1994; Ridley and Hall, 1992). Activation of RhoA can be achieved through many different mechanisms; specific ones that directly impact Kv1.2 include lysophosphatidic acid (Stirling et al., 2009), pyrimidine nucleotides (Luykenaar et al., 2004), and the M3 acetylcholine receptor (Keller et al., 1997). Dephosphorylation is promoted by phosphorylated receptor protein tyrosine phosphatase α (Tsai et al., 1999).  This phosphorylation system regulating cell surface expression of Kv1.2 has been shown to influence excitability in CA3 pyramidal cells, where activity drives plasticity via long term 26  potentiation (Hyun et al., 2013, 2015). This occurs via calcium-driven back-propagating APs into the dendrite, which in turn activates tyrosine kinases that phosphorylate Kv1.2, resulting in endocytosis and increased synaptic excitability (Hyun et al., 2013).   Glycosylation  Kv1.2 channels, along with all other Kv1s except Kv1.6, are N-glycosylated with complex sugar groups in the S1-S2 linker in heterologous cells as well as hippocampal neurons (Fig. 1.3, green Figure 1.3. Kv1.2 channel modulating interactions and modifications. Kv1.2 channel phosphorylation and glycosylation sites highlighted in green. Interacting proteins Sigma 1 receptor (PDB 5HK1) (Schmidt et al., 2016) and Kvβ (with bound NADP cofactor, PDB 2A79) (Long et al., 2005) are shown in teal and blue, respectively. Interacting phospholipids, PI(4,5)P2 and phosphatidic acid (PA) are shown with phosphates highlighted in red. Binding is only shown for Kvβ, location of other binding sites is not known. Kv1.2 channel crystal structure from PDB 3LUT (Chen et al., 2010). 27  residue) (Shi and Trimmer, 1999; Zhu et al., 2003).  Glycosylation plays an important role in promoting trafficking and membrane stability of Kv1.2 channels (Thayer et al., 2016). Preventing glycosylation by mutating the glycosylated residue, Asn207Gln, depolarizes the activation curve and decelerates kinetics of activation, while introducing additional glycosylation sites in the S1-S2 linker has the opposite effect (Watanabe et al., 2007). Several factors regulate glycosylation, including certain amino acid residues in the C-terminus (Li et al., 2000) and the pore (Fujita et al., 2006; Utsunomiya et al., 2010), as well as association of the Kvβ subunit (Shi et al., 1996). Interacting proteins Sigma 1 receptor  The sigma 1 receptor (Sig-1R) (Fig. 1.3 teal) affects Kv1.2 membrane expression through a direct interaction, where activation of the receptor causes an increase in Kv1.2 trafficking to the membrane (Kourrich et al., 2013). Sig-1R is a transmembrane chaperone protein located in the mitochondrial associated ER membrane (MAM) and is associated with interorganellar signalling. Upon stimulation, Sig-1R translocates to the endoplasmic reticulum and the cell membrane (Su et al., 2010). Cocaine activates Sig-1R, mediating cocaine-related behaviours including conditioned place preference (Romieu et al., 2000, 2002) and psychomotor effects, among others (Kourrich et al., 2012a; Matsumoto et al., 2002). Kourrich et al. showed how cocaine stimulation of Sig-1R leads to increased Kv1.2 trafficking to the cell surface in the nucleus accumbens, mediated by a direct Sig-1R interaction (Kourrich et al., 2013). This increase in Kv1.2 leads to hypoactivity in nucleus accumbens neurons, which is thought to facilitate the cocaine-triggered reward circuit (Kourrich et al., 2012b; Taha and Fields, 2006). Therefore, this interaction may contribute to drug-seeking behaviour.  28  Kvβ  Kvβ proteins are the best recognized auxiliary subunits of Kv1 family channels. There are three genes encoding Kvβ subunits, with numerous alternatively spliced variants (Kvβ1.1-1.3, Kvβ2.1-2.2, and Kvβ3.1-3.2) (Pongs and Schwarz, 2010). Kvβ assembles with a 1:1 stoichiometry to Kv1α (Gulbis et al., 1999; Parcej et al., 1992), associating with the cytoplasmic T1 domain of Kvα, where they form a ‘hanging gondola’ structure on the intracellular side (Fig. 1.3, blue) (Gulbis et al., 2000; Kobertz et al., 2000; Long et al., 2005a). Assembly of Kvβ1 and Kvβ3 with Kv1.2 confers N-type inactivation on the channel through the Kvβ N-terminus, and increases Kv1.2 expression (Heinemann et al., 1996; Rettig et al., 1994). Kvβ2 is the most abundant subunit in the brain (Rhodes et al., 1995) and lacks the residues in the N-terminus to confer inactivation (Rettig et al., 1994), however it promotes Kv1 expression and cell surface stability. Kvβ2 associates with Kv1.2 in the endoplasmic reticulum (Nagaya and Papazian, 1997; Shi et al., 1996) where it promotes N-linked glycosylation and decreases the turnover rate of Kv1.2 channels five fold to promote cell surface expression (Shi et al., 1996). Interestingly, each Kvβ has a similar core structure that is homologous to the aldo-keto reductase family (McCormack and McCormack, 1994). This core structure is composed of a TIM barrel which is eight parallel beta sheets with surrounding alpha helices and a catalytic C-terminus (Gulbis et al., 1999, 2000), which has active catalytic function in converting between NADPH and NADP+ (Weng et al., 2006). The catalytic function can affect channel function. When coexpressed with Kv1.5, Kvβ3 confers inactivation only in mammalian cells, not in Xenopus oocytes, and this effect is dependent on the catalytic residue of Kvβ3 (Bähring* et al., 2004; Heinemann et al., 1995; Morales et al., 1995). If the catalytic residues are replaced with those of Kvβ1 (which always 29  confers N-type inactivation on Kv1.5), then Xenopus oocytes also show N-type inactivation (Bähring et al., 2001). Furthermore, inactivation can be enhanced in mammalian cells if the substrate NADPH is added (Tipparaju et al., 2007). How this catalytic function influences other Kv1 channels is not known.  The physiological effect of the different Kvβ subunits has been examined through knockout mouse models. Kvβ1 null mice have impaired learning ability (Giese et al., 1998) and recordings from CA1 pyramidal neurons show reduction in action potential spike widening and decreased slow afterhyperpolarization (Giese et al., 1998). The effects of Kvβ1 knockout on mice have been disputed, however (Murphy et al., 2004). Kvβ2 null mice have more severe symptoms that include occasional seizures, motor dysfunction under stress and a reduced lifespan (Connor et al., 2005; McCormack et al., 2002). When both Kvβ subunits are knocked out, there is an increase in mortality (Connor et al., 2005). Despite these severe effects, and a well-researched role for Kvβ in promoting expression of Kv1, Kv1.1/Kv1.2 expression and localization in knockout mice is indistinguishable from WT (Connor et al., 2005; Giese et al., 1998), indicating that Kvβ1 and 2 subunits are not absolutely required for Kv1 channel expression, but rather have some other important physiological function.  Lipids Phosphatidyl inositol bis(4,5)phosphate (PIP2) – Kv1.2 channels are sensitive to PIP2 at levels present in the plasma membrane (Fig. 1.3). PIP2 depletion causes a ~30% current inhibition and a hyperpolarizing shift in the activation, either -4mV in tSA cells (Kruse and Hille, 2013) or -14 mV in oocytes (Rodriguez-Menchaca et al., 2012). PIP2 modulation appears to play an important 30  regulatory role in spiral ganglion neurons (SGNs) where depletion of PIP2 inhibits the low voltage-activated current in SGNs, likely carried by Kv1.1/Kv1.2 heteromeric channels. This increase in excitability leads to a loss in fidelity of acoustic information travelling to the brainstem (Smith et al., 2015). In contrast to the Kv1.2, certain other potassium channels require PIP2 for channel function. These channels include the Kv7 family and inwardly rectifying potassium channels (Hilgemann et al., 2001). In contrast, most of the Kv1 family is relatively insensitive to PIP2 (Kruse and Hille, 2013). One potential explanation for this diversity in PIP2 sensitivity is that while PIP2-sensitive channels may be silenced by low levels of PIP2, other Kv1 channels will remain active, with Kv1.2 being even more active due to the hyperpolarized V1/2, to be able to contribute to RMP and membrane repolarization (Kruse and Hille, 2013). Phosphatidic acid (PA) – PA (Fig. 1.3) has profound effects on Kv1.2, causing a depolarization of the activation curve (Hite et al., 2014). Part of this shift is through an alteration of the surface charge. When PA is exclusively on the extracellular leaflet, this causes a hyperpolarizing shift in activation, whereas when it is present in the intracellular leaflet, it causes a depolarizing shift. When it is distributed on both leaflets there is still a 30 mV hyperpolarizing shift, which is a result of PA in the intracellular leaflet interacting with an Arg residue close to the bottom of S4, stabilizing the voltage sensor in its open state (Hite et al., 2014). Despite the significant shifts in activation, the mole fraction of PA required to produce this shift is supra-physiological at ~10%, with a 5% mole fraction producing one third of the shift. These values are quite high compared to the 1-2% present in cell membranes (Vance, 2015), so it is likely that the shift in mammalian cells is smaller.  31   AUXILIARY PROTEINS AND THEIR NON-CANONICAL FUNCTIONS The previous section detailed various reported interacting partners of the Kv1.2 channel. This view of auxiliary partners is quite restrictive, limiting their function to ion channel modulation. In this section, I provide a broader view of four auxiliary proteins that highlights their pleiotropic roles beyond ion channel regulation. As a consequence, they have the potential to link ion channel function to other signaling systems mediated by the ‘auxiliary’ partner. This section highlights the richness of auxiliary subunit functions, and highlights that we have a lot left to learn about many of the auxiliary proteins of Kv1 channels.  a. Sodium channel β subunit (Navβ)  Sodium channel auxiliary subunits are named Navβ because they were initially isolated in association with the pore forming Navα subunits (Isom et al., 1994). There are four Navβ subunits, β1-β4, each of which has an N-terminal extracellular immunoglobulin (Ig) domain, a single pass transmembrane domain, and a short C-terminal fragment (Hull and Isom, 2017). Navβ has profound effects on sodium channel function, producing faster activation and inactivation kinetics, increased current density, and modulation of activation and inactivation (Isom et al., 1994; Kruger and Isom, 2016). Since these early discoveries it has increasingly become clear that Navβ subunits have additional roles. Firstly, Navβ can regulate a variety of potassium channels. They modulate the activation of Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv7.2 (Nguyen et al., 2012). They play a prominent role in expression of Kv4 channels, where Navβ increases Kv4.3 current 4-fold (Deschênes and Tomaselli, 2002). 32  Secondly, the Ig component of Navβ acts as an immunoglobulin superfamily cell adhesion molecule (CAM) (Isom et al., 1995). This function is important for neuronal outgrowth during development (Davis et al., 2004), whereby homophilic interactions between Navβ1 subunits on different neurons promotes neurite growth (Brackenbury et al., 2008; Davis et al., 2004). While the molecular mechanisms have not been fully detailed, the functional outcomes are very clear. Navβ1 knockout mice have defective architecture of the dentate gyrus, CA1 (Brackenbury et al., 2013), corticospinal tract and cerebellum due to aberrant pathfinding (Brackenbury et al., 2008). This may be the result of the interplay between the Navβ1 subunit and Nav1.6 channel, which is localized to the axon initial segment starting at postnatal day 14 (Brackenbury et al., 2010, 2013). The Nav1.6 channel requires the Navβ1 subunit to traffic efficiently to the membrane of the axon initial segment to help sustain high frequency action potentials, whereas Navβ1 requires the electrical activity of Nav1.6 to promote neuronal growth (Brackenbury et al., 2010). Thirdly, Navβ subunits are subject to proteolytic cleavage through which they regulate gene transcription. Each β subunit has β secretase 1 (BACE) and γ-secretase cleavage sites. BACE cleavage releases the extracellular domain to act as a ligand for cell adhesion (Hull and Isom, 2017) and γ-secretase cleavage releases the C-terminal domain which functions as a regulator of transcription, increasing Nav1.1 mRNA up to 22-fold (Kim et al., 2007). Taken together, Navβ1 is critical to normal neuronal development and function and regulation of many ion channel types. b. Potassium channel interacting protein (KChIP) The potassium channel interacting protein (KChIP) has four known subunits KChIP1-4, each of which has four C-terminal calcium-binding EF hand motifs, and a variable N-terminus (An et al., 2000; Birnbaum et al., 2004). It interacts with Kv4 channels on the cytoplasmic N-terminus to 33  promote cell surface expression and activation, and delay inactivation (An et al., 2000). Binding is independent of Ca2+, although Ca2+ is required for its full effect on the Kv4 channel (An et al., 2000). In neurons, Kv4 channels are bound to KChIP and a dipeptidyl peptidase protein, DPPX to generate IKA, a current characterized by rapid activation and inactivation (Nadal et al., 2003).  Prior to its recognition as a potassium channel binding protein, KChIP was known to have many other calcium-dependent effects in the cell. It was first discovered as a presenilin binding protein, and it was termed calsenilin for its ability to bind calcium and presenilin (Buxbaum et al., 1998). Presenilin has many functions, and one of the best known is as the catalytic component of γ secretase that processes amyloid precursor protein (APP), and it also plays an important role regulating internal calcium stores (Duggan and McCarthy, 2016). Calsenilin binds the C-terminus of presenilin (Buxbaum et al., 1998; Choi et al., 2001) where it regulates presenilin-mediated changes in intracellular calcium (Leissring et al., 2000). Presenilin mutants can lead to dysregulation of calcium signalling, which is likely involved in the progression of Alzheimer’s disease, and calsenilin may attenuate these effects (Guo et al., 1999; Leissring et al., 1999). In addition to its role in presenilin binding, KChIP was also identified as a DNA binding transcription regulator termed DREAM (direct response element (DRE) antagonist modulator). Calcium binding to the EF hands prevents DREAM binding to the direct response element, and allows transcription of prodynorphin (involved in pain and memory acquisition) and c-fos (Carrión et al., 1999).  The interplay between KChIP binding to Kv4 channels and the role of KChIP as a presenilin binding protein or transcriptional regulator is not fully understood, however it would be interesting to test whether these mechanisms are linked and regulate different physiological processes.  34  c. Fragile X Mental Retardation Protein (FMRP)  Fragile Mental Retardation Protein (FMRP) is encoded by the gene Fmr1 and is involved in regulating nervous system development. Its most widely recognized function is as an RNA binding protein that shuttles mRNA within neurons and localizes it to dendrites (Antar et al., 2005; Bhakar et al., 2012) and it also associates with polyribosomes to negatively regulate protein expression (Brown et al., 1998). This protein derives its name from Fragile X Syndrome, which arises from a triplet codon (CGG) expansion within the CpG island upstream of the Fmr1 gene, leading to hypermethylation and silencing of Fmr1 expression (Pieretti et al., 1991; Verkerk et al., 1991). FMRP has many more effects, including regulation of three different ion channels.  Firstly, FMRP interacts with Slack, a sodium-activated potassium channel, at the C-terminus. FMRP binding eliminates subconductance states of the Slack channel and increases its open probability (Brown et al., 2010; Zhang et al., 2012). In the sea slug Aplysia, Slack associated with FMRP is expressed in abdominal bag cell (BC) neurons which influence egg laying frequency (Kupfermann and Kandel, 1970). These neurons exhibit a 30 min after-discharge in response to a brief stimulus, followed by an ~18 hour recovery (Zhang et al., 2012). Slack firing during the after-discharge triggers a series of events that culminates in egg laying, one of which is FMRP-mediated changes in protein translation that are necessary for Aplysia to elicit subsequent prolonged discharges to lay another round of eggs (Zhang et al., 2012). In humans, Slack mutations lead to intellectual disability, developmental delay and hyperactivity (Lee et al., 2014). Most mutations are in the Slack C-terminus where FMRP binds. Interestingly, both Slack and nicotinic acetylcholine receptor channelopathies lead to nocturnal frontal lobe epilepsy, however only people with mutant Slack channels also have developmental disabilities. This indicates that Slack, 35  in addition to its ion channel function, may also play a role in activity-dependent regulation of protein expression through FMRP (Lee et al., 2014).  Secondly, FMRP enhances BK channel calcium sensitivity through an interaction with the BK channel β4 subunit (Deng et al., 2013). This leads to a shortening of the AP, limiting calcium entry and neurotransmitter release in cortical and CA3 pyramidal neurons (Deng et al., 2013). Thirdly, FMRP regulates Cav2.2 channel expression. FMRP binds to the DII-III linker and C-terminal domain to target Cav2.2 to the proteasome for degradation, thereby limiting its presence at the soma and presynaptic membranes (Ferron et al., 2014). In support of these two interactions, Deng et al. showed that Fragile X syndrome mouse exhibits abnormal short-term plasticity and information processing (Deng et al., 2011). The severity of Fragile X Mental Retardation can begin to be understood when taking into consideration the diverse roles that FMRP carries out in neurons. d. Calmodulin (CaM) Calmodulin (CaM) is a multi-functional calcium-binding protein containing two lobes, each with an EF hand motif that binds calcium, and regulates many cellular functions. It binds to Cav1 (L-type calcium channels) via an isoleucine-glutamine (IQ) motif at the C-terminus (Dolmetsch et al., 2001). Influx of calcium through Cav1 channels activates CaM, which leads to a cascade of events that ends in the phosphorylation of the cAMP response element binding protein (CREB) (Dolmetsch et al., 2001). In neurons, this chain of events translates calcium influx into a nuclear signal that affects long-term protein expression via CREB (Cheung, 1970; Kakiuchi and Yamazaki, 36  1970) which drives long term potentiation (McCleskey et al., 1987) and activity-dependent survival (Franklin and Johnson, 1992).  CaM also regulates Kv7 channels by interacting with the C-terminus of the channel (Sun and MacKinnon, 2017). Binding of CaM has been implicated in the stability and membrane expression of Kv7 channels, and mutations in the C-terminus of Kv7 where CaM binds can lead to long-QT syndrome in KCNQ1 channels or benign familial neonatal seizures in KCNQ2/KCNQ3 (Haitin and Attali, 2008). The specific functional effects of CaM on KCNQ channels is controversial, and it is not known whether Kv7 channels are able to regulate the activity of CaM (Haitin and Attali, 2008). V. MOMENT-TO-MOMENT REGULATION OF ION CHANNELS   There are only a handful of mechanisms of regulation of the Kv1.2 channel that affect its function in a reversible manner (ie. phosphorylation, lipid modulation). Most forms of regulation are permanent and determined soon after translation of Kv1.2 (ie. glycosylation, assembly with Kvβ, heteromerization). Reversible changes in channel activity are important in allowing neurons to adapt to changing conditions, including changing synaptic inputs and changes in activity (Misonou et al., 2004). The focus of chapters 3 and 4 of this thesis is on a mechanism of moment-to-moment regulation of Kv1.2 channels, proposed to be driven by one or more state-dependent auxiliary binding partners. This type of activity-dependent channel regulation is evident in other channel types. In this section two such mechanisms are outlined: use-dependent disinhibition of Cav2.2 channels from Gβγ, and stargazin facilitation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor currents.  37  N-type Voltage-gated calcium channels Gαi/o coupled G-protein coupled receptor (GPCR) activation inhibits N- and P/Q-type calcium channels (Cav2.1, Cav2.2 respectively) (Bourinet et al., 1996), both of which are important presynaptic channels that mediate calcium-dependent neurotransmitter release (Zamponi and Currie, 2013). Activation of GPCRs releases the Gβγ subunit, which binds to and inhibits Cav2 channels (Herlitze et al., 1996; Ikeda, 1996) in an activity dependent manner. Cav2 channels bound to Gβγ show slow kinetics of activation and a depolarized activation curve. A large depolarization removes inhibition, and channels are able to gate normally (Bean, 1989). Using gating current measurements, Hernandez-Ochoa et al. showed that Gβγ exerts its effect through deterring voltage sensor movement (Hernández-Ochoa et al., 2007). There are two binding sites for Gβγ in the DI-II linker, one of which is a consensus Gβγ binding site QXXER and another site within the second domain of the linker (Waard et al., 1997; Zamponi et al., 1997). The Gβγ interaction is therefore modulated by other regulatory factors that require the DI-II linker. One interaction is with the Cavβ subunit which binds at the α interaction domain (AID) that overlaps with the Gβγ binding site (Herlitze et al., 1997).  These two have an unusual relationship where the Cavβ subunit does not affect Gβγ inhibition of calcium channels, but it is required for activity-dependent disinhibition (Dresviannikov et al., 2009; Zhang et al., 2008), suggesting that both subunits can interact with the linker at the same time, but relief from Gβγ inhibition requires the Cavβ subunit to exert its full effect (Zamponi and Currie, 2013). Another modulatory factor of Gβγ is Cav2 phosphorylation by PKC at the consensus site of Gβγ binding. Phosphorylation at this site causes an increase in surface expression and excludes Gβγ binding. As a consequence, PKC and Gβγ compete, allowing Cav2 channels to be precisely tuned by a fine balance between Gβγ 38  inhibition and PKC-mediated phosphorylation surface expression (Hamid et al., 1999; Zamponi et al., 1997).  Cav2 channels can associate with GPCRs, and this interaction helps to facilitate GPCR-mediated calcium current inhibition (Zamponi and Currie, 2013). For example, Cav2.2 channels interact with nociceptin receptors (ORL1) (Beedle et al., 2004). When both are co-expressed, Cav2.2 shows tonic inhibition which can be relieved by depolarization (Beedle et al., 2004).  Overall, changes in Cav2.1 and 2.2 currents through interaction with Gβγ, either inhibition, or facilitation with depolarization, provides a mechanism through which neurotransmitter release may be fine-tuned. Indeed, Bourinet et al. (1994) speculate that morphine and opioid medications that act on Gαi/o inhibit Cav2.1 and 2.2 channels, decreasing neurotransmitter release to produce analgesic effects (Bourinet et al., 1996). AMPA receptor and stargazin AMPA receptors (AMPARs) are located at excitatory synapses on the post-synaptic membrane where they are critical for synaptic transmission, long-term changes in synaptic activity and therefore learning and memory (Wollmuth and Sobolevsky, 2004). AMPARs consist of four GluA subunits, with many known interacting proteins, including the transmembrane AMPA receptor regulatory proteins (TARPs). The most well studied TARP is stargazin, which links AMPAR with PSD-95 to localize and stabilize the receptor at synapses (Chen et al., 2000). Stargazin also affects AMPAR gating through stabilization of the open state. This increased open state stability is achieved by converting the activated state of AMPAR to a ‘superactivated’ state through an allosteric mechanism that stabilizes the M2 helices and pore loops of the AMPA receptor (Chen 39  et al., 2017; Priel et al., 2005; Riva et al., 2017). Therefore, stargazin endows the AMPAR with use-dependent increases in activity (Carbone and Plested, 2016). Repetitive ligand application on the AMPAR are expected to elicit use-dependent desensitization and consequent current decrease. However, this effect is overcome by coexpression with either stargazin or another TARP, ϒ8. That is, with repetitive depolarizations at 200 Hz, AMPARs that are associated with stargazin initially exhibit some use-dependent desensitization, followed by a use-dependent increase in current due to stargazin-mediated stabilization of the open state (Carbone and Plested, 2016). Use-dependent activation of Kv1.2 channels Kv1.2 channels are subject to a unique and poorly-understood mechanism of regulation that is dependent on pulse history (Grissmer et al., 1994; Ishida et al., 2015; Nguyen et al., 2012; Rezazadeh et al., 2007). This mechanism of regulation is apparent in heterologous cells transiently transfected with Kv1.2. Different cells exhibit very different channel properties where some channels gate in a ‘slow’ mode, others in a ‘fast’ mode, and some in between. Channels in the ‘slow’ mode exhibit slow kinetics of activation and a depolarized activation curve, whereas cells in the ‘fast’ mode show fast kinetics of activation and hyperpolarized activation curves (Rezazadeh et al., 2007). With a depolarizing prepulse, all channels become uniformly ‘fast’, and channels that were originally ‘slow’ can be restored by holding channels at a negative potential where they are closed. This effect is dependent on a residue in the intracellular S2-S3 linker, Thr252. When mutated to a Lys, all channels gate in the ‘fast’ mode. Rezazadeh et al. postulated that this mechanism of regulation is due to a regulatable extrinsic protein that inhibits channel function a little in some cells (fast), much more in others (slow), and in between these two 40  extremes (Rezazadeh et al., 2007). I will expand on the underlying mechanism in Chapter 3 and show how the variability in regulation can be modulated by extracellular redox in Chapter 4. THESIS OVERVIEW The research presented in this thesis explores the regulation of a prominent neuronal potassium channel, Kv1.2, by previously unrecognized signals and accessory proteins. Chapter 3 describes use-dependent activation of Kv1.2 channels in neurons and heterologous systems, and persistence of this regulatory mechanism in heteromeric channels containing at least one Kv1.2 subunit. Chapter 4 describes powerful regulation of Kv1.2 use-dependent activation by the extracellular redox environment, and that altering the redox environment can restore wild-type like function to disease-causing Kv1.2 channel mutants. In Chapters 5 and 6, I describe a novel Kv1.2 interacting partner, Slc7a5, with profound effects on Kv1.2 expression and gating, leading to a novel mechanism of Kv channel inhibition, an ‘inactivation trap’. I demonstrate that this direct physical association alters the gating phenotype of epilepsy-linked Kv1.2 mutations even more dramatically than WT Kv1.2. Overall, my research has identified previously unrecognized mechanisms of regulation of Kv1.2 and opens the door to studying how these impact neuronal physiology and pathophysiology.     41  CHAPTER 2: METHODS   Constructs and expression cell lines Kv1 channel, Slc7 transporter and Slc3a2 cDNAs and dimeric constructs were expressed using the pcDNA3.1(-) vector (Invitrogen). Monomeric constructs were subcloned into the EcoRI and HindIII sites. Constructs were all verified by diagnostic restriction digestions and Sanger sequencing (Genewiz, Inc.). Kv1.1 or Kv1.4 (ΔN19) homotetramers did not express well in the mouse ltk- fibroblast (LM) cell line. Therefore, we generated chimeras with the Kv1.5 N terminus, replacing sequence up to the N-terminal boundary of the T1 domain (these chimeras maintain the native Kv1.1 or Kv1.4 sequence in the S2–S3 linker that is important for mediating use-dependent activation). These chimeric constructs expressed well and were used to test for use-dependent activation in Figure 3.2. Native Kv1.1 was used in Chapter 6. For dimer construction, the leading protomer was subcloned into the NheI and XhoI (or EcoRI) sites in the pcDNA3.1(-) MCS. The trailing protomer was subcloned into the EcoRI and HindIII sites. For tetramer construction, the leading two protomers were constructed similarly to the dimers, except an NheI site was introduced at the 5′ end of the trailing protomer. This construct was subcloned into the NheI site of a dimeric construct (assembled as above) containing the trailing two protomers. Chimeric Kv1 channels and Slc7 transporters were constructed at the indicated breakpoints, and site-specific point mutations were constructed by overlapping PCR. All constructs were verified by diagnostic restriction digestions and Sanger sequencing (Genewiz). 42  Heterologous cell line culture Mouse ltk-fibroblast cells were maintained in culture in a 5% CO2 incubator at 37°C in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were split onto sterile glass coverslips and, 12 hrs later, were transfected with channel cDNAs using jetPRIME transfection reagent (Polyplus) or Lipofectamine 2000 (Invitrogen). Cells were co-transfected with fluorescent proteins to allow identification of cells for recording by epifluorescence. Recordings were done 24-48 hr following transfection. Electrophysiology Patch pipettes were manufactured from soda lime capillary glass (Fisher), using a Sutter P-97 puller (Sutter Instrument). For Figures 3.1-3.5, when filled with standard recording solutions, pipettes had a tip resistance of 3-5 MΩ. This pipette size is somewhat smaller than we usually use for generation of high fidelity recordings, but we found this to be helpful in maintaining the use-dependent activation phenotype of Kv1.2 (which is lost more rapidly in recordings that dialyze cellular contents) (Rezazadeh et al., 2007). For all other figures, when filled with standard recording solutions, pipettes had a tip resistance of 1-3 MΩ. Whole cell capacitance was between 5-30 pF. Recordings were filtered at 5 kHz, sampled at 10 kHz, with manual capacitance compensation and series resistance compensation between 60-90%, and stored directly on a computer hard drive using Clampex software (Molecular Devices). Bath solution had the following composition: 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and was adjusted to pH 7.4 with NaOH. Pipette solution had the following composition: 135 mM KCl, 5 mM K-EGTA, 10 mM HEPES and was adjusted to pH 7.2 using KOH. Chemicals were purchased from Sigma-Aldrich or Fisher. Voltage protocols are described in each chapter. 43  Unless otherwise indicated, recordings in reducing conditions were carried out by incubating cells in the indicated reducing agent (diluted in serum-free DMEM media) for 5-20 min prior to recording. During the recordings, cells were continuously bathed in extracellular solution with the indicated reducing agent. All reducing agents were stored as stock solutions at -20⁰C and diluted just prior to experimental use to minimize spontaneous oxidation. For intracellular application of reducing agents, cells were held for five minutes after whole-cell break-in before recording, to allow equilibration with the pipette solution.  MTSET delivery MTSET was dissolved in standard recording solution at a concentration of 5 mM at the beginning of each experimental day and stored on ice. A small amount of MTSET was delivered to a known volume of extracellular solution (containing no KCl) in the recording chamber, resulting in a final concentration of 100 µM. MTSET was purchased from Toronto Research Chemicals (Toronto, Canada). Hippocampal cell culture preparation and electrophysiology Hippocampi from embryonic day 18 Sprague-Dawley rats of either sex were prepared as previously described (Xie et al., 2000) and plated at a density of 75 cells per mm2. Neurons were used for electrophysiology experiments at 7-16 DIV. Patch pipettes were manufactured from soda lime glass and pulled to a resistance of 1.5-2.5 MΩ. Whole-cell patch clamp of neurons was performed, with an access resistance of 2-4 mΩ and a capacitance of 10-40 pF. Tityustoxin sensitive currents were isolated by applying tityustoxin at 100 or 300 nM and subtracting toxin-insensitive current from total current. Recordings were filtered at 5 kHz, sampled at 10 kHz, with 60-80% series resistance compensation, and stored directly on a computer hard drive using 44  Clampex software (Molecular Devices). Bath and pipette solutions were as described above, except NaCl was replaced by 135 mM NMDG in the bath solution. Western blot analysis Cell lysates from transfected ltk- fibroblasts were harvested in NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl) 3 days after transfection, separated using 8% SDS-PAGE gels, and transferred to nitrocellulose membranes using standard methods. Kv1.2 was detected using a mouse monoclonal Kv1.2 antibody (clone #K14/16 75-008; NeuroMab) at a 1:10 000 dilution, and HRP-conjugated goat anti-mouse antibody (SH023; Applied Biological Materials) at a 1:30 000 dilution. Slc7a5 was detected using a rabbit polyclonal Slc7a5 antibody (KE026; Trans Genic Inc.) at a 1:500 dilution and HRP-conjugated goat anti-rabbit antibody (SH012; Applied Biological Materials) at a 1:15 000 dilution. Chemiluminescence was detected using SuperSignal West Femto Max Sensitivity Substrate (Thermo Fisher Scientific) and a FluorChem SP gel imager (Alpha Innotech). Grx1-roGFP To track the intracellular redox potential of mouse Itk- fibroblast cells, we transiently transfected the pLPCX cyto Grx1-roGFP2 (Gutscher et al., 2008) construct using jetPRIME transfection reagent. After 24 hours, cells were placed in the imaging chamber with bath solution flowing. Fluorophores were excited with wavelengths of either 408 nm or 480 nm (Mightex LED light source, filtered; Mightex), and emission intensity at 510 nm was determined by capturing images (Hamamatsu ORCA Flash 2.8, HC Image software, Hamamatsu) and subsequent processing. Images were background subtracted, and the ratio of emission with 408 nm and 480 nm excitation was calculated on a cell by cell basis. Using ImageJ, the intensity within a region of 45  interest drawn around one cell was subtracted from the background. This corrected intensity at the 408 nm excitation was divided by that at 488 nm excitation to calculate the 408/488 ratio. pLPCX cyto Grx1-roGFP2 was a gift from Tobias Dick (Addgene plasmid # 64975). Redox potential measurements Two membrane impermeant redox couples were used to clamp the extracellular environment at different redox potentials: reduced/oxidized glutathione (GSH:GSSG) and cysteine/cystine (Cys:CySS). For the GSH:GSSG couple, different ratios of the oxidized and reduced forms were mixed to a final concentration of 100 µM to generate redox potentials between -210 mV and +115 mV. The redox potential was calculated using equation (1), where Eh is the redox potential, Eo is the standard reduction potential for glutathione (-240 mV), R is the gas constant, T is the temperature, z is the number of electrons transferred and F is Faraday constant.  (1) 𝐸ℎ = 𝐸𝑜 −𝑅𝑇𝑧𝐹ln⁡([𝐺𝑆𝐻][𝐺𝑆𝑆𝐺]2)  The Cys:CySS couple was treated in a similar manner, with calculated redox potentials between -220 mV to +105 mV. The redox potential was calculated with equation (2), where the standard reduction potential for cysteine, Eo, is -250 mV. (2) 𝐸ℎ = 𝐸𝑜 −𝑅𝑇𝑧𝐹ln⁡([𝐶𝑦𝑠][𝐶𝑦𝑆𝑆]2)  Data analysis Describing the variability of Kv1.2 gating is central to our findings, and so throughout the text we have displayed data for all individual cells collected, in addition to reporting mean ± SD or a box plot with the median. Dose-response curves to different reducing agents were generated using 46  the Hill equation (3), where max and min use-dependent activation are the top and bottom of the dose-response curve and are fitted values, Kd is the dissociation constant, D is the concentration and n is the Hill coefficient.   (3) %⁡𝑈𝐷𝐴 =⁡max𝑈𝐷𝐴⁡−⁡min𝑈𝐷𝐴𝐾𝑑 𝐷𝑛⁄ ⁡+⁡1+⁡min𝑈𝐷𝐴  Activation curves were fit with the Boltzmann equation (4), where I/Imax is the normalized current, V is the voltage applied, V1/2 is the half activation voltage, and k is a fitted value reflecting the steepness of the curve. (4)  𝐼𝐼𝑚𝑎𝑥=⁡11+𝑒−(𝑉−𝑉1 2⁄ )/𝑘  Activation curves from individual cells were fit, followed by statistical calculations for individual fit parameters. The time constant of recovery of inhibition was calculated by fitting an exponential decay equation (5) to the peak currents elicited by the -10 mV test pulse every 2 sec, where y is the % inhibited current at the given time (t), y0 is the total % inhibition in that cell, τ is the time constant of recovery of inhibition, and yss is the % current remaining after the cell returns to its maximal inhibition. (5)  𝑦 = 𝑦0⁡𝑒−𝜏 𝑡⁄ + 𝑦𝑠𝑠  P-values shown throughout the text are calculated using an unpaired t-test, unless otherwise stated. Co-immunoprecipitation  We generated a bait construct (Kv1.2-1D4) in pcDNA3.1(-) comprising the N-terminus of Kv1.5 (residues 1-121), and the transmembrane domains and C-terminus of Kv1.2[S371T] to increase 47  cell surface and overall expression (Utsunomiya et al., 2010). This hybrid channel construct also included a C-terminal 1D4 epitope tag (Molday and Molday, 2014). 10 cm dishes of HEK cells were transfected with Kv1.2-1D4 and harvested after 72 hours of growth in a 37⁰C 5% CO2 incubator. Cells were then either incubated in 1 mM DTT or ambient redox PBS (10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) for 1 hour. To harvest protein, cells were washed with PBS, then incubated for 30 minutes in 250 µM ethylene glycol bis(succinimidyl succinate), a 16 Å long bifunctional crosslinker to link nearby free amines, diluted in PBS. Cells were lysed with 20 mM HEPES, 0.1 M NaCl, 2 mM MgCl2, 20mM CHAPS, and protease inhibitor at 4oC for 20 min. Lysates were centrifuged at 15000 rpm for 5 min.  1D4 antibody-coated beads were prepared for affinity purification by incubating CNBr activated sepharose 4B beads (GE Healthcare) with 1D4 monoclonal antibody (Rho-1D4 purified monoclonal antibody, Flintbox) according to the manufacturer’s instructions. 60 µL of beads were washed with 500 µL of wash buffer (20 mM HEPES, 0.1 M NaCl, 2 mM MgCl2, 10 mM CHAPS). Supernatant from cell lysates was added to the beads and incubated at 4oC for 20 min. Beads were then washed with wash buffer 6 times. Proteins were then eluted with 60 µL of elution buffer (wash buffer + 2 mg/mL 1D4 peptide) at 10oC for 10 min. All chemicals were purchased from Sigma-Aldrich or Fisher.  Mass spectrometry Cross-linked protein samples were analyzed by mass spectrometry at the Proteomics Core Facility at the University of British Columbia (Vancouver, Canada). Briefly, protein precipitates were in-gel digested and post-tryptic peptides from three different conditions (Kv1.2-1D4, Kv1.2-1D4 + 1 mM DTT, untransfected) were labelled with stable isotopic dimethyl labels for quantitation of 48  relative peptide abundance in each sample (Boersema et al., 2008).  Samples were mixed and analyzed by a quadrupole–time of flight mass spectrometer (Impact II; Bruker Daltonics) coupled to an Easy nano LC 1000 HPLC (ThermoFisher Scientific), as previously described (Gibbs et al., 2017).  Mass spectrometry data analysis and selecting candidate interacting proteins Analysis of mass spectrometry data was performed using MaxQuant 1.5.3.30. The search was performed against a database comprised of the protein sequences from Uniprot’s Homo sapiens sequence entries plus common contaminants with variable modifications of methionine oxidation, and N-acetylation of the proteins, in addition to the isotopes of dimethyl modifications for quantitation. Only those peptides exceeding the individually calculated 99% confidence limit (as opposed to the average limit for the whole experiment) were considered as accurately identified (Gibbs et al., 2017). The amount of each protein was quantified relative to the others based on the relative abundance of each protein tagged with the three different dimethyl tags. Candidate interacting proteins were selected and prioritized manually based on abundance relative to untransfected control samples, and screening against the CRAPome (Mellacheruvu et al., 2013). cDNAs for candidate interactors were purchased from the DNASU plasmid repository, and subcloned into pEGFP-C1 using PCR to introduce compatible restriction sites.  Bioluminescence resonance energy transfer Nanoluc was amplified from pcDNA3.1-ccdB-Nanoluc (gift from Mikko Taipale, Addgene plasmid # 87067), and fused to the Kv1.2 C-terminus in pcDNA3.1(-) using EcoRI and HindIII restriction sites. Other cDNAs were tagged at the N-terminus with mEGFP using standard subcloning methods. HEK cells were transiently transfected with cDNAs encoding BRET donors and acceptors 49  for 48 hours, then replated onto white polystyrene 96 well plates (Thermo Fisher). After 24 hours, cells were washed with PBS, and incubated with Nano-Glo live cell assay reagent (Promega). Emission spectra were measured between 400-700 nm in 2 or 5 nm increments, for 2 s at each interval, with a Synergy H4 Hybrid Reader (BioTek). Spectra were normalized to the peak nanoluc emission, and the normalized Kv1.2-nanoluc spectrum (measured in parallel) was subtracted to obtain the mEGFP emission. Integrated mEGFP emission (‘area under the curve’) was normalized to the integrated mEGFP emission from Kv1.2-nanoluc + mEGFP-Kv1.2 in each experiment.  Fluorescence-activated flow cytometry HEK cells were transfected with the indicated constructs for 72 hours. Cells were washed with PBS, trypsinized for 5-10 min, resuspended in DMEM then spun down, resuspended in PBS and spun down and finally resuspended in PBS + 2% FBS + 2 µM EDTA. Samples were run at the Flow Cytometry core at the University of Alberta (Edmonton, Canada) on the Attune NxT Flow Cytometer and analyzed using the FlowJo program.   50  CHAPTER 3: USE-DEPENDENT ACTIVATION OF NEURONAL Kv1.2 CHANNEL COMPLEXES   INTRODUCTION Voltage-gated potassium (Kv) channels in the nervous system influence threshold firing properties and propagation of action potentials. In the axon initial segment, clustered Kv channels integrate dendritic electrical signals and regulate neuronal excitability (Rasband, 2010), with this region sometimes referred to as a ‘hot spot’ for seizure generation (Robbins and Tempel, 2012; Wimmer et al., 2010). Kv channels also exhibit marked clustering in the juxtaparanodal region around nodes of Ranvier (Rasband et al., 1998). Supporting the general physiological importance of the Kv1 family, numerous reports have linked mutations in Kv1 channels to movement disorders and epilepsy (Adelman et al., 1995; Kullmann and Hanna, 2002; Xie et al., 2010). Knockout models of certain Kv1 channels suffer neurological consequences, depending on which Kv1 subunit is deleted. Defects are especially pronounced with genetic deletion of Kv1.2, which results in complete mortality within weeks of birth, and contrasts with mild consequences of knocking out most other Kv1 channels (Brew et al., 2007; London et al., 1998; Smart et al., 1998). Overall, these findings imply distinct roles for Kv1 subtypes in neuronal excitability, although it is not known what features of Kv1.2 gating and regulation underlie its stringent physiological requirement.  Most biophysical studies of Kv channels focus on homotetrameric channels. However, it is recognized that Kv channels achieve functional diversity via their ability to assemble as 51  heteromeric complexes (Plane et al., 2005; Po et al., 1993; Sheng et al., 1993; Wang et al., 1993a, 1994). Heteromerization of Kv1 channels can involve assembly with other Kv1 subtypes, and auxiliary proteins such as the Kvβ subunits (Bixby et al., 1999; Li et al., 1992). This diversity is reflected in the biophysical details (eg. kinetics and voltage-dependence) of channel gating. But perhaps more interestingly, combination of different Kv1 subtypes may harness regulatory pathways unique to each (Cachero et al., 1998; Holmes et al., 1996a, 1996b; Huang et al., 1994; Nitabach et al., 2001; Tsai et al., 1997). Thus, each subtype in a heteromeric channel may contribute sensitivity to specific cellular signals, generating a wide range of control mechanisms. For most regulatory mechanisms identified, it is poorly understood whether and/or how their effects are translated into altered function of heteromeric channel complexes.  Previous reports have described a unique regulatory mechanism of Kv1.2 channels, sometimes described as ‘prepulse potentiation’ (Grissmer et al., 1994; Rezazadeh et al., 2007). Although the signaling molecule(s) involved are not yet identified, this mechanism has been localized to a threonine residue in the intracellular S2-S3 linker of Kv1.2 (Rezazadeh et al., 2007). In this study, we address several questions related to the phenomenon of Kv1.2 prepulse potentiation. Firstly, we have tested whether prepulse potentiation can lead to use-dependent behavior of Kv1.2 channels during repetitive stimulus trains. Secondly, we asked how assembly of Kv1.2 with other Kv1 subtypes influences use-dependent gating, and what stoichiometry of channel assembly is required for this phenomenon to occur. Thirdly, we have investigated the presence of Kv1.2 mediated currents that exhibit use-dependent activation in primary neuronal cultures. Lastly, we have investigated the duty cycle (frequency and pulse-length) dependence and the essential sequence determinants of this regulatory process. Our findings demonstrate that Kv1.2 channels 52  are unique in their ability to generate marked use-dependent activation and can confer this property upon heteromeric Kv1.2-containing channel complexes in mammalian cell lines and hippocampal neurons. Furthermore, we propose that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulatory molecule with state-dependent affinity for the channel.  RESULTS Prepulse potentiation of Kv1.2 channels Previous reports have described a gating ‘mode shift’ in homomeric Kv1.2 channels expressed in mammalian cell lines (Rezazadeh et al., 2007). This manifests as a significant acceleration of activation kinetics, and a leftward shift in the V1/2 of activation following prepulses that activate channels. These features are summarized in Fig. 3.1 using a double pulse protocol (Fig. 3.1A) in which cells were pulsed to either a low voltage (-60 mV, very few channels open) or a high voltage (+100 mV, maximal channel opening), followed by a test pulse to +20 mV. Cells exhibit a wide range of responses to this protocol. On one extreme, ‘fast’ cells (Fig. 3.1A, bottom) are typified by rapid activation at +20 mV, regardless of whether channels were opened during the P1 pulse. In contrast, ‘slow’ cells (Fig. 3.1A, top) exhibit variable kinetics of channel opening at +20 mV, depending on whether the prepulse was sufficient to activate channels. In ‘slow’ cells, if the P1 prepulse activates large numbers of channels, the P2 pulse exhibits potentiation (faster activation kinetics and larger currents). The marked variability between cells suggests that this reflects a regulatory process, rather than an intrinsic property of the channels. However, the mechanisms underlying this variability are not yet known. Using a Kv1.2-selective channel toxin, tityustoxin 53  (Werkman et al., 1993), we demonstrated that currents in cells exhibiting either the ‘fast’ or ‘slow’ gating phenotypes could both be inhibited (data not shown), confirming that both behaviors originate from the same channel type.   As previously reported, the prepulse-dependent shift in activation kinetics is accompanied by a shift in the V1/2 of activation (Fig. 3.1B). In the absence of a prepulse (empty symbols), ‘slow’ cells (red) exhibit a V1/2 that that is displaced by 32 ± 3 mV to the right compared to ‘fast’ cells (blue). This shift is normalized when the activation curves are collected after a prepulse. That is, when channels are potentiated by a prepulse, they exhibit a similar left-shifted V1/2 (filled symbols), indicating that the ‘slow’ mode channels have been shifted to a ‘fast’ gating mode. Figure 3.1C Figure 3.1. Prepulse potentiation of Kv1.2 channels. (A) Whole-cell patch clamp recordings of Kv1.2 channels expressed in ltk- mouse fibroblasts. Recordings are representative of currents elicited from cells exhibiting a ‘fast’ gating phenotype with no prepulse potentiation (bottom), and a ‘slow’ gating phenotype (top) in which activation of channels during a prepulse causes accelerated activation in a second pulse. (B) Conductance-voltage relationships elicited before and after a 500 ms potentiating prepulse to +60 mV (protocol depicted in sample currents in (C)), in cells that exhibit either ‘slow’ mode or ‘fast’ mode gating. Cells with prominent ‘slow’ mode gating (red symbols) exhibit a marked leftward shift of the V1/2 of activation after potentiation. (C) Activation curves of non-potentiated and potentiated channels were measured by pulsing cells in 10 mV steps between -80 mV and +60 in two families of sweeps (P1, P2), separated by a pulse to +60 mV. Currents from a representative cell exhibiting the ‘slow’ gating phenotype are highlighted in red, and a cell exhibiting the ‘fast’ gating phenotype in blue (sweeps with test pulses to -30 mV are bolded). Activation kinetics are plotted against the shift in activation V1/2 measured between the P1 and P2 families of sweeps. 54  (right) highlights this behavior using a protocol to collect activation curves before and after a potentiation pulse to +60 mV. Non-potentiated channels require strong depolarizations to open, but their V1/2 of activation shifts to more negative voltages after a potentiation pulse. In the red exemplar traces, (from a cell with marked prepulse potentiation) we have bolded voltage steps to -30 mV before and after a depolarization to +60 mV. It is apparent that the -30 mV pulse elicits very little current in the ‘non-potentiated’ first pulse, but large, rapidly activating currents in the ‘potentiated’ second pulse. The shift in V1/2 due to prepulse potentiation is plotted in Fig. 3.1C, along with the activation kinetics measured at +10 mV (in the P1 pulse). Cells that exhibit slow activation in P1 (highlighted as red symbols, and the red sample sweeps), are prone to strong leftward shifts in V1/2 after a potentiation pulse, often larger than 30 mV. In contrast, cells that exhibit ‘fast’ gating of Kv1.2 (highlighted in blue), do not exhibit prepulse potentiation. It is noteworthy that we have observed Kv1.2 channel activation with a wide range of V1/2 (from -20 mV to nearly +40 mV). Within this palette of variable behavior, cells with slow activation kinetics (red symbols, traces) exhibit the most prominent prepulse-dependent potentiation (shifts of V1/2). Prepulse potentiation generates use-dependent activation Previous work has not addressed whether prepulse potentiation can affect channel function over short time scales that reflect bursting action potentials in the CNS, or whether long pulses (hundres of milliseconds, as in Fig. 3.1) are required to elicit the potentiated state. We hypothesized that the prepulse potentiation phenomenon would generate use-dependent increases in current during trains of repetitive brief depolarizations due to progressive accumulation of channels in the potentiated state. Indeed, a unique behavior of channels in the 55  ‘slow’ mode emerges in response to repetitive brief depolarizations (10 ms, 20 Hz), highlighted in Fig. 3.2A (top trace). In this protocol, each depolarizing stimulus is too brief to activate a large number of ‘slow’ mode channels (Fig. 3.2A). However, each depolarization activates a small fraction of channels, and causes them to switch to a ‘potentiated’ gating mode. With a long sequence of pulses, this pattern results in gradual recruitment of channels to the potentiated mode, and a dramatic increase in the K+ current that can be activated. For the remainder of the study, we refer to this process as ‘use-dependent activation’.  56   Figure 3.2. Use-dependent activation is observed in Kv1.2 but not other Kv1 channel subtypes. (A) Mouse ltk- cells expressing WT Kv1.2 or Kv1.2[T252R] channels (as indicated) were subjected to 20 Hz trains of repetitive depolarizations (+60 mV for 10 ms, -80 mV for 40 ms). Exemplar currents generated from 50 pulses illustrate the ‘slow’ and ‘fast’ Kv1.2 phenotypes, along with exclusively ‘fast’ currents generated by Kv1.2[T252R] channels. (B) Similar protocols were carried out with numerous Kv1 family channels. % use-dependent activation was calculated (inset) as the difference in peak current between the first pulse to +60 mV, and currents elicited at the end of the depolarizing train (n=176 for WT Kv1.2, >20 for Kv1.2[T252R] and other Kv1 subtypes). Sample sweeps show pulses 1 (colored) and 50 (black) from the pulse train. Scatter/box plots illustrate the variability in % use-dependent activation for each channel type. Overall, significant use-dependent activation is only observed for Kv1.2 channels. (C) Structural localization of Kv1.2 residue T252 in the intracellular S2-S3 linker is highlighted on the Kv1.2 paddle chimera structure. (D) Sequence alignment of the S2-S3 linker for numerous Kv1 channel subtypes, illustrating that a Thr at this position is only present in Kv1.2. 57  We quantified the ‘% use-dependent activation’ as the percentage of peak current that develops between the first depolarizing pulse, and the fully potentiated steady state (Fig. 3.2B, left). Thus, ‘slow’ cells with prominent increases of peak current exhibit a large % use-dependence, while ‘fast’ cells with little change in current magnitude between the first and last pulses (Fig. 3.2A, middle trace) exhibit small % use-dependence. Between these two extremes, there is considerable variability in cells transfected with WT Kv1.2 (Fig. 3.2B, each data point represents a measurement from a different cell). Previous work illustrated the importance of Kv1.2 residue T252 for prepulse potentiation, and demonstrated that transplantation of the Kv1.2 S2 segment and S2-S3 linker into Kv1.5 could introduce prepulse potentiation (Rezazadeh et al., 2007). As expected, the T252R mutation also abolishes Kv1.2 use-dependent activation, confirming that use-dependent activation and prepulse potentiation reflect the same regulatory mechanism (Fig. 3.2A bottom trace, 2B).  We surveyed numerous other Kv1 channel types using identical protocols to detect use-dependent activation. Due to the large cell to cell variability of Kv1.2, we tested at least 20 cells per channel type. While a large fraction of Kv1.2 recordings exhibited prominent use-dependent activation, no other Kv1 channels exhibited this property (Fig. 3.2B). It should be noted that Kv1.1 or Kv1.4 (with a short 19aa N-terminal deletion to remove N-type inactivation) (Hoshi et al., 1990) did  not express functional currents in the cell line we used for this experiment. However, when fused to the N-terminus of Kv1.5 (replacing sequence preceding the conserved T1 domain), these channels exhibited robust currents. Importantly, these experiments left the S2-S3 linker (that contains Kv1.2 residue T252, Fig. 3.2C) unchanged. Further illustrating the importance of Kv1.2 58  residue T252, no other Kv1 channel exhibited use-dependent activation (all have either a Lys or Arg at the equivalent position, highlighted in Fig. 2D).  Use-dependent activation is transferred to heteromeric channels We aimed to determine whether use-dependent activation gating of Kv1.2 could be observed in heteromeric Kv1 channel complexes. In native cells, it is common for Kv1 channel subtypes to assemble as heteromeric complexes, yet few studies have explored how various regulatory/signalling mechanisms can impact function of channels with mixed composition (Nitabach et al., 2001; Plane et al., 2005; Po et al., 1993; Sheng et al., 1993). To ensure assembly of heteromeric channels, we generated tandem dimer constructs of Kv1.2 with several other Kv1 channel subtypes and used repetitive trains of depolarizations to measure use-dependent activation (Fig. 3.3A-C). Currents could be detected from dimeric combinations of Kv1.2 with Kv1.1, Kv1.4, and Kv1.5, but not Kv1.3. Notably, each of the mixed dimer constructs exhibited pronounced (but variable) use-dependent activation, similar to Kv1.2 (Fig. 3.3A-C). These findings suggest that use-dependent activation conferred by Kv1.2 can persist in heteromeric complexes with other Kv1 subunits. 59   Certain previous reports have questioned the appropriate assembly of concatenated K+ channel subunits (McCormack et al., 1992). This prompted us to rule out the possibility that monomeric Kv1.2 subunits were generating the use-dependent currents observed. For example, monomeric Kv1.2 subunits could conceivably be synthesized from spurious internal translation start sites in the dimeric constructs. It has also been suggested that inappropriate assembly of concatenated subunits can lead to unpredictable channel stoichiometry. Thus, we tested dimeric constructs with both orientations of the protomers (ie. for the Kv1.1-Kv1.2 dimer, we used a dimer with Kv1.1 in the protomer A position, and a second dimer with Kv1.2 in the protomer A position). Figure 3.3. Use-dependent activation persists in Kv1.2-containing heteromeric channel complexes with other Kv1 channel subtypes. (A-C) Dimeric channel constructs were built with Kv1.2 and various other Kv1 channel subtypes, with protomers arranged in both orientations. Use-dependent activation was measured with trains of repetitive depolarizations (10 ms, +60 mV, 20 Hz), as described in Figs 3,4. (D-F) For each dimeric construct, transfected cell lysates were probed with a monoclonal anti-Kv1.2 antibody, to confirm and validate synthesis of full-length dimer constructs. 60  Dimers generated in both orientations exhibited similar use-dependent activation. Importantly, we also performed western blots of lysates from transfected cells to confirm the generation of dimeric channels, and absence of Kv1.2 monomeric subunits (Fig. 3.3D-F). Typically, lysates from cells transfected with monomeric Kv1.2 generated two bands (Fig. 3.3D), reflecting mature and immature glycosylated forms (Fujita et al., 2006). Transfection with dimeric constructs generated protein with approximately double the molecular weight of these characteristic Kv1.2 bands (Fig. 3.3D-F), in all cases but one (the A:Kv1.4,B:Kv1.2 dimer, Fig. 3.3E). More importantly, virtually no monomeric Kv1.2 protein was observed in dimer lysates, negating the possibility that the use-dependence of mixed dimers arises from unexpected expression of monomeric Kv1.2. However, an important exception to these findings was the A:Kv1.4,B:Kv1.2 dimer, which yielded fairly weak Kv1.2 immunoreactivity in the expected dimeric molecular weight range, and some signal slightly larger than monomeric Kv1.2 (Fig. 3.3E). These observations suggest that use-dependent currents detected in cells transfected with this particular dimer (A:Kv1.4,B:Kv1.2) arise from spurious translation of subunits missing a large amount of Kv1.4 sequence, rather than the full-length dimer construct. These findings indicate that in most cases, full length dimers (containing Kv1.2) are synthesized as expected, with use-dependent activation conferred to these heteromeric channels by Kv1.2. However, these observations also highlight the importance of controls to ensure appropriate translation of dimer constructs. Functional demonstration of dominance of Kv1.2 use-dependence in dimeric channels Uncertainty related to appropriate synthesis and assembly of transfected dimeric channel constructs (Fig. 3.3E) prompted further investigation to confirm Kv1.2 in heteromeric channels. We adopted a functional approach to demonstrate that Kv1.2 can confer use-dependent 61  activation to other Kv1 subunits, based on cysteine modification in the channel pore mouth. C-type inactivation of Shaker family channels is associated with a conformational change in the outer pore mouth that changes the accessibility of substituted cysteines (Liu et al., 1996). We generated dimeric channels comprised of Kv1.2[V381T] (to match the Shaker T449 position), in tandem with Kv1.5[P488C] (equivalent to the Shaker P450 position), bearing a cysteine substitution that enables pronounced current inhibition after MTSET modification (Liu et al., 1996). With this design (Fig. 3.4A), we tested for channels that exhibited use-dependent activation (arising from the presence of Kv1.2) in combination with MTSET sensitivity (arising from the presence of Kv1.5[P488C]). Cells were first exposed to a train of depolarizations, demonstrating use-dependent activation due to the presence of Kv1.2 in the functional channels (Fig. 3.4B,i). The presence of a cysteine at position P488 does not impact the observation of use-dependent activation (Fig. 3.4C). Next, cells were depolarized for 400 ms at 4 s intervals, in the presence of extracellular MTSET (100 µM, Fig. 3.4B,ii). In channels lacking a modifiable cysteine in the Kv1.5 subunit, no rundown was observed (Fig. 3.4C, black symbols). However, the P488C substitution in the Kv1.5 protomer rendered channels highly sensitive to MTSET, and complete channel rundown (Fig. 3.4C, red symbols). Since cysteine modification was sufficient to abolish all use-dependent currents, we conclude that virtually all of the channels that exhibit use-dependent activation (arising from Kv1.2) also contain a modifiable cysteine (contributed by Kv1.5[P488C]). These experiments demonstrate that both subunits in the dimeric construct contribute to functional channels, and that use-dependent activation features of Kv1.2 persist in heteromeric channels.  62   The dimeric constructs with trafficking and synthesis issues were the Kv1.4-Kv1.2 dimers (Fig. 3.3E). Therefore, we carried out similar experiments using Kv1.2[V381T]-Kv1.4[P521C] tandem Figure 3.4. Functional contribution of Kv1.2 and Kv1.5 to heteromeric channel complexes that exhibit use-dependent activation. (A) Dimeric channel constructs were generated comprising Kv1.2[V381T] and Kv1.5 (with or without a substituted cysteine at position P488), or Kv1.4 (cysteine substitution at P521). This design enables us to test whether channels that exhibit use-dependent activation (due to Kv1.2) also contain Kv1.5/Kv1.4 subunits in the functional channel. (B) Cells transfected with A:Kv1.5[P488C]; B:Kv1.2[V381T] dimeric constructs were subjected to i) a series of 100 repetitive brief depolarizations (‘potentiation pulses’ of 10 ms, +60 mV, 20 Hz) to demonstrate use-dependent activation. Next, in panel ii) (‘MTSET modification’) cells were exposed to 100 µM MTSET in the bath solution, and pulsed to +60 mV for 400 ms, every 4 s. (C) Mean data describing the experiments in panel (B), with the distribution of % use-dependent activation for both constructs (left panel), and the MTSET sensitivity of both constructs (right panel). In dimers containing WT Kv1.5 (with no modifiable cysteines), no rundown is observed (black symbols). However, dimers comprising Kv1.5[P488C] exhibit virtually complete channel rundown, n=4-5 per construct. (D,E) Similar experiments as in panels (B,C) were carried out using A:Kv1.2[V381T]; B:Kv1.4[P521C] dimeric channels, n=4-5 per construct. 63  dimers, first confirming the presence of Kv1.2 by demonstrating use-dependent activation, and next confirming the contribution of Kv1.4 to the functional dimer by triggering rundown by MTSET modification of residue P521C. We observed similar results as described for the Kv1.5[P488C]-Kv1.2[V381T] dimer (Fig. 3.4D,E), thereby demonstrating the presence of use-dependent activation in heteromeric channels containing Kv1.2 and Kv1.4.  Use-dependent activation in heteromeric channels containing one Kv1.2 subunit Heteromeric channel assemblies containing two subunits of Kv1.2 are able to recapitulate strong use-dependent activation. We also investigated the stoichiometry of this effect and asked whether subunits with only one Kv1.2 subunit could also exhibit use-dependent activation. We generated a concatenated tetrameric channel containing three subunits of Kv1.5 and one Kv1.2 subunit (Protomer A:Kv1.5, B:Kv1.5, C:Kv1.5, D:Kv1.2), and used repetitive depolarizations to elicit use-dependent activation (Fig. 3.5A). These tandem constructs generate robust ionic currents and some cells exhibited prominent use-dependent activation. However, the majority of cells exhibited less use-dependent activation relative to parallel experiments on Kv1.2 monomeric channels (Fig. 3.5A). Western blots of cells transfected with the tetrameric construct exhibited two bands at approximately four times the weight of the monomeric construct (Fig. 3.5B). The lack of spurious bands at weights lower than expected indicates appropriate expression of the tetrameric construct. Overall, the presence of one subunit of Kv1.2 appears to be sufficient to confer use-dependent activation in a heteromeric channel. However, there may be a dosing effect in which increased numbers of Kv1.2 subunits renders channels more susceptible to use-dependent activation. 64   Use-dependent activation is evident in a primary culture of hippocampal neurons Use-dependent activation can arise in homomeric and heteromeric channel assemblies, even those containing just one Kv1.2 subunit. Furthermore, this behaviour has been observed in all mammalian cell lines tested including mouse Itk- fibroblasts, COS, and HEK cells, prompting us to ask whether use-dependent activation might be observed in native cell types. To this end, we isolated currents generated from Kv1.2-containing channels in cultured primary rat hippocampal neurons (embryonic day 18) using tityustoxin (Werkman et al., 1993). We validated the specificity of this toxin by applying 100 or 300 nM to heterologously expressed Kv1.2 and found that it was fully sensitive, whereas Kv1.1, Kv1.4 and Kv1.5 were insensitive (Fig. 3.6A). It should be noted that Kv1.3 has been reported to be susceptible to tityustoxin, with a KD of 19.8 nM (Rodrigues et al., 2003), although the more prominent inactivation and absence of use-dependent activation of Kv1.3 can be clearly distinguished from Kv1.2. We also tested whether dimeric and tetrameric Figure 3.5. Use-dependent activation persists in channels containing a single Kv1.2 subunit. (A) Use-dependent activation was measured in mouse ltk- cells as described in Fig. 2B, comparing monomeric Kv1.2 channels with a tandem-linked tetramer comprising three Kv1.5 subunits and one Kv1.2 subunit. Sample currents for the tandem tetramer construct illustrate the variability of use-dependent activation observed for these channels. (B) Lysates for cells transfected with monomeric, dimeric, or tetrameric constructs (as indicated) were probed using a monoclonal anti-Kv1.2 antibody, illustrating the faithful generation of tetrameric channels from the tandem-linked construct. 65  constructs containing two or one Kv1.2 subunits, respectively, were tityustoxin-sensitive. The effect of tityustoxin was variable depending on subtype composition, with the Kv1.1-Kv1.2 dimer being especially sensitive. Nevertheless, each of these constructs was partially inhibited by 100 nM tityustoxin (Fig. 3.6A). Overall, these findings demonstrate that tityustoxin inhibits homomeric and heteromeric (partially) channels containing Kv1.2 subunits, indicating this is an effective tool to isolate currents generated by Kv1.2-containing channels.   To test for the presence of use-dependent activation in dissociated hippocampal neurons, cells were stimulated with short (10 ms, 10 Hz) repetitive depolarizing voltage steps before and after addition of tityustoxin (Fig. 3.6B). Typically, the tityustoxin-sensitive current made up a significant proportion of the total voltage-gated K+ current, although this was quite variable from cell to cell and ranged from 10 to 80%. Much like in mammalian cell lines, a variety of current phenotypes were observed (Fig. 3.7A,B). Interestingly, we frequently observed use-dependent activation in the tityustoxin-sensitive current, highlighted in the upper traces in Fig. 3.7B. We also often Figure 3.6. Isolation of tityustoxin sensitive currents in dissociated hippocampal neurons. (A) The tityustoxin (100 nM) sensitivity of Kv1.2 and various Kv1.2-containing heteromeric channels was measured for channels expressed in mouse ltk- cells. (B) K+ currents were measured from dissociated hippocampal neurons in the presence and absence of 100 or 300 nM tityustoxin, in order to isolate tityustoxin-sensitive currents by subtraction. 66  observed currents with a prominent inactivating component (lower traces, Fig. 3.7B), which might emerge from co-assembly of Kv1.2 with Kv1.4 or a Kvβ subunit. The % use-dependence was calculated as described in previous figures and is summarized in Fig. 3.7A, illustrating the wide range of biophysical properties emerging from Kv1.2-containing channels in these cells. Please note that only cells that exhibited use-dependent activation in either the 10 Hz or 2 Hz stimulation protocols (discussed below) are included in Fig. 3.7A (some degree of use-dependent activation was observed in 45% of the neurons tested). Overall, cells that exhibit use-dependent activation in neurons closely mimic the effect seen in mammalian cells and so it seems reasonable to speculate that the underlying mechanism (although unknown at present) is likely the same.   67   We also used longer (100 msec) repetitive depolarizations to reveal other features of elicited K+ currents (Fig. 3.7A,C), and this was helpful in revealing use-dependent activation in cells with a prominent inactivating component in the tityustoxin-sensitive current. We have presented several sample traces to contrast the variable phenotypes that were observed (Fig. 3.7C).  In cells with very a small A-type component, use-dependent activation was frequently observed in trains of both short (10 ms) and/or long (100 ms) depolarizations. However in some cells, particularly Figure 3.7. Variability of gating properties of tityustoxin sensitive channels in dissociated hippocampal neurons. (A) Summary of use-dependent properties of neurons subjected to repetitive depolarizations at indicated frequencies. Cells exhibit a range of use-dependent activation and/or inactivation. (B) Sample traces of 10 Hz recordings of tityustoxin-sensitive currents from hippocampal neurons, illustrating extreme cases of either use-dependent activation (upper panel) or inactivation (lower panel). (C) Sample traces from longer pulse durations, illustrating the cell-to-cell variability of inactivation, and the emergence of use-dependent potentiation in the sustained current component of some cells with pronounced inactivation (middle and right panels). 68  those with a prominent A-type component, use-dependent activation was not observed with repetitive 10 ms pulses, but emerged with 100 ms pulses as an increase in the sustained current (Fig. 3.7C, middle and right traces). Other phenotypes resulting from a mixture of A-type current and use-dependent activation were observed between these extremes. Taken together, these data illustrate that Kv1.2 can assemble with other Kv1 subunits to generate marked cell-to-cell diversity in current properties (combining inactivation and use-dependent activation). As with mammalian cells lines expressing Kv1.2, use-dependent activation is one of the predominant phenotypes. Model for Kv1.2 regulation To synthesize the information presented thus far, we will outline the fundamental features of Kv1.2 activation gating being investigated, as well as a conceptual model that frames our interpretation. The current record in Fig. 3.8A illustrates use-dependent activation/potentiation of WT Kv1.2 channels in response to a train of depolarizing pulses. The first pulse elicits very little current, but subsequent pulses elicit larger and larger currents. The extent of this phenomenon is highly variable in cells expressing Kv1.2. This behavior can be completely eliminated with a point mutation in the S2-S3 linker (T252R, Fig. 3.8B). We have conceived a hypothetical mechanism for use-dependent activation, dependent on an extrinsic regulatory subunit that may bind Kv1.2 and modulate its gating properties (Fig. 3.8A, lower panel). We speculate that this regulatory subunit interacts preferentially when channels are closed, and hinders channel opening upon depolarization (slowing activation kinetics, and shifting the voltage-dependence of activation). Upon channel opening, interaction with this 69  extrinsic factor is lost or altered leading to ‘disinhibition’ of the channel (moving to the lower tier of the gating scheme). Upon repolarization, sufficient time will allow the regulator to bind channels, causing them to again gate slowly. However, channels that have failed to re-associate with the regulatory subunit will remain in a potentiated state, and re-open quickly in a subsequent depolarization. In this way, rapid repetitive stimuli will cause channels to accumulate in the potentiated tier of the model, allowing for larger currents to be generated later in the train of depolarizations. In the context of this model, we speculate that the Thr252Arg mutation alters the binding site for the regulator, thereby weakening its interaction with the channel and causing channels to preferentially occupy the potentiated (unbound) tier of the model (Fig. 3.8B, lower panel highlighted by blue arrow). Kv1.2[T252R] channels are thus much less susceptible to the effects of the regulatory subunit and exhibit little use-dependence (Fig. 3.8B). 70   Duty-cycle dependence of Kv1.2 potentiation A prediction of this model is that use-dependent activation/potentiation will depend on the frequency and duration of channel stimulation and recovery intervals. For these experiments, we restricted our analysis to cells that exhibited prominent use-dependent activation (>80 %), and normalized currents during each repetitive pulse to the peak current observed during a long depolarization to +60 mV. To explore duty cycle dependence, we first varied the test pulse duration, while maintaining a 10 Hz stimulus frequency (thereby varying the recovery interval between each depolarization, Fig. 3.9A). Brief depolarizations were not sufficient to elicit the maximal amount of current in Kv1.2 channels (for example, 5 ms depolarizations spaced by 95 ms Figure 3.8. Hypothetical model for interpretation of use-dependent activation of Kv1.2 channels. (A) Hypothetical model for interpretation of use-dependent activation of Kv1.2 channels. (A,B) Traces in the upper panels illustrate behavior of Kv1.2 and Kv1.2[T252R] channels in response to repetitive 10 ms depolarizations between −80 mV and +60 mV, with a pulse-to-pulse interval of 50 ms. (Lower panels) The gating scheme depicts a proposed model depicting an extrinsic gating regulator (blue) which binds Kv1.2 preferentially in the closed state (potentially in the S2-S3 linker). Upon depolarization, binding affinity for the channel is reduced, causing it to unbind, leading to disinhibition of channels. In Kv1.2[T252R] channels, we propose that binding of the regulator is reduced or abolished, causing channels to largely dwell in the potentiated state (unbound, lower tier of gating scheme). 71  resting intervals). In contrast, as the length of the depolarization stimulus was increased, full current potentiation could be achieved with fewer sweeps (for example, 80 ms depolarizations spaced by 20 ms resting intervals). These observations are consistent with channel opening being required for potentiation/disinhibition.  Subsequently, we tested the effects of varying the interpulse interval, while maintaining a consistent depolarizing pulse duration (10 ms), thereby changing the frequency of stimulation (Fig. 3.9B). Longer recovery intervals elicited submaximal Kv1.2 potentiation whereas shorter (≤50 ms) recovery intervals elicited full potentiation (Fig. 3.9B). These observations are consistent with the notion that longer resting intervals increase the fraction of channels that can re-associate with the regulator. In the gating scheme presented in Figure 3.8A, this would correspond to channels moving from the lower ‘potentiated/unbound’ gating tier, to the upper Figure 3.9. Frequency and pulse duration effects on use-dependent activation. Cells transfected with Kv1.2 exhibiting prominent use-dependent activation were subjected to repetitive depolarizing steps from −80 mV to + 60 mV, with variable depolarization or resting intervals. Peak current in each pulse was normalized to the peak current observed during a long pulse to +60 mV. In panel (A), the depolarization duration was altered while maintaining a constant pulse frequency of 10 Hz. In panel (B), the interpulse interval was altered while maintaining a constant 10 ms depolarization duration. 72  gating tier. Taken together, these data are consistent with the prediction that increased channel stimulation (either by long pulses, or more frequent stimulation) relieves Kv1.2 from the influence of an inhibitory regulatory factor. Structure-function analysis of the S2-S3 linker region Previous work highlighted the importance of Kv1.2 residue Thr252 for use-dependent activation. Numerous mutations of Thr252 and two upstream phenylalanines (Phe251 and Phe250), have been reported to eliminate use-dependent activation. To extend previous investigation of the molecular determinants of this regulatory mechanism, we tested the effects of additional Thr252 mutations, including the isosteric Thr252Val substitution, on channel responses to high-frequency stimulation (Fig. 3.10A). We also highlight that we have adopted a different stimulation protocol than that used in previously published studies of Thr252 mutants, opting for a sequence of repetitive 10 ms depolarizations delivered at 20 Hz. This protocol enables more detailed quantification of the extent of use-dependent potentiation, based on the percent change in current between the 1st and 100th pulse in a train. Also, this parameter (% use-dependent activation) allows us to illustrate the cell-to-cell variability that is inherent in this regulatory mechanism. In previous studies, potentiation of Kv1.2 was examined with a sequence of 2 significantly longer (∼400 ms) pulses, and cells were classified as ‘slow’ (i.e. sensitive to the use-dependent regulatory mechanism) or ‘fast’ (i.e., insensitive) based on whether the initial depolarization altered the kinetics of the second test pulse. 73   Overall, we observed that smaller, uncharged substitutions including Ser, Val and (to a lesser extent) Met, are permissive for use-dependent activation, while bulkier or charged amino acids eliminated the effect. Importantly, the Thr252Val substitution exhibited use-dependent activation that was indistinguishable from WT Kv1.2 channels, strongly indicating that phosphorylation of Thr252 does not regulate use-dependence. Also noteworthy was that despite its steric similarity to Ser, Cys substitution at residue 252 did not preserve use-dependence. An important difference from previously published work was that Asp and Glu substitutions for Thr252 largely eliminated use-dependent activation, although previously reported to preserve channel potentiation (Rezazadeh et al., 2007). We suspect that this discrepancy is due to different methods of measuring this regulatory mechanism (as highlighted above). Specifically, we have observed that introduction of either Asp or Glu intrinsically slows the kinetics of Kv1.2 activation, Figure 3.10. Effects of S2-S3 linker mutations on use-dependent activation. Use-dependent activation of a panel of S2-S3 linker mutations was quantified using trains of repetitive depolarizations (20 Hz) from −80 mV (40 ms) to +60 mV (10 ms), as described in . Each data point represents the extent of use-dependent activation observed in a single cell. Prominent use-dependence was observed with Thr, Val, Ser, or Met in position 252 (n = 15–26 for each construct). 74  and this may have led to their classification as ‘slow’ (sensitive to use-dependent activation). However, using the repetitive pulse protocol, we observed very little pulse-to-pulse potentiation. We also attempted to transplant use-dependent activation from Kv1.2 into several other Kv1 family channels. Replacement of the native Lys of Kv1.5 with Thr (at the Thr252 equivalent position) has been previously studied, and was not sufficient to restore use-dependent potentiation (Rezazadeh et al., 2007). We extended these experiments by introducing a Thr at the equivalent position in Kv1.1, Kv1.3 and ΔN19-Kv1.4 (Fig. 3.11). Similar to findings in Kv1.5, Thr substitution in the S2-S3 linker was not sufficient to reintroduce use-dependence, indicating that there are additional channel elements that influence use-dependent activation.  Figure 3.11. Substitution of Thr at the 252 equivalent position of Kv1.2 into use-dependent activation insensitive channels. Substitution of Thr at the 252 equivalent position of Kv1.2 into use-dependent activation insensitive channels. Use-dependent activation of indicated mutants of Kv1.1, 1.3, and 1.4 (ΔN19 background to eliminate N-type inactivation), was measured using repetitive 10 ms depolarizations (20 Hz) from −80 mV to +60 mV, as described in previous Figures. In each of these channels a Thr (present in the Kv1.2 S2-S3 linker at position Thr252) was substituted at the equivalent position, and mutant channels were expressed in ltk- fibroblasts. Kv1.2 Thr252 is necessary for use-dependent activation, however introduction of this residue in either Kv1.1, Kv1.3, or Kv1.4 was not sufficient to transfer this use-dependent property (n = 14–20 for each construct). 75  Effects of S2-S3 linker mutations on surface expression We investigated whether there was a relationship between channel expression and use-dependence of Kv1.2, by measuring current density and cell surface expression (by western blot) of the same panel of Thr252 mutants (Fig. 3.12). These experiments were motivated by a lingering uncertainty that large channel expression might overwhelm the expression of an endogenous negative regulator, leading to a stoichiometric mismatch in which many channels would be unbound and therefore appear ‘potentiated’. In some cases (i.e. Thr252Cys, Thr252Lys and Thr252Arg), increased current density was observed together with weaker use-dependence. However, changes in current density relative to WT Kv1.2 were not especially large, and some channels with little or no use-dependent activation (i.e., Thr252His and Thr252Phe) had comparable current density to WT Kv1.2 (Fig. 3.12C). This finding was corroborated by western blot analysis, in which we quantified the ratio of the intracellular core-glycosylated band (highlighted by ‘IC’ in Figure 3.12A) and the more heavily glycosylated band corresponding to the cell surface fraction (highlighted by ‘CS’) (Fujita et al., 2006). Overall, there were no marked differences in surface expression between strongly use-dependent mutants and those that are resistant to the regulatory mechanism (Fig. 3.12A,B). One noteworthy S2-S3 linker mutation was Phe251Arg, which abolished use-dependence while dramatically weakening current density and cell surface expression. Overall, these findings suggest that mutations that alter use-dependent activation of Kv1.2 do so by influencing channel interaction with a regulatory factor, rather than a secondary consequence of dramatically increased channel expression leading to stoichiometric mismatch with an extrinsic regulatory subunit.  76   Although channel expression cannot account for the loss of use-dependent activation in certain mutants, we have observed a relationship between peak current and use-dependence among Kv1.2 channel mutants with strong use-dependence. For example, in cells transfected with WT Kv1.2, the most prominent use-dependence tended to appear in cells with smaller currents (Fig. 3.13). This feature was most dramatic in cells co-transfected with Kv1.2 and a pool of Kv1.2 siRNA oligonucleotides that diminished channel expression. Most of the siRNA-treated cells had relatively small currents with very prominent use-dependence (Fig. 3.13A). Similar observations were made in other Kv1.2 mutants that retained sensitivity to use-dependent activation (Fig. 3.13 Figure 3.12. Cell surface expression and current density for a panel of different Kv1.2 S2-S3 linker mutants. (A) Representative western blot of a panel of S2-S3 linker mutants of Kv1.2. Kv1.2channels typically runs as multiple bands on the gel, characterized by a heavier ˜95 kDa glycosylated cell surface band (highlighted by ‘CS’) and a smaller ˜67 kDa intracellular ‘core’ glycosylated band (highlighted by ‘IC’). (B) Densitometry was used to measure the ratio of the cell surface band to the intracellular band (small ratio indicates weak surface expression, n = 3). No significant difference was detected between different mutations, with the exception of Kv1.2[F251R] where relative surface expression was reduced. (C) Current density at 10 mV was measured for cells expressing the panel of Kv1.2 mutant channels. In panels B and C, the dashed horizontal line is a reference for comparison to WT Kv1.2 levels. 77  B,C). This trend further supports our conclusion that use-dependent activation is mediated by an extrinsic regulator, and the relative abundance of channel and regulator within a cell determines the strength of use-dependence. If there are less Kv1.2 channels on the membrane, it is more likely that the SGR will be able to bind all of the channels, and that the cell will exhibit strong use-dependent activation. If there are many Kv1.2 channels, however, there may be a stoichiometric mismatch leading to markedly less use-dependent activation.  DISCUSSION Expression of Kv1.2 in mammalian cell lines generates remarkable plasticity in the kinetic and thermodynamic properties of channel activation. From cell to cell, varying degrees of prepulse potentiation are observed, but a consistent feature is that channels can be shifted from a ‘slow’ Figure 3.13. Comparison of peak current to use-dependent activation in Kv1.2, Kv1.2[T252S] and Kv1.2[T252V]. (A) Relationship between peak current and % use-dependent activation, with each data point representing the current magnitude and use-dependence for a single cell. Data points distinguish between cells transfected with Kv1.2 (black circles) vs. cells transfected with Kv1.2 plus siRNA oligonucleotides (white circles). Exemplar traces illustrating pronounced use-dependence in a cell with small currents (top, co-transfected with siRNA), and weak use-dependence in a cell with large currents (bottom) are shown. Similar data were collected for Thr252 mutants that retain sensitivity to use-dependent activation: (B) Kv1.2[T252V], and (C) Kv1.2[T252S]. Solid lines show best linear fit. 78  gating mode to a ‘fast’ gating mode by delivering a depolarizing stimulus (Rezazadeh et al., 2007). In this study, we have demonstrated that this prepulse potentiation translates into a mechanism of use-dependent activation in trains of repetitive short pulses designed to more closely reflect the brief stimulations that might be encountered by Kv1.2-containing channels in the CNS. We extend earlier findings several important ways, by demonstrating that Kv1.2 channel potentiation can occur over very brief time scales, can persist in heteromeric Kv1 channels with even a single Kv1.2 subunit, and can be observed in cultured hippocampal neurons. Use-dependent activation is clearly unique to Kv1.2 subunits and relies on the presence of a threonine in the intracellular S2-S3 linker (it is a Lys or Arg in all other Kv1 channel types, and most other Kv channels). Moreover, this property can be transferred to the ‘fast’ gating Kv1.5 subtype by substituting the S2 segment and S2-S3 linker of Kv1.2. The regulatory mechanism involved has not been identified, although this property appears to be due to a diffusible cellular signal rather than an intrinsic property of the channel (Rezazadeh et al., 2007). The model of use-dependent activation that we propose is that a binding site for the regulator exists when Kv1.2 channels are in a closed conformation, with the interaction becoming much weaker in open channels (Fig. 3.8A). During a train of depolarizations, channels that have opened (and unbound from the regulator) will accumulate in a ‘potentiated’ state, so long as the interpulse interval is sufficiently short to allow little restoration of the interaction. Kv1.2 regulation of excitability in the hippocampus Kv1.2 plays a key role in regulating excitability in the hippocampus. At the neuronal level, its presence provides a strong dampening force to regulate action potential firing (Palani et al., 2010). Pathologies associated with abnormal Kv1.2 function and expression also highlight its 79  importance. In a seizure-prone gerbil model, Kv1.2 and other Kv1 channels are down-regulated whereas neuronal Kv2, 3 and 4 channels are unaffected (Lee et al., 2009). As mentioned earlier, complete knockout of Kv1.2 out in mice leads to death by generalized seizure within 2 weeks of birth (Brew et al., 2007). Also, limbic encephalitis and Morvan Syndrome, both of which lead to increased seizure susceptibility, are associated with autoantibodies to Kv1, particularly anti-Kv1.1 and anti-Kv1.2 antibodies (Kleopa et al., 2006). At the other end of the spectrum, hypothyroidism in rats leads to increased Kv1.2 (and Kv4) expression, with associated action potential shortening (Buckley et al., 2001). These gross changes in regulation and expression of Kv1.2 are associated with severe diseases of excitability, hinting that more subtle and controlled changes in gating and/or expression could provide a powerful mechanism for tuning cellular excitability.  Ion channel responses to repetitive stimuli Effects of repetitive or prolonged stimuli of Kv channels have normally been considered in the context of inactivation mechanisms (Kurata and Fedida, 2006). Central to this is the concept of ‘cumulative inactivation’, in which a series of repetitive stimuli causes the accumulation of channels in an inactivated state (Aldrich, 1981; Klemic et al., 1998; Kurata et al., 2001). This phenomenon arises because the time between stimuli is insufficient to allow complete recovery of channels that have inactivated in a preceding depolarization. Accumulation of inactivation is especially important in the context of Kv channel contributions to cellular excitability, because it will determine the number of channels ‘available’ to activate during an action potential or at subthreshold voltages, and thereby influence the strength of the repolarizing influence that can develop from these channel types.  80  Our study highlights an entirely different response of a Kv channel to repetitive depolarizations, in which channels accumulate into a potentiated state. Rather than progressive loss of Kv channel availability, use-dependent activation enhances Kv channel activity later in trains of depolarizations. Potential physiological consequences of this behavior might be to suppress cellular excitability in a use-dependent manner during bursts of action potentials in certain cell types. One report has described use-dependent activation of a Kv channel (KVS-1) cloned from C. elegans (Rojas et al., 2008), however this does not appear to share similar structural determinants or cellular mediators (for example, use-dependent activation mechanisms of KVS-1 persists in Xenopus laevis oocytes, but is absent for Kv1.2 in this expression system (data not shown). Use-dependent activation of Kv1.2 also appears to be highly contextual, as it varies dramatically from cell to cell in the same preparation, and thus may depend on cell cycle stage or other unidentified regulatory processes. Several noteworthy studies have proposed signaling pathways that control gating of Kv1.2 channels, including regulation by the sigma receptor, cortactin, and the M1 ACh receptor (Kourrich et al., 2013; Tsai et al., 1997; Williams et al., 2007). However, channel properties influenced by these signaling mechanisms do not appear similar to the use-dependent activation features that we observe. Most recently, phosphatidic acid was shown to induce a marked rightward shift in the activation curve of the Kv1.2/2.1 paddle chimera, although the role of phosphatidic acid in use-dependent activation of Kv1.2 has not been tested explicitly (Hite et al., 2014). Notable similarities are also apparent between the prepulse potentiation observed for Kv1.2, and prepulse-dependent relief of Gβγ inhibition of N- and P/Q-type Cav channels (Zamponi and Snutch, 1998). However, preliminary experiments in our lab 81  suggest that these candidate regulatory mechanisms do not affect use-dependent activation of Kv1.2. Integration of signaling mechanisms in heteromeric Kv channels Although it is widely recognized that different Kv1 channel subtypes assemble into heteromeric channels in numerous tissues (Vacher et al., 2008), it is not commonplace to study the behavior of heteromeric channels, or to examine the mechanisms by which regulation of one Kv1 channel subtype influences other subunits in a functional channel. In the case of use-dependent activation of Kv1.2, this gating behavior clearly persists in channels of mixed subtype composition, and can appear with even a single Kv1.2 subunit in the tetrameric channel (Figs. 3.3,3.5). Moreover, in primary hippocampal neurons, use-dependent activation was frequently observed in combination with fast inactivation in a given cell, demonstrating the ‘co-mingling’ of these regulatory mechanisms. A previous study used a variety of recording modes to infer that use-dependent activation relied on interaction of the channel with a diffusible cellular component. For example excision of membrane patches or prolonged dialysis of intracellular contents led to a loss of this effect (Rezazadeh et al., 2007). Our findings suggest that interaction of this hypothetical regulator with a single channel subunit is enough to decelerate activation kinetics and shift the V1/2 of channel activation. Thus, an interesting possibility is that Kv1.2 may act to recruit this regulatory mechanism to heteromeric channels, enabling a mechanism for use-dependent modulation of neuronal excitability. Whether this unique gating mechanism plays a role in the stringent requirement of Kv1.2 in animal models is not established, but could be tested in knock-in models using a T252 mutation to ablate use-dependent activation. 82  Conclusion We report that Kv1.2 channels exhibit a unique mechanism of use-dependent activation. This mechanism acts in a ‘dominant’ manner, in that it confers use-dependent activation to heteromeric channels comprising Kv1.2 and other (non use-dependent) Kv1 subtypes, with as few as one Kv1.2 subunit. This regulatory mechanism persists across mammalian cell lines as well as primary cultures of hippocampal neurons. We postulate that this mechanism arises from state-dependent binding of an extrinsic regulatory protein that binds to the S2-S3 linker.    83  CHAPTER 4: EXTRACELLULAR REDOX SENSITIVITY OF Kv1.2 POTASSIUM CHANNELS   INTRODUCTION Kv1.2 is a voltage-gated potassium channel subtype that is most prominently expressed in the central nervous system, where it assembles with other members of the Kv1 channel subfamily, auxiliary β-subunits, and potentially other interacting partners that may regulate expression and function (Sheng et al., 1993; Wang et al., 1993a, 1994). Kv1.2 has become a valuable model for investigation of ion channel gating mechanisms, as it was the first eukaryotic voltage-gated ion channel for which an atomic resolution structure was reported (Long et al., 2005a). Thus, it has served as an essential structural template for the interpretation of functional data in a variety of ion channel types, and for the generation and simulation of in silico models of ion channels. Despite the understanding implied by the precision of an atomic resolution structure, there is remarkable variability of Kv1.2 gating behavior in different experimental reports and expression systems, suggesting a regulatory mechanism that has yet to be described (Baronas et al., 2015; Grissmer et al., 1994; Ishida et al., 2015; Rezazadeh et al., 2007; Scholle et al., 2004).   We have recently reported a detailed description of the cell-to-cell variability of Kv1.2 regulation, by characterizing its property of ‘use-dependent activation’ (Baronas et al., 2015, 2016). This describes potentiation of Kv1.2 channel activity in response to prior stimuli (either long depolarizing prepulses, or repetitive trains of brief depolarizations), and is a reflection of rapid switching of the channel between gating modes with different voltage sensitivity. We have 84  observed this behavior in Kv1.2 currents recorded in heterologous mammalian cell lines and in primary neuronal cultures, and its marked cell-to-cell variability has been interpreted to suggest the involvement of an extrinsic mechanism (Baronas et al., 2015, 2016; Rezazadeh et al., 2007). Use-dependent activation can be abolished by various mutations of Thr252 in the S2-S3 linker. However, it has remained unclear what cellular variables promote occupancy of the diverse gating modes of Kv1.2. In comparison to the inner working of voltage sensitivity, regulation of ion channels by extrinsic regulators has received less attention, although auxiliary protein and lipid regulators clearly have important functional and physiological effects (Heinemann et al., 1994; Hite et al., 2014; Oliver et al., 2004). The most widely recognized auxiliary subunits of Kv1.2 are the family of Kvβ subunits (Gulbis et al., 2000; Heinemann et al., 1994; Rettig et al., 1994), although other proteins (such as PSD-95, cortactin, RhoA) and lipids have been suggested to interact and regulate expression and/or gating of Kv1.2 (Cachero et al., 1998; Tiffany et al., 2000; Tsai et al., 1997, 1999). The importance of understanding regulatory mechanisms is highlighted be the recognition that heteromeric assembly of ion channel subunits often enables recruitment of sensitivity to diverse signaling pathways (Nitabach et al., 2001; Ruppersberg et al., 1990). This is also true of Kv1.2, which assembles in heterotetrameric complexes with other Kv1 channels, and can recruit sensitivity to use-dependent activation (Baronas et al., 2015). In this study, we report the surprising finding that use-dependent activation of Kv1.2 is regulated by the redox environment. Exposure of Kv1.2 to reducing conditions causes these channels to exhibit pronounced use-dependent activation as channels ‘escape’ from the inhibited gating mode upon membrane depolarization. Channels can be shuttled on the time scale of seconds, 85  between an inhibited gating mode (favored by reducing agents) and a potentiated gating mode (populated after strong or repetitive depolarizations). Using membrane-impermeant reducing agents (tris(2-carboxyethyl)phosphine (TCEP), glutathione (GSH) and cysteine (Cys)), we demonstrate that this effect is exclusively controlled by the extracellular redox potential, and can be recruited to heteromeric Kv1 channels with one or more Kv1.2 subunits. Overall, we demonstrate a novel mechanism of regulation of Kv1.2 channel complexes by the extracellular redox potential.  RESULTS Redox conditions strongly regulate voltage-dependence of Kv1.2  Despite being a member of the well-characterized Shaker-related Kv1 family, and described structurally at atomic resolution (Long et al., 2005a, 2005b), there is poor understanding of the dramatic variability of Kv1.2 channel gating when expressed in mammalian cell lines and neurons (Baronas et al., 2015). Under ambient redox conditions, we observed that cells expressing Kv1.2 channels exhibit a wide range of V1/2 of activation, from -1.7 mV to +43 mV, illustrated by data collected from individual cells (Figure 4.1A, gray lines). This variability reflects a distribution between two extreme gating modes that can been favored by either strong depolarizing prepulses (Baronas et al., 2016; Rezazadeh et al., 2007), or as we show here, redox environment. In the presence of dithiothreitol (DTT) (Fig. 4.1A, blue), channel activation clusters towards a strongly depolarized V1/2 of +64 ± 11 mV – we describe these channels as operating in an ‘inhibited’ gating mode that resists opening. In contrast, exposure of channels to a strong depolarization prior to measuring channel activation normalizes the conductance-voltage 86  relationship to a much more hyperpolarized V1/2 of -11 ± 3 mV, reflecting the voltage-dependence of activation of channels in a ‘potentiated’ gating mode (Fig. 4.1A, black). Additionally, there is a change in the steepness of the activation curve reflected in a slope factor (k) value of 16.5 ± 3.0 mV for DTT-treated cells and 8.5 ± 1.6 mV for cells exposed to a strong depolarization. These data demonstrate a much larger dynamic range of activation properties of Kv1.2 than has been previously recognized, with a difference of ~75 mV between the extreme gating modes. These findings also illustrate how channels can be manipulated to gate nearly uniformly in either mode, using strong depolarizing prepulses (potentiated mode) or reducing conditions (inhibited mode).  87   Figure 4.1. Reducing conditions promote use-dependent activation. (A) Conductance-voltage relationships were recorded from tail current amplitudes at -30 mV (see inset) in Itk- fibroblast cells expressing Kv1.2. Grey lines are conductance-voltage relationships from multiple individual cells in ambient redox conditions (spread of V1/2 is from -1.7 to +43 mV). Mean conductance-voltage relationships (± S.D.) are shown for cells incubated in 666 µM DTT (N = 13, blue symbols, V1/2 = +64 ± 11 mV), or collected with a modified protocol that delivers a 500 ms depolarization to + 60 mV before each voltage sweep (black symbols, N = 11, V1/2 = -11 ± 3 mV). (Inset) Currents elicited with a +40 mV depolarization illustrate the suppression of current in DTT. (B, i-iii) Kv1.2 expressing cells were stimulated with repetitive 10 ms depolarizations from a holding potential of -80 mV to +60 mV (20 Hz frequency).  Different sample sweeps reflect the variability of use-dependence in ambient redox (i, ii), and a shift towards strong use-dependence after incubation in DTT (iii). (C) % use-dependent activation (UDA) is calculated as the fraction of activating current during the pulse train: (pulse 100 peak current – pulse 1 peak current)/(pulse 100 peak current), (blue symbols: mean ± S.D., grey symbols: data for individual cells in each condition; N = 87 for ambient redox, N = 44 for 666 µM DTT, and N = 12-14 for other DTT concentrations). DTT dependence of average UDA was fit with a Hill equation (EC50 = 1.2 µM and a Hill coefficient of 1.0). (D) Cell-by-cell correlation between % use-dependent activation and V1/2 measured in ambient redox (gray, N = 21), DTT (blue, N = 13), or after potentiation by strong prepulses (black, N = 11). 88  The bi-modal gating of Kv1.2 gives rise to a unique regulatory property that we have described as use-dependent activation, characterized by pronounced current potentiation during trains of repetitive depolarizations (Fig. 4.1B,i). Upon depolarization, channels initially in the inhibited mode will resist opening, but upon opening they appear to switch into the potentiated mode. In this way, channels slowly accumulate in the potentiated mode that is permissive to opening leading to a progressive pulse-by-pulse increase in current (Baronas et al., 2016). We assessed the variability of Kv1.2 use-dependent activation by delivering trains of 10 ms depolarizations (+60 mV, 20 Hz), and measuring the percent difference between the current elicited by the first and last pulse of the train (‘% use-dependent activation’ or UDA). Under ambient redox conditions, some cells exhibit a dramatic increase in current (Fig. 4.1B,I), whereas other cells exhibit negligible use-dependence (Figure 4.1B,ii). However, in the presence of the reducing agent DTT, cells are shifted quite uniformly to a pronounced use-dependent phenotype (Fig. 4.1B,iii, 1B). In a concentration-response experiment, 10-30 μM DTT was sufficient to strongly bias use-dependent behavior (EC50 of 1.2 μM DTT, Fig. 4.1C). Note that the use-dependent activation phenotype is a functional reflection of the bi-modal gating properties of Kv1.2 and is a good surrogate measure for V1/2 of channel activation (Fig. 4.1D). Shown on a cell-by-cell basis, V1/2 correlates with the extent of use-dependent activation in ambient redox (Fig. 4.1D, grey circles), and both phenotypes are shifted together upon exposure to DTT (Fig. 4.1D, blue circles). We also tested the effects of a variety of redox species on use-dependent activation (Fig. 4.2). In the presence of oxidizing agents, use-dependent activation is not abolished, but rather retains the substantial variability observed in ambient redox conditions.  89    DTT accelerates recovery of use-dependent gating Use-dependent activation is reversible within seconds if channels are held closed with hyperpolarization, as potentiated channels can spontaneously revert to the inhibited gating mode. To quantify the rate and extent of recovery, we first potentiated Kv1.2 channels with repetitive depolarizations, then returned the voltage to -80 mV and delivered 15 ms depolarizations to -10 mV every 2 s (Fig. 4.3A). At -10 mV, only channels in the potentiated gating mode can open significantly (see activation curves in Figure 4.1A). Immediately after the depolarizing train, most channels are potentiated, yielding large currents at -10 mV. Channels then gradually revert to the inhibited gating mode, causing test pulse currents to decline (Fig. 4.3A). The rate of recovery was highly variable in ambient redox conditions, illustrated by the grey area delimiting the extreme boundaries of recovery kinetics in ambient redox (data from Figure 4.2. Oxidizing agents do not affect use-dependent activation. (A) Cells expressing Kv1.2 were incubated with various oxidizing agents, 2-aldrithiol (100 or 500 µM, N = 10), copper(II) phenanthrolene (2 µM/100 µM or 150 µM/500 µM, N = 14)), oxidized DTT (0.5 or 1 mM, N = 18), oxidized glutathione (GSSG) (1 mM, N = 14) and cystine (500 µM, N = 14), or in ambient redox (N = 57) and % use-dependence was calculated as described in Figure 4.1. (B) Use-dependent activation was also quantified in cells over a range of H2O2 concentrations (10 µM – 1 mM) with no effect observed. 90  individual cells are presented in Fig. 4.4B,C). To minimize variability and avoid complications arising from oxidation of DTT in solutions, we used a high concentration of DTT to test recovery in reducing conditions. The rate of return of current inhibition in 666 µM DTT was nearly 2.5-fold faster and more uniform (τ = 2.2 ± 1.0 s in DTT vs 5.1 ± 2.9 s in ambient redox, Fig. 4.3A, see also Fig. 4.4). We measured the % recovery of inhibition as the % difference between the first and final recovery test pulses. On a cell-by-cell basis, there was a close correlation between the extent of use-dependent activation and recovery of inhibition (Fig. 4.3B, gray circles). This illustrates that cells recover robustly to the level of use-dependence observed initially after whole cell break-in. After exposure to 666 μM DTT, recovery of use-dependence is accentuated, with cells predominantly exhibiting pronounced use-dependent activation and rapid complete recovery of inhibition (Fig. 4.3B, blue circles). These findings reinforce that use-dependent activation reflects a reversible shift between inhibited and potentiated gating modes and is strongly influenced by redox conditions. 91   Figure 4.3. Time dependent recovery of inhibited gating mode. (A) Cells expressing Kv1.2 were stimulated with a train of repetitive depolarizations to +60 mV, followed by a sequence of 15 ms pulses to -10 mV (0.5 Hz) to assess recovery of the inhibited gating mode. Mean test pulse magnitude (± S.D) for ambient redox (grey, 5.1 ± 2.9 s, N = 51) and DTT incubation (blue, 2.2 ± 1.0 s, N = 46) are presented, along with surfaces illustrating the range of variability observed in individual cells. (B) Cell-by-cell correlation of % use-dependent activation and % recovery of inhibition. Each data point reflects an individual cell (grey: ambient redox, blue: 666 µM DTT). 92   Kv1.2[T252R] weakens use-dependent activation and redox sensitivity Previous studies identified residue 252 in the Kv1.2 S2-S3 linker as an important regulator of use-dependent activation, with mutations such as T252R causing apparent loss of use-dependent activation (Baronas et al., 2015, 2016; Rezazadeh et al., 2007). We tested the sensitivity of Kv1.2[T252R] channels to increasing concentrations of DTT and observed a blunted response to reducing conditions. In Kv1.2[T252R] channels, saturating DTT generated mean use-dependent activation of ~50% (Fig. 4.5A), with an EC50 of 1.0 μM DTT. In ambient redox conditions, V1/2 of individual cells were closely clustered, ranging from -9.0 to +1.4 mV (grey traces, Figure 4.3B, mean V1/2 = -5.3 ± 3 mV). Delivering potentiating prepulses prior to recording activation curves Figure 4.4. Quantification of recovery of inhibited gating mode in ambient redox and reducing conditions. (A) Detailed protocol for assessing recovery of inhibition. Cells were pulsed repetitively to +60 mV to populate the potentiated gating mode, then pulsed every 2 s to -10 mV to assess recovery to the inhibited gating mode. (B,C) Exemplar traces illustrate a sequence of recovery pulses in ambient redox (B) or 666 µM DTT (C). The time course of recovery is illustrated for all individual cells collected to illustrate the variability in ambient redox vs. DTT conditions. 93  yielded a V1/2 of -14 ± 2 mV, suggesting that under ambient redox conditions Kv1.2[T252R] channels are predominantly in a potentiated gating mode, consistent with previous reports (Baronas et al., 2015; Rezazadeh et al., 2007). However, cells pre-incubated with 2 mM DTT exhibited a biphasic activation curve (Figure 4.4B) that was reasonably well fit with a sum of two Boltzmann functions constrained by fit parameters derived for the extremes of Kv1.2 gating (Fig. 4.5A). This observation suggests that the Kv1.2[T252R] mutant retains some sensitivity to redox, but channels are not all shifted to the inhibited mode in the experimental conditions we have tested. Kv1.2[T252R] channels exhibited very little recovery of inhibition under ambient redox, mirroring the small use-dependent activation of this mutant (Fig. 4.5C,D, also see Fig. 4.6). When incubated with 2 mM DTT, the recovery becomes more pronounced (τ = 5.3 ± 1.1 s). These findings are consistent with the T252R mutation reducing susceptibility of the channel to use-dependent activation. However, some use-dependence can be rescued by exposing cells to reducing conditions (see Discussion for further consideration). 94   Figure 4.5. Weakened use-dependence and redox sensitivity in Kv1.2[T252R] channels. (A) The DTT concentration response of use-dependent activation was assessed in cells expressing Kv1.2[T252R] mutant channels, as described in Figure 4.1B (N = 53 for ambient redox, and N = 11-24 for each DTT concentration). A fit to a Hill equation yields an EC50 of 1.0 µM and a Hill coefficient of 2.8. (B) Conductance-voltage relationships for Kv1.2[T252R] channels were measured as described in Figure 4.1A, in ambient redox conditions (grey traces for individual cells, V1/2 ranging from -9.0 mV to +1.4 mV, N = 10), after strong depolarizing prepulses (V1/2 = -14 ± 2 mV, N = 10, black), or after incubation with 2 mM DTT (blue, N = 13). The biphasic activation curve in DTT was fit with a double Boltzmann equation with V1/2 and k values by the potentiated (-11 mV and 8.5, respectively) and inhibited (+64 mV and 16.5, respectively) gating behaviors (described in Fig. 2A). (C) Recovery of inhibition in ambient redox (grey, N = 33) and DTT (blue, N= 15) conditions was assessed as described in Figure 3 (data are mean ± S.D., with surfaces showing the range of values observed in individual cells). (D) Cell-by-cell correlation of % use-dependent activation and % recovery of inhibition in ambient redox (N = 30) and DTT (N = 15) conditions. (E) Exemplar traces illustrating a modest onset of use-dependent activation in Kv1.2[T252R] channels after incubation in 2 mM DTT. 95   Kv1.2 recruits redox sensitivity to heteromeric channel complexes Kv1.2 channels frequently assemble with other Kv1 subunits to generate heteromeric channels. However, only Kv1.2 has been reported to exhibit use-dependent activation among the Kv1 family, and all but Kv1.2 possess a Lys or Arg at the critical Thr252 position previously show to be essential for the Kv1.2 gating mode shift (Rezazadeh et al., 2007). Figure 3.3 shows robust use-dependent activation in Kv1.2-containing heteromeric complexes, with one or two Kv1.2 subunits being sufficient to impart use-dependence. We tested redox sensitivity of use-dependence in other Kv1 channels and heteromeric complexes, as we were uncertain whether the accentuated use-dependent behavior in reducing conditions would evoke a latent use-dependent phenotype in closely related channels. Kv1.1 homomeric channels exhibit no apparent use-dependent activation in ambient redox conditions, or after incubation in up to 2 mM DTT (Figure 4.7A). Figure 4.6. Quantification of recovery of inhibited gating mode in Kv1.2[T252R] channels. (A,B) Recovery of the inhibited gating mode was assessed as described in Figure 3. Exemplar traces of recovery pulses to -10 mV are show in ambient redox (grey) and DTT (blue) conditions. Time course of recovery of the inhibited gating mode is illustrated for all cells tested, in the lower panels (N = 33 in ambient redox, grey and N = 15 in 2 mM DTT, blue).  96  However, using concatenated Kv1.1-Kv1.2 heterodimeric constructs (1:1 Kv1.1:Kv1.2 stoichiometry), modest use-dependent activation was apparent in ambient redox, together with a profound shift to use-dependent behavior in reducing conditions (Figure 4.7A, B).  Kv1.2-Kv1.5 heterodimers showed some redox sensitive use-dependence, although not as prominent as WT Kv1.2. Additionally Kv1.2-1.5-1.5-1.5 heterotetrameric constructs (1:3 Kv1.2:Kv1.5 stoichiometry) responded sub-maximally to 2 mM DTT (Fig. 4.7A). These findings illustrate that Kv1.2 can act as a module that recruits use-dependent activation and strong redox sensitivity to heteromeric Kv1 channel complexes, although the extent of these effects may depend on subunit composition.  Figure 4.7. Redox-sensitive use-dependence is transferable in heteromeric channels containing Kv1.2 subunits. (A) Repetitive depolarizations were used to assess % use-dependent activation, as described in Figure 4.1B, for a variety of Kv1 channel subtypes and concatenated combinations with Kv1.2 as indicated. Each data point reflects the % use-dependence recorded from an individual cell in ambient redox conditions (gray) or in the presence of either 666 µM or 2 mM DTT (blue). N = 10-17 for each construct and condition. (B) Exemplar current tracings illustrating use-dependent activation of Kv1.1-Kv1.2 dimeric channels in (i) ambient redox conditions or (ii) 666 µM DTT. 97  Extracellular redox environment controls Kv1.2 channel gating Since DTT is membrane permeable, it does not reveal the sidedness of redox sensitivity. DTT application either extracellularly (in the external solution) or intracellularly (in the internal pipette solution) is sufficient to accentuate use-dependent activation (Fig. 4.8A,B, blue). The membrane impermeable reducing agent (TCEP) also potentiated use-dependent activation, much like DTT, when applied extracellularly (pH was adjusted to 7.4 after TCEP dilution in external potassium solution). In contrast, internally applied TCEP failed to promote use-dependent activation (Fig. 4.8A,B, orange). Other extracellularly applied membrane impermeable reducing agents (GSH and Cys) mimicked the effects of extracellular TCEP and DTT. We measured the concentration-response of use-dependence to each of these membrane impermeable reducing agents and found that each had an EC50 of ~2-4 uM (Fig. 4.9).    98   Figure 4.8. Extracellular redox environment modulates Kv1.2 use-dependent activation. (A) Use-dependent activation was assessed using repetitive depolarizations as described in Figure 1. DTT (membrane permeant, blue) or TCEP (membrane impermeant, orange) reducing agents were applied intracellularly at 500 µM (through the pipette solution) or extracellularly at 200 µM, as indicated (External agents: N = 41 for ambient redox, N = 12 for DTT and N = 15 for TCEP; internal agents: N = 12 for ambient redox, DTT and TCEP). (B) Exemplar current traces from cells expressing Kv1.2 channels in various reducing agents illustrate the membrane sidedness of TCEP effects. (C) Mouse Itk- fibroblasts transfected with the genetically encoded redox sensor Grx1-roGFP were perfused with a range of redox active compounds, as indicated (H2O2 at 1 mM, DTT and TCEP at 666 µM). Extracellular TCEP provides minimal recovery of intracellular redox potential, while DTT rapidly reduces the intracellular compartment (black symbols = mean ± S.D., grey symbols are data from individual cells, N = 10). Pseudo-colouring was generated by the ‘Fire’ lookup table in ImageJ. (D) Use-dependent activation was assessed under ambient redox conditions (time = 0) with repetitive depolarizations as described in Figure 1B, followed by perfusion with 200 µM extracellular TCEP (N = 6).  TCEP produces a maximal shift in use-dependent activation within 1-2 min, also illustrated with exemplar current traces in lower panels. 99  To confirm that TCEP was exclusively controlling Kv1.2 via the extracellular compartment, we tracked the intracellular redox potential using a genetically encoded ratiometric redox sensor, Grx1-roGFP. In ambient redox conditions, the intracellular redox potential is strongly reducing, reflected by a low 408/488 ratio (Figure 4.8C). Perfusion with 1 mM H2O2 (membrane permeable) quickly leads to oxidation of the redox potential, reflected in an increased 408/488 ratio (see methods for ratio calculation). Subsequent addition of extracellular TCEP (666 µM) for up to 5 min did not markedly restore the intracellular redox potential, while addition of DTT (666 µM) rapidly reduced the intracellular redox potential. These slow/absent effects of TCEP on the intracellular redox potential contrast with the time course of extracellular TCEP effects on Kv1.2 gating, which are nearly complete within one minute of exposure to 200 µM TCEP (Fig. 4.4D). These findings confirm that the gating effects described in Figures 4.1 and 4.3 are strongly controlled by changes to the extracellular redox potential.  100   Cysteine residues in Kv1.2 do not control redox sensitivity Use-dependent activation of Kv1.2 has been reported to show significant cell type variation. It is apparent in all mammalian cell lines we have tested to date, but is not reported when Kv1.2 is expressed in Xenopus laevis oocytes, suggesting it may not be an intrinsic property of the channel (Horne et al., 2010; Ishida et al., 2015; Peters et al., 2009; Rezazadeh et al., 2007). To investigate potential determinants of redox sensitivity in Kv1.2, we systematically mutated all cysteines that had the potential to become accessible to the extracellular solution to alanine (Figure 4.10A). None of these mutations abolished use-dependent activation properties (Figure 4.10B, black), and all Cys→Ala mutants retained sensitivity to reducing agents (Figure 4.10B, blue). Based on these findings, the redox sensitivity of Kv1.2 does not appear to arise from modification or formation of a disulfide involving a cysteine that is native to the channel. Our current findings Figure 4.9. Dose-dependent shift in use-dependence with membrane impermeant reducing agents. (A-C) Concentration-response of use-dependent activation was measured with increasing concentrations of extracellularly applied (A) TCEP (EC50 of 3.4 µM and a Hill coefficient of 1.1), (B) reduced glutathione (GSH, EC50 of 2.1 µM and a Hill coefficient of 1.2), or (C) cysteine (EC50 of 2.8 µM and a Hill coefficient of 1.1). N = 10-20 for each condition. 101  seem consistent with this hypothesis of an extrinsic inhibitory regulator mediating use-dependent activation presented in Figure 3.8, and lead to a further suggestion that the extrinsic regulator (or its interaction with the channel) is sensitive to extracellular redox conditions. In our model, Kv1.2 is depicted as having a high affinity for the inhibitory regulator in its reduced state (perhaps when a key disulfide is broken), leading to stabilization of the channel resting state, and a weaker interaction with the regulator in its oxidized state (Figure 4.10C). The interaction between the regulator and the channel can also be weakened by depolarization leading to channel opening, likely due to a binding site which is only present when channels are closed and is altered when channels open. Based on our observations that oxidizing agents do not alter use-dependent activation beyond the ambient redox condition (Fig. 4.2), we would predict that the oxidized state of the regulator has a weakened (but non-zero) affinity for the channel. 102   Figure 4.10. Systematic mutagenesis of transmembrane cysteine residues in Kv1.2. (A) Cysteine residues are highlighted in red on the Kv1.2 structure (PDB 3LUT). C181, C229, C244, and C394 in the transmembrane domains were mutated to alanine. (B) Use-dependent activation of each cysteine mutant was assessed with trains of repetitive depolarizations, as described in Figure 1, in ambient redox and after incubation in 666 µM DTT (N = 10-22 for each condition). (C) Schematic model depicting redox dependent interactions of Kv1.2 with a postulated extrinsic binding partner (blue) that mediates use-dependent activation. We propose that the reduced state of the regulatory partner has a high affinity for the Kv1.2 channel that promotes an inhibited gating mode by stabilizing the resting conformation. In more oxidizing conditions, the regulatory partner has weakened or altered interactions with Kv1.2, causing the inhibited gating mode to be less prominent. 103  Calibrated redox potential measurements of Kv1.2 use-dependent gating Using membrane impermeant redox couples, we clamped the extracellular redox potential to calibrate the redox sensitivity of the use-dependent effect. We measured use-dependence after incubation in a range of cysteine:cystine (Cys:CySS) ratios, the most prominent physiological extracellular redox couple (Figure 4.11A). At more positive redox potentials (ie. more oxidized conditions, low Cys:CySS ratio), there was wide cell-to-cell variability of use-dependent activation. At negative redox potentials (more reducing conditions, high Cys:CySS ratio), use-dependent activation became far more pronounced between -50 mV and -100 mV. The approximate redox potential of the Cys:CySS couple in plasma (-80 mV) is plotted as a dashed line for reference, suggesting that use-dependent activation is sensitive to extracellular redox potentials in this range, but distant from typical cytoplasmic redox potentials (~-270 mV) (Jones et al., 2000; Moriarty-Craige and Jones, 2004). We performed a similar set of calibrating experiments using the reduced glutathione:oxidized glutathione (GSH:GSSG) ratio to clamp the extracellular redox potential, and observed that use-dependent activation was also strongly shifted in the range of 0 mV to -100 mV (Figure 4.11B), similar to the Cys:CySS redox buffer.  104   Rescue of normal use-dependence with redox in an epilepsy-causing mutant of Kv1.2 Kv1.2 channelopathies have recently been found to lead to epileptic encephalopathy, ataxia and developmental delay (Syrbe et al., 2015). We tested whether a gain-of-function mutation, Kv1.2[Arg297Glu], exhibits disregualtion of use-dependence. First we examined its electrophysiological properties and found that it hyperpolarizes the activation curve by 43 mV to -62 ± 7 mV and slows deactivation kinetics by ~4-7 fold at every voltage tested (Fig. 4.12A,B). Next, we recorded use-dependent activation to determine whether the shift in activation and slowing of deactivation kinetics perturbed its regulation. We found that the rate at which Kv1.2[R297Q] channels become potentiated following a 20 Hz train of depolarizations occurs more rapidly than Kv1.2wt (Fig. 4.13A, black). The raw traces show that Kv1.2[R297Q] channels cannot close as quickly or completely as Kv1.2wt channels at a holding voltage of -80 mV. This results in an increase in the time Kv1.2[R297Q] channels spend in the open state, and therefore faster onset of channel potentiation (Fig. 4.13A, teal).  Figure 4.11. Calibrated effects of redox potential on Kv1.2 use-dependent activation. (A,B) Extracellular redox potential was clamped using membrane impermeant redox couples ((A), cysteine and cystine (Cys:CySS), or (B),reduced and oxidized glutathione (GSH:GSSG)) by varying the ratio of the two species while maintaining a total concentration of 100 µM. N = 13-15 for each redox potential. 105  We added 200 µM DTT to the external solution to determine whether we could restore wt-like use-dependent activation. Kv1.2 channels were highly susceptible to redox, showing full use-dependent activation. Kv1.2[R297Q] channels were similarly sensitive to a reducing environment, with most cells exhibiting high use-dependent activation, similar to WT (Fig. 4.13B, gray). Raw traces show that Kv1.2[R297Q] channels are now able to fully close at a similar rate to wt channels during the -80 mV interpulse interval (Fig. 4.13B, light teal). This demonstrates that a reducing environment can restore wt-like response to repetitive stimuli to a gain of function disease-causing mutant Kv1.2[R297Q]. Figure 4.12. Kv1.2[R297Q] hyerpolarizes the activation curve and slows deactivation kinetics. (A) Cells expressing Kv1.2[R297Q] exhibit a V1/2 of -62 ± 7 mV (teal), and cells expressing Kv1.2 a V1/2 of -20 ± 7 mV (black). (B) Measurement of deactivation kinetics at voltages from -150 to -70 mV in wt (black) and mutant (teal) channels. Values are average ± SD, n=6. 106    DISCUSSION Variability of use-dependent activation of Kv1.2 channels  Despite evolving into an archetype of voltage-dependent gating, and providing the rare luxury of directly comparing ion channel function with atomic resolution structure, there is a lack of understanding of regulatory mechanisms that generate considerable variability in the voltage-Figure 4.13. Use-dependent activation of Kv1.2[R297Q] in ambient and reducing extracellular conditions. Cells expressing Kv1.2[R297Q] and Kv1.2 wt were subjected to 20 Hz depolarization train in the absence (A) or presence (B) of 200 µM DTT. Pulses were normalized to the final pulse and plotted on the graph (left) for Kv1.2 wt (black) and Kv1.2[R297Q] (teal) (n=6), and raw traces are shown on right. 107  dependent activation of Kv1.2. There is marked inter-report variation in the parameters of voltage-dependent activation of Kv1.2, including significant dependence on expression system (Hite et al., 2014; Ishida et al., 2015; Rezazadeh et al., 2007). Moreover, other reports have described significant ‘intra-experimental’ or ‘pulse-to-pulse’ variation in Kv1.2 that has been challenging to explain (Grissmer et al., 1994; Rezazadeh et al., 2007). These past studies clarified that depolarizing voltages could reversibly populate Kv1.2 channels in a potentiated/facilitated mode, although it remained unclear what signals governed the inhibited gating mode. We report here that occupancy of the inhibited gating made is strongly favored by mild reducing conditions in the extracellular compartment. The dramatic effect of reducing conditions has allowed us to demonstrate that Kv1.2 channel activation can be modulated reversibly over a far greater range of voltages (between V1/2 of -11 mV and +64 mV) than was previously recognized. This basic observation will hopefully pave the way to unraveling molecular details of this poorly understood regulatory mechanism of Kv1.2. Role of Kv1.2 in diseases of electrical hyperexcitability  Beyond its importance as a biophysical model of ion channel gating, Kv1.2 appears to be an essential contributor to neurological function in humans and animal models. Severe effects of Kv1 gene deletions in mice demonstrates that loss-of-function of Kv1.2 is very poorly tolerated compared to knockout of other Kv1 channel subtypes, as Kv1.2-deficient mice die within weeks of birth due to severe generalized seizures (Brew et al., 2007; London et al., 1998b; Smart et al., 1998). A growing number of reports have linked Kv1.2 mutations to neurological diseases (Pena and Coimbra, 2015; Syrbe et al., 2015). Mutations causing either gain- or loss-of-function phenotypes of Kv1.2 have been linked to epileptic encephalopathy and ataxia (Brew et al., 2007; 108  Robbins and Tempel, 2012; Xie et al., 2010), and there is a notable low frequency of predicted loss-of-function Kv1.2 mutants (along with Kv1.4) in exome aggregation databases (ExAC consortium) (Lek et al., 2016). It should be recognized that a role for use-dependent activation of Kv1.2 has not yet been tested in these mutants when exposed to physiological extracellular redox potential. However, use-dependent activation is a unique property of Kv1.2 among the Kv1 family, we have also demonstrated previously that use-dependent activation can be detected in toxin-subtracted Kv1.2 currents recorded in primary neuronal cultures, and the redox-dependence we have observed falls in the physiological range of extracellular redox potential (Baronas et al., 2015; Jones et al., 2000; Moriarty-Craige and Jones, 2004). Taken together, it is fair to say that Kv1.2 appears to be critical for normal neurological function and other Kv1 channels cannot compensate for its loss. Redox regulation of use-dependent activation  ‘Moment-to-moment’ variation of Kv1.2 activation properties is hypothesized to reflect channel regulation by an extrinsic binding partner that stabilizes the resting channel conformation, and thereby causes channels to exhibit the ‘inhibited’ gating mode (Baronas et al., 2015, 2016). We propose that this binding partner is sensitive to extracellular redox conditions, which influence its interactions with Kv1.2 channels. Several experimental observations suggest this is a reasonable mechanism. Firstly, there is considerable cell-to-cell variability under ambient redox conditions (even when redox is buffered at mild oxidized potentials, Fig. 2, Fig. 11), which may be explained by variable stoichiometric ratios of Kv1.2 and its regulatory partner(s) (Rezazadeh et al., 2007). We speculate that in reducing conditions, the affinity of Kv1.2 interactions with its binding partner is sufficiently high that most/all channels are bound. However, in 109  ambient/oxidizing conditions (weaker channel:regulator interaction), only cells with high expression of the putative binding partner will exhibit prominent use-dependent activation. A second critical observation is that mutation of all transmembrane cysteine residues in the channel (as potential intrinsic redox sensors) fails to abolish use-dependent activation or redox sensitivity (Figure 10). We have not exhaustively tested other possible redox sensitive side chains such as methionine, although it is noteworthy that concentrations of DTT reported to be required for reduction of methionine sulfoxide are far greater than the mild DTT concentrations (and short durations) that appear to be sufficient for a dramatic gating effect in Kv1.2 (Figure 4.1C) (Houghten and Li, 1979). Also, a redox sensor intrinsic to the channel does not readily explain the large cell-to-cell variability of this phenomenon. Thirdly, expression of Kv1.2 in certain common expression systems like Xenopus laevis oocytes fails to reconstitute use-dependent activation or redox sensitivity, suggesting that these properties are not intrinsic to the channel (Horne et al., 2010; Peters et al., 2009). Lastly, it is intriguing that the only mutations identified thus far that can attenuate use-dependent activation (one of which is Kv1.2 residue T252) lie at the intracellular side of the channel (on the S2-3 linker), while the gating effect is clearly modulated by an extracellular signal. Due to this apparent transmembrane cross-talk, our ongoing investigation of use-dependence is primarily focused on the identification of interacting proteins, although we do not have absolute evidence to rule out the possibility that a lipid or other class of signaling molecule is involved. Many ion channels are regulated by extracellular redox  Other ion channel types have also been reported to be sensitive to the external redox environment, although this has generally been ascribed to intrinsic redox-sensing mechanisms. 110  TRPC5 channels contain a disulfide in the S5-6 linker which is sensitive to extracellularly applied thioredoxin (Trx) (Xu et al., 2008). Similarly, Cav3.2 channels are modulated by extracellular Trx, although in this case the extracellular redox sensor remains unidentified (Boycott et al., 2013). The NMDA (NR1/NR2A) receptor (Köhr et al., 1994), Orai1 (Bogeski et al., 2010), and ASIC channels (Chu et al., 2006) all have intrinsic redox-sensitive elements that alter channel function. Perhaps the mechanism of extracellular redox sensitivity that is most analogous to the mechanism we have proposed is the regulation of β3 subunit effects on BK channels by extracellular redox. This appears to be related to the formation of disulfides in the extracellular loop of β3 (Zeng et al., 2003), thus extracellular redox sensitivity is imparted by disulfide formation/breakage on an auxiliary binding partner, similar to our proposed model (Figure 4.10C), albeit with different functional outcomes. While the physiological roles of redox sensitivity of ion channels including Kv1.2 remain unclear, it is apparent that multiple channel types have evolved diverse mechanisms of redox responsiveness.  We now have tools in hand to continue to investigate physiological roles of use-dependent activation of Kv1.2 and its regulation by redox. Furthermore, alteration of redox state appears to be an effective way to regulate Kv1.2, and could prove to be a novel drug target in the treatment of epilepsy, especially those caused by gain of function mutations in the Kv1.2 channel. The importance of Kv1.2 for normal neurological function in humans and mice, together with the transferability of Kv1.2 use-dependent activation into heteromeric Kv1 complexes, suggests potentially important roles for this incompletely understood mode of regulation. Conclusion  111  In summary, we have demonstrated redox conditions and patterns of voltage stimulation that bias homo- and heterotetrameric Kv1.2 channels into previously unrecognized extreme modes of channel gating. This variability of Kv1.2 gating relative to its close Kv1 family relatives underlies its unique but variable use-dependent activation properties. Ongoing investigation of the molecular basis for this process will hopefully reveal previously unrecognized binding partners and signaling mechanisms that control ion channel function.  112  CHAPTER 5: SLC7A5 REGULATION OF Kv1.2 POTASSIUM CHANNELS BY AN INACTIVATION GATING TRAP MECHANISM   INTRODUCTION Kv1.2 is a prominent voltage-gated potassium channel in the central nervous system, where it influences cellular excitability and action potential propagation (Bean, 2007; Bekkers and Delaney, 2001; Guan et al., 2007). As the first eukaryotic voltage-gated channel with a reported atomic resolution structure (Long et al., 2005a), it has been used as a template for understanding and investigating voltage-dependent regulation of ion channels, perhaps at the expense of understanding its function in a physiological context. Early mouse knockout models showed a particular requirement for Kv1.2 among the Kv1 subfamily, as Kv1.2 knockout fail to survive beyond 3 weeks of life due to severe generalized seizures (Brew et al., 2007). Even mildly perturbative mutations of Kv1.2 have been linked to an ataxic phenotype in mice (Xie et al., 2010). More recently, the advent of next-generation sequencing has accelerated the correlation of genetic mutations with rare phenotypes, and several Kv1.2 mutations have been identified in patients with severe epilepsies (Corbett et al., 2016; Hundallah et al., 2016; Masnada et al., 2017; Syrbe et al., 2015). Molecular phenotyping of these genetic defects in heterologous systems yields basic information that may partly inform the link between the mutation and the disease, but missing from these interpretations is a more complete understanding of interactions between channels and extrinsic regulators such as accessory proteins. Although our study focuses on Kv1.2, this shortcoming is likely true for many investigations of disease-linked ion channel or neurotransmitter receptor mutations.  113  In the specific case of Kv1.2, several previous reports have described a poorly understood dynamic regulation in heterologous systems and primary dissociated neurons, generating wide cell-to-cell variability of Kv1.2 gating that likely depends on extrinsic regulatory mechanisms (not directly encoded by the primary sequence of the channel) (Baronas et al., 2015; Rezazadeh et al., 2007). The canonical accessory proteins for Kv1.2 and other Kv1 subtypes are Kvβ subunits, which promote cell surface maturation and (in some cases) inactivation (Heinemann et al., 1996; Rettig et al., 1994; Shi et al., 1996). Kv1.2 subunits also bind to cytoskeletal anchors including cortactin, in a tyrosine phosphorylation dependent manner that influences Kv1.2 endocytosis (Nesti et al., 2004; Williams et al., 2007). The sigma-1 receptor is another associated protein of Kv1.2, reported to assemble with Kv1.2 and promote trafficking to the cell membrane in response to cocaine exposure  (Kourrich et al., 2013). Certain lipids, including phosphatidic acid, can alter the voltage-dependence of Kv1.2 activation (Hite et al., 2014). Despite the variety of extrinsic factors reported to regulate Kv1.2 channel gating, none of these processes can account for dramatic moment-to-moment alteration of Kv1.2 activity that has been observed. In order to address this gap in our understanding of ion channel regulation, we investigated the potential assembly of Kv1.2 with previously unrecognized accessory proteins. We used a mass spectrometry approach to generate candidate genes, followed by screening of their effects on Kv1.2 function and expression. We report the surprising finding that Slc7a5, a neutral amino acid transporter, associates with Kv1.2 channels and dramatically alters their gating and expression. Several aspects of this regulatory complex stand out as novel and interesting. Firstly, Slc7a5 mutations have been linked to recessively-inherited neurodevelopmental delay (Tărlungeanu et al., 2016), and while this has been attributed to its role as an amino acid transporter, the 114  pleiotropy we report suggests other possible mechanisms that may lead to a severe neurological phenotype. Secondly, the assembly of an ion channel and transporter is part of an emerging trend of functional interactions between complex transmembrane proteins (channels, transporters, GPCRs) (Abbott, 2017; Doupnik, 2008; Zamponi, 2015). Thirdly, the gating effects of Slc7a5 are far more dramatic than any previously reported accessory subunit of Kv1 channels, and we also describe a novel ‘inactivation trap’ mechanism of current suppression involving compounded effects of accelerated inactivation and a pronounced hyperpolarizing shift of channel activation. Lastly, we report that gain-of-function Kv1.2 mutations identified in patients with severe human epilepsy are particularly susceptible to suppression by the Slc7a5 inactivation trap, and this may underlie the paradoxical observation that both gain- and loss-of-function Kv1.2 mutations lead to severe epilepsy (Syrbe et al., 2015). RESULTS Identification of novel Kv1.2-associated proteins We have reported marked variability of Kv1.2 gating parameters between commonly used expression systems, suggesting these channels are subject to unrecognized regulatory mechanisms (Baronas et al., 2015, 2017). To identify interacting proteins, we used 1D4 affinity purification of cross-linked Kv1.2 channel complexes, followed by quantitative LC-MS/MS mass spectrometry. Several previously reported Kv1.2 regulatory proteins appeared in the screen, including Kvβ (Shi et al., 1996), several phosphatases and kinases (Cachero et al., 1998; Nesti et al., 2004), and a RhoA guanine nucleotide exchange factor (Keller et al., 1997). Based on criteria including abundance relative to pulldowns from untransfected cells, and cross-referencing against the CRAPome (Mellacheruvu et al., 2013) we selected 30 candidate proteins for further 115  screening by electrophysiology in ltk- fibroblasts (Fig. 5.1A). Our previous study highlighted a prominent effect of redox conditions on Kv1.2 (Baronas et al., 2017), so we also included abundant proteins previously identified to contain labile extracellular disulfide bonds (Metcalfe et al., 2011). Electrophysiology recordings were performed in ambient redox conditions (Fig. 5.1B,C) and we parameterized effects on channel function using the half-activation voltage (V1/2, Fig. 5.1B) and current density (Fig. 5.1C). Most tested proteins had no discernible impact on Kv1.2 electrical function, although they may impact Kv1.2 in ways that are not detected by our screening protocol. Among the candidate proteins tested, the neutral amino acid transporter Slc7a5 (LAT1) had a pronounced impact on Kv1.2, greatly reducing Kv1.2 currents and shifting the conductance-voltage relationship by approximately -40 mV (Fig. 5.1B,C).  116   Suppression of Kv1.2 currents by Slc7a5 We further explored the effects of Slc7a5 on Kv1.2 using electrophysiology (Fig. 5.2A) and western blot (Fig. 5.2B-D). Co-expression of Kv1.2 and Slc7a5 (2:1 transfection ratio) markedly decreased Kv1.2 current density, recapitulating the findings of our initial screen. We also tested Slc3a2, known to form a heterodimer with Slc7a5 and influence cell surface maturation (Napolitano et al., 2015). Co-expression with Slc3a2 did not affect Kv1.2 current density. Figure 5.1. Mass spectrometry and screening potential Kv1.2 interacting proteins. Kv1.2-1D4 was expressed in HEK cells, and EGS-crosslinked complexes were immunoprecipitated and analyzed by LC-MS/MS. 30 proteins were chosen for further screening. (A) Abundance of proteins relative to untransfected cells, with cross-linking performed in ambient redox conditions (black) or 1 mM DTT (gray). Proteins containing labile extracellular disulfides are indicated by the black bar. (B) V1/2 (mean ± S.D.) was determined for Kv1.2 when co-expressed with each candidate gene (mEGFP-tagged) in Itk-mouse fibroblasts. (C) Kv1.2 current densities at +60 mV after co-expression with each candidate gene. Data from individual cells are superimposed on bars that represent mean ± S.D. (n=5-15, n=31 for WT Kv1.2). 117  However, the combination of Kv1.2, Slc3a2, and Slc7a5 (2:2:1) partially rescued the Slc7a5 mediated suppression of Kv1.2 currents. We chose this ratio because there is better rescue of Kv1.2 currents with increasing amounts of Slc3a2 (data not shown). We also measured Kv1.2 protein expression in cell lysates after transient transfection with Kv1.2 and various combinations of Slc7a5 and Slc3a2 (Fig. 5.2B-D). Kv1.2 generates two prominent bands on SDS-PAGE: a high molecular weight mature cell surface band, and a low molecular weight immature core glycosylated band (Fujita et al., 2006). Slc7a5 + Kv1.2 (1:1) co-expression diminished total Kv1.2 expression by 50 ± 15 % relative to Kv1.2 alone (p=0.0003). Slc3a2 + Kv1.2 (1:1) did not significantly affect overall expression (116 ± 43 %, p=0.2), and co-expression of Slc3a2 with Slc7a5 + Kv1.2 (1:1:1) partially rescued Kv1.2 expression from the Slc7a5 effect, to 68 ± 3 % (p<0.01) (Fig. 5.2C). In all cases, surface expression as a fraction of total protein was not affected (Fig. 5.2D).  118   We noted an inconsistency between Kv1.2 current density versus protein expression, depending on the expression of Slc7a5 and Slc3a2. For example, co-expression of Kv1.2 + Slc7a5 + Slc3a2 did not dramatically rescue protein expression (relative to Kv1.2 + Slc7a5, Fig. 5.2B,D), but had much greater current relative to the Kv1.2 + Slc7a5 condition (Fig. 5.2A). Thus, it was unclear why Slc7a5 caused such pronounced current suppression. We resolved this inconsistency when we recognized that hyperpolarized holding voltages lead to significant disinhibition of Kv1.2 current when co-expressed with Slc7a5. We measured Kv1.2 current at 0.5 s intervals, with various holding potentials between -80 mV and -120 mV (Fig. 5.3A).  We observed that with a holding potential of -120 mV, Kv1.2 currents increase significantly (~5-fold) during a 30 s pulse train, but Figure 5.2. Effects of Slc7a5 on Kv1.2 expression and current density. (A) Combinations of Kv1.2, mCherry-Slc7a5, and mEGFP-Slc3a2 were expressed in Itk-mouse fibroblasts. Current density was measured at + 60 mV. Transfection ratios were: Kv1.2:Slc7a5 (1:0.5); Kv1.2:Slc3a2 (1:1); Kv1.2:Slc7a5:Slc3a2 (1:0.5:1), n = 14-19 cells. (B) Exemplar anti-Kv1.2 Western blot of Itk-mouse fibroblasts transfected as in panel (A) except Kv1.2+Slc7a5+Slc3a2 (1:1:1) for 72 hours. (C) Densitometry measurements of the cell surface:total Kv1.2 protein. No significant differences from Kv1.2 were observed (p > 0.05). (D) Densitometry of total Kv1.2 protein, normalized to WT Kv1.2 alone. Data from individual experiments are superimposed on bars that depict mean ± S.D. (n=3-5). 119  not with -80 or -100 mV holding potentials (Fig. 5.3B,C). We are uncertain whether significantly more disinhibition can be achieved with more negative holding potentials or a longer time at -120 mV, as the long application of strong hyperpolarized voltages was a technical challenge that led to seal breakdown.    Slc7a5 induces a prominent hyperpolarizing shift of Kv1.2 activation  The recognition of hyperpolarization-mediated disinhibition in the presence of Slc7a5 allowed us to examine effects on channel activation in more detail. Kv1.2 + Slc7a5 co-expression (2:1 transfection ratio) leads to a hyperpolarized V1/2 of Kv1.2 activation of -58 ± 3 mV, compared to -11 ± 3 mV in Kv1.2 channels expressed alone. Co-expression of Slc3a2 with Kv1.2 (1:1 transfection ratio) does not affect the V1/2 (-11 ± 10 mV). However, similar to the effects on Figure 5.3. Hyperpolarization disinhibits Kv1.2 co-expressed with Slc7a5. (A) ltk-mouse fibroblast cells transfected with Kv1.2+Slc7a5 (1:1) were held at a range of voltages: -80 mV (n=7), -100 mV (n=12) and -120 mV (n=25). Currents were measured at +10 mV (50 ms pulses, every 300 ms), and normalized to the peak current after 30 s at -120 mV (mean ± S.D.). (B) Cell-by-cell current density of the first (gray) and final (green) pulses (+10 mV) of a train of depolarizations with a -120 mV holding voltage. (C) Fold change between the initial and final test pulses of a 30 s pulse train with the indicated holding voltages (mean ± S.D.: 1.4 ± 0.1 at -80 mV; 1.6 ± 0.3 at -100 mV; 5 ± 2-fold at -120 mV).  120  current density, co-expression of Kv1.2 with both Slc7a5 and Slc3a2 rescues the Slc7a5-mediated gating shift, generating a V1/2 of -16 ± 3 mV (Fig. 5.4 A, B). Although we have not exhaustively tested the entire Slc7 transporter family, the effects of Slc7a5 appear to be quite specific, based on our observation that Kv1.2 is unaltered by co-expression with Slc7a6, a closely-related amino acid transporter that also heterodimerizes with Slc3a2 (Pineda et al., 1999). Also, the Slc1a5 amino acid transporter has no effect on Kv1.2 gating or current magnitude (Fig. 5.5A). Lastly, Slc7a5 appears to exhibit subtype specificity within the Kv1 subfamily, as co-expression of Slc7a5 with Kv1.5 did not alter gating or expression (Fig. 5.5B).  Overall, these findings suggest the interaction between Kv1.2 and Slc7a5 exhibits subtype specificity, and this may be a useful observation to identify critical elements underlying the interaction.  Figure 5.4. Slc7a5 co-expression hyper-polarizes the Kv1.2 activation curve. (A) Indicated combinations of Kv1.2, Slc7a5, and Slc3a2 were transfected in ltk- mouse fibroblasts. Kv1.2+Slc7a5 cells were given a hyperpolarization to -120 mV for 30 sec prior to recording. Conductance voltage relationships were measured by stepping between -120 and +60 mV (100 ms in 10 mV steps) followed by a tail current voltage of -30 mV. In addition, a 100 ms depolarization to +60 mV was delivered before each sweep to relieve any use-dependent activation. Conductance-voltage relationships were generated from the tail current amplitudes and  fit with a Boltzmann function (Kv1.2 V1/2 = -11 ± 3, k = 11 ± 3; Kv1.2 + Slc7a5 V1/2 = -58 ± 3, k= 10 ±2; Kv1.2 + Slc3a2 V1/2 = -11 ± 10, k = 10 ± 3; and Kv1.2 + Slc7a5 + Slc3a2 V1/2 = -16 ± 3 mV, k = 11 ± 2). (B) Representative traces of the activation curves measured in A, and the pulse to -20 mV is highlighted. 121   Slc7a5 promotes Kv1.2 inactivation The hyperpolarization-induced current disinhibition led us to speculate that Slc7a5 might induce Kv1.2 inactivation, which may have an especially prominent effect in heterologous cell lines due to their relatively high resting membrane potential (~-35 mV) (Chemin et al., 2000; Ince et al., 1984). To investigate the effects of Slc7a5 on Kv1.2 inactivation, we used the Kv1.2[Val381Thr] mutant, which replaces the outer pore residue equivalent to Shaker Thr449, making channels more prone to C-type inactivation (López-Barneo et al., 1993). Kv1.2[V381T] channels exhibit a similar Slc7a5-mediated shift in voltage-dependence as WT Kv1.2 (Fig. 5.6A), along with disinhibition induced by hyperpolarization to -120 mV (Fig. 5.6B). Most strikingly, the greater susceptibility to C-type inactivation revealed that Slc7a5 co-expression markedly accelerates the rate of inactivation (Fig. 5.6C) and shifts the steady state inactivation curve from -31 ± 3 mV to -69 ± 6 mV (Fig. 5.7).  Figure 5.5. Other Slc7 and Kv1 subtypes do not alter gating. A) Kv1.5 channels were expressed alone or with Slc7a5 (1:1), Slc3a2 (1:1), or Slc7a5+Slc3a2 (1:1:1), and activation curves recorded and fit with Boltzmann showed a V1/2 of -2.7 ± 2.6, -5 ± 4.6, -1.7 ± 5.0, and -3.2 ±3.9 mV, respectively, and k values of 9.0 ± 1.8, 8.7 ± 1.8, 9.3 ± 1.6 and 8.9 ± 1.4, respectively. B) Kv1.2 channels co-expressed with Slc7a6 (1:1) or Slc1a5 (1:1) did not alter activation curve, with a V1/2 of -15 ± 7 and -14 ± 5 mV and a k value of 9.7 ± 2.3 and 9.3 ± 2.0, respectively (n=4, values are average ± SD). 122   Figure 5.6. Slc7a5 influences inactivation of Kv1.2. (A) Kv1.2[V381T] channels were co-expressed alone or with Slc7a5 (1:1). Conductance-voltage relationships were generated with the same protocol as Fig. 5.4, after disinhibition by a 30 s hyperpolarization to -120 mV (Kv1.2[V381T] V1/2 = -16 ± 4 mV, k = 8.5 ± 0.7; + Slc7a5 V1/2 = -55 ± 11 mV, k = 13.1 ± 3.2 (n=6-8)). B) Current density at +60 mV was measured at the beginning and of a pulse train with -120 mV holding potential (identical to Fig. 3; Kv1.2[V381T] n = 8; + Slc7a5 n = 16). (C) Exemplar traces of Kv1.2[V381T] ± Slc7a5 (green) currents elicited by a 1 s depolarization to +60 mV (after current disinhibition by holding at -120 mV). (D,E) Identical experiments as in (A,B) were performed with Kv1.2 ‘LT’ channels (Kv1.2[Ile304Thr][Ser308Thr]) ± Slc7a5 (Kv1.2 ‘LT’ V1/2 = +80 ± 10 mV, k = 18 ± 2; +Slc7a5 V1/2 = +40 ± 11 mV,  k = 17 ± 2) (n=7). (F) Current disinhibition (fold change in current after a -120 mV pulse train) for Kv1.2[V381T] or Kv1.2’LT’. Bars represent mean ± S.D. 123  We also tested Slc7a5 effects on the Kv1.2[Ile304Thr][Ser308Thr] (Kv1.2 ‘LT’) mutant, which shifts channel activation to depolarized voltages by dissociating voltage sensor movement from channel activation (Ledwell and Aldrich, 1999). We hypothesized that Kv1.2 ‘LT’ would be less prone to current suppression by Slc7a5 because very few channels would activate at resting membrane voltages. Kv1.2 ‘LT’ co-expression with Slc7a5 caused a hyperpolarizing shift of activation relative to Kv1.2 ‘LT’ expressed alone (Fig. 5.6D). More importantly, hyperpolarization to -120 mV did not generate any consistent disinhibition of current, regardless of the presence of Slc7a5 (Fig. 5.6E,F). Taken together, these findings suggest that Slc7a5 promotes C-type inactivation (coupled to channel opening) of Kv1.2 channels, which underlies the Slc7a5-mediated current suppression (Fig. 5.2A). In combination with the shift in voltage-dependence of channel activation, this effect leads to a requirement for strong hyperpolarizing voltages to recover from inactivation.    Kv1.2 and Slc7a5 are in close physical proximity Slc7a5 has profound functional effects on Kv1.2 function and expression. We further investigated the physical nature of this interaction using a bioluminescence resonance energy transfer (BRET) Figure 5.7. Slc7a5 shifts steady state inactivation of Kv1.2[V381T]. Kv1.2[V381T] channels were expressed alone or with Slc7a5 to speed inactivation kinetics, and steady state inactivation was recorded by pulsing to various voltages for 6 sec and allowing channels to recover for 7 s at -100 mV. Curves were fit with a Boltzmann function and we found a V1/2 of inactivation of -31 ± 3 mV, and a k value of 11.1 ± 2.1 for Kv1.2[V381T] alone and a V1/2 of -69 ± 6 mV, a k value of 6.7 ± 1.8 with Slc7a5. 124  approach to assess their proximity in a recombinant system. We fused the Nanoluc bioluminescent donor protein to Kv1.2 and used mEGFP as an acceptor fluorophore fused to various test constructs. We collected emission spectra between 450 and 700 nm after addition of furimazine, resulting in a large emission peak centered at ~440 nm corresponding to Nanoluc bioluminescence, and variable levels of a ‘shoulder’ with a peak centered at ~510 nm corresponding to mEGFP emission. Emission spectra were well fit by the sum of weighted components of the Nanoluc and mEGFP spectra (Fig. 5.8A). We tested a variety of mEGFP-tagged BRET acceptor constructs, which resulted in variable acceptor emission when co-expressed with Kv1.2-Nanoluc. Kv1.2 can assemble as a homotetramer, therefore co-expression with mEGFP-Kv1.2 generates mEGFP emission when the donor and acceptor are brought into proximity by channel assembly. Although not quite as pronounced, mEGFP-Slc7a5 generated a clearly discernable acceptor emission signal (Fig. 5.8A), whereas the Slc1a5 negative control generated a much smaller emission. To quantify these signals, we calculated the area under the curve (AUC) of the mEGFP emission component between 480-600 nm (Fig. 5.8B) and normalized each AUC to a matched mEGFP-Kv1.2 positive control (run in parallel with each experiment, Fig. 5.8C). We also tested whether co-expression of Slc3a2 with EGFP-Slc7a5 would influence the BRET signal. In the presence of Slc3a2, the BRET signal generated by Kv1.2-Nanoluc and mEGFP-Slc7a5 was attenuated, but not fully eliminated, to 0.42 ± 0.2. These findings suggest that Slc3a2 can influence the interaction between Kv1.2 and Slc7a5, but may not completely prevent association of the two proteins. 125   Variable responses of a tripartite Kv1.2:Slc7a5:Slc3a2 complex We further investigated the nature of mutual interactions between Kv1.2, Slc7a5, and Slc3a2, using a flow cytometry approach to determine whether expression of one protein might influence assembly of the other two. We used complementation of split YFP fragments fused to two subunits as a crude assessment of protein interaction, and tested whether the third subunit could influence YFP reconstitution (Michnick et al., 2000). For example, we tested whether YFP reconstitution between Kv1.2-YFPC and YFPN-Slc7a5 was altered by expression of Slc3a2-LSSmOrange (or LSS-mOrange alone as a control). For all permutations tested, we found only modest effects of the LSS-mOrange tagged subunit on reconstitution of YFP (Fig. 5.9A), suggesting that assembly of Slc7a5 with Slc3a2 does not preclude assembly with Kv1.2, and similarly, assembly of Slc7a5 with Kv1.2 does not prevent assembly with Slc3a2 (Fig. 5.9A,B). In Figure 5.8. Measurement of the proximity of Slc7a5 and Kv1.2 with bioluminescence resonance energy transfer (BRET). (A) Emission spectra were collected from HEK293 cells transfected with Kv1.2-Nanoluc + EGFP-Slc7a5 (green line normalized to peak). The Kv1.2-nanoluc (donor) spectrum was subtracted o yield the mEGFP component. Weighted components of the nanoluc (1.0) and mEGFP (0.1) spectra were used to fit the experimental spectrum. (B) mEGFP fluorescence (nanoluc-subtracted) was measured for Kv1.2-Nanoluc co-expressed with various acceptor constructs, as indicated. (C) The area under the curve (AUC) for each BRET acceptor in B was normalized to the positive control AUC (Kv1.2-EGFP). Data are shown as mean ± SD (n=4). P-values calculated using a paired t-test. 126  the representative experiment depicted in Fig. 5.9B, it is noteworthy that cells with the brightest LSS-mOrange fluorescence (from Slc3a2) also tended to have the brightest YFP signal (from the assembly of Kv1.2-YFPC and YFPN-Slc7a5), suggesting that Slc3a2 does not prevent assembly of Slc7a5 and Kv1.2. This finding was supported by patch clamp experiments testing Slc3a2 effects when assembly of the Kv1.2:Slc7a5 complex was enhanced using Kv1.2-YFPC and YFPN-Slc7a5 constructs (driving irreversible formation of the 1.2:Slc7a5 complex). In these experiments, co-expression of Kv1.2-YFPC and YFPN-Slc7a5 mimicked the gating shift and current suppression observed with WT constructs (Fig. 5.9C). Co-expression of Slc3a2-LSS-mOrange generated a wide range of gating phenotypes despite the irreversible fusion of Slc7a5 and Kv1.2, suggesting that Slc3a2 can alter the effects of Slc7a5 on Kv1.2 even when they are in close proximity (Fig. 5.9C,D). Although further investigation of a possible tripartite complex will be required, these findings together suggest that Slc3a2 may modulate the Slc7a5:Kv1.2 interaction without a requirement for direct competition with Kv1.2.  127   Figure 5.9. Using split YFP constructs to measure competition for Slc7a5 between Kv1.2 and Slc3a2. GFP was split into two fragments between positions Q157 and K158, and the C-terminal portion was attached to Kv1.2, Kv1.2-YFPC and the N-terminal portion to either Slc7a5 or Slc3a2, YFPN-Slc7a5 and YFPN-Slc3a2. A) Fluorescence-activated cell sorting was performed on Kv1.2-YFPC+YFPN-Slc7a5 (middle) with mOrange or with mOrange-Slc3a2, Kv1.2-YFPC+YFPN-Slc3a2 (right) with mOrange or mOrange-Slc7a5, and Slc7a5-YFPC+YFPN-Slc3a2 (left) with mOrange or mOrange-Kv1.2. Each bar represents the total fraction of transfected cells, the green bar represents cells with only YFP fluorescence, the orange bar cells with only mOrange fluorescence and the grey bars fluorescence from both. B) Graphs show a scatter plot of all cells in the Kv1.2-YFPC+YFPN-Slc3a2+mOrange or +mOrange-Slc7a5 runs. C) Kv1.2-YFPC produced a current density of 390 ± 410, Kv1.2-YFPC+YFPN-Slc7a5 (1:1) 11 ± 4, Kv1.2-YFPC+YFPN-Slc3a2 (1:1) 280 ± 160, and Kv1.2-YFPC+YFPN-Slc7a5+mOrange-Slc3a2(1:1:1) 420 ± 514 pA/pF. Individual data and average ± SD are shown, n=7-20. D) Activation curves were recorded for each construct after a 30 sec -120 mV hyperpolarization, fit with a Boltzmann and the V1/2 was -8.4 ± 7, -49 ± 2, -11 ± 4, and -36 ± 16 mV, respectively, with the exception that Kv1.2-YFPC+YFPN-Slc7a5 ratio was 1:0.5 to have enough current to record activation curves. Boltzmann fit and average ± SD are shown, n= 4-11. 128  Disease-linked mutants of Slc7a5 attenuate the WT-dependent current reduction and activation shift Slc7a5 mutations that impair amino acid transport have recently been linked to recessively-inherited forms of autism spectrum disorder (Tărlungeanu et al., 2016). We tested the effects of these disease-linked mutants on Kv1.2 gating and expression. Slc7a5[A246V] had no measurable effect on Kv1.2 gating, current density, or disinhibition at -120 mV (Fig. 5.10A,C dark blue). In addition, Slc7a5[A246V] did not reduce total Kv1.2 protein levels (Fig. 5.10D). Co-expression of Slc7a5[P375L] had attenuated effects on gating relative to WT Slc7a5, and also some suppression of current expression (Fig. 5.10A,C light blue). Neither Slc7a5 mutant generated a BRET signal above the Slc1a5 negative control when coexpressed with Kv1.2-Nanoluc (Fig. 5.10B). Overall, these disease-linked Slc7a5 mutants lead to weakened or abolished effects on Kv1.2 gating and expression.     129   Disease-linked mutants of Kv1.2 are extremely susceptible to Slc7a5 We tested the effect of Slc7a5 on two disease-linked mutations in Kv1.2, Arg297Gln and Leu289Phe (Syrbe et al., 2015). When expressed alone, these mutations cause a gain-of-function phenotype involving a hyperpolarizing shift of the conductance-voltage relationship relative to WT Kv1.2 (Fig. 5.11C,D), and modest or absent disinhibition after hyperpolarization to -120 mV (Fig. 5.11A,B). However, both mutants are extremely susceptible to Slc7a5, leading to dramatic Figure 5.10. Disease-linked Slc7a5 mutations have attenuated effects on Kv1.2. (A) Kv1.2 channels were co-expressed with Slc7a5[A246V] or Slc7a5[P375L] (1:1) in Itk-mouse fibroblast cells. Conductance-voltage relationships were gathered as described in Fig. 5.4 (+Slc7a5[A246V] V1/2 = -10 ± 6 mV, k = 11 ± 3; +Slc7a5[P375L] V1/2 = -32 ± 8 mV, k = 16 ± 8). Dashed lines illustrate conductance-voltage relationships for WT Kv1.2 (black) and Slc7a5 (green) as a reference (n=6-10). (B) BRET signals were calculated for mEGFP-tagged Slc7a5 mutants co-expressed with Kv1.2-nanoluc, as described in Fig. 6. (C) Current density at +60 mV was measured from Kv1.2 co-expressed with various Slc7a5 mutants, before and after a 30 s train of -120 mV hyperpolarizations to -120 mV (D) Cell lysates were probed for Kv1.2 and Slc7a5 expression using western blot for cells transfected with Kv1.2 plus each of the Slc7a5 mutations. No significant changes were detected for Kv1.2 expression when co-expressed with the Slc7a5 mutants (n=3, p=0.3). 130  current reduction, and hyperpolarizing shifts of channel activation (Kv1.2[R297Q] V1/2 = -143 ± 6 mV; Kv1.2[L298F] V1/2 < -200 mV; Fig. 5.11C,D, bottom). This is especially evident in the exemplar traces where current inhibition coupled with the shift in activation can be better appreciated (Fig. 5.11C,D, top). The large shift in voltage-dependent activation provides a rationale for the low current density and very weak extent of disinhibition observed at -120 mV (Fig. 5.11A,B).  Since the activation curve of the mutant channels is so dramatically shifted, significantly more negative voltages would be required for recovery from inactivation to occur. Using Western blots, we demonstrated that expression of the mutant channels was slightly reduced relative to WT Kv1.2 channels and decreased further by co-expression of Slc7a5 (fraction of WT Kv1.2 expression: 33 ± 3% for Kv1.2[R297Q] and 35 ± 6% for Kv1.2[L298F]), indicating that mutant Kv1.2 channels are still present at the membrane. Overall, disease-linked Kv1.2 mutants categorized as a gain-of-function phenotype, appear to be strongly suppressed by Slc7a5 due to diminished protein expression and hypersensitivity to the gating. 131    Figure 5.11. Kv1.2 disease-linked mutations are strongly suppressed by Slc7a5. (A,B) Current density at +60 mV was measured before and after a 30 s hyperpolarizing train to -120 mV for Kv1.2[R297Q] or Kv1.2[L298F] ± Slc7a5, as indicated. (C,D) Conductance-voltage relationships were generated for Kv1.2[R297Q] (V1/2 = -57 ± 4 mV, k = 8 ± 1, n=6) and  Kv1.2[R297Q]+Slc7a5 (2:1 transfection, V1/2 = -143 ± 6 mV, k = 17 ± 2, n=9), Kv1.2[L298F] (V1/2 = -56 ± 1, 7 ± 1, n=4) mV and Kv1.2[L298F]+Slc7a5 (4:1 transfection, V1/2 < -200 mV, estimated to be -210 ± 5 mV, k = 35 ± 6, n=9). Exemplar traces (C,D) recorded from -200 to +30 mV, followed by a -30 mV tail current, demonstrate the shift in activation and reduction in current caused by Slc7a5, with highlighted sweeps at -120 mV. (E,F) Western blot analysis confirms expression of Kv1.2[R297Q] (76 ± 26% relative to WT Kv1.2), Kv1.2[L298F] (71 ± 12%). Expression decreased by ~2-fold for both mutants after co-expression with Slc7a5 (n=3). 132  DISCUSSION In this study we demonstrate profound regulation of many aspects of Kv1.2 gating and function by Slc7a5, an unexpected regulatory partner with a previously described function as a neutral amino acid transporter (Kanai et al., 1998; Mastroberardino et al., 1998).  We demonstrate that Slc7a5 suppresses Kv1.2 function by a combination of diminished Kv1.2 expression (Fig. 5.2B-D), and compounded effects of accelerated inactivation and a hyperpolarized voltage-dependence of activation that shifts the voltage dependence of recovery to very hyperpolarized voltages (Figs. 5.3, 5.4, 5.6). Recently reported Slc7a5 mutations linked to autosomal recessive inherited neurodevelopmental delay/autism spectrum disorder (A246V and P375L) have attenuated effects on Kv1.2 gating and current density. In contrast, epileptic encephalopathy-linked mutations of Kv1.2 (R297Q and L298F) enhance susceptibility to the gating effects of Slc7a5. Relative to other known accessory subunits of Kv1 channels, these gating effects are unique and large in magnitude, and reveal a previously unrecognized regulatory protein of Kv1.2 (Kourrich et al., 2013; Rettig et al., 1994; Tsai et al., 1999; Williams et al., 2007). Identification of novel interactors of the Kv1.2 channel Kv1.2 was the first eukaryotic voltage-gated ion channel with a reported atomic resolution structure, and has served as a valuable template for understanding mechanisms of voltage-dependent gating of ion channels (Long et al., 2005a). Beyond the core structure and function of Kv1.2, a variety of putative regulatory proteins have been reported. In search of novel regulatory mechanisms, we adopted a mass spectrometry approach of cross-linked protein complexes, followed by screening to identify proteins with clear functional effects. Our rationale was primarily to ensure detection of proteins that may have transient or lower affinity interactions 133  with the channel, or that are lost with detergent solubilization and co-immunoprecipitation. We used 16 Å-long bifunctional crosslinker, leading to identification of a large number of candidate interactors, so using a quantitative mass spectrometry approach was useful to filter this list and focus on the most abundant candidates. Generally speaking, we weighted our approach to be less stringent at the immunoprecipitation stage, at the expense of more laborious screening of candidate genes. A more ‘stringent’ (non-crosslinked, more detergent washes, etc.) would certainly yield less candidate genes, but might also miss important transient or low affinity interactions. ‘Inactivation trap’ is a novel mechanism of Kv1.2 regulation We refer to the gating effects of Slc7a5 on Kv1.2 potassium channels as an ‘inactivation trap’. The hyperpolarizing shift of channel activation, coupled with suppression of current, initially seemed to be counteracting effects. However, these are related and the hyperpolarizing shift contributes to current suppression by making the channels more prone to inactivate. This effect of Slc7a5 is accomplished by the combination of opening at more negative voltages (stabilization of channel activation) and enhancing the inactivation rate (accentuated in the V381T mutant). In the presence of Slc7a5, channel activation and inactivation are sufficiently shifted (Fig. 5.12C, green lines) that some inactivation of Kv1.2 may occur even when cells are at rest. Perhaps most importantly, due to the prominent gating shift, extremely negative voltages are required to allow recovery from inactivation (Fig. 5.3A, holding potential to -120 mV). In a physiological system where such extreme voltages are never reached, this molecular complex would act as a ‘trap’ that could alter excitability by silencing Kv1.2 channels. Although the stoichiometry of the Kv1.2:Slc7a5 interaction is not yet known, it is important to note that assembly with different 134  numbers of Slc7a5 subunits, or the possible assembly of channels with Slc7a5 sensitive (e.g. Kv1.2) and insensitive (e.g. Kv1.5) subunits, may lead to intermediate instances of the effects described here. Further exploration of the entire Kv1 family, other Kv subtypes, and the Slc7 family will likely reveal functional diversity of these effects. Reconciling heterogeneity of Kv1.2 disease mutants that lead to the same disease phenotype  The inactivation trap effect of Slc7a5 is noteworthy the context of an apparent paradox that has emerged in the characterization of epilepsy-associated Kv1.2 mutations. Recent findings have reported similar epileptic phenotypes for both gain- and loss-of-function Kv1.2 mutants (Masnada et al., 2017; Syrbe et al., 2015). In addition, an observation lacking a good explanation has been that Kv1.2 mutants with powerful gain-of-function (increased current magnitude and ~50 mV hyperpolarizing gating shifts) lead to a phenotype of hyperexcitability, rather than suppression of excitation. Although many factors may contribute to these outcomes, it is noteworthy that gain of function mutations of Kv1.2 are much more sensitive to the Slc7a5-mediated silencing, and this could lead to a loss-of-function effect when expressed in the presence of Slc7a5. The hypersensitivity of Kv1.2 gain-of-function mutations arises from two main differences that we have observed. Firstly, there is an intrinsic gating shift of the mutant channels (Fig. 5.12D, black lines), and for reasons that are not clear, the Slc7a5-mediated gating shift is even more extreme than seen with WT Kv1.2 (Fig. 5.12D, green lines). Therefore, a greater fraction of Kv1.2 channels would activate and subsequently inactivate at typical resting voltages. Secondly, this extreme gating shift induced by Slc7a5 causes the gain-of-function mutants to require extreme hyperpolarized voltages in order to recover from inactivation. The unique gating 135  effects of Slc7a5 provide an interesting example of how the outcome of a disease-linked ion channel mutant might be fundamentally altered by assembly with an accessory protein.  Pleiotropic functions of the Slc7a5 transporter Slc7a5 is a fairly large transmembrane protein (Fotiadis et al., 2013), a feature that stands out from previously described accessory proteins of Kv1 channels, which are predominantly soluble cytoplasmic proteins, and often associated with the cytoskeleton (Hattan et al., 2002; Shi et al., Figure 5.12. Model demonstrating the effect of Slc7a5 on Kv1.2. A) Gating of Kv1.2 cycles between closed, activated, and inactivated. B) Slc7a5 increases the propensity for channel activation and inactivation, and slows recovery from inactivation.  C) Activation curves from Fig. 5.4 and inactivation curves from Fig. 5.7 are shown, illustrating the greater propensity for inactivation of Kv1.2 when assembled with Slc7a5. D) Disease-linked gain-of-function mutants (GOF Kv1.2) exhibit greatly exaggerated sensitivity to Slc7a5 gating effects, such that Kv1.2 channels in complex with Slc7a5 are predominantly inactivated at resting voltages. Activation curves from Fig. 5.11, inactivation curves are hypothetical, as they could not be measured due to the extreme voltages required. 136  1996; Williams et al., 2007). Slc7a5 is studied in the context of the blood brain barrier to transport amino acids, neurotransmitters and small drugs into the brain (Duelli et al., 2000; Matsuo et al., 2000; Verrey et al., 2004). While it is enriched in the epithelial cells of blood vessels in the brain it is also abundant in the midbrain structures, cortex and hippocampus where its function is less well characterized (Kageyama et al., 2000a). Additionally, this new interaction of Slc7a5 opens the question of whether Kv1.2 has any effects on Slc7a5 transport activity. This complex may be an emerging theme, as other transporter subunits have recently been reported to influence ion channels. One example is a functional interaction of the sodium chloride cotransporter (NCC) pump and the endothelial sodium channel (ENaC) at the distal convoluted tubule (Mistry et al., 2016). Also, the sodium-coupled myoinositol transporter SMIT1 has been shown to affect expression of the KCNQ1 and KCNQ2/3 potassium channels (Manville et al., 2017; Neverisky and Abbott, 2015). We add the Kv1.2-Slc7a5 complex to this growing list of ion channel-transporter interactors and suggest that this interaction may help to shape potassium currents in excitable cells.    137  CHAPTER 6: DISSECTING THE SPECIFICITY OF Kv1.2 AND SLC7A5 INTERACTIONS   INTRODUCTION Kv1.2 is a prominent and essential voltage-gated potassium channel subunit in the CNS, where it plays an important role in regulating neuronal excitability. Kv1.2 subunits can coassemble with other Kv1 subunits, along with a variety of accessory subunits including Kvβ (Shi et al., 1996), the sigma 1 receptor (Kourrich et al., 2013) and PIP2 (Kruse and Hille, 2013). We have identified the amino acid transporter Slc7a5 as a novel regulatory protein of Kv1.2, with dramatic effects on Kv1.2 activation and inactivation. The net gating effect of Slc7a5 is a prominent suppression of Kv1.2 current due to trapping of channels in an inactivated state at physiological membrane potentials. In addition, Slc7a5 effects can be modulated by Slc3a2, a previously known auxiliary partner of Slc7a5 (Napolitano et al., 2015). However, it is unknown whether Slc7a5 influences other members of the Kv1 subfamily, or whether other Slc7 subtypes can influence Kv1.2.  Kv1.2 channels rarely assemble as homomers, rather they mix with other Kv1 subunits to generate channels with mixed subunits. For instance, in the central nervous system, Kv1.1 and Kv1.2 channels often co-assemble in the hippocampus and the cerebellum, as well as at juxtaparanodes in myelinated central and peripheral axons (Monaghan et al., 2001; Sheng et al., 1994; Wang et al., 1993a). In the cardiovascular system, Kv1.5 and Kv1.2 subunits have been reported to coassemble in atrial myocytes (Bertaso et al., 2002). Given that Kv1.2 often coassembles with other Kv1 subunits, we asked whether Slc7a5 effects are unique to the Kv1.2 138  channel and, if so, whether Kv1.2-containing heteromeric channels will transfer this Slc7a5 sensitivity to the channel complex.  Similarly, regulation of ion channels by other Slc7 transporters has not been systematically explored. The Slc7 family has eleven members, of which Slc7a1-4 make up the cationic amino acid transporter (CAT) family and Slc7a5-11 the hetero-dimeric amino acid transporter (HAT) family. The HAT family is so named because each subunit coassembles with an auxiliary subunit, Slc3a2, via a disulfide bond, to increase trafficking of the transporter to the cell membrane (Fotiadis et al., 2013). Each member of the family has distinct properties in terms of the amino acids that they transport with highest efficiency, and whether they are sodium-coupled or passive transporters (Verrey et al., 2004). We therefore asked whether other Slc7 transporters influence Kv1.2 activation and current density. We have addressed these questions of specificity here, demonstrating that Kv1.1 is extremely susceptible to Slc7a5-mediated current inhibition, but lacks the shift of voltage-dependent activation observed with Kv1.2. In contrast, Kv1.5 is completely insensitive to Slc7a5. Kv1.2-Kv1.5 chimeric constructs allowed us to identify the Slc7a5-sensitive region to the S1 segment, with the most prominent effects attributed to Ile164 near the cytoplasmic side. We also tested all other Slc7 family members for effects on Kv1.2 and found that, with the exception of Slc7a8, other Slc7 subtypes do not strongly alter Kv1.2 current density or activation. Using a chimeric approach between Slc7a5 and the closely related (but inert to Kv1.2) Slc7a6, we have identified a critical transmembrane segment of the transporter that influences functional effects on Kv1.2. Overall, we have extended our knowledge on the mechanism by which Slc7a5 mediates its effect on Kv1.2 and expand this interaction to include Kv1.1.  139   RESULTS Slc7a5 inhibits Kv1.1 but does not affect Kv1.5 Kv1.2 channels are extremely susceptible to Slc7a5, which hyperpolarizes the activation and inactivation gating of Kv1.2, leading to accumulation of inactivated channels at physiological membrane voltages and therefore low current density. This effect results in a pronounced disinhibition of current when cells are held at very hyperpolarized voltages (-120 mV or more negative), which allows for recovery from inactivation. We further explored whether these effects of Slc7a5 persist in other Kv1 subtypes. We found that Kv1.1 channels exhibit very small currents (Fig. 6.1A) which become disinhibited in response to a -120 mV hyperpolarization by 3-fold (Fig. 6.1B). This effect is more pronounced upon co-expression with Slc7a5, which further decreases current density, and leads to a 6-fold current disinhibition in response to hyperpolarization.  Furthermore, co-expression of Slc3a2 increased Kv1.1 current density, and combining Slc7a5 and Slc3a2 restored Kv1.1 current density (Fig. 6.1A). Neither Slc7a5, Slc3a2 or the combination affect the voltage-dependence of Kv1.1 activation (Fig. 6.1C), in contrast to the Slc7a5-mediated ~-55 mV shift in Kv1.2 activation. Taken together, these findings suggest that Kv1.1 is inhibited by endogenous levels of Slc7a5, and this inhibition can be enhanced by overexpression of Slc7a5 and relieved by strong hyperpolarization or overexpression with Slc3a2. 140   In contrast to Kv1.1 and Kv1.2, Kv1.5 channels are insensitive to Slc7a5. Kv1.5 co-expression with Slc7a5, Slc3a2 or both did not change the current density (Fig. 6.2A), response to a hyperpolarization to -120 mV (Fig. 6.2B) or the V1/2 (Fig. 6.2C). Figure 6.1. Kv1.1 channels are highly susceptible to Slc7a5 regulation. A) Kv1.1 channels alone, or co-transfected with Slc7a5 (1:1), Slc3a2 (1:1) or Slc7a5+Slc3a2 (1:1:1) were co-transfected into Itk-mouse fibroblast cells. Current densities were 60 ± 34 for Kv1.1, 26 ± 45 +Slc7a5, 210 ± 195 +Slc3a2 and 36 ± 40 pA/pF +Slc7a5+Slc3a2. B) Fold change following a -120 mV hyperpolarization for 30 s.  C) Activation curves were measured with steps from -130 - +110 mV, followed by a tail current to -30 mV, after a -120 mV 30 s pulse. The activation curves were extracted from the tail currents and fit with a Boltzmann function. The measured values were V1/2 = -35 ± 1, k = 6.9 ± 0.9 for Kv1.1, V1/2 = -38 ± 4, k = 7.3 ± 1.0 for Kv1.1+Slc7a5, V1/2 = - 33 ± 3, k = 8.3 ± 1.6 for Kv1.1+Slc3a2, and V1/2 = -39 ± 3 mV, k = 7.8 ± 0.8 for Kv1.1+Slc7a5+Slc3a2. Data represented as average ± S.D. for B, C and as box plots with median ± 95% C.I. in A (n=10-12). 141    Slc7a5 sensitivity depends on heteromeric channel composition Kv1.2 subunits rarely assemble as homotetrameric channels. In the central nervous system, they commonly mix with Kv1.1 or Kv1.4 (Rhodes et al., 1997; Sheng et al., 1994) and in the cardiovascular system with Kv1.5 (Dixon and McKinnon, 1994). To determine the influence of Slc7a5 on heteromeric channels containing Kv1.2, we built dimeric constructs that force a 1:1 stoichiometry of Kv1.2 with other Kv1.X subunits. Kv1.2-Kv1.1 channels exhibited a -31 mV shift in the presence of Slc7a5 (Fig. 6.3A). Similar to homomeric expression of Kv1.1 and Kv1.2, current density decreased with Slc7a5 co-expression (Fig. 6.3B). Kv1.2-Kv1.1 expressed alone did not exhibit pronounced disinhibition with a -120 mV holding voltage. However, when co-expressed with Slc7a5, we observed ~6-fold disinhibition (Fig. 6.3C).   Figure 6.2. Kv1.5 channels are insensitive to Slc7a5 regulation. A) Kv1.5 channels were transfected alone or with Slc7a5 (1:1), Slc3a2 (1:1), or Slc7a5+Slc3a2 (1:1:1) into Itk-mouse fibroblast cells. Current density measurements were 1300 ± 600 for Kv1.5, 1400 ± 600 +Slc7a5, 1000 ± 400 +Slc3a2 and 1000 ± 300 pA/pF +Slc7a5+Slc3a2. B) Fold change in current after a -120 mV hyperpolarization is shown. C) Activation was measured as in Figure 6.1 (Kv1.5: V1/2 = -2.7 ± 2.6, k = 9.0 ± 1.8, Kv1.5+Slc7a5: V1/2 = -5.1 ± 4.6, k = 8.7 ± 1.8, Kv1.5+Slc3a2: V1/2 = -1.6 ± 5.0, k = 9.2 ± 1.6, Kv1.5+Slc7a5+Slc3a2: V1/2 = -3.2 ± 3.9 mV, k = 8.9 ± 1.4). Data represented as average ± S.D. for B, C and box plots with median ± 95% C.I. in A (n=10-12). 142    Figure 6.3. Heteromeric channels containing Kv1.2 can restore some Slc7a5 sensitivity. A) Kv1.2-Kv1.1 dimers were transfected in the absence or presence of Slc7a5 (1:1 transfection ratio) and activation curve was measured as in Fig. 6.1 (V1/2 = -24 ± 1.7 and -55 ± 3 mV +Slc7a5). B) Current density was 580 ± 360 for Kv1.2-Kv1.1 and 61 ± 74 pA/pF +Slc7a5. C) Fold change after a -120 mV pulse. D) Kv1.2-Kv1.5 dimers were expressed with or without Slc7a5 (1:1 transfection ratio) and the activation curve was measured (V1/2 = -9 ± 2 and -26 ± 6 mV +Slc7a5). E) Current density was 700 ± 290 for Kv1.2-Kv1.5 and 540 ± 320 pA/pF +Slc7a5. F) Fold change after a -120 mV pulse. G) The 3XKv1.5-Kv1.2 tetrameric construct was transfected in the absence or presence of Slc7a5 (1:1 transfection ratio) and activation curve was measured (V1/2 = -17 ± 3 mV and -24 ± 4 mV +Slc7a5). H) Current density was 320 ± 64 for 3XKv1.5-Kv1.2 and 99 ± 120 pA/pF +Slc7a5. I) Fold change following a -120 mV pulse. Average ± S.D. shown in A,C,D,F,G and box plot with median ± 95% C.I. is shown in B, E, H (n=5-8). Black dashed line represents Kv1.2 and green dashed line Kv1.2+Slc7a5 activation curves from figure 5.4 in A, D, G. 143  Kv1.2-Kv1.5 dimeric channels exhibit a smaller V1/2 shift (~-17 mV) when co-expressed with Slc7a5 (Fig. 6.3D). In addition, current density was only modestly reduced (Fig. 6.3E), and little or no disinhibition was observed at -120 mV, independent of Slc7a5 overexpression (Fig. 6.3F). We also tested Slc7a5 sensitivity of a concatenated construct 3XKv1.5-Kv1.2 (3 Kv1.5 subunits + 1 Kv1.2 subunit). This channel exhibited even weaker Slc7a5 sensitivity, with a small V1/2 shift of -7 mV (Fig. 6.3G). There was also a small reduction of current density, and a small degree of current disinhibition at -120 mV (Fig. 6.3H,I). Taken together, these findings demonstrate that Slc7a5 sensitivity depends on channel composition, where mixed Kv1.1/Kv1.2 channels are much more sensitive to Slc7a5 than Kv1.5/Kv1.2. It is likely that multiple Slc7a5-sensitive subunits are required for a pronounced channel response.   The S1 segment mediates channel interaction with Slc7a5 We exploited the differential Slc7a5 sensitivity of Kv1.2 and Kv1.5 by generating a series of chimeras to isolate channel segments important for Slc7a5-mediated effects. An initial chimera screen where we replaced increasing segments of the Kv1.2 channel into Kv1.5 at the N terminal portion starting at either the S1, S3, S4 or S5 segments were created, and we found that channels require the N-terminal portion of Kv1.2 up to the S2-3 linker to preserve Slc7a5 sensitivity (Fig. 6.4).  144   Next, we refined these chimeras by substituting the S1, S1-2 linker, S2 and S2-3 linker of Kv1.5 into Kv1.2 (Fig. 6.5). Of these, the S1 chimera Kv1.2[Kv1.5 S1] was the most perturbative, leading to a loss of Slc7a5 effects on channel activation (Fig. 6.5B). Similarly, Slc7a5 prominently suppressed currents from most chimeras except Kv1.2[Kv1.5 S1] (Fig. 6.5A).  Activation of Kv1.2[Kv1.5S2] and suppression of current from the Kv1.2[Kv1.5 S1-2L] were both variable, for reasons that are not clear. These findings suggest that the S1 segment of Kv1.2 is of primary importance for Slc7a5 sensitivity, although other segments may also have some role.  Figure 6.4. Testing chimeric Kv1.2/Kv1.5 channels to introduce Slc7a5 sensitivity into Kv1.5. Current densities recorded at +60 mV were 330 ± 140 for Kv1.2, 90 ± 80 pA/pF +Slc7a5, and 650 ± 330 for Kv1.5, 610 ± 570 pA/pF +Slc7a5. Chimeras had current densities of 450 ± 290 for Kv1.2[Kv1.5 S1->], 30 ± 50 for Kv1.2[Kv1.5 S3->], 10 ± 20 for Kv1.2[Kv1.5 S4->] and 10 ± 10 pA/pF for Kv1.2[Kv1.5 S5->] expressed with Slc7a5. Chimeras indicate N-terminal Kv1.2 with C-terminal portions of Kv1.5 which start at the indicated segment. Values are box plot showing median ± 95% CI (n=4-6, n=20 for Kv1.2 alone). 145     Ile164 is a key residue in mediating the interaction between Kv1.2 and Slc7a5 Next, we explored the five residue differences in the S1 segments of Kv1.2 and Kv1.5 with point mutations (Fig. 6.6G). Most Kv1.2 mutations (Kv1.2[P161S], Kv1.2[M171L], Kv1.2[V178I] and Kv1.2[S179T]) exhibited full sensitivity to Slc7a5, in terms of the gating shift (Fig. 6.6A,C-E) and current suppression (Fig. 6.6F). However, Kv1.2[I164A] was only weakly affected by Slc7a5 showing very little shift in activation and little current reduction (Fig. 6.6B,F).  Figure 6.5. Testing Kv1.2-Kv1.5 chimeric channels for Slc7a5 effects. A) Various chimeras were constructed where the S1, S1-S2 linker, S2 and S2-S3 linker of Kv1.5 were replaced into Kv1.2. Current densities recorded at +60 mV were 770 ± 460 for Kv1.2[Kv1.5 S1] and 710 ± 460 +Slc7a5; 810 ± 490 for Kv1.2[Kv1.5 S1-S2L] and 480 ± 650 +Slc7a5; 680 ± 340 for Kv1.2[Kv1.5 S2] and 80 ± 75 +Slc7a5 and 410 ± 165 for Kv1.2[Kv1.5 S2-S3L] and 62 ± 61 pA/pF + Slc7a5. C) The activation curves for the chimeras in B were measured as in Fig. 6.1 (Kv1.2[Kv1.5 S1] V1/2 = -10 ± 4 and -18 ± 3 mV +Slc7a5; Kv1.2[Kv1.5 S1-S2L] V1/2 = -3.2 ± 6 and -52 ± 3 mV +Slc7a5; Kv1.2[Kv1.5 S2] V1/2 = -7.9 ± 3 and -38 ± 16 mV; and Kv1.2[Kv1.5 S2] V1/2 = -9.7 ± 1.7 and -47 ± 11 mV +Slc7a5). Box plots with median ± 95% is shown in A, B and average ± SD in C (n=4-5).  146   Co-transfecting increasing amounts of Slc7a5 with Kv1.2[I164A] partially restored the Slc7a5 effect. While WT Kv1.2 exhibits a maximal Slc7a5-mediateed gating shift when transfected in a 1:0.3 ratio (Kv1.2:Slc7a5), Kv1.2[I164A] channels require transfection with ~7-fold more Slc7a5 DNA to generate a gating shift of only -33 ± 12 mV (Fig. 6.7A). We observed a similar trend for current density, where Kv1.2[I164A] co-transfection with large amounts of Slc7a5 were required to mimic the extent of current suppression observed for WT Kv1.2 (Fig. 6.7B). All ratios of Slc7a5 lead to some degree of current disinhibition in response to a -120 mV hyperpolarization (Fig. Figure 6.6. Testing S1 mutations of Kv1.2 to isolate a key residue in mediating Slc7a5 sensitivity. Five residue differences in the S1 segment were identified between Kv1.2 and Kv1.5, and the corresponding Kv1.5 residues were substituted into Kv1.2. The activation curves were measured as in Fig. 6.1. A) Kv1.2[P161S] V1/2 = -13 ± 3, and -56 ± 4 mV +Slc7a5; B) Kv1.2[I164A] V1/2 = -14 ± 2, and -20 ± 10 mV +Slc7a5; C) Kv1.2[M171L] V1/2 = -13 ± 2, and -50 ± 4 mV +Slc7a5; D) Kv1.2[V178I] V1/2 = -8.9 ± 2, and -53 ± 7 mV +Slc7a5; E) Kv1.2[S179T] V1/2 = -12 ± 2 and -52 ± 4 mV +Slc7a5. In all cases, Slc7a5 was transfected at a 3:1 ratio channel+Slc7a5. F) Current density was measured to be Kv1.2[P161S] 1300 ± 620, and 160 ± 390 +Slc7a5; Kv1.2[I164A] 1400 ± 540, and 590 ± 380 +Slc7a5; Kv1.2[M171L] 870 ± 760, and 43 ± 22 +Slc7a5; Kv1.2[V178I] 880 ± 700, and 97 ± 110 +Slc7a5; Kv1.2[S179T] 800 ± 670 pA/pF and 32 ± 18 pA/pF +Slc7a5 (n=5-10). Box plots with median ± 95% C.I. are shown in F and average ± S.D. in A-E. G) Kv1.2 structure viewed from the top, with the S1 residues highlighted in this figures shown in blue. Inset is a side view of the S1 segment. 147  6.7D). We also tried replacing the I164 residue with valine, the residue found in Kv1.1, and found that the Kv1.2 channel retained sensitivity to Slc7a5 (Fig. 6.7B-D).   Mutations of Kv1.1[Val168] affect its sensitivity to Slc7a5 Kv1.1 channels seem to be exquisitely sensitive to Slc7a5-mediated current inhibition. We tested whether this current inhibition is due to endogenous Slc7a5 by mutating the Kv1.1[Val168] Figure 6.7. Mutation of Kv1.2 Ile164Ala abolishes Slc7a5 sensitivity in a dose-dependent manner. A) Kv1.2[I164A] was co-transfected with increasing Slc7a5 amounts at a 1:0.3, 1:1 and 1:2 ratio, and the activation curve was measured as in Fig. 6.1 (V1/2 = -10 ± 4, k = 8.4 ± 1.3 for Kv1.2[I164A], V1/2 = -20 ± 10, k = 11 ± 4.0 for +1:0.3 ratio, V1/2 = -30 ± 6, k = 13 ± 4.4 for +1:1 ratio and V1/2 = -33 ± 12 mV, k = 13 ± 3.3 for +1:2 ratio). B) Current densities were 1400 ± 540 for Kv1.2[I164A], 440 ± 420 +1:0.3 ratio, 91 ± 80 +1:1 ratio and 160 ± 130 pA/pF +1:2 ratio. For Kv1.2[I164V] current density was 900 ± 240 pA/pF and 54 ± 35 pA/pF +Slc7a5 (1:1). C) Activation curves were measured for Kv1.2[I164V] and the V1/2 = -8.8 ± 1, k = 8.4 ± 1.1 and V1/2 = -52 ± 5 mV, k = 9.6 ± 0.8 +Slc7a5 (1:1). D) Fold change after a 30 sec -120 mV hyperpolarization. Values in A, C and D are average ± S.D. and box plot with median ± 95% C.I. in B (n=4-10). 148  residue (equivalent to Kv1.2[Ile164]) to Ala, the residue present in the Slc7a5-insensitive Kv1.5 channel. Kv1.1[V168A] channels had a higher current density than Kv1.1 channels, and a -120 mV pulse elicited a smaller, 2-fold increase in current compared to 3-fold for WT Kv1.1. Overexpression of Slc7a5 restored WT Kv1.1 behaviour, causing smaller current density and a more pronounced disinhibition of current at -120 mV (Fig. 6.8B,C). In contrast, Kv1.1[V168I] (equivalent to Kv1.2) retained strong sensitivity to Slc7a5, a small current density even in the absence of exogenous Slc7a5, and large current disinhibition (Fig. 6.8B,C). The activation curve again was not changed (Fig. 6.8A). Therefore, Kv1.1[V168A] channels have a decreased sensitivity to Slc7a5 whereas Kv1.1[V168I] retains sensitivity, suggesting that Kv1.1 channels are sensitive to endogenous levels of Slc7a5. Interestingly, the Kv1.1[V168I] mutant is not sufficient to recapitulate the Slc7a5-mediated shift in voltage-dependence observed in Kv1.2.    Figure 6.8. Kv1.1 Val168Ala diminishes Slc7a5 sensitivity. A) Kv1.1[V168A] or Kv1.1[V168I] was expressed alone and with Slc7a5 (1:1 transfection ratio) and the activation curve was measured as in Fig. 6.1 (Kv1.1[V168A] V1/2 = -33 ± 3 mV, k =7.7 ± 1.1 and -34 ± 2 mV, k = 8.0 ± 2.0 + Slc7a5; Kv1.1[V168I] V1/2 = -35 ± 5, k = 9.0 ± 1.5 and V1/2 = -38 ± 3 mV, k = 13.1 ± 2.8 + Slc7a5). B) Current density for Kv1.1[V168A] is 210 ± 120 and 70 ± 35 pA/pF +Slc7a5, and for Kv1.1[V168I] 20 ± 15 and 9.6 ± 9 pA/pF +Slc7a5. C) Fold change after a 30 sec -120 mV hyperpolarization. Average ± S.D. shown in A, C, and box plot with median ± 95% CI shown in B (n=4-11). 149  Kv1.5 Ala251 mutations cannot reconstitute Slc7a5 sensitivity Kv1.5 channels are insensitive to Slc7a5. We examined whether mutation of the residue Ala251, (equivalent Kv1.2[Ile164]) to either Ile or Val, would increase its sensitivity to Slc7a5. Neither mutation produced a shift in activation when expressed with Slc7a5 (Fig. 6.9A,D), current inhibition (Fig. 6.9B,E) or an increase in current following a -120 mV hyperpolarization (Fig. 6.9C,F).   Although this point mutation is not sufficient to confer Slc7a5 sensitivity to Kv1.5, it is noteworthy that a chimera containing the N-terminus and the first two transmembrane segments of Kv1.2 was sufficient to transplant Slc7a5 sensitivity to Kv1.5 (Fig. 6.4). Therefore the I164 residue in Kv1.2, while necessary for the Slc7a5 effect, is not sufficient to confer Slc7a5 sensitivity. Slc7 family screen for functional effects on Kv1.2 We further investigated effects of other Slc7 family members, which are broadly categorized in two groups. Slc7a1-4 are homomeric cationic amino acid transporters, whereas Slc7a5-11 are Figure 6.9. Kv1.5 Val168 mutation cannot introduce Slc7a5 sensitivity. A) Kv1.5[A251I] or Kv1.5[A251V] alone or with Slc7a5 (1:1 transfection ratio) activation curves were recorded as in Fig. 6.1 (Kv1.5[A251I] V1/2 = -14 ± 6, k = 8.5 ± 1.2 and V1/2 = -14 ± 0.4 mV, k = 9.0 ± 1.8 +Slc7a5, and Kv1.5[A251V] V1/2 = -12 ± 1, k = 9.6 ± 0.9 and V1/2 = -14 ± 4 mV and k = 11.0 ± 1.5 + Slc7a5). B) Current density for Kv1.5[A251I] is 1100 ± 240 and 470 ± 440 pA/pF +Slc7a5 and for Kv1.5[A251V] 800 ± 320 and 320 ± 260 pA/pF +Slc7a5. C) Fold change after a 30 sec -120 mV hyperpolarization. Average ± S.D. shown in A, C, and box plot with median ± 95% CI shown in B (n=4-11).  150  heteromeric amino acid transporters that assemble with Slc3a2. We found that none of the transporters consistently reduced Kv1.2 current density to the same degree as Slc7a5 (Fig. 6.10A). In addition, most transporters did not appreciably affect Kv1.2 activation. The only exception was Slc7a8, which produced a large but inconsistent hyperpolarization of V1/2 from -3.9 ± 2 mV to -32 ± 16 mV (Fig. 6.10B), although we have not investigated this effect any further. Taken together, these findings suggest that Slc7a5 is fairly unique within the Slc7 family, in terms of the novel ion channel regulatory properties that we have identified.  Slc7a5-Slc7a6 chimeras isolate the key regions that determine the Slc7a5 effect on Kv1.2 We took advantage of the observation that a closely related heterodimeric transporter to Slc7a5, Slc7a6, had no effect on Kv1.2. We built Slc7a5:Slc7a6 chimeras by replacing the N-terminus, TM1, TM1-2 linker and TM2 of Slc7a5 with Slc7a6 sequence. The Slc7a5[Slc7a6 Nterm] and Slc7a5[Slc7a6 TM2] chimeras retained the Slc7a5 gating effects on Kv1.2 whereas Slc7a5[Slc7a6 Figure 6.10. Screening Slc7 family for effects on Kv1.2 activation and current. A) Kv1.2 channels were cotransfected with Slc7a1-11 (1:1), we measured current density and B) V1/2 values. Slc7a5 and Slc7a8 were the only members that affected Kv1.2 function. Slc7a5 hyperpolarized the V1/2 and decreased current as described in Fig. 5.2, 5.4, and Slc7a8 hyperpolarized the V1/2 to -32 ± 16 mV. Values are box plot showing median ± 95% CI in A and average ± SD in B (n=4-6, n=20 for Kv1.2 alone). 151  TM1] and Slc7a5[Slc7a6 TM1-2L] did not reconstitute this shift (Fig. 6.11A). Current density measurements were consistent with this finding, as Slc7a5[Slc7a6 Nterm] and Slc7a5[Slc7a6 TM2] strongly inhibited Kv1.2 current, whereas Slc7a5[Slc7a6 TM1] and Slc7a5[Slc7a6 TM1-2L] lost this effect (Fig. 6.11B). These findings suggest that TM1 and TM1-2L are essential contributors to the function effects of Slc7a5 on Kv1.2.   Slc7a5 alters redox sensitivity of the Kv1.2 channel As demonstrated in chapter 4, Kv1.2 channels are exquisitely sensitive to external redox conditions, and this effect is proposed to arise from interaction with an unidentified accessory subunit. We tested how the effects of Slc7a5 might interact with redox-induced changes in Kv1.2 Figure 6.11. Slc7a5-7a6 chimeric channels isolate the TM1 and TM1-2 linker as the effectors of Kv1.2 gating shift. A) Chimeric transporters were constructed by replacing the N-terminus, TM1, TM1-TM2 linker and TM2 of Slc7a6 into Slc7a5. Each chimera was coexpressed with Kv1.2 (1:1) and the activation curves were measured and fit with Boltzmann functions which yielded V1/2 of -52 ± 5 for Slc7a5[Slc7a6 Nterm], -23 ± 7 for Slc7a5[Slc7a6 TM1], -20 ± 9 for Slc7a5[Slc7a6 TM1-2L], and -47 ± 7 mV for Slc7a5[Slc7a6 TM2]. Values are average ± S.D. B) Current density measurements were 31 ± 52 for Slc7a5[Slc7a6 Nterm], 160 ± 160 for Slc7a5[Slc7a6 TM1], 200 ± 180 for Slc7a5[Slc7a6 TM1-2L], and 79 ± 61 pA/pF for Slc7a5[Slc7a6 TM2], and 440 ± 350 pA/pF for Kv1.2 alone measured in parallel. Values are box plot with median ± 95% C.I. (n=6-11). 152  gating. We recorded conductance-voltage relationships after ‘potentiation’ in ambient redox, by delivering a +60 mV depolarizing pulse before each sweep. We also measured channel activation in 200 µM extracellular TCEP, allowing 10 seconds between each sweep to fully recover any use-dependent activation. These two conditions are designed to reveal the maximal dynamic range of Kv1.2 voltage-dependence. WT Kv1.2 exhibits a 71 mV shift between these conditions, from a ‘potentiated’ V1/2 of -10 ± 3 mV, to +64 ± 11 mV in the presence of TCEP (Fig. 7.12A).  Co-expression of Slc7a5 shifts the potentiated V1/2 to -58 ± 3 mV, and in the presence of TCEP generates a bimodal activation curve that was well fit by the sum of two Boltzmann functions (hyperpolarized V1/2 of -57 ± 4 mV, 45%; depolarized V1/2 of +45 ± 12 mV, 55%; Fig. 7.12B). This result suggests that redox and Slc7a5 regulate Kv1.2 gating by distinct, mutually exclusive mechanisms.  153    Consistent with the previous demonstration that Slc3a2 attenuates Slc7a5 effects, we also observed that Slc3a2 overexpression restored WT Kv1.2-like redox sensitivity, when co-expressed with Slc7a5 and Kv1.2 (Fig. 6.13A,B).  Figure 6.12. Slc7a5 attenuates the redox sensitivity of Kv1.2 channels. Kv1.2 channel alone or with transporter and the activation curve was recorded in the absence (filled circles) or presence of 200 µM TCEP in the external solution (empty circles). The V1/2 at ambient redox conditions was recorded by delivering a 100 ms depolarization to +60 mV prior to each sweep of the activation curve to record a ‘potentiated GV’, which is also reported in Fig. 5.4. A) In the presence of TCEP, Kv1.2 channels had a V1/2 of 64 ± 11 mV and a k value of 19 ± 4 (empty circles). The potentiated GV yielded a V1/2 = -11 ± 3 and k = 11 ± 3 (filled circles). B) Kv1.2+Slc7a5 (1:0.5 transfection ratio) activation was measured in the presence of TCEP after a -120 mV hyperpolarization and yielded a biphasic activation curve which was fit with a sum of two Boltzmann functions to yield a hyperpolarized V1/2 of -57 ± 4 mV and a k value of 7.1 ± 1.7, and a depolarized V1/2 of 45 ± 12 mV and a k value of 21 ± 4. The potentiated GV yielded a V1/2 = -58 ± 3 and k= 10 ±2. The proportion of the hyperpolarized section was 0.45 and that of the depolarized section was 0.55. Values are average ± S.D. (n=10). 154   DISCUSSION Structural insights into the interaction between Kv1.1/Kv1.2 and Slc7a5  The findings in this chapter expand our understanding of the diversity of effects of Slc7a5 and the Kv1 channel types that are sensitive to this enigmatic transporter. For example, Kv1.1 exhibits increased sensitivity to Slc7a5, but lacks the voltage-dependent shifts observed for Kv1.2. This raises the question of how Slc7a5 exerts its inhibitory effect on the Kv1.1 channel; specifically whether it is through accelerating C-type inactivation. Making the Kv1.1 channel more permissive to C-type inactivation through mutagenesis, and measuring the effects of Slc7a5 would shed some light on this question. In contrast to Kv1.1 and Kv1.2, Kv1.5 channels are insensitive to Slc7a5. These differences in sensitivity of closely related channels enabled identification of the S1 segment, and particularly Kv1.2 residue I164, as a critical determinant of Slc7a5 sensitivity. Similarly, chimeras of Slc7a5:Slc7a6 suggest that TM1 and the TM1-2 linker is an especially important determinant of Slc7a5 gating effects. Taken together, these findings highlight that this Figure 6.13. Slc3a2 and Slc3a2+Slc7a5 leave redox sensitivity of Kv1.2 unaffected. Kv1.2 channels were transfected with the indicated transporters and treated as in Fig. 11. A) Kv1.2+Slc3a2 (1:1) in the presence of TCEP yielded a V1/2 of 65 ± 13 mV and a k value of 19 ± 4 (potentiated V1/2 = -11 ± 10, k = 10 ± 3). B) Kv1.2+Slc7a5+Slc3a2 (1:0.5:1) in the presence of TCEP yielded a V1/2 of 65 ± 20 mV and a k value of 28 ± 9 (potentiated V1/2 = -16 ± 3 mV, k = 11 ± 2). Values are average ± S.D. (n=10-11). 155  new mode of Kv1 channel regulation is mediated by interactions in the transmembrane domain, likely involving the voltage sensing domain of the channel. Channel modulation by a transmembrane protein is a relatively unusual finding. Many of the canonical regulators of Kv1 channel function are soluble proteins, for instance Kvβ, KChIP, and cytoskeletal anchoring proteins (Deschênes and Tomaselli, 2002; Poliak et al., 1999; Shi et al., 1996). The profound effects that Slc7a5 has on Kv1.1 and Kv1.2 are therefore unique in the magnitude, their effect on C-type inactivation and that it is mediated by a transmembrane protein. Slc7a5 has partial gating effects on Kv1.2-containing heteromeric channels Our findings also highlight the influence of channel composition on Slc7a5 sensitivity. Although the exact physiological role of Slc7a5 in regulation of channel function is not yet known, it is clear that the extent of the gating shift and current suppression vary widely depending on the number of Kv1.1, Kv1.2, and Kv1.5 subunits. Moreover, this finding hints that multiple Slc7a5 subunits likely interact with a tetrameric channel, in order to generate the maximal gating shift observed in homomeric Kv1.2 channels. More detailed biophysical approaches would be helpful to identify the stoichiometric details of channel assembly with Slc7a5.  Use-dependent activation competes with Slc7a5 on Kv1.2 We were intrigued to find that the Slc7a5 interaction competes with the inhibitory process that underlies use-dependent activation. We have not yet identified the molecular partner of Kv1.2 that generates the use-dependent gating phenotype. However, our findings suggest that binding of Slc7a5 competes with this putative regulator, and thus attenuates the redox sensitivity that is normally observed. The mixed effects of redox and Slc7a5 result in a clear separation of the conductance-voltage relationship, suggesting these regulatory processes are mutually exclusive. 156  That is, interaction with Slc7a5 prevents association with the putative use-dependent regulator, and attenuates the degree of use-dependence observed in reducing conditions. This bi-modal split suggests that, in ambient redox conditions ~100% of Kv1.2 channels are bound to Slc7a5. Upon addition of TCEP, the use-dependence regulator increases its affinity for the Kv1.2 channel. As a consequence, only 50% of channels remain bound to Slc7a5 and show a hyperpolarized activation, and the other 50% unbind Slc7a5 and become available to bind the use-dependence regulator to produce a depolarizing shift in activation. This suggests that Slc7a5 and the regulator of use-dependence cannot exert their opposing effects on the channel at the same time and therefore compete for binding. Looking forward, our finding that S1 residues contribute to channel interaction with Slc7a5 leads us to speculate that a similar/overlapping region of the channel will govern interaction with the putative use-dependent regulator, perhaps in the crevice between voltage-sensing domains where both the I164 (Fig. 6.14, green residue and green circle as the putative binding domain) and T252 residues (Fig. 6.14, blue residue and blue circle as putative binding domain) are nearby.  Figure 6.14. Slc7a5 and use-dependence regulator likely have overlapping binding sites. Kv1.2 structure shown from the top of the channel, with the voltage sensing domain in light gray (S1-S4) and the pore domain in dark gray (S5-S6). Slc7a5 interacting residue I164 is shown in green and residue important in mediating the use-dependent activation, T252, effect shown in blue. Hypothetical interacting site for Slc7a5 shown by the green circle and for use-dependence regulator shown by the blue circle. 157  Conclusion Altogether, findings presented in this chapter expand our understanding of the effects of the Slc7 family on the Kv1 family, and the underlying molecular determinants of this effect. An emerging trend arising from this data is that multiple transmembrane proteins may interact with the channel and regulate its function. These fundamental insights into the molecular basis of the interaction will provide the basis for future manipulation of this regulatory process in biophysical studies and possibly model organisms.   158  CHAPTER 7: DISCUSSION   This thesis has focused on discovery of new mechanisms of potassium channel regulation and identification of novel regulatory proteins. I have shown the dramatic variability in gating that extrinsic regulators can endow the Kv1.2 channel, and how this has the potential to impact neuronal physiology. One mechanism of regulation (use-dependent activation) allows Kv1.2 channels to dynamically respond to changes in electrical excitability, giving a neuron the ability to change its electrical properties on a moment-to-moment basis. This is done simply by changes in its binding to auxiliary subunits in a voltage-dependent manner. The Slc7a5-mediated regulatory mechanism silences Kv1.2 channels through an ‘inactivation trap’, however this can be modulated by increasing expression of a third competitive partner (Slc3a2). These two novel mechanisms highlight the importance of extrinsic ion channel regulators, which can have profound consequences on ion channel function. In contrast, the field has generally put a greater focus on studying intrinsic mechanisms of channel gating, using mutagenesis and functional characterization of channels as independent units. As a consequence, we have a very good understanding of the mechanism of voltage sensing and gating, but much less on how these properties can be modified by external regulatory proteins, lipids and other molecules. The aim of my thesis has therefore been to broaden the perspective of ion channels to include novel extrinsic partners that influence ion channel gating.   159  REGULATORY FACTOR MEDIATING USE-DEPENDENT ACTIVATION  Kv1.2 channels are uniquely susceptible to a mechanism of regulation which we have described as ‘use-dependent activation’. In this thesis I demonstrated that repetitive depolarizations of Kv1.2 channels leads to a gradual increase in current due to channel accumulation in a potentiated gating mode. We argue that use-dependence is mediated by an extrinsic, inhibitory, redox-sensitive transmembrane protein. In this section I provide evidence for each of these postulations and reconcile my findings with previously published work to develop a coherent model. Evidence for an extrinsic regulatory protein Although there have been scattered previous descriptions of use-dependent potentiation of Kv1.2 (Baronas et al., 2015; Grissmer et al., 1994; Nguyen et al., 2012; Rezazadeh et al., 2007; Scholle et al., 2004), this phenomenon has not been broadly appreciated in the field of ion channel biophysics and lacks a cogent mechanistic description.  Based on my findings and previously published work, I have postulated that use-dependent regulation is mediated by an extrinsic regulatory factor, such as an accessory protein. A variety of distinct pieces of evidence support this assertion. Firstly, there is wide variability in the amount of use-dependent activation observed on a cell-by-cell basis in heterologous cells and hippocampal neurons (Fig. 3.2, 3.7). Therefore, it is unlikely that an intrinsic ion channel property gives rise to use-dependence, because each cell would be expected to behave similarly. Secondly, I observed a relationship between current magnitude and the degree of use-dependence, such that cells with large currents tended to have less use-dependence than cells with less current (Fig. 3.13). This finding suggests the stoichiometric ratio between the putative regulatory protein and Kv1.2 influences 160  the degree of use-dependent activation. For example, a high level of Kv1.2 expression would overwhelm the endogenous expression of the putative regulator, causing many Kv1.2 channels to be unbound, leading to gating in the potentiated mode. However, there is not a perfect relationship between current density and use-dependence, so there are likely other regulatory factors at play. A final piece of evidence suggesting an extrinsic regulatory partner is that use-dependent activation is highly redox sensitive, and this sensitivity cannot be ascribed to any individual cysteines in Kv1.2 (Fig.4.10). Therefore, it is likely that an extrinsic protein is conferring redox sensitivity on the Kv1.2 channel. Taken together, these findings suggest an extrinsic regulator of Kv1.2 channel activation. Evidence for an inhibitory regulatory protein Use-dependent activation phenomena in ion channels is normally explained by models requiring state dependent modulation of ion channel function. One example is a model of a facilitatory regulatory factor that acts by stabilizing the open state. In this model, use-dependent potentiation arises because binding of the regulatory partner causes a hyperpolarizing shift of the activation curve, and increased macroscopic conductance, or some combination of both. A train of repetitive depolarizations allows a facilitatory molecule to bind, leading to potentiation of current through the channel with every depolarization, until channels are fully bound by the regulator. A prominent example of this is AMPAR interaction with stargazin (Carbone and Plested, 2016). In this system, the ligand binding domain in AMPAR and the first extracellular loop of stargazin interact when channels are open, and thereby stabilize the open configuration (Dawe et al., 2016). This results in a hyperpolarization of the activation curve (Fig. 7.1A), and increased current with repetitive trains of glutamate exposure (following an initial glutamate-161  induced desensitization). This is exemplified by the green trace in Fig. 7.1A, with the potentiation indicated by the arrow. In contrast, AMPAR alone will only desensitize in response to repetitive glutamate applications (Fig. 7.1A, black trace).  An alternative explanation for use-dependent activation is through an inhibitory regulatory factor that preferentially binds and stabilizes the closed state of the channel. In this instance, interaction with the regulator would generate a depolarizing shift of the conductance-voltage relationship and repetitive depolarizations would generate disinhibition of channel current due to unbinding of the inhibitory factor. One well known example of this mode of regulation is the inhibitory effect of Gβγ on Cav2.1 and 2.2 channels which results in depolarization of the activation curve (Fig. 7.1C). When channels are subjected to a train of depolarizations, Gβγ unbinds from channels that open during depolarization, leading to a use-dependent increase in current. This is shown by the pink trace in Fig. 7.1C, where potentiation elicited by a train of depolarizations is indicated by the arrow.   162    Both of these examples of state-dependent interactors elicit use-dependent increases in channel current. Our model for use-dependent increases of Kv1.2 current argues for a model of an inhibitory binding partner, similar to Gβγ-regulation of Cav channels, which stabilizes the closed channel state (Fig. 7.1B). The primary evidence for this is that the rate at which channels activate is dependent on pulse history whereas deactivation occurs with similar kinetics independent of pulse history (Rezazadeh et al., 2007). This finding suggests that the putative regulatory partner acts by stabilizing the closed channel conformation (rather than stabilizing the activated state). A second piece of evidence is that channels with high use-dependence exhibit slow activation kinetics at the beginning of the train, but speed up with more depolarizing pulses and eventually gate like channels that lack any use-dependence. Therefore, the interacting partner is inhibiting Figure 7.1. Comparison of use-dependent potentiation of AMPA receptors and disinhibition of voltage-gated potassium channels with Kv1.2. (Left box) AMPA receptor composed of GluR1 subunits in complex with stargazin hyperpolarizes the activation curve and causes a use-dependent increase in AMPA receptor currents in response to glutamate application. Adapted from (Carbone and Plested, 2016; Tomita et al., 2005). (Middle box) DTT addition to Kv1.2 depolarizes the activation curve relative to Kv1.2 with a potentiating pulse. Use-dependent increase in Kv1.2 current is maximized in the presence of DTT. (Right box) Voltage gated calcium channel (Cav2), when expressed with Gβγ, depolarizes the activation curve, and leads to a use-dependent increase in current. Adapted from (Bean, 1989; Brody and Yue, 2000). 163  channel function, and this inhibition is relieved by depolarization. Thirdly, many mutations in the S2-S3 linker eliminate this regulatory effect (Rezazadeh et al., 2007), causing channels to gate uniformly in the fast/potentiated mode. My interpretation of this observation is that mutations (such as T252R) disrupt interaction with the putative regulator, thereby preventing the appearance of the slow gating mode. These observations suggest that the gating regulator is an inhibitory molecule that preferentially binds and stabilizes the closed state of Kv1.2 channels.  Evidence for a transmembrane, redox sensitive regulatory protein Findings presented suggest that use-dependent activation is mediated by a transmembrane protein. This mechanism appears to be dependent on several residues located in the intracellular S2-S3 linker, including T252, F251, and F250 (Rezazadeh et al., 2007). However, based on my findings, it remains possible that the linker is required for the transduction of the effect rather than binding. In addition, I demonstrated unequivocally that that use-dependence is sensitive specifically to the extracellular redox environment (Fig.4.8). I attribute this property to direct redox modification of the putative regulatory protein. This seems to be a reasonable explanation because redox-mediated effects on use-dependence have a rapid onset, similar to other ion channels that are modified by extracellular redox conditions. Examples include suppression of Cav3.2 currents by carbon monoxide (Boycott et al., 2013), DTT potentiation of ASIC channels (Andrey et al., 2005), and H2O2 inhibition of BK channels (Tang et al., 2004), all reported to occur within minutes. However, oxidizing the extracellular environment with up to 1 mM H2O2 does not eliminate use-dependence. These two observations indicate that the reduced state of the regulator adopts a higher affinity configuration leading to increased use-dependence in reducing conditions, whereas the oxidized state retains sub-maximal, regulatable affinity for Kv1.2. 164  Another interesting observation was that Slc7a5 competes with redox regulation of use-dependence. While this does not directly demonstrate that a transmembrane protein controls use-dependent activation, its competition with a transmembrane protein, Slc7a5, does suggest involvement of a membrane-spanning protein in regulating use-dependence. Our proposed model differs somewhat from an earlier report describing prepulse potentiation of Kv1.2. Rezazadeh et al. suggested that this gating behavior is mediated by a soluble cytoplasmic regulator which transiently binds to the S2-S3 linker (Rezazadeh 2007). This suggestion was supported primarily by the observation that prepulse potentiation became attenuated and often lost over the course of minutes of whole-cell recording. In my recordings, I have observed some loss of use-dependent activation, though not as extreme (Fig. 7.2, top tier). Even recordings in the inside-out patch mode, which might be expected to accelerate the dissociation of a soluble accessory protein, as reported by Rezazadeh, retained prominent use-dependent activation and slow kinetics of activation for >10 minutes when incubated with DTT (Fig. 7.2, bottom tier). We do not have a confirmed explanation for these differences, and we suspect that further investigation of this regulatory process and identification of the molecular players involved will eventually clarify underlying reasons. Overall, my findings are consistent with a model of Kv1.2 regulation by a transmembrane protein that is sensitive to extracellular redox. 165   What is left to learn about use-dependent activation? First and foremost, we have yet to discover the identity of the regulatory protein that influences Kv1.2 function. The main approach we have taken is crosslinking and immunoprecipitation of Kv1.2 channel complexes, identification of the crosslinked proteins via mass spectrometry, and various screening approaches, as described in chapter 5. In fact, this led to discovery of the Slc7a5 and TMEM33 proteins as novel interactors. Continued efforts in this area have already shown promise that the identity of this regulatory factor is LMAN2, a vesicular chaperone protein. Secondly, there have been many suggestions throughout this thesis that use-dependent activation is a highly regulated phenotype, both in heterologous cells and in hippocampal neurons. While I have identified a few factors that can regulate use-dependent activation (stoichiometry, extracellular redox), I am still not able to predict a priori how much use-dependence any given cell will have until I record from it. This variability likely arises from other Figure 7.2. Comparison of time-dependent changes in use-dependent activation. Comparison of data collected for this thesis and data in (Rezazadeh et al., 2007). (Data from thesis, top) Use-dependent activation in cells immediately after obtaining whole-cell access and 5 min after. Individual cells linked by a line. (Data from thesis, bottom) Cell-attached recording (black) followed by patch excision in 200 µM DTT for 10 min. (Data from Rezazadeh et al. 2007, top) Time dependent loss of ‘slow’ cell phenotype, and conversion to ‘fast’ cells. (Data from Rezazadeh et al. 2007, bottom). Cell attached recording (black) followed by patch excision for 10 min (teal).   166  unidentified factors that regulate expression of the regulatory factor on a cell-by-cell basis, even in a seemingly homogeneous population of cells. This assumption of homogeneity is likely not true as there are many factors that may vary on a cell-by-cell basis. For instance, individual cells may be at different cell cycle stages. In addition, some cells produce Kv1.2 currents that are beyond the dynamic range of the amplifier, while others produce little or no Kv1.2 currents, despite having been transfected at the same time and treated in the same way. Additionally, use-dependence seems to be regulated by an unidentified soluble molecule as we and Rezazadeh et al. have observed some loss of use-dependent activation with time (Rezazadeh et al., 2007). Further investigation of this mechanism will hopefully reveal what processes influence the expression of the regulatory protein and whether there are other regulatory molecules, and thus allow us to predict and modulate the extent of use-dependent activation. Thirdly, we have yet to learn what the physiological consequences of use-dependent activation are. One possibility is that repetitive firing of neurons could lead to increasing activation of Kv1.2, and this increased potassium conductance could act as a self-silencing mechanism to shut off further neuronal activity. To learn more about the physiological implications of use-dependence we have generated a conditional transgenic knock-in mouse model with the Kv1.2[T252R] mutation to specifically eliminate use-dependence without deleting Kv1.2. My hypothesis is that this mouse model will recapitulate the severity of the Kv1.2 knockout mouse model, given that there are still other potassium channels (ie. Kv1.1, Kv1.5) that heteromerize with Kv1.2 and whose intrinsic function is very similar to Kv1.2, and can therefore replace its ion channel function, but lack use-dependent activation as a regulatory mechanism. 167  Lastly, we do not have a good grasp on why use-dependent activation is regulated by the extracellular redox environment, and what the potential physiological consequences are, partly because not much is known about how, when or why the extracellular redox environment changes. There are two predominant redox couples that regulate extracellular redox potential: GSH/GSSG, and CyS/CySS. While glutathione (GSH/GSSG) is the prominent intracellular redox species, the extracellular redox potential is primarily set by the CyS/CySS couple (total concentration ~60 µM, redox potential of -80 mV), with the GSH/GSSG couple making a smaller contribution (total concentration ~5 µM, redox potential of -137 mV) (Yi and Khosla, 2016). Changes in redox potential have been shown to affect many physiological processes including catecholamine synthesis (Zheng et al., 2014), metabolic processes in hepatocytes (Nocito et al., 2015), and cell proliferation and apoptosis (Jones, 2006). With age and disease, these redox couples are thought to become more oxidized. The CyS/CySS redox couple can become as oxidized as -10 mV (Yi and Khosla, 2016), and this perturbation has been associated with several neurological diseases including Alzheimer’s disease (Ghosh et al., 2012; Islam and Tabrez, 2017), cerebrovascular disease (Cai et al., 2016) and Parkinson’s disease  (Zhu et al., 2012), among others. The precise mechanism through which extracellular redox potential is controlled, and the mechanism underlying the shift towards more oxidized redox potentials with age, has not yet been elucidated (Jones, 2006). = Based on our findings, the dynamic range of redox regulation of Kv1.2 is near the reported range of extracellular redox potential in vivo (Fig. 4.11). Therefore, there is a strong possibility that Kv1.2 gating properties may be regulated by redox in physiological systems and perturbed in pathological systems where extracellular environment becomes oxidized, but the potential consequences are unclear.  168  SLC7A5 AND KV1.2 INTERACTION AND PHYSIOLOGICAL CONSEQUENCES  Expanding the role of Slc7a5 In our search for novel accessory partners of Kv channels, we have identified Slc7a5 as a prominent gating modifier with effects on activation, inactivation, and expression of Kv1.2. Slc7a5 also exerts marked effects on inactivation and expression of Kv1.1. Slc7a5 has not been previously identified or considered as a protein that can impact electrical activity. Rather, research has focused on its canonical role as a transport protein, often in the context of the blood brain barrier. Slc7a5 (LAT1) and Slc3a2 (4f2hc) were first recognized for their amino acid transport capability nearly twenty years ago (Kanai et al., 1998; Mastroberardino et al., 1998). Slc7a5 constitutes the amino acid transporting partner, whereas Slc3a2 assembles with Slc7a5 to increase its surface expression (Napolitano et al., 2015). This complex is an obligate exchanger that transports large neutral amino acids into the cell in exchange for Met, Leu and Ile, and other amino acids at a 1:1 ratio (Fotiadis et al., 2013; Meier et al., 2002). Therefore, there is no net uptake of amino acids with Slc7a5, but rather equilibration of amino acids across the membrane (Verrey et al., 2004). Additionally, it also regulates the intake of several neurotransmitters and drugs, including L-DOPA (Kageyama et al., 2000a) and gabapentin (Dickens et al., 2013), and has been investigated as a mechanism of transporting drugs into the brain (Puris et al., 2017). Slc7a5 is also involved in activation of the mTOR signalling pathway in conjunction with Slc1a5. Slc1a5 imports glutamine while Slc7a5 then couples glutamine export to import of essential amino acids including Leu, Trp, Phe, and Arg, leading to mTOR activation (Nicklin et al., 2009). The focus of research on this transporter is almost exclusively on its role in the blood brain barrier as a transporter of amino acids, small molecules and drugs (Duelli et al., 2000; Matsuo et al., 2000), 169  as well as its role in cancer (Fotiadis et al., 2013). However the recent discovery of its involvement in neurodevelopmental disorders and epilepsy suggests that its effect on Kv1.1 and Kv1.2 may be significant. Slc7a5 expression is not limited to microvessels in the brain. In mice, it is also present in many structures in the brain where Kv1.1 and Kv1.2 co-exist (Figure 7.3) (Kageyama et al., 2000b).   Figure 7.3. mRNA expression patterns of Slc7a5, Kv1.2, and Kv1.1 in the central nervous system in mice. Expression of mRNA in isocortex (isoctx), olfactory areas (OLF), hippocampal formation (HPF), cortical subplate (CTXsp), striatum (STR), pallidum (PAL), thalamus (TH), hypothalamus (HY), midbrain (MB), pons (P), medulla (MY), cerebellum (CB) of mice brains. Data shown for Slc7a5 (top), Kv1.2 (middle) and Kv1.1 (bottom). Data extracted from Allen mouse brain atlas (Lein et al., 2007). 170  Significance of the Slc7a5-Kv1.2 interaction Slc7a5 is remarkable in its effect on Kv1.2 gating. It has profound effects on activation and inactivation, far beyond the effects of any other auxiliary protein on a Kv1 channel. In fact, its effect on inactivation is so pronounced that it effectively silences Kv1.2 channels in the membrane, not allowing them to recover at physiologically plausible voltages. This is the first instance of an auxiliary protein influencing Kv1.2 inactivation, and it does so in a profound and virtually irreversible fashion. Furthermore, it provides a rationale for why patients with very diverse mutations of the Kv1.2 channel ranging from extreme gain of function to loss of function all result in epilepsy and movement disorders. What is left to learn about the Slc7a5-Kv1.2 interaction? We now have a good understanding of the effects of Slc7a5 on the Kv1.2 channel, however there are still many aspects of this interaction that are still unanswered. The first question is to elucidate the mechanism by which Slc7a5 affects Kv1.2 gating. This includes effects on voltage sensor movement, voltage sensor coupling to the pore, as well as the mechanism through which Slc7a5 decreases Kv1.2 expression. The second is how Slc7a5 and Kv1.2 affect each other’s function. This involves determining whether Kv1.2 affects Slc7a5 transport function, whether different blockers/modulators of channel and transporter function can affect the interaction, and whether there is competition between different regulatory molecules of each protein. For instance, we have shown that Slc3a2 affects the interaction of Slc7a5 with the Kv1.2 channel, and Slc7a5 competes with the regulator of use-dependent activation. It would be interesting to see whether other regulatory molecules were also impacted by this interaction. Lastly is the question of the physiological significance of this interaction. A transgenic knock-in mouse model with a 171  Kv1.2[I164A] mutation to eliminate binding to Slc7a5 would answer this question definitively, as this mutation does not affect channel function. While Tărlungeanu et al. recapitulated some of the disease phenotype of the Slc7a5 autism spectrum disorder mutants specifically by induction of these mutations in the blood brain barrier epithelial cells, this could be expanded with whole-body or brain-specific induction (Tărlungeanu et al., 2016). A third possibility would be to determine whether this interaction is present in cultured neurons by delivering a hyperpolarizing pulse to see whether current through Kv1.2- or Kv1.1-containing channels increases. Additionally, overexpression of Slc7a5 in cultured neurons would allow us to observe whether Slc7a5 affects Kv1.2/Kv1.1 localization and expression by immunohistochemistry, and whether it has any effect on neuronal physiology. Overall, while we have elucidated many aspects of the Kv1.2-Slc7a5 interaction, many steps remain to fully map out the mechanism of interaction and its physiological significance.  SUMMARY AND CONCLUSION  In this thesis I have described two unique mechanisms of Kv1.2 regulation: use-dependent activation by a redox-sensitive pathway, and an inactivation trap mediated by association with Slc7a5. The first mechanism activates Kv1.2 current with repetitive stimuli, and thus endows a neuron with the ability to alter its electrical activity in response changing stimuli. The second mechanism involves Slc7a5-mediated gating effects that lead to more prominent inactivation of Kv1.2, and this may generate a mechanism for fine-tuning neuronal excitability. Remarkably, both mechanisms of regulation appear to interact in a competitive manner.  172  There are likely many more proteins to be discovered that impact Kv1 channel function. 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