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Regulation of the IKs current by PKA phosphorylation Thompson, Emely Rose McKinley 2019

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Regulation of the IKs Current by PKA Phosphorylation by  Emely Rose McKinley Thompson  B.Sc., University of Hertfordshire, 2012 M.Sc., University of London, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (PHARMACOLOGY & THERAPEUTICS)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  November 2019  © Emely Rose McKinley Thompson, 2019 ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Regulation of the IKs current by PKA phosphorylation  submitted by Emely Rose McKinley Thompson in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacology and Therapeutics  Examining Committee: Dr. David Fedida Supervisor  Dr. Eric Accili Supervisory Committee Member  Dr. Michael Walker Supervisory Committee Member Dr. Kathleen MacLeod University Examiner Dr. Ismail Laher University Examiner   iii  Abstract The IKs current, which is composed of KCNQ1 and KCNE1 subunits, increases in size as a result of b-adrenergic stimulation. This response is a result of the KCNQ1 subunit being phosphorylated by protein kinase A (PKA). This is an important physiological response at high heart rates that allows the ventricles adequate time to fill. Mutations in either of these subunits can cause impaired cardiac repolarization and result in long and short QT syndromes, as well as familial atrial fibrillation. The mechanism by which the channel complex reacts to this stimulation has not been fully elucidated.   To investigate the mechanism behind this, both total internal reflection florescence microscopy (TIRF) and single-channel recording were used. 8-CPT-cAMP, a membrane-permeant analog of cAMP, was used to induce PKA phosphorylation of KCNQ1. TIRF studies found no significant change in the number of channels at the cell surface. Single-channel recordings of IKs had a reduced first latency to opening showing that the channel opened more quickly in response to 8-CPT-cAMP. The IKs current has multiple open states and, in the presence of 8-CPT-cAMP, occupied the higher subconducting states more frequently. An increase in the open probability of the channel complex was also seen. In response to phosphorylation triggered by 8-CPT-cAMP, the first latency to opening of the channel is reduced and the channel opens more quickly, more often and passes more current by increasing the higher conducting open states. This results in an increase in IKs current at the macroscopic level. Using enhanced gating mutant KCNQ1 channels, it is shown that the effect of phosphorylation is likely through further activation of the voltage sensor. KCNE1 is required for a functional response to PKA iv  phosphorylation. Both whole-cell and single-channel recordings show that as the number of KCNE1 subunits is reduced, a graded effect is seen in response to 8-CPT-cAMP; the less KCNE1 subunits, the smaller the response. However, in single-channel recordings, there was also a KCNE1-independent effect of phosphorylation as the first latencies for all KCNQ1-KCNE1 complexes were reduced in a non-graded manner. This suggests that phosphorylation may have both KCNE1-dependent and independent effects on the channel. v  Lay Summary The heart muscle contracts and relaxes in response to an electrical current generated by the flow of ions across ion channels in the cell membrane. Potassium ions are important for the heart to relax. At high heartrates, more potassium is needed to ensure the heart relaxes properly before it beats again. One particular potassium ion channel involved at high heart rates is IKs, which is composed of two different proteins: KCNQ1 and KCNE1. At high heart rates, the size of the IKs current increases and the heart muscle relaxes more quickly. The research conducted in this thesis investigates how this increase IKs current occurs at high heart rates. We found that the channel opens more often, more quickly, and allows more potassium to flow through. We also established that the number of KCNE1 proteins present in the channel made a difference in the channel’s response to high heart rate.  vi  Preface All of the work shown in this thesis was conducted in Dr. David Fedida’s laboratory at the University of British Columbia in collaboration with Dr. Jodene Eldstrom, Dr. Maartje Westhoff, and Dr. Donald McAfee.  Chapter 2: cAMP-Dependent Regulation of IKs Single-Channel Kinetics I was involved in the acquisition of both single-channel and whole cell data, analysis and interpretation of data, figure production and drafting and revising the manuscript. Dr. Jodene Eldstrom was involved in conception and design of experiments, acquisition of single channel data, analysis and interpretation of data and drafting and revision of the manuscript. Dr. Maartje Westhoff was involved in the acquisition of TIRF microscopy data and its analysis and interpretation of data. She was also involved in the drafting and revision of the manuscript. Dr. Donald McAfee was involved in the acquisition of whole cell data. Dr. Elise Balse was involved in the acquisition of TIRF microscopy data and its analysis and interpretation of data. Dr. David Fedida was the supervisory author and was involved in the conception and design of experiments, acquisition of single-channel data, analysis and interpretation of data, and drafting and revising the manuscript.   A version of Chapter 2 has been published:   Thompson, E., J. Eldstrom, M. Westhoff, D. McAfee, E. Balse, and D. Fedida. 2017. cAMP-dependent regulation of IKs single-channel kinetics. The Journal of General Physiology. 149:781. vii  Chapter 3: Single-Channel Kinetic Analysis of the cAMP Effect on IKs Mutants, S209F and S27D/S92D. I was involved in the conception and design of experiments, acquisition of both single-cell and whole cell data, interpretation and analysis of data, production of figures as well as drafting and revising the manuscript. Dr Jodene Eldstrom was involved in both the conception and design of experiments, the acquisition of single channel data, analysis and interpretation of data and drafting and revising the manuscript. David Fedida was the supervisory author and was involved in the conception and design of experiments, analysis and interpretation of the data, and drafting and revising the manuscript.  A version of Chapter 3 has been published:   Thompson, E., J. Eldstrom, and D. Fedida. 2018. Single channel kinetic analysis of the cAMP effect on IKs mutants, S209F and S27D/S92D. Channels. 12:276-283.   Chapter 4: The IKs Current Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry I was involved in the conception and design of experiments, acquisition of both single-channel and whole-cell data, analysis and interpretation of data, figure production, and drafting and revising the manuscript. Dr. Jodene Eldstrom was involved in conception and design of experiments, acquisition of single channel data, analysis and interpretation of data, and drafting and revising the manuscript. Dr. Maartje Westhoff was involved in the acquisition of whole-cell data, analysis and interpretation of data. Dr. Donald McAfee was viii  involved in the acquisition of whole-cell data. Dr. David Fedida was the supervisory author and was involved in conception and design of experiments, analysis and interpretation of data, and drafting and revising the manuscript.   A version of Chapter 4 has been published:  Thompson, E., J. Eldstrom, M. Westhoff, D. McAfee, and D. Fedida. 2018. The IKs Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry. Biophys J.  ix  Table of Contents Abstract .......................................................................................................................... iii Lay Summary .................................................................................................................. v Preface ............................................................................................................................ vi Table of Contents .......................................................................................................... ix List of Tables ................................................................................................................ xv List of Figures .............................................................................................................. xvi List of Abbreviations ................................................................................................. xviii Acknowledgements .................................................................................................... xxv Chapter 1: Introduction .............................................................................................. 1 1.1 Voltage-gated potassium (Kv) channels ......................................................... 1 1.2 Role of potassium channels in cardiac action potentials ................................ 1 1.2.1 Electrical conduction system of the heart ................................................ 1 1.2.2 Resting membrane potential, phase 4 ..................................................... 3 1.2.3 Phase 0 ................................................................................................... 4 1.2.4 Phase 1 ................................................................................................... 4 1.2.5 Phase 2 ................................................................................................... 4 1.2.6 Phase 3 ................................................................................................... 5 1.3 Structure of voltage-gated potassium channels ............................................. 5 x  1.3.1 KCNQ potassium channels ..................................................................... 9 1.3.1.1 Requirement of phosphatidylinositol 4,5-biphosphate (PIP2) .............. 9 1.3.1.2 Calmodulin (CaM) requirement ......................................................... 10 1.3.1.3 Stoichiometry of IKs ............................................................................ 11 1.4 Physiology and pathophysiology of KCNQ1 and KCNQ1:KCNE channels .. 13 1.4.1 KCNQ1 alone ........................................................................................ 13 1.4.2 KCNE1 .................................................................................................. 14 1.4.3 KCNE2 .................................................................................................. 17 1.4.4 KCNE3 .................................................................................................. 18 1.4.5 KCNE4 .................................................................................................. 19 1.4.6 KCNE5 .................................................................................................. 21 1.5 Hormonal modulation of IKs ........................................................................... 21 1.5.1 Norepinephrine ...................................................................................... 22 1.5.2 Insulin .................................................................................................... 25 1.5.3 Estrogen ................................................................................................ 28 1.5.4 Testosterone ......................................................................................... 32 1.5.5 Thyroid hormone ................................................................................... 34 1.5.6 Gastrin ................................................................................................... 36 1.6 Scope of the thesis ....................................................................................... 37 1.7 Patch clamp and single-channel recording methodologies .......................... 39 1.7.1 Single-channel recording ....................................................................... 40 1.7.2 Patch-clamp recording .......................................................................... 42 1.7.3 Single-channel analysis ......................................................................... 43 xi  1.7.4 Single-channel models .......................................................................... 45 1.7.5 Single-channel recordings of IKs current to date .................................... 46 Chapter 2: cAMP-Dependent Regulation of IKs Single-Channel Kinetics ............ 48 2.1 Introduction ................................................................................................... 48 2.2 Materials and methods ................................................................................. 51 2.2.1 Reagents ............................................................................................... 51 2.2.2 Molecular biology .................................................................................. 51 2.2.3 Cell culture and transfections ................................................................ 52 2.2.4 Patch-clamp electrophysiology .............................................................. 52 2.2.5 Microscopy ............................................................................................ 53 2.2.6 Solutions ................................................................................................ 53 2.2.7 Data analysis ......................................................................................... 54 2.2.8 Statistics ................................................................................................ 55 2.2.9 Supplemental material ........................................................................... 55 2.3 Results .......................................................................................................... 56 2.3.1 The 8-CPT-cAMP–induced increase in IKs current is seen at room temperature. ........................................................................................................ 56 2.3.2 Response of IKs to cAMP in cell-attached recordings ............................ 58 2.3.3 IKs single-channel kinetics in the presence of 8-CPT-cAMP .................. 59 2.3.4 Changes in substate occupancy in the presence of 8-CPT-cAMP are primarily VSD activation effects ........................................................................... 64 2.3.5 8-CPT-cAMP effects on IKs mutants described as having fixed and activated VSDs .................................................................................................... 69 xii  2.3.6 Effect of 8-CPT-cAMP on the pseudo-phosphorylated KCNQ1 mutants S27D and S27D-S92D ......................................................................................... 74 2.3.7 Effect of 8-CPT-cAMP on the surface expression of WT IKs ................. 79 2.4 Discussion .................................................................................................... 81 2.4.1 Single-channel studies of IKs ................................................................. 82 2.4.2 Effects of cAMP on the E1R/R4E and S209F channels with augmented VSD function ........................................................................................................ 84 2.4.3 Effects of cAMP on phosphomimetic mutants ....................................... 85 2.4.4 Trafficking and expression of IKs ............................................................ 87 2.5 Conclusion .................................................................................................... 88 Chapter 3: Single-Channel Kinetic Analysis of the cAMP Effect on IKs Mutants, S209F and S27D/S92D. ............................................................................................. 90 3.1 Introduction ................................................................................................... 90 3.2 Methods ........................................................................................................ 92 3.2.1 Reagents ............................................................................................... 92 3.2.2 Molecular biology .................................................................................. 92 3.2.3 Cell culture and transfections ................................................................ 92 3.2.4 Patch-clamp electrophysiology .............................................................. 93 3.2.5 Solutions ................................................................................................ 93 3.2.6 Data analysis ......................................................................................... 93 3.2.7 Statistics ................................................................................................ 93 3.3 Results and discussion ................................................................................. 93 xiii  3.3.1 Kinetic analysis of 8-CPT-cAMP on an enhanced gating mutant, S209F     .............................................................................................................. 93 3.3.2 Single-channel analysis of KCNQ1 double-phosphomimetic mutant S27D/S92D .......................................................................................................... 98 3.4 Conclusion .................................................................................................. 103 Chapter 4: The IKs Current Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry ............................................................................... 104 4.1 Introduction ................................................................................................. 104 4.2 Methods ...................................................................................................... 107 4.2.1 Reagents ............................................................................................. 107 4.2.2 Molecular biology ................................................................................ 107 4.2.3 Cell culture and transfections .............................................................. 108 4.2.4 Patch-clamp electrophysiology ............................................................ 108 4.2.5 Solutions .............................................................................................. 109 4.2.6 Data analysis ....................................................................................... 110 4.2.7 Statistics .............................................................................................. 111 4.3 Results ........................................................................................................ 112 4.3.1 The effect of cAMP on IKs in whole-cell recordings ............................. 112 4.3.2 The effect of cAMP on IKs single-channel openings ............................ 119 4.4 Discussion .................................................................................................. 126 4.4.1 Mechanisms for the KCNE1-dependent action of 8-CPT-cAMP ......... 131 4.4.2 IKs in the heart ...................................................................................... 133 4.5 Conclusions ................................................................................................ 133 xiv  Chapter 5: Discussion ............................................................................................ 135 5.1 Summary of thesis findings ........................................................................ 135 5.2 Different pharmacological sensitivity of IKs when PKA phosphorylated ...... 140 5.3 Species variation of IKs in the heart ............................................................ 141 5.4 Variation in stoichiometry of IKs in the diseased heart ................................ 143 5.5 Enhanced PIP2 affinity in PKA phosphorylated channels ........................... 144 5.6 Limitations .................................................................................................. 146 5.7 Future directions ......................................................................................... 148 5.8 Conclusion .................................................................................................. 148 Bibliography ................................................................................................................ 150 Appendices ................................................................................................................. 180 Appendix A: Supplementary Figures for Chapter 2. ............................... 180 Appendix B: Supplementary Figures for Chapter 4. ............................... 188   xv  List of Tables Table 2.1: First latency data of EQ, S209F, S27D, S27D/S92D, and KCNQ1 (Q1) + KCNE1 (E1) before and after 200µM 8-CPT-cAMP/0.2 µM OA ..................................... 63 Table 4.1: V1/2 of Activation Before and After 8-CPT-cAMP/OA ................................... 114 Table 4.2: First Latency Data for KCNQ1 (Q1) + KCNE1 (E1), EQ, EQQ, and EQQQQ, Before and After 200 μM 8-CPT-cAMP/0.2 μM OA ...................................................... 124  xvi  List of Figures Figure 1.1: Structure of KCNQ1, voltage-gated potassium channel. ................................ 6 Figure 1.2: Cryo-EM structure of KCNQ1-calmodulin binding. ....................................... 10 Figure 1.3: IKs Stoichiometry ........................................................................................... 12 Figure 1.4: β-adrenergic activation mechanism leading to phosphorylation of the IKs channel complex. ............................................................................................................ 22 Figure 1.5: Estrogen inhibits the KCNQ1-KCNE3 current and reduces chloride secretion. ........................................................................................................................ 29 Figure 2.1: Phosphorylation of IKs by PKA. ..................................................................... 49 Figure 2.2: Increase in whole-cell IKs current at room temperature after 8-CPT-cAMP addition. .......................................................................................................................... 57 Figure 2.3: 8-CPT-cAMP hyperpolarizes EQ activation and can increase the number of active channels in cell-attached patches. ....................................................................... 59 Figure 2.4: Figure 2.4. 8-CPT-cAMP increases the ensemble average current and shortens the first latency in EQ. ...................................................................................... 61 Figure 2.5: Subconductance analysis of EQ before and after 8-CPT-cAMP/OA. ........... 65 Figure 2.6: EQ closed dwell times and burst analysis. ................................................... 68 Figure 2.7: The mutant E1R/R4E + KCNE1 channel is unaffected by 8-CPT-cAMP. .... 70 Figure 2.8: 8-CPT-cAMP has a small kinetic effect on the high Po mutant KCNQ1 S209F coexpressed with E1. .......................................................................................... 73 Figure 2.9: Effects of 8-CPT-cAMP on first latency of KCNQ1 S27D + KCNE1 and Yotiao. ............................................................................................................................ 76 xvii  Figure 2.10: Effects of 8-CPT-cAMP on currents in KCNQ1 S27D/S92D + KCNE1 and Yotiao. ............................................................................................................................ 78 Figure 2.11 200 µM 8-CPT-cAMP does not alter overall surface expression of KCNQ1 and KCNE1 subunits in CHO cells. ................................................................................ 80 Figure 3.1: Subconductance analysis of KCNQ1 S209F + KCNE1 before and after 8-CPT-cAMP/Okadaic acid (OA). ...................................................................................... 95 Figure 3.2: KCNQ1 S209F + KCNE1 closed dwell times and burst analysis. ................ 97 Figure 3.3: Subconductance analysis of KCNQ1 S27D/S92D + KCNE1 before and after 8-CPT-cAMP/OA. ......................................................................................................... 100 Figure 3.4: KCNQ1 S27D/S92D + KCNE1 closed dwell times and burst analysis. ...... 102 Figure 4.1: KCNQ1 does not respond to 8-CPT-cAMP in whole-cell recordings. ........ 113 Figure 4.2: EQQ V1/2 is hyperpolarized in response to 8-CPT-cAMP addition. ............ 116 Figure 4.3: EQQQQ V1/2 is slightly hyperpolarized in response to 8-CPT-cAMP addition. ...................................................................................................................................... 118 Figure 4.4: 8-CPT-cAMP shortens the first latency of EQQ single-channels. .............. 120 Figure 4.5: Subconductance dwell time analysis of EQQ before and after 8-CPT-cAMP/OA. ..................................................................................................................... 121 Figure 4.6: 8-CPT-cAMP shortens the first latency of EQQQQ single-channels. ......... 122  xviii  List of Abbreviations g - Gamma a – Alpha b – Beta t – tau, Time constant b-AR – Beta-adrenergic receptor °C – Degrees in Celsius 17 b-estradiol – 17-beta-isomer of estradiol 8-CPT-cAMP - 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt A.U. - Fluorescence Arbitrary Unit aa – Amino acid AC9 – Adenyl cyclase 9 Akt – Protein kinase B ANOVA – Analysis of variance APD – Action potential duration ATP – Adenosine triphosphate AUC – Area under the curve AV – Atrial-ventricular node C-terminus – Carboxy-terminus  Ca2+ – Calcium CaCl2 – Calcium dichloride CaM – Calmodulin xix  cAMP – Cyclic adenosine monophosphate CFTR – Cystic fibrosis transmembrane conductance regulator CHO – Chinese hamster ovary cell CiVSP – Voltage sensitive lipid phosphatase from Ciona intestinalis Cl- – Chloride CREB – cAMP response element-binding protein Cryo-EM – Transmission electron cryomicroscopy d – Day DHT – Dihydrotestosterone DMEM – Dulbecco's modified Eagle's medium DMSO – Dimethyl sulfoxide E1 – KCNE1 E1R/R4E – KCNQ1 with two mutations E160R and R237E EGTA – Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid EQ – KCNE1 linked to one KCNQ1 (KCNE1-KCNQ1) EQQ – KCNE1 linked to two KCNQ1s (KCNE1-KCNQ1-KCNQ1) EQQQQ – KCNE1 linked to four KCNQ1s (KCNE1-KCNQ1-KCNQ1-KCNQ1-KCNQ1) ER – Estrogen receptor FBS – Fetal bovine serum G – Conductance G-V – Conductance-Voltage relationship GFP – Green fluorescent protein GTP – Guanosine triphosphate xx  GWAS – Genome-wide association study HCl – Hydrogen chloride HEK – Human embryonic kidney cells HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hESC-CMs – Cardiomyocytes derived from human embryonic stem cells Hr – Hour Hz – Hertz I-V – Current-Voltage relationship If – pacemaker current or funny current IK1 – Inward-rectifier potassium current IKr – Rapid delayed rectifier potassium current IKs – Slow delayed rectifier potassium current IKur - Ultrarapid delayed rectifier potassium current JLNS – Jervell and Lange-Nielson Syndrome JTc – QRS duration is subtracted from the entire QT interval duration k – Slope factor K+ – Potassium  KCa – Calcium-gated potassium channel KCl – Potassium chloride kHz – Kilohertz Kir – Inwardly rectifying potassium current KOH – Potassium hydroxide Kv – Voltage-gated potassium channel xxi  LM – Mouse ltk- fibroblast cells LQT – Long QT LQTS – Long QT Syndrome M – Molar MW – Megaohm MEM – Minimal Essential Medium mER – membrane estrogen receptor MES – 2-(N-Morpholino) ethanesulfonic acid MgCl2 – Magnesium chloride min – Minute MIN6 – insulin secreting pancreatic tumor mouse cell line ml – Milliliter mM – Millimolar ms – Millisecond mV – Millivolts N-terminus – Amino-terminus  nA – Nanoampere Na+ – Sodium Na2-ATP – Adenosine 5’-triphosphate disodium salt NaCl – Sodium chloride NaOH – Sodium hydroxide Nav – Voltage-gated sodium ion channel NKCC1 – Sodium-potassium-chloride co-transporter 1 xxii  NMDG – N-Methyl-D-glucamine NMR – Nuclear magnetic resonance OA – Okadaic Acid pA – Picoampere PBS – Phosphate-buffered saline PD – Pore domain PDB – Protein Data Bank PDE4D3 – cAMP-specific 3’,5’-cyclic phosphodiesterase type 4D PI3K – Phosphoinositide 3-kinase PIP2 – Phosphatidylinositol 4,5-bisphosphate PIP3 – Phosphatidylinositol 3,4,5-trisphosphate PKA – cAMP-dependent protein kinase A PKCd - Protein kinase C delta type Po – Open probability PP1 – Protein phosphatase 1 PP2 – Protein phosphatase 2 pS – Picosiemens Q1 – KCNQ1 QTc – QT interval corrected for heart rate RFU – Relative Fluorescence Unit ROI – Regions of Interest RWS – Romano-Ward syndrome s – Seconds xxiii  SA – Sinoatrial node SCD – Sudden cardiac death SE – Standard error of the mean SiRNA – Small interfering ribonucleic acid SQT – Short QT SQTS – Short QT Syndrome T3 – Triiodothyronine T4 – Thyroxine TEA+ – Tetraethylammonium chloride  TIRF – Total internal reflection fluorescence microscope TRs – Thyroid hormone receptors TSA – tsA201 transformed human embryonic kidney 293 cells V – Voltage V1/2 – Voltage at half maximal activation VCF – Voltage clamp fluorometry VS – Voltage sensor VSD – Voltage sensor domain WT – Wild-type Yotiao – A-kinase anchoring protein 9 µM – Micromolar    xxiv  Amino Acid Name and Abbreviations Ala (A) – Alanine Arg (R) – Arginine Asn (N) – Asparagine Asp (D) – Aspartate Cys (C) – Cysteine Glu (E) – Glutamate Gln (Q) – Glutamine Gly (G) – Glycine His (H) – Histidine Ile (I) – Isoleucine Leu (L) – Leucine Lys (K) – Lysine Met (M) – Methionine Phe (F) – Phenylalanine Pro (P) – Proline Ser (S) – Serine Thr (T) – Threonine Trp (W) – Tryptophan Tyr (Y) – Tyrosine Val (V) – Valine  xxv  Acknowledgements  I would like to express my sincere gratitude to my supervisor, Dr. David Fedida. Thank you for all of your support, advice, and supervision during my Ph.D. studies.  I would also like to thank Dr. Jodene Eldstrom for all of her support and guidance over the past five years. It was a pleasure to learn from and work with both of you.            Thank you to both Dr. Eric Accili and Dr. Michael Walker for agreeing to be a part of my supervisory committee, all of your helpful advice over the years, and for reviewing my thesis.  I would like to also thank my wonderful labmates and the other members of the Fedida lab. Thank you for making this experience a fun and friendly one. To Dr. Maartje Westhoff, and Dr. Logan Macdonald, you both have made this experience extra special.   To my friends and family, thank you for putting up with me over the last few years. Thank you for all the wonderful advice, pick me up texts and phone calls. I appreciate all of you very much. To my boyfriend, Jordan, thank you for being there for me and supporting me through all the craziness over the past couple of years. And finally, to my parents, thank you both for encouraging me every step of the way and for being the most supportive parents I could ever ask for. 1  Chapter 1: Introduction 1.1 Voltage-gated potassium (Kv) channels  Potassium ion channels can be placed into four major classes: calcium-activated (Kca), inwardly rectifying (Kir), two-tandem pore domain (TWIK, TREK, TASK, THIK, and TALK), and Kv channels. Voltage-gated ion channels in particular are proteins which respond to changes in the membrane potential. The pores of these channels open and close in a voltage-dependent manner, which makes them immensely important in excitable cells. Kv channels not only form the largest class of voltage-gated ion channels, but they are also the most diverse with twelve known types of Kv channels (Kv1-12).  Kv channels are essential regulators of cell excitability and signaling. They are expressed in both excitable and non-excitable cells. They play a crucial role in maintaining the membrane potential, as well as resetting the membrane potential following the initiation of an action potential in excitable cells such as cardiomyocytes, neurons, and muscle cells (Hille, 2001). In non-excitable cells, such as interstitial and endothelial cells, Kv channels have also been found to be involved in cell proliferation, enzyme and hormone secretion (Hebert and Andreoli, 1984; Lin et al., 1993; Koo et al., 1997; Grahammer et al., 2001; Urrego et al., 2014).   1.2 Role of potassium channels in cardiac action potentials 1.2.1 Electrical conduction system of the heart The heart’s electrical conduction system is responsible for generating and propagating electrical signals throughout the heart, which results in heart muscle contraction. These 2  electrical signals are referred to as action potentials. Cardiac action potentials are elicited as a result of the movement of ions such as, sodium, calcium, and potassium across the membrane through ion channels. In the heart, the action potential is initiated in the sinoatrial (SA) node and spreads through atrial cells causing atrial contraction, before passing through the atrioventricular (AV) node. The AV node delays the propagation of the signal to allow for complete contraction of the atria before contraction of the ventricles can commence, which ensures proper blood flow.  The signal is then passed from the AV node to the His bundle, which splits into the left and right branches, before eventually reaching the Purkinje fibers. The Purkinje fibers transmit the signal received from the AV node to the ventricular cardiomyocytes, which results in ventricular contraction. These different cell types make up the heart’s conduction system. The form of the action potential differs between these types of cardiac cells due to the varied expression of ion channels in each cell-type (Bartos et al., 2015).   Action potentials are elicited when the membrane is depolarized, made more positive, and reaches the threshold of activation, which in SA and AV nodal cells is ~ -40 mV and in atrial and ventricular myocytes is ~-70 mV (DiFrancesco, 1993; Nerbonne and Kass, 2005). Once this threshold is reached, the action potential is initiated. The number of phases in the cardiac action potential vary between nodal (SA and AV nodes) and non-nodal (atrial and ventricular) cells. In non-nodal cells, the action potential follows five phases, phase 0-4, whereas in nodal cells, only three phases occur; phases 1 and 2 are not seen.  3  1.2.2 Resting membrane potential, phase 4 At rest, cellular permeability of potassium is much greater than that of other ions, such as sodium. Potassium constitutive leak currents play a major role in determining the resting membrane potential of the cell. The resting membrane potential differs between cell type.   In the SA and AV nodes, the resting potential is ~ -60 mV, whereas in atrial and ventricular myocytes it is more negative at ~ -80 mV. The reason for this is due to a larger potassium leak current in atrial and ventricular myocytes, predominately due to the potassium selective, inward rectifier channel, IK1 (Dhamoon and Jalife, 2005). The resting membrane potential in nodal cells is not as stable, which allows these cells to spontaneously trigger action potentials. In these cells, the funny current, If, produced by the HCN channel, passes both potassium and sodium ions (Baruscotti and Robinson, 2007; Pinnell et al., 2007). If current produces the leak potassium current needed for the resting membrane potential, but also allows sodium ions to pass into the cell, slowly depolarizing the membrane. As the membrane potential increases, calcium channels become activated, first the transient-type calcium channels (T-type Ca2+) and then upon further depolarization the long-lasting, L-type calcium channels (Baruscotti and Robinson, 2007; Pinnell et al., 2007). The If allows these cells to spontaneously generate action potentials. In atrial and ventricular cells, the action potential is passed from cell to cell by gap junctions.  4  1.2.3 Phase 0 Once the threshold of activation is reached, further depolarization of the membrane occurs, this is phase 0. Phase 0 in atrial and ventricular myocytes sees a rapid influx of sodium ions into the cell through the activation of voltage-gated sodium (Nav) channels. As the Nav channels open, the sodium permeability increases allowing more Nav channels to open resulting in rapid depolarization of the membrane potential between 0 and 20 mV (Bartos et al., 2015).   In nodal cells, phase 0 is slower. Nav channels are not expressed in these cells and phase 0 is reliant on the slower to activate voltage-gated L-type calcium channels instead (Mesirca et al., 2015).   1.2.4 Phase 1 In atrial and ventricular myocytes, the Nav channels quickly start to inactivate and the sodium current is reduced. The outward potassium current, Ito, starts to activate (Santana et al., 2010). This results in the membrane potential becoming slightly more negative (repolarization) due to the efflux of potassium from the cell creating a ‘notch’ in the waveform of the action potential. This ‘notch’ is not seen in the waveform of SA and AV action potentials.  1.2.5 Phase 2 During phase 2 or the plateau phase, the potential across the membrane remains constant as the transient outward Ito current effluxes potassium ions and L-type calcium 5  channels bring calcium ions into the cell (Pinnell et al., 2007; Santana et al., 2010). This balance of ion movement allows the membrane potential to remain relatively constant. Atrial myocytes have a narrower plateau phase than ventricular myocytes as the calcium current in these cells is smaller and they also express the delayed rectifying current, IKur, which is not found in ventricular cells (Bootman et al., 2006). Phase 2 is not seen in AV or SA action potential waveforms.  1.2.6 Phase 3 During phase 3, the repolarization phase, the membrane potential is returned to its resting state (phase 4). This is a result of the calcium ion channels undergoing inactivation, reducing the influx of positive calcium ions. At the same time, Kv channels are being activated. In ventricular and atrial cells, the delayed rectifying potassium currents, IKr and IKs are activated (Nerbonne and Kass, 2005; Pinnell et al., 2007) and in the nodal cells, the delayed rectifying potassium current, IK. The efflux of potassium ions returns the membrane to its resting potential (phase 4).   1.3 Structure of voltage-gated potassium channels Four Kv subunits need to co-assemble to form a functioning tetrameric Kv channel (Fig. 1.1A). All Kv channel subunits have the same principal structure of six transmembrane regions per subunit: S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD) (Fig. 1.1B-C) (Jiang et al., 2003; Long et al., 2005).  The PD from each of the four subunits, when assembled, form the channel’s aqueous pore through which ions flow.   6    Figure 1.1: Structure of KCNQ1, voltage-gated potassium channel. (A) shows the top and side view of the tetrameric KCNQ1EM channel structure (PDB: 5VMS) (Sun and MacKinnon, 2017) illustrated using Pymol. Each α-subunit is displayed in a different color. (B) shows a cartoon of a single subunit of a voltage-gated potassium (Kv) channel. Shown in blue are the four transmembrane regions, segments 1-4 (S1-S4) composing the voltage-sensing domain (VSD). S5-S6 form the pore domain (PD) and are shown in red. The positive signs illustrate the positive residues found in the S4 region (voltage-sensor), which can move in response to changes in the membrane potential. Negative signs show negative residues in S2 and S3. (C) only shows the side view of three KCNQ1 α-subunits. The S1-S6 transmembrane domains of one subunit are labeled along with three of the four C-terminal helices (A-C).   In Kv channels, the VSD and PD are arranged in two configurations: the domain-swapped and non-domain swapped. In the domain-swapped configuration, the VSD of one subunit is packed against the PD of its neighboring subunit. In non-domain-swapped 7  configuration, the VSD is in contact with the PD of its own subunit. Typically, Kv1-9 are domain-swapped and Kv10-11 are found in non-domain swapped configurations (Barros et al., 2019). Kv1-9 have longer S4-S5 linkers, which allow them to arrange themselves in such a way.  Many Kv channels are homotetramers, but heterotetrametric channel assembles are possible. For example, within the Kv7 ion channels (Kv7.1-7.5), which are encoded by KCNQ genes (KCNQ1-5) and express α-subunits that form tetrameric ion channels, KCNQ1 subunits do not co-assemble with KCNQ2-5 subunits; they co-assemble as homotetramers, but KCNQ2-5 can co-assemble with each other to form heterotetramers (Schwake et al., 2003).   The potassium pore contains a highly conserved amino acid sequence, TVGYG, which is known as the selectivity filter. The selectivity filter allows these channels to be highly selective for potassium ions (Doyle et al., 1998). When ions enter the pore, they become dehydrated. These dehydrated potassium ions are now able to interact with the selectivity filter, specifically the four sets of carbonyl oxygen atoms which line the pore. The dehydrated potassium ions and the carbonyl oxygen atoms interact and form energetically favorable bonds similar to those of potassium ions and water (Zhou et al., 2001). Once these ions pass through the selectivity filter, they bind to water molecules outside the cell. Other ions such as sodium are smaller in size and do not as efficiently interact with the carbonyl oxygen ions and therefore, do not easily flow through these channels (Choe, 2002). 8  The VSD detects variations in the membrane potential, in particular, S4, which contains positively charged amino acid residues, making the channel sensitive to electrical changes (Long et al., 2005; Kuang et al., 2015). When the cell membrane becomes depolarized (more positive), these positive charges move causing a conformational rearrangement of the channel complex and the opening of the channel pore.   Most Kv channels will undergo inactivation if the membrane is continually depolarized.  There are three types of channel inactivation: N-type, C-type, and U-type inactivation, all of which are non-conducting states for potassium ions (Kurata and Fedida, 2006; Bähring et al., 2012). N-type inactivation also referred to as ball and chain inactivation, is a result of the pore becoming blocked by the N-terminus of the channel subunit (Kurata and Fedida, 2006; Bähring et al., 2012). The open channel very quickly transitions to this non-conducting state where the channel is still in its open configuration, but no ions can flow through. C-type inactivation results from structural rearrangements to the pore, specifically the selectivity filter, which no longer allows potassium ions to pass through (Kurata and Fedida, 2006; Bähring et al., 2012; Kuang et al., 2015). U-type inactivation is also a slow form of inactivation that has been identified in several Kv channels (Kv2.1, 3.1, 1.5, and Shaker) (Klemic et al., 2001; Kurata and Fedida, 2006; Cheng et al., 2011; Bähring et al., 2012). This form of inactivation gets its name from its inactivation-voltage relationship, which is U-shaped. The greatest amount of U-type inactivation is seen at more negative membrane potentials, where only a fraction of the channels are open (Kurata and Fedida, 2006; Bähring et al., 2012). As the membrane is further depolarized and more channels open, less inactivation is seen. 9  1.3.1 KCNQ potassium channels KCNQ channels are known to produce several types of current; two of the most well-known are the slow-delayed rectifying potassium current (IKs) and the M-current (Wang and Li, 2016). KCNQ1 along with the β-subunit KCNE1 make up IKs, which plays a vital role during repolarization of cardiomyocytes (Barhanin et al., 1996). KCNQ2-5 subunits form channels which produce a current known as the M-current that is important in neuronal excitability (Wang and Li, 2016).  1.3.1.1 Requirement of phosphatidylinositol 4,5-biphosphate (PIP2) PIP2 is a membrane lipid that binds to many types of ion channels, including all KCNQ subunits and is found in the inner leaflet of the membrane. For KCNQ channels, PIP2 acts as a gating modulator and is a required co-factor for channel opening (Zaydman and Cui, 2014). Experiments where PIP2 levels were depleted resulted in current abolition (Zaydman et al., 2013; Royal et al., 2017). A voltage-clamp fluorometry (VCF) experiment using a voltage-sensitive phosphatase from Ciona intestinalis (CiVSP) found that in the absence of PIP2, KCNQ1’s voltage sensor (VS) was still able to move to its activated state as normal, but pore opening did not occur, which suggests that PIP2 is needed for coupling voltage-sensing and pore opening (Li et al., 2011b). The recent cryogenic electron microscopy (Cryo-EM) study of KCNQ1, which was performed in the absence of PIP2, revealed a channel with a VS in an activated confirmation, but a closed pore (Fig. 1.1A & C) (Sun and MacKinnon, 2017). This provides further evidence that PIP2 is required for the coupling of the VS and the PD.  10  There have been a number of putative PIP2 binding sites suggested including residues in the S2-S3 loop, S4-S5 linker, and S6 C-terminus. Several studies across the KCNQ channels have found that mutating residues in these regions have altered the channel’s affinity of PIP2 suggesting that these areas are of importance for KCNQ-PIP2 interaction (Dvir et al., 2014; Kasimova et al., 2015; Choveau et al., 2018). The cryo-EM study further supports the idea that PIP2 can bind in the S4-S5 linker and facilitate coupling of the VSD and PD.  1.3.1.2 Calmodulin (CaM) requirement The calcium binding protein, CaM, is a well-known regulator of many ion channels. All KCNQ channels require CaM binding. CaM is involved in channel assembly and trafficking and is also important for channel gating (Shamgar et al., 2006).    Figure 1.2: Cryo-EM structure of KCNQ1-calmodulin binding. KCNQ1EM channel structure bound with calmodulin (PDB: 5VMS) (Sun and MacKinnon, 2017) illustrated using Pymol. Shows sideview of two KCNQ1 subunits bound with two calmodulin (colored in orange) subunits. Each α-subunit is displayed in a different color. The S1-S6 transmembrane domains of one subunit are labeled along with three of the four C-terminal helices (A-C). 11  CaM has two sites to which it is thought to bind in KCNQ channels. The first interface is in helix A in the C-terminal domain of KCNQ channels. The N- and C-lobes of the CaM complex act as a type of clamp around helix A and helix B (Fig. 1.2). The second was first described following the resolution of the KCNQ1-CaM cryo-EM structure by Sun & MacKinnon (2017). They found that CaM interacts with KCNQ1 through residues in the S2-S3 loop (Fig. 1.2), in particular, a nine amino acid stretch that is conserved and unique to KCNQ channels. They suggest that CaM being able to bind to both the VSD and the PD could potential provide a link between the two not involving the S4-S5 linker (Sun and MacKinnon, 2017).    1.3.1.3 Stoichiometry of IKs KCNE1 b-subunits are believed to reside in the exterior clefts between the VSDs of neighboring KCNQ1 subunits (Kang et al., 2008). As each channel is made up of four KCNQ1 subunits, there are four available exterior clefts for KCNE1 subunits to occupy (Fig. 1.3).    12   Figure 1.3: IKs Stoichiometry Cartoon shows four KCNQ1 α-subunits (light blue) and four KCNE1 β-subunits (dark blue) residing in the exterior clefts (A) shows top-down view and (B) shows side view. The red circle represents a potassium ion in the channel pore. (B) shows that each KCNQ1 subunit (light blue) has six transmembrane regions and KCNE1 subunit (dark blue) only has one.    The number of KCNE β-subunits that co-assemble with the tetrameric KCNQ1 channel has been the subject of much debate and investigated by several groups (Plant et al., 2014; Murray et al., 2016). While some have proposed a strict ratio of two KCNE1 subunits to four KCNQ1 subunits (Wang and Goldstein, 1995; Chen et al., 2003a; Morin and Kobertz, 2007; Kang et al., 2008; Morin and Kobertz, 2008; Plant et al., 2014), others have shown it is possible for all four exterior clefts to be occupied by KCNE1 (Fig. 1.3) (Cui et al., 1994; Wang et al., 1998; Morokuma et al., 2008; Nakajo et al., 2010; Zheng et al., 2010; Strutz-Seebohm et al., 2011; Wang et al., 2011; Yu et al., 2013; Murray et al., 2016), and the number of KCNE1s present is variable and dependent on the expression level of KCNE1 (Wang et al., 2011). This may provide another important regulatory mechanism of KCNQ1.   While the majority of research focuses on KCNE1, some studies have suggested that other KCNE subunits such as KCNE2, are able to co-assemble with KCNQ1-KCNE1 channel complexes and alter the current’s kinetics in cardiac myocytes (Wu et al., 2006).  13  1.4 Physiology and pathophysiology of KCNQ1 and KCNQ1:KCNE channels 1.4.1 KCNQ1 alone KCNQ1 is widely distributed throughout the body, from the inner ear to the large intestine (Lundquist et al., 2006). When KCNQ1 is expressed alone in vitro, a small, rapidly activating potassium-selective current is produced (Bendahhou et al., 2005; Lundquist et al., 2006). However, it is thought that in vivo, KCNQ1 is more likely to form a channel complex with at least one of five known KCNE β-subunits (KCNE1-E5) (Bendahhou et al., 2005; Lundquist et al., 2006) rather than exist as a homomeric complex of KCNQ1 alone (Abbott et al., 2001b).  KCNEs have a single transmembrane domain and cannot produce currents when expressed alone (McCrossan and Abbott, 2004), but each produces drastically different currents upon assembly with KCNQ1 (Bendahhou et al., 2005).   KCNQ1 mutations have been linked to several disorders, particularly cardiac, such as Long QT (LQT) syndrome type 1, short QT (SQT) syndrome, and familial fibrillation (Moss et al., 1991; Chen et al., 2003c; Bellocq et al., 2004). As well, several KCNQ1 single nucleotide polymorphisms have been identified in genome-wide association studies in diabetic populations (Unoki et al., 2008; Yasuda et al., 2008). KCNQ1 knockout mice exhibit many different phenotypes, supporting the idea that KCNQ1 has many roles throughout the body. These knockout mice exhibit deafness, inner ear and cardiac abnormalities, abnormal thyroid hormone levels, gastric hyperplasia, and changes in insulin and glucose sensitivity (Lee et al., 2000; Casimiro et al., 2004; Rivas and Francis, 2005; Boini et al., 2009; Song et al., 2009; Frohlich et al., 2011).  14  1.4.2 KCNE1 KCNQ1 and KCNE1 co-assemble to produce a slow-activating K+ current called IKs. KCNE1 alters the kinetics of KCNQ1 alone from a small, rapidly activating and inactivating current, to one that is much larger, which activates and deactivates much more slowly (Barhanin et al., 1996), and is no longer seen to inactivate (Tristani-Firouzi and Sanguinetti, 1998).  In the heart, both KCNQ1 and KCNE1 subunits are expressed in the atria and ventricles, where IKs is important during phase 3, repolarization, of the cardiac action potential particularly at high heart rates. The amplitude of IKs at high heart rates is increased, which results in a larger ‘repolarization reserve’. The repolarization reserve is composed of several repolarizing currents, including IKr and IKs. An increase in the repolarization reserve results in faster repolarization, shortens the duration of the cardiac action potential and allows the ventricles adequate time to fill (Roden and Yang, 2005; Terrenoire et al., 2005).   In the inner ear, KCNQ1 and KCNE1 subunits are expressed in marginal cells of the stria vascularis and create a flow of potassium ions into the endolymph to maintain the high potassium concentration of the fluid surrounding the hair cells necessary for normal hearing transduction to occur (Wrobel et al., 2012).   15  In the pancreatic acinar cells, where digestive enzymes are synthesized and secreted, the current produced by IKs complexes has been suggested to be important for the release of electrolytes and enzyme secretion (Warth et al., 2002).   In the kidney, KCNE1 and KCNQ1 co-assemble in the proximal tubular cells of the nephron. Their role is to allow potassium ions to flow into the lumen from the proximal tubular cells, repolarizing the cell membrane to offset the membrane depolarization caused by sodium-coupled transport of glucose as well as amino acids (Vallon et al., 2001).  In the heart, mutations in either KCNQ1 or KCNE1 can lead to improper repolarization of the cardiac action potential, and cause diseases such as LQT and SQT syndromes as well as familial atrial fibrillation (Moss et al., 1991; Chen et al., 2003c; Bellocq et al., 2004).   Mutations in KCNQ1 leading to LQT syndrome are known as LQT type 1 and those in KCNE1, type 5. Jervell and Lange-Nielsen syndrome (JLNS) is one type of LQT syndrome that can be caused by mutations in KCNQ1 or KCNE1 (Jervell and Lange-Nielsen, 1957; Schulze-Bahr et al., 1997; Murray et al., 2002; Rivas and Francis, 2005; Faridi et al., 2018). JLNS is an autosomal recessive disorder meaning that the loss-of-function is severe, as both copies of either KCNQ1 or KCNE1 contain the JLNS mutation (Rivas and Francis, 2005). JLNS is characterized not just by a prolonged QT interval, but also profound deafness due to IKs’s role in the inner ear (Lee et al., 2000; Rivas and Francis, 2005). Romano-ward syndrome (RWS) is an autosomal dominant form of LQT 16  syndrome similar to JLNS, but where no hearing impairment is seen (Faridi et al., 2018). Typically, KCNQ2-5 are thought to be expressed neuronally and not KCNQ1. However, a link between LQT1 and seizures has been suggested (Goldman et al., 2009; Tiron et al., 2015).   Knockout KCNE1 mice display several different phenotypes. They exhibit profound deafness, as well as Shaker-Waltzer behavior (circling, hyperactivity, head-bobbing, and tilting), which is a sign of inner ear impairment. The vestibular membrane, which alongside the basilar membrane compartmentalizes the endolymph, collapses shortly after birth in KCNE1 knockout mice. This results in impaired endolymph production, loss of the hair cells, and loss of hearing in these mice. These knockout mice also exhibit hypokalemia and dehydration. KCNE1 deletion results in a loss of driving force provided by the potassium current, leading to impaired absorption of sodium and glucose (Charpentier et al., 1998; Vallon et al., 2001; Casimiro et al., 2004; Temple et al., 2005). KCNE1 knockout mice have only a mild cardiac phenotype, which is thought to be due to low expression of IKs in mouse heart (Charpentier et al., 1998). The QT interval is unchanged under normal conditions, but when the heart rate changes, the QT interval adapts to change of rate in KCNE1 knockout mice differently than that of the control mice. A prolonged QT interval is seen at slow heart rates in these knockout mice as well as a shorter QT interval at fast heart rates (Warth and Barhanin, 2002). Another knockout mouse study found that in atrial myocytes, the APD shortened, increasing the risk of atrial fibrillation (Temple et al., 2005).   17  1.4.3 KCNE2 When KCNQ1 co-assembles with KCNE2, the resultant current has very different kinetics to IKS. KCNQ1 and KCNE2 produce a small constitutively active potassium current, also referred to as a leak current (Tristani-Firouzi and Sanguinetti, 1998). KCNE2 is expressed in cardiac tissue as well as in epithelial cells of the thyroid, choroid plexus and the stomach (Roepke et al., 2006; Roepke et al., 2008; Roepke et al., 2009; Roepke et al., 2010; Roepke et al., 2011). The effect that KCNQ1-KCNE2 has on the cardiac action potential is not fully understood. KCNE2 can co-assemble with other potassium currents expressed in the heart (Eldstrom and Fedida, 2011), which makes understanding its role more challenging. It has also been suggested that KCNE2 can co-assemble with KCNQ1-KCNE1 (Wu et al., 2006; Jiang et al., 2009) and form a KCNQ1-KCNE1-KCNE2 channel complex. The resultant current kinetics resemble that of IKs, but with smaller current amplitudes, suggesting that KCNE2 may act as a regulatory subunit.   KCNQ1-KCNE2 complexes in epithelial cells of the thyroid, stomach, and perhaps the choroid plexus, are essential for potassium recycling, which helps maintain the electrochemical gradient of these cells (Schroeder et al., 2000; Roepke et al., 2006; Roepke et al., 2009). KCNQ1-KCNE2’s ability to produce functional currents at low pH is essential for its role in the stomach, where cells are exposed to gastric acid secretions.   Mutations in KCNE2 (R27C, M23L, and I57T) have been implicated in atrial fibrillation (Yang et al., 2004). These mutations eliminate the suppressive effect KCNE2 has on KCNQ1 and KCNQ1-KCNE1 currents. Mutant KCNE2 subunits (T10M and V65M) have 18  been found to cause LQT syndrome type 6 (Jiang et al., 2004; Tester and Ackerman, 2009).  KCNE2 knockout mice have prolonged QT intervals (Roepke et al., 2008). This, however, may not be due to KCNE2 and KCNQ1 complexes, but most likely Kv4.2, Kv4.3, Kv2.1, and Kv1.5, which are responsible for ventricular repolarization in mice (Mazhari et al., 2001; Roepke et al., 2008; Wu et al., 2010b). Apart from cardiac abnormalities, KCNE2 knockout mice have hypothyroidism, alopecia, and dwarfism (Roepke et al., 2008; Roepke et al., 2009; Roepke et al., 2010). These knockout mice also exhibit gastric hyperplasia.  1.4.4 KCNE3 KCNQ1-KCNE3 complexes produce a constitutively active potassium current similar to KCNQ1-KCNE2. However, KCNQ1-KCNE3 generates a larger current (Bendahhou et al., 2005). KCNE3 is thought to directly interact with the voltage sensor and stabilize the activated state (Lundquist et al., 2006). Recently, it was shown that KCNQ1-KCNE3 is not voltage independent, but the voltage-dependence is shifted to such hyperpolarized potentials that the channel complex is active at physiological voltages (Barro-Soria et al., 2017). This channel complex is expressed in the stomach, lungs, kidney and the intestine (Preston et al., 2010), where it is involved in K+ recycling similar to KCNE2. In the intestine and the lungs, KCNQ1-KCNE3 current is needed to hyperpolarize the membrane, which leads to an increase in the chloride driving force and facilitates increased chloride secretion (Preston et al., 2010). KCNQ1-KCNE3 current’s role is in salt and fluid 19  homeostasis by recycling potassium ions and driving the secretion of chloride ions from the gut lumen into epithelial lining cells. KCNE3 is also known to form complexes with Kv3.4, Kv4.2, and Kv4.3 (Abbott et al., 2001a; Abbott, 2017).  In KCNE3 knockout mice, cAMP-activated chloride secretion was drastically reduced. KCNQ1 localization was unaffected, which reinforces the importance of KCNE3 in epithelial cells of the lungs and the intestine for fluid homeostasis (Preston et al., 2010).   Higher KCNQ1-KCNE3 expression in colorectal cancer tumors is associated with a good survival outcome (Rapetti-Mauss et al., 2017). KCNQ1 itself is a tumor suppressor gene in mice and analysis of tissue samples from colorectal cancer patients showed that lower expression of KCNQ1 has a negative outcome on survival (Than et al., 2014). Elevated KCNQ1-KCNE3 expression levels has been suggested to be beneficial for patient survival (Rapetti-Mauss et al., 2017). The suggested mechanism is that KCNQ1-KCNE3 current is able to maintain a hyperpolarized membrane potential in the colorectal cancer cells, which is thought to slow cell proliferation - which requires more depolarized membrane potentials (Yang and Brackenbury, 2013; Rapetti-Mauss et al., 2017).   1.4.5 KCNE4 KCNE4 and KCNE5 both exhibit an inhibitory effect on KCNQ1 (Angelo et al., 2002; Lundquist et al., 2006); currents are similar to KCNQ1-KCNE1 but at physiological voltage ranges these subunits cause inhibition of the KCNQ1 current, shifting the voltage-dependence of the complex by +140 mV (Angelo et al., 2002; Lundquist et al., 2006). 20  KCNE4 is expressed in the heart, brain, uterus, testis, placenta, kidney, liver, and skeletal muscle (Abbott, 2016). Calmodulin binds to both KCNQ1 and KCNE4, which is suggested to be a mechanism of inhibition (Ciampa et al., 2011). KCNE4 has also been shown to reduce IKs currents when it is co-expressed with KCNQ1 and KCNE1 (Manderfield and George, 2008), indicating that it may have a role in modulating IKs.  KCNE4 and KCNE5 are the two least understood KCNE β-subunits. KCNE4 has the highest transcript expression levels in the heart of all the KCNE subunits, but not a lot is known about its function (Radicke et al., 2006). One study found that in the left ventricles of male mice, the Kv current density was greater than that seen in pre-menopausal female mice and that this may be a result of 5α-dihydrotestosterone (DHT) upregulating KCNE4 expression. This was further supported in postmenopausal female mice. After menopause, DHT levels are increased. This study also found these mice to have increased KCNE4 expression levels compared with their premenopausal counterparts (Crump et al., 2016). Similar to other KCNE subunits, KCNE4 has also been found to modulate other potassium ion channels such as Kv1.1, Kv1.3, Kv1.5, Kv2.1, Kv4.2, and Kv4.3 (Grunnet et al., 2003; Levy et al., 2010; David et al., 2015; Crump et al., 2016; Abbott, 2017).   There are not many known disease-causing mutations in KCNE4; one known example is the E145D mutation found in a Chinese population that has been linked to atrial fibrillation (Ma et al., 2007), the mechanism of which is not fully understood.   21  1.4.6 KCNE5 Similar to KCNE4, not too much is known about this β-subunit. KCNE5 is expressed in the heart, brain, skeletal muscle, and the placenta. KCNE5 modulates Kv1.5, Kv4.2, Kv4.3 channels as well as KCNQ1 (David et al., 2019).  Mutations in KCNE5 have been implicated in several disorders; Brugada syndrome (Y81H and D92E/E93X), LQT syndrome, and atrial fibrillation (P33S, L65F, and R85H) (Ravn et al., 2005; Ravn et al., 2008; Ohno et al., 2011; Mann et al., 2012; Palmer et al., 2012).   In a knockout mouse model of KCNE5, an increase in voltage-sensitive potassium current was seen in ventricular myocytes, which supports KCNE5 having an inhibitory effect on Kv channels. KCNE5 knockout mice also exhibited an increase in Kv2.1, which was suggested to be as a result of reduced intracellular sequestration of Kv2.1 subunits by KCNE5 (David et al., 2019).  1.5 Hormonal modulation of IKs Given the range of different phenotypes that occur as a result of mutations or knockouts in KCNQ1 or KCNE1-5 and the wide range of tissue types they are expressed in, we could expect there to be many different regulators of these potassium currents. There have been numerous studies investigating the roles of different hormones and how they affect these different KCNQ1-KCNE channel complexes and their function in different tissues.  22  1.5.1 Norepinephrine The most well studied hormonal regulation of KCNQ1 to the present day is that of the IKs current by norepinephrine. During periods of high heart rates in cardiac tissue, IKs plays an important role during the ventricular repolarization phase (phase 3) of the cardiac action potential (Sanguinetti and Jurkiewicz, 1990; Stengl et al., 2003; Silva and Rudy, 2005; Terrenoire et al., 2005).   Figure 1.4: β-adrenergic activation mechanism leading to phosphorylation of the IKs channel complex. The G-coupled β-adrenergic receptor (β-AR) is activated by an agonist, such as norepinephrine, and the α-subunit of the G-protein is released. The α-subunit activates adenyl cyclase 9 (AC-9), which increases 3',5'-cyclic adenosine monophosphate (cAMP) levels. Yotiao is an anchoring protein that brings together proteins to form a complex on the c-terminus of KCNQ1. Protein kinase A (PKA) is activated in response to cAMP, which phosphorylates two residues on the KCNQ1 n-terminus, serine 27 and serine 92 (denoted by *). PDE4D3 is cAMP-specific phosphodiesterase that degrades cAMP. Protein phosphatase 1 (PP1) 2 is also brought into the complex by Yotiao and dephosphorylates residues phosphorylated by PKA.  Norepinephrine binds to β-adrenergic receptors in the membranes of the cardiomyocytes, which are coupled to heterotrimeric G-proteins and triggers a signaling cascade, leading to an increase in IKs current as a result of phosphorylation of KCNQ1 (Terrenoire et al., 23  2005). This mechanism is illustrated in Figure 1.4. Once the β-adrenergic receptor is activated, the GTP-bound α-subunit of the G-protein coupled receptor dissociates and activates adenyl cyclase 9. Adenyl cyclase 9 increases the intracellular concentration of 3’-5’-cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) (Marx et al., 2002; Cumbay and Watts, 2004) (Fig. 1.4). Yotiao, a scaffolding protein, coordinates the assembly of PKA, phosphodiesterase (PDE4D3), and phosphatase (PP1) together on the C-terminus of KCNQ1 (Marx et al., 2002; Terrenoire et al., 2009; Zheng et al., 2010). This complex is essential for the regulation of IKs and its response to sympathetic activation. Yotiao brings the C- and N-termini of KCNQ1 closer, giving PKA the ability to phosphorylate residues S27 and S92 of the N-terminus (Marx et al., 2002; Lopes et al., 2007; Zheng et al., 2010; Lundby et al., 2013). Once these residues are phosphorylated, a hyperpolarizing shift in the V½ of activation and slower deactivation kinetics are observed and IKs current is increased (Dilly et al., 2004; Terrenoire et al., 2005).  Though not a target of PKA phosphorylation, KCNE1 must be present in the channel complex for any functional effect of phosphorylation, (Kurokawa et al., 2003), specifically, the C-terminus of KCNE1. When it is removed, IKs no longer responds to phosphorylation (Kurokawa et al., 2009). PKA can still phosphorylate KCNQ1 alone, but no changes in the current are seen as a result (Kurokawa et al., 2003).   During periods of stress, fear or exercise, the sympathetic nervous system is activated, and the heart rate increases. To allow sufficient time for the ventricles to fill with blood 24  between contractions, the cardiac action potential must terminate more quickly at these higher heart rates (Jost et al., 2005). This is accomplished by the aforementioned enhancement of the IKs current (Stengl et al., 2003). Because IKs is now quicker to activate but slow to deactivate, it starts to accumulate from beat to beat. This increase in potassium current shortens the cardiac action potential and allows for complete repolarization of the membrane potential and an adequate pause for chamber filling before another action potential commences (Hund and Rudy, 2004), which is essential for the cardiac action potential to adapt to an increase in heart rate.   Mutations in either KCNQ1 or KCNE1 can cause a loss-of-function leading to LQT syndrome, type 1 and 5, respectively (Wang et al., 1996; Splawski et al., 1997). LQT missense mutations, such as KCNE1 D76N, which is found in the C-terminus, have an impaired response to β-adrenergic stimulation (Kurokawa et al., 2003; Kurokawa et al., 2009). Mutations in Yotiao have been found to cause LQT syndrome and are referred to as LQT type 11. Yotiao mutant S1570L reduces the interaction of KCNQ1 and Yotiao causing a reduction KCNQ1 phosphorylation and removes the phosphorylation induced response of IKs (Chen et al., 2007). The prolonged cardiac APD in LQT syndrome (Wang et al., 1996) increases the risk of early afterdepolarizations and the development of arrhythmias, such as ventricular tachycardia, which can lead to sudden death (Schwartz et al., 1991; Chen et al., 2003b).   At present, patients are typically treated with β-blockers, but KCNQ1 is of interest as a potential therapeutic target. However, the stoichiometry of the channel complex appears 25  to greatly impact the efficacy of potential modulators. IKs inhibitors such as Chromanol-293B and HMR-1556 are greatly affected by the amount of KCNE β-subunits that are present in the channel complex and have a greater potency when KCNE1 subunits are present (Lerche et al., 2000; Barrese et al., 2018). IKs activators such as ML277 and mefenamic acid are also sensitive to the stoichiometry. The potency of ML277 is drastically reduced as the number of KCNE1 subunits in the channel complex increases (Yu et al., 2013). Mefenamic acid activates IKs but does not affect KCNQ1 alone (Busch et al., 1997). To date, it is not clear what the IKs stoichiometry is in the human heart, although there is some evidence to suggest that the complex is not fully saturated with KCNE1 subunits as ML277 has an effect on cultured human myocytes (Yu et al., 2013). It is not known if stoichiometry is developmentally, regionally or physiologically regulated, therefore, this represents an important area for further research in the context of the development of IKs activators.    1.5.2  Insulin Insulin, a vital hormone in the regulation of blood glucose levels, is produced in the pancreatic islets by beta cells (Wu et al., 2014; Wu et al., 2017). It is released from the pancreas as circulating glucose levels rise, which then allows glucose to move into tissues. When insulin levels are high, the body stores excess glucose as glycogen for use during periods when the glucose levels are low. However, if this process is impaired, diabetes can occur (Wilcox, 2005; Röder et al., 2016). Type 1 diabetes occurs as a result of a lack of insulin and is typically treated via insulin replacement therapy (Atkinson et al., 2014; Katsarou et al., 2017). Whereas in type 2 diabetes mellitus, the body becomes 26  resistant to insulin, which leads to high blood glucose levels and further possible complications such as neuropathy, blindness, and kidney damage (DeFronzo, 2004).   Both type 1 and 2 diabetics are reported to have an increased risk of sudden cardiac death (SCD) (Whitsel et al., 2005; Vasiliadis et al., 2014). One proposed mechanism behind this increased risk in the diabetic population is due to insulin induced hypoglycemia, low blood glucose levels. Hypoglycemia is a common symptom exhibited by both type 1 and 2 diabetics. It can be caused by insulin replacement therapy as well as other diabetic medications such as sulfonylureas. Several studies have shown that hypoglycemia prolongs the QTc (QT interval corrected for heart rate)  (Lo et al., 1993; Heller, 2002; Suys et al., 2002; Veglio et al., 2002; Robinson et al., 2003; Kobayashi et al., 2018), which can lead to a higher risk of ventricular tachycardia and SCD. Hyperinsulinemia has also been associated with QTc prolongation in non-diabetics (van Noord et al., 2010).   In a canine model of type I diabetes, the repolarization reserve was diminished, due to a reduction in KCNE1 expression (Lengyel et al., 2007). This decrease in IKs current density was not seen in diabetic dogs treated with insulin, which suggests that the lack of insulin could play a role in the increased risk of SCD seen in type I diabetic patients by decreasing the IKs current and reducing ventricular repolarization. Interestingly, in vitro studies of both acute and chronic insulin exposure on IKs has shown opposing results. Acute insulin application on KCNQ1-KCNE1 resulted in a suppression of the IKs current as a result of reduced KCNE1 expression. However, chronic exposure to insulin lead to 27  an increase in KCNE1 expression and IKs current. Insulin exerts effects on KCNE1, in particular residues in the distal C-terminus of KCNE1 (amino acids 111-118), which were found to be essential through mutagenesis experiments (Wu et al., 2014; Wu et al., 2017). Insulin's effect on the IKs current is thought to be mediated through the PI3K/Akt pathway, as wortmannin, a PI3K inhibitor, removes the effect (Wu et al., 2014). As mentioned earlier, PIP2 is a requirement for IKs current activity with low or depleted PIP2 leading to much lower IKs current density (Ishii et al., 2014; Wu et al., 2014). While KCNQ1 has a relatively low sensitivity to PIP2, sensitivity is greatly enhanced when KCNE1 is part of the channel complex (Loussouarn et al., 2003).  PI3K reduces PIP2 levels by converting it to PIP3, which may be part of the mechanism behind the IKs current decreases in response to acute insulin exposure (Wu et al., 2014). The response to chronic insulin exposure is thought to be due to increased protein synthesis and/or increased trafficking from the endoplasmic (Wu et al., 2017). Insulin has a complex effect on the IKs current and further investigation is required to elucidate the mechanism behind it.  In the MIN6 mouse β-cell line, overexpression of KCNQ1 resulting in an increase in potassium current led to significantly reduced insulin secretion and hyperglycemia (Yamagata et al., 2011). Inhibition of KCNQ1 using siRNA increased insulin secretion (Torekov et al., 2014). Thus, insulin secretion is, at least in part, regulated by the expression level of KCNQ1, and this is supported by the results of several genome-wide association studies (GWAS).  Many KCNQ1 polymorphisms in different ethnic groups, specifically in intron 15, have been identified by GWAS (Unoki et al., 2008; Yasuda et al., 2008; Balakrishnan et al., 2018) to be associated with susceptibility to type II diabetes. In 28  addition, KCNQ1 was found to be hypermethylated in type 2 diabetic patients compared to normal individuals, suggesting that the expression of KCNQ1 could also be altered in diabetics (Zhou et al., 2018). Cardiomyocytes from cardiac-specific insulin receptor knockout mice have a prolonged APD caused by a reduction in potassium current (Lopez-Izquierdo et al., 2014).  1.5.3 Estrogen Estrogen is one of the main female sex hormones. It is also present in males but does not have such a vital role. Estrogen levels fluctuate during the female reproductive cycle, and when levels are high, women experience bloating and fluid retention, which is required for proper implantation of fertilized blastocysts into the engorged uterus (Ma et al., 2003). However, estrogen receptors are not only found in reproductive tissues but also other tissue types such as the colon, kidney and the lungs (Kuiper et al., 1997).   KCNQ1 and KCNE3 play a vital role in colonic crypt cells producing a constitutive potassium current at physiological voltages. Together with KCa3.1, the KCNQ1-KCNE3 current is important in ‘potassium recycling’, which allows cells to maintain a hyperpolarized membrane potential for continuous chloride secretion (Preston et al., 2010). Chloride secretion is essential for regulating fluid homeostasis and ensuring the mucosal lining of the colon is well hydrated. This balancing act primarily involves three ion types: sodium, potassium, and chloride. Potassium ions are brought into the cell basolaterally by the Na+/K+/Cl- co-transporter, NKCC1, and chloride ions are transported out of the cell by the cystic fibrosis transmembrane conductance regulator (CFTR) 29  (Preston et al., 2010) (Fig. 1.5). Deficiencies in potassium recycling and chloride secretion can cause cystic fibrosis, pulmonary edema, and are also important in the pathogenic effects of cholera toxins (Kroncke et al., 2016).   Figure 1.5: Estrogen inhibits the KCNQ1-KCNE3 current and reduces chloride secretion. KCNQ1-KCNE3 complexes are expressed in the basolateral membrane of colonic endothelial cells and allow for efflux of potassium (K+). Na+ /K+ /Cl- co-transporter NKCC1 allows for the influx of sodium (Na+), K+, and chloride (Cl-) into the cell. The Na+ /K+ ATPase exchanges Na+ in the cell for K+. The cystic fibrosis transmembrane conductance regulator (CFTR) allows Cl- efflux from the cell. These transporters maintain a balance of Na+, Cl- and K+ inside the cell. α membrane-bound estrogen receptors, mERα, are activated when estrogen binds, which triggers protein kinase C δ (PKCδ), to phosphorylate KCNE3 and promote dissociation of the KCNQ1-KCNE3 subunits. This leads to a loss of K+ efflux and Cl- influx, which inhibits Cl- secretion via CFTR.  Estrogen acts via two different pathways. The first is by binding to intracellular (nuclear) estrogen receptors (ER) that act as ligand-inducible transcription factors and regulate the expression of certain genes, such as membrane-bound ERs (mER) (O'Mahony et al., 2009). For the second pathway, estrogen binds to mER and produces a rapid response 30  through the activation of protein kinase signaling cascades. Both PKA and protein kinase C δ (PKCδ) phosphorylate the KCNQ1-KCNE3 complex in female rats, but not in male rats (O'Mahony et al., 2007). When estrogen activates the PKCδ cascade leading to phosphorylation of KCNE3 at S82 (Abbott et al., 2006; Alzamora et al., 2011a), KCNQ1-KCNE3 subunits dissociate from each other (Alzamora et al., 2011a) (Fig. 1.5). Thus, as levels of estrogen fluctuate throughout the cycle, so too does the association between KCNQ1 and KCNE3, which reduces the potassium current and the resultant chloride current, leading to fluid retention. This is further supported by experiments conducted using colonic crypt cells isolated from female and male Sprague-Dawley rats. Using co-immunoprecipitation studies, Alzamora et al. (2011a) found that the number of KCNQ1-KCNE3 channel complexes in the female rat crypt cells were reduced after being exposed to 17β-estradiol, an estrogen steroid hormone. This effect, however, was not seen in male crypt cells (Alzamora et al., 2011a).   Estrogen, in addition to decreasing the KCNQ1-KCNE3 currents by causing dissociation of subunits, has also been found to stimulate clathrin-mediated endocytosis of KCNQ1 (Rapetti-Mauss et al., 2013), for prolonged inhibition of the current.  Once these KCNQ1 subunits are endocytosed, they are sequestered into early endosomes where they can be recycled back to the cell membrane via Rab4 and 11 (Rapetti-Mauss et al., 2013). If cells were pre-treated with inhibitors of PKC or PKCδ, KCNQ1 failed to be endocytosed (Rapetti-Mauss et al., 2017). Phosphorylation by PKCδ which leads to rapid dissociation of subunits is, therefore, also thought to be involved in the endocytosis of KCNQ1 channels. 31  Females produce more estrogen than males such that a ‘gender gap’ is seen in response to estrogen regulation. The sexually dysmorphic regulation of fluid retention/secretion would suggest that in diseases where fluid secretion is affected in these tissues, a difference in severity may be seen between the sexes. For example, the disease cystic fibrosis, is a genetic condition in which CFTR is mutated or deleted. This reduces the ability of the channel to secrete chloride in the lungs, intestine, pancreas and other tissues (Kreda et al.) As estrogen inhibits chloride secretion via KCNQ1-KCNE3, females with cystic fibrosis may experience more severe phenotypes due to estrogen further reducing chloride secretion in tissues where secretion in already impaired (Kroncke et al., 2016).   However, estrogen may be beneficial in some disease states, such as diarrheal disorders, for example during infections with the Vibrio cholerae and Escherichia coli (Alzamora et al., 2011b). KCNQ1 plays an important role in potassium recycling in the colon, which is important for chloride secretion. During infection with Vibrio cholerae and Escherichia coli, chloride secretion from the crypt cells in the intestine is thought to be important. 17β-estradiol, a derivative of estrogen, was found to inhibit the enterotoxin-induced chloride secretion in female crypt cells (Alzamora et al., 2011b). KCNQ1-KCNE3 current is thought to allow the enterotoxins to enhance chloride secretion and thereby cause diarrhea (Alzamora et al., 2011b). Estrogen is therefore protective for women with secretory diarrhea.    32  1.5.4 Testosterone In both males and females, testosterone production is stimulated by luteinizing hormone. In men, the majority of the hormone is produced and secreted by the Leydig cells in the testes. In women, testosterone is primarily produced by the interstitial cells of the ovaries. Overall, men produce far more testosterone and DHT, its more potent metabolite, than women (Vierhapper et al., 2001). Testosterone and DHT are important in the development of male sexual characteristics as well as in regulating bone density, hair growth, and fat distribution.   Testosterone also has effects on non-reproductive tissues, such as the heart. Men and women have different incidence rates for many cardiovascular diseases including arrhythmias (Mosca et al., 2011). In men, testosterone levels typically start to decrease after the age of forty (Wang et al., 2017). This decline in testosterone is associated with an increase in cardiovascular risk and all-cause mortality (Khaw et al., 2007). In females and prepubescent boys, the QTc interval lengths are typically longer than men as they produce less testosterone (Rautaharju et al., 1992; Lehmann et al., 1997). Similarly, in men who have hypogonadism, a condition where testosterone levels are low, there is an increased risk of developing a prolonged QT interval and cardiac arrhythmias (Charbit et al., 2009). To further reinforce testosterone’s effect on repolarization, in castrated males, who typically have reduced testosterone levels, a longer JTc interval (after subtraction of the QRS duration from the entire QT interval duration) is seen than in ‘normal’ adult males, but once these castrated males started testosterone replacement therapy their JTc intervals were no longer different than control males (Bidoggia et al., 2000). This 33  reinforces testosterone’s role in repolarization and shortening the QT interval. This was shown in castrated rats and the mechanism behind further investigated.   Ventricular myocytes of castrated rats expressed significantly less KCNQ1 than those of control. Upon DHT administration, KCNQ1 expression actually increased. Kinetic studies of the IKs current produced by these ventricular cardiomyocytes suggest that testosterone does not seem to affect the current-voltage relationship, but increases the current density (Masuda et al., 2018). Testosterone has been shown to affect cardiovascular ventricular repolarization by increasing the potassium repolarization current, IKs, and may be one of the reasons why there is a gender difference in QT interval lengths.  Isoproterenol is a β-adrenoceptor agonist that triggers PKA phosphorylation of KCNQ1 and increases IKs current. Masuda et al. (Masuda et al., 2018) showed that both isoproterenol and DHT upregulate KCNQ1 in cardiomyocytes when applied for 24 hours. H89, an inhibitor of PKA, blocked the effect of isoproterenol but had no effect on DHT’s ability to increase KCNQ1 expression. Through further studies using specific inhibitors for transcription factors and siRNAs, they suggested that isoproterenol is upregulating KCNQ1 via the nuclear transcription factor, cAMP response element binding (CREB) protein. However, DHT seems able to upregulate KCNQ1 independently of CREB. Upregulation of IKs by testosterone may, at least in part, explain gender differences in QT intervals.   34  Male KCNE4 knockout mice have impaired ventricular repolarization as compared to females, which worsened with age in these males (Crump et al., 2016). Prolonged QTc intervals were seen in the males as well as in post-menopausal female mice. One study suggested that KCNE4 expression levels were increased by DHT, which is why deletion is exacerbated in male mice, but how DHT is regulating KCNE4 is not understood (Crump et al., 2016).   1.5.5 Thyroid hormone The thyroid gland produces two hormones, triiodothyronine (T3) and thyroxine (T4). T3 is the more active hormone, to which T4 can be converted by deiodinases.  They affect many different systems, including the metabolic and cardiovascular systems, and are active at different stages during growth and development. Thyroid hormones increase not only heart rate but also cardiac contractility and output (Vargas-Uricoechea et al., 2014).   KCNQ1-KCNE2 form a constitutively active potassium current and are expressed in the thyroid glands of humans and mice (Roepke et al., 2009). KCNQ1, particularly, is highly expressed in the thyroid. KCNQ1-KCNE2 is upregulated by thyroid stimulating hormone (Roepke et al., 2009). Targeted deletion of KCNQ1 or KCNE2 has been shown to cause hypothyroidism (Lee et al., 2000; Roepke et al., 2008). KCNE2 deletion mice have a reduced ability to accumulate iodide ions in the thyroid (up to 8-fold), which results in female mice producing less milk for their offspring. The milk that is produced has reduced T4 levels and results in the offspring developing hypothyroidism (Roepke et al., 2009). The pups grow to be much smaller in size and develop dwarfism, as well as alopecia. 35  Cardiac defects were also noticeable, such as cardiac hypertrophy and fibrosis. If T3/T4 was given to the KCNE2-/- pups or the KCNE2-/- pups were weaned by KCNE2+/+ mothers, these defects were not seen. Also, if KCNE2-/- mothers weaned KCNE2+/+ pups, they developed these abnormalities also (Roepke et al., 2009). This suggests that KCNE2 is needed to produce the thyroid hormones, T3 and T4. Positive emission tomography (PET) showed that when KCNE2 was deleted, I- uptake into the thyroid was reduced, and in wild-type (WT) cells when KCNQ1-KCNE2 is inhibited, thyroid function could be altered (Purtell et al., 2012). This further supports the idea that KCNQ1-KCNE2 current is required for thyroid hormone biosynthesis.   Typically, patients with hyperthyroidism exhibit an increased heart rate, as well as contractility (Vargas-Uricoechea et al., 2014). In a rat model of hypothyroidism, the ventricular APD is significantly shortened by T3 administration which results in an increase in potassium current (Sun et al., 2000). T3 binds to nuclear thyroid hormone receptors (TRs) and regulates the expression of a number of genes in the cardiac tissue. There are two types of TRs, TRα, and TRβ and each has two isoforms, TRα1, TRα2, TRβ1, and TRβ2. A number of potassium channels and accessory subunits have been shown to be regulated by thyroid hormones, including KCNE1. TRα1 receptors are thought to regulate both heart rate and repolarization. TRβ knockout mice exhibit a higher heart rate and have higher thyroid hormone concentrations in their serum, which activates the TRα1 receptors (Weiss et al., 1998; Johansson et al., 1999). In a TRα1 receptor knockout mouse model, heart rate is decreased, and the QT interval is increased (Mansen et al., 2010). Interestingly, in the TRα1 deficient mice, KCNE1 expression in the 36  heart was between 4 to 10-fold greater than that of WT mice and mice with other TR isoform knockouts, which suggests that TRα1 may negatively regulate KCNE1 expression in the mouse heart (Mansen et al., 2010). The lengthening of the QT interval may not be caused by KCNE1, but due to thyroid hormones also regulating the expression of other genes such as the Na+/K+-ATPase, which when impaired causes lengthening of the QT interval (Mansen et al., 2010). In hypothyroid environments, the reduced levels of T3 and T4 would decrease inhibition of KCNE1 expression caused by TRα1 when bound with thyroid hormone.  1.5.6 Gastrin Gastrin is secreted by gastrin cells in the stomach, duodenum and the pancreas (Shulkes and Baldwin, 1997), which stimulates the parietal cells of the stomach to produce gastric acid required for digestion. Gastrin binds to membrane G protein-coupled receptors known as cholecystokinin CCK receptors (Shulkes and Baldwin, 1997). These receptors bind both gastrin and cholecystokinin.  Gastric acid is made up of hydrochloric acid, potassium chloride and sodium chloride and its main role is in the digestion of proteins. The low pH of gastric juice in the lumen of the stomach is maintained by the proton H+/K+ ATPase pump in the mucosal cells (Hirst, 2002). For balance, potassium needs to be recycled back into the gastric lumen across the apical membrane to maintain optimal H+/K+-ATPase function (Hirst, 2002). There are at least three types of potassium channels expressed in the membrane of parietal cells that could be involved in potassium recycling, KCNQ1, Kir4.1, and Kir2.1. KCNQ1 is 37  expressed in both human and mouse gastric mucosa and has also been shown to co-localize with the H+/K+ ATPase (Grahammer et al., 2001). Knockout mice models of KCNQ1 show gastric hyperplasia (Lee et al., 2000) and knockout mouse models of KCNE2 show reduced proton secretion from the parietal cells (Roepke et al., 2010). The distribution of KCNQ1 throughout these cells is also affected in the KCNE2 knockout mice (Roepke et al., 2010). In in vivo experiments done in both rats and dogs to measure gastric acid secretion, chromanol-293B, an IKs blocker, inhibits gastric acid secretion. Interestingly, some JLNS patients (KCNQ1 mutation) are reported to have gastric hyperplasia and increased gastrin levels (Rice et al., 2011), suggesting that potassium current produced by KCNQ1 along with its accessory subunit KCNE2 could be important for the secretion of gastric acid.      1.6 Scope of the thesis  Under β-adrenergic stimulation, the IKs current amplitude is increased, which allows the duration of the cardiac action potential to be shortened and normal repolarization to occur prior to another action potential commencing. The aim of the research in this thesis was to elucidate the mechanism behind this increase in IKs at the single-channel level, which to this point has not been fully understood. Central to this work is an appreciation of the use of the cell-attached patch variation of the suction electrode-recording technique as it allows an examination of the kinetic properties of a single ion channel complex.  The aim of Chapter 2 was to investigate the response of the IKs current to PKA phosphorylation from a microscopic point of view using the single-channel recording 38  technique by using a membrane permeant analog of cAMP (8-CPT-cAMP). We also investigated the effect that 8-CPT-cAMP had on the overall surface expression of IKs using total internal reflection fluorescence (TIRF) microscopy.   Single-channel kinetic analysis was performed in Chapter 3 on two mutant KCNQ1 subunits to further investigate their response to cAMP. From experiments conducted in Chapter 2, the mechanism behind this increase in IKs was suggested to be due to increased activation of the voltage sensors. We further investigated the effects of cAMP on an IKs mutant channel complex with enhanced gating. The second mutant channel analyzed in this chapter was on a double phosphomimetic channel to see if cAMP had any further effect as there are two possible residues that have been suggested to be phosphorylated in KCNQ1 by cAMP. We wanted to investigate if mutating these residues to mimic their phosphorylated state by replacing both serine residues with aspartic acid would have any further effect on the channel upon the addition of cAMP.  The aim in Chapter 4, was to study the effect that the number of KCNE1 subunits present in the channel complex had on the channel’s response to phosphorylation. KCNQ1 alone does not respond to cAMP and KCNE1 must be present for an effect to be seen. Using fusion constructs of KCNQ1-KCNE1 subunits with fixed stoichiometries, we investigated how these complexes responded to cAMP using whole-cell and single-channel recording.  39  1.7 Patch clamp and single-channel recording methodologies As mentioned above, since the essential experiments contained in this thesis comprise single channel recordings it is appropriate to spend a little time considering the background and the advantages and disadvantages of this technique compared with recordings from the whole cell.  In the 1940s and 1950s, Hodgkin and Huxley published a series of papers investigating the roles sodium, potassium, and chloride ions have on the action potential of the squid axon. For these classical experiments a wire was inserted down the inside of the axon to pass currents across the membrane (Hodgkin and Huxley, 1952). At about the same time thin, sharp, glass pipettes were introduced by others to impale cells directly, and this allowed investigators to record membrane potentials and eventually voltage clamp them in order to investigate the voltage-sensitivity and kinetics of the membrane currents in the cells. The cells used for this technique were relatively large in size as large currents were needed for these experiments to offset the inherent leak currents caused by the impalement electrodes.   Another major limitation of this technique is the large signal-to-noise ratio seen in the recordings, which means that single-channel currents cannot be observed. In the 1970’s, Neher and Sakmann perfected the patch-clamp technique, which greatly improved the resolution of recordings and allowed for the recording of single-channel activity (Neher and Sakmann, 1976, 1992). Patch-clamp recording differs from the voltage-clamp technique mentioned above as instead of impaling the cell, the tip of the glass pipette is 40  tightly sealed onto a small patch of the cell membrane. The tight-seal reduces leak currents and lessens damage to small cells, and the high seal resistance allows low noise single channel recordings to be obtained. This technique can be used for both whole-cell and single-channel recording with several different configurations based on the experimental conditions. Tight-seal whole-cell and perforated patch recording are two variations that are used to record whole-cell currents. Cell-attached, inside-out and outside-out are used to record single-channels. It should be noted that single channel recording allows a number of analyses to be performed (see below) that are not available to data from whole-cell and perforated patch recordings.  1.7.1 Single-channel recording For single-channel recording, the glass electrodes have higher resistances than those used for whole-cell recording, >50 MW. There are three configurations of single-channel recording: cell-attached, inside-out and outside-out (Conforti, 2012). The cell-attached configuration is achieved once the electrode has sealed onto the cell with a high series resistance. In this configuration, all the channels under the patch pipette can be recorded from. The intracellular solution and components remain intact and so it is a useful system to study second messenger systems in. Intracellular co-factors are not dialyzed out of the cell and current does not usually run down. The patch pipette solution mimics the extracellular solution (low potassium). The bath solution mimics the intracellular solution (high potassium) (Conforti, 2012).  A disadvantage is that in order to study how intracellular signaling mechanisms affect the channel, the intracellular components must 41  be able to diffuse across the membrane or have analogues that can, as the intracellular solution cannot be manipulated.  The inside-out patch requires another step after the cell-attached configuration has been achieved. Once the high resistance seal between the pipette and cell membrane is achieved, the pipette is pulled off the cell. The part of the membrane under the electrode comes with it. The bath solution now becomes the cell’s intracellular solution and can now be manipulated.  The outside-out patch is achieved after the whole-cell configuration has occurred. Once the membrane is perforated or ruptured by the application of suction, the pipette is slowly pulled off the cell. Part of the membrane breaks off from the cell on either side of the pipette, which attach to each other to form a vesicle (Conforti, 2012). This configuration is similar to whole-cell, but the newly created tiny cell only has a few channels, ideally a single-channel. In the other two single-channel variations, the external solution (pipette) solution cannot be readily changed during experiments, which makes experiments requiring changes to the solution or the addition of drugs or other soluble factors that act externally very difficult. This configuration allows for the external (bath) solution to be easily and rapidly changes as the channel is not directly under the pipette. The disadvantage of this configuration is that it involves many steps and is often difficult to achieve.    42  1.7.2 Patch-clamp recording Patch-clamp allows for the membrane of a cell or part of a cell, as defined by the modes described above, to be held or ‘clamped’ at a specific voltage. The amplifier uses a feedback circuit to do this. The head stage, which acts as a probe, is connected to both the bath electrode and the pipette holder. The bath electrode relays to the amplifier changes in current, which allows the amplifier to inject current, if needed, through the electrode to maintain the desired membrane voltage, which is controlled by the experimenter.   For both whole-cell and single-channel recordings, amplification and filtering of the data at acquisition is necessary. During acquisition, data are typically filtered using a low-pass Bessel filter and then converted to a digital form using an AD converter. However, AD converters have a limited sampling frequency, much less than the intrinsic frequencies present in noise and current signals, which is why Bessel filters are used to remove high frequency events. If these events are under-sampled, the signal is distorted, which is a phenomenon known as ‘aliasing’ (Sakmann and Neher, 1985). The filter also improves the signal-to-noise ratio during recording as noise generated from the electrical components of the system or thermal noise generated by the movement of atoms are removed.  For single-channels, further filtering is typically required as the currents are much smaller and require that the signal-to-noise ratio be improved before data can be analyzed. Filtering reduces the number of data points and can result in the omission of brief events. 43  Thus, it is important to find an appropriate balance between the amount of filtering to minimize the number of brief events missed. The rise time of the filter is the time it takes for the filter to pass between 10 and 90% of the full-amplitude of the event (Sakmann and Neher, 1985). Typically, events that are shorter than twice the rise time are deemed ambiguous and are often excluded from dwell-time analysis (Colquhoun and Sigworth, 1995).   1.7.3 Single-channel analysis After filtering, single-channel sweeps with a good signal-to-noise ratio can be used for further analysis. Sweeps where no channel activity occurs (blank sweeps) are subtracted from sweeps where the channel opens to correct for seal leak, reduce baseline noise, and remove capacity transients. These baselined sweeps can be used to generate all-points amplitude histograms, which were used extensively in the work presented in this thesis (See Fig. 2.3).  Events are counted and binned by amplitude (bin size usually ~0.01 pA), and histograms show peaks, which reflect the amplitude at which the majority of the events occur. Typically, there is a peak around 0 pA, which reflects all the closed channel events and another peak that shows the main open amplitude of the channel. The amplitude can be derived by fitting these peaks using a Gaussian distribution (Colquhoun and Sigworth, 1995). The amplitude bin with the most open events is fairly reproducible across multiple single-channel patches of the same channel type. The all-points histogram shows all events, raw data, including those events that are noise.   44  Single-channel records can then be idealized using an automated half-amplitude threshold-detection search. The amplitude for each state is defined in the program before running the analysis. Typically, level 0 is the baseline and is 0 pA and level 1 would be the amplitude at which the channel opens. If this channel has multiple open states, additional levels can be added with each open level representing one of the open states.  The threshold detection uses a 50% threshold between levels (Colquhoun and Sigworth, 1995). Every time the current crosses this threshold between each level, a transition to a different level occurs. The amplitude and duration of this event are recorded. This analysis can be done manually, where each transition is decided by the operator or it can be done automatically by the computer. At the end of the analysis, a table of each event, the level it opened to and for how long, is produced. These are known as the idealized events. Due to filtering, very brief openings and closings may result in transitions being missed by this type of analysis.   The idealized events are used to analyze how long the channel spends open and closed, the open and closed dwell times. The open and closed dwell times can be plotted as separate histograms. If the channel has multiple open states those open levels can also be plotted separately. These histograms are then fit with exponentials. These dwell times provide a detailed view on kinetics of channel opening and closing. In Shaker channels as well as IKs, exponential fits of the closed dwell times have shown three different closed states; fast, intermediate, and slow (Hoshi et al., 1994; Werry et al., 2013). The dwell times can provide details on the number of states and rate constants for each state, which is important for modelling the channel’s kinetic behavior. One limitation of this analysis is 45  that brief opening and closing events are probably missed, which may cause some closed or open events to appear longer than they actually are.   Channel openings can occur very quickly one after another separated by brief channel closings. These are called a burst of channel openings. The very short periods of channel closings are referred to as inter-burst closings. A burst of channel activity ends when the channel closes for longer and a new burst commences upon the next channel opening. The exact length of time required for a closing to be considered a longer closing is called the critical time (tc) and it is typically at least 50 times longer than the length of the typical short closings (Qin and Li, 2004). Burst analysis is likely to be less affected by the missing brief events than dwell time analysis.   1.7.4 Single-channel models Werry et al. (2013) adapted a Markov model of IKs from Silva and Rudy to account for their single-channel findings (Silva and Rudy, 2005). In the Silva-Rudy model, four independent subunits must go through two transitions for the channel to open. Werry et al. changed this to include the multiple open states they had seen in their single-channel recordings. All four subunits need to undergo the first transitions, but only one of the subunits needs to undergo the second transition for the channel to open. This creates multiple different conducting states. As another subunit undergoes the second transition, a larger current is passed, which reflects the subconducting states seen with IKs. IKs also displays rapid closing events during burst of channel activity, so Werry et al. (2013) added a fast-closed state (Cf), which allows the open channel to pass to these Cf states and 46  return directly back to its conducting state without passing through the initial closed states once again. For all their subconducting states they increased the forward rates between the conducting states and made them equal. They also did the same for the backward rates, which allowed the channel to move between the sublevels easily. These modification to the model, allowed the model to reproduce the activation kinetics and burst behavior of their single-channel recordings.   1.7.5 Single-channel recordings of IKs current to date KCNQ1 and KCNE1 were first identified to underlie the IKs current in 1996 by two groups (Barhanin et al., 1996; Sanguinetti et al., 1996). Obtaining single-channel recordings of IKs subsequently proved to be quite difficult. The channel tends to cluster together in surface membranes, which makes the probability of isolating a single-channel quite low. However, multichannel patches were obtained, which allowed groups to perform noise-variance analysis to try to determine the conductance of the channel. Yang and Sigworth determined the conductance of IKs to be 4.5 pS (Yang and Sigworth, 1998). They also recorded from a patch with only a handful of channels, which allowed them to see IKs flickery behavior and its long first latency.   In 2013, Werry et al. first consistently recorded single-channels of IKs. The channel complex has multiple open-state configurations and transitions through five or six open levels (Werry et al., 2013; Murray et al., 2016). At room temperature IKs is slow to activate, with a first latency to opening of ~1.5 s at +60 mV. The channel also has a low open probability (Po) of 0.15 after four seconds at +60 mV and undergoes long periods of 47  quiescence (Werry et al., 2013; Murray et al., 2016). Once a stable current level is reached, the channel preferentially occupies two or three of the higher subconductance sublevels (Werry et al., 2013). This is reflected in macroscopic currents where the current activates and reaches the peak amplitude slowly. Single recordings also show IKs’s flickery behavior as the channel moves quickly between the closed (Cf) and open states (Werry et al., 2013; Eldstrom et al., 2015; Murray et al., 2016). This flickery phenotype along with the channels multiple subconducting states, low Po and long first latency to opening are some key defining characteristics of the IKs current.  48  Chapter 2: cAMP-Dependent Regulation of IKs Single-Channel Kinetics 2.1 Introduction The repolarization phase of the cardiac action potential is dominated by the activity of two potassium currents, IKs and IKr (Sanguinetti and Jurkiewicz, 1990). At normal heart rates, IKs plays the lesser role, but at high heart rates and during stress, the current is upregulated, and the rate of deactivation is slowed (Terrenoire et al., 2005). This increase in IKs leads to a repolarization reserve, which shortens the action potential duration (APD) and allows for adequate ventricular filling (Stengl et al., 2003; Jost et al., 2005; Silva and Rudy, 2005).  IKs is the product of the coassembly of the Kv7.1 voltage-gated potassium channel (KCNQ1) with the KCNE1 β-subunit. The coassembly of KCNE1 with KCNQ1 causes notable changes in channel kinetics, converting a rapidly activating current into one with very slow activation and deactivation kinetics, and in which inactivation is altogether abolished. In addition, overall current amplitude is increased upon KCNE1 coassembly (Takumi et al., 1988; Barhanin et al., 1996; Sanguinetti et al., 1996; Tristani-Firouzi and Sanguinetti, 1998). Mutations in Q1 or KCNE1 can cause cardiac arrhythmias such as long QT and short QT syndromes and familial atrial fibrillation (Moss et al., 1991; Chen et al., 2003c; Bellocq et al., 2004). Long QT mutations lead to a lengthening of the action potential because the amount of repolarizing IKs is reduced (Wang et al., 1996; Splawski et al., 2000). Because of the importance of IKs at high heart rates, many individuals with these mutations display symptoms when their heart rates are elevated—for example, while exercising or when under stress (Wit et al., 1975). 49  Response to stressful situations—for example, fight or flight—involves sympathetic nervous system activation via β-adrenergic receptors (β-ARs). This triggers a signaling cascade, which leads to phosphorylation of Q1 and an increase in IKs current. The β-ARs are coupled to heterotrimeric G-proteins that release GTP-bound α-subunits to activate the downstream effector, adenylyl cyclase 9 (Cumbay and Watts, 2004; Piggott et al., 2008; Dessauer, 2009). Activating adenylyl cyclase 9 increases the level of intracellular cAMP, which in turn activates PKA. PKA is brought into the macromolecular complex and in close proximity to Q1 by Yotiao (A-kinase anchoring protein 9 [AKAP-9]), which allows PKA to phosphorylate the residue Ser27 (Fig. 2.1) (Chen et al., 2005; Piggott et al., 2008; Li et al., 2012). Yotiao also anchors protein phosphatase 1 (PP1), which is key for regulation of β-AR stimulation of IKs (Fig. 2.1) (Marx et al., 2002).  Figure 2.1: Phosphorylation of IKs by PKA.  Once the G protein–coupled β-adrenergic receptor is activated, GTP binds to the α-subunit of the G protein, which allows for the release of active α-GTP subunits. This complex binds and activates adenylyl cyclase (AC), producing cAMP, which activates cAMP-dependent PKA. Yotiao anchors PKA to the KCNQ1 channel, which allows the activated PKA to phosphorylate the N terminus of the KCNQ1 channel. Protein phosphatase 1 can dephosphorylate cAMP-mediated phosphorylation and is blocked by OA. 50  The full range of mechanisms behind the increase in IKs current after β-adrenergic stimulation is not fully understood. Phosphorylation of channels is known to cause a hyperpolarizing shift of the half-activation potential (V1/2), where the IKs current activates at more negative membrane potentials (Dilly et al., 2004; Terrenoire et al., 2005). Along with this is an increase in the rate of activation and a decrease in the rate of deactivation (Terrenoire et al., 2005), which increases IKs by allowing a buildup of open channels during successive depolarizations. As well, increases in current could be caused by a change in the number of channels at the cell surface. This has been seen to be the case for other ion transporters, such as the Na+/K+-ATPase pump, which shows increased translocation to the plasma membrane during cAMP stimulation (Gonin et al., 2001).  Here, we aimed to study the mechanisms by which PKA phosphorylation increases the IKs current at the microscopic ion channel level. From our prior work, we now understand that IKs complexes do not occupy a single open state but transition through five or six open levels during activation of the channel complex, and only reach a Po of ∼0.15–0.2 at 60 mV, even during prolonged depolarizations (Werry et al., 2013; Eldstrom et al., 2015; Murray et al., 2016). Once current reaches a stable level, channel activity is characterized by preferential occupancy of two or three relatively higher subconductance states while the channel pore opens in long bursts with frequent brief closings. By using a membrane-permeant cAMP analog, 8-CPT-cAMP, we can look for modulation of the single-channel kinetics in cell-attached patches. Up-regulation of VSD function is expected to cause changes in the first latency to opening or an increased Po that would underlie reported whole-cell findings such as the hyperpolarizing shift of the 51  conductance–voltage (G-V) relationship. In addition, we hypothesize that PKA phosphorylation can facilitate single-channel gating and induce changes in substate occupancy that further increase single-channel currents.  2.2 Materials and methods 2.2.1 Reagents To activate PKA inside cells, membrane-permeable cAMP, 8-CPT-cAMP (Sigma-Aldrich). To maintain activation of PKA after stimulation, okadaic acid (OA) was used to inhibit PP1 and PP2A protein phosphatases (EMD Millipore). To ensure that channels under study were indeed IKs, chromanol 293B was used as a selective blocker (Tocris Bioscience).  2.2.2 Molecular biology KCNQ1 and KCNE1 constructs were bought from OriGene. KCNQ1 S209F was constructed from the KCNQ1 gene using a two-step PCR reaction (Eldstrom et al., 2010). KCNQ1 S27D was a gift from the laboratory of N. Schmitt of the University of Copenhagen (Copenhagen, Denmark). Yotiao (AKAP-9) was a gift of R. Kass of Columbia University (New York, NY) and was inserted into a pcDNA3 vector. The E160R/R237E (E1R/R4E) and S27D/S92D KCNQ1 mutants were gifts from J. Cui of Washington University (St. Louis, MO). The EQ construct forces a 4:4 KCNQ1:KCNE1 stoichiometry to IKs and was constructed by tethering the C terminus FLAG tag on KCNE1 and a V5 epitope on KCNQ1, plus an additional 21 aa (SRGGSGGSGGSGGSGGSGGRS) after the FLAG-tag sequence (Murray et al., 2016). Sequencing confirmed all mutations. 52  2.2.3 Cell culture and transfections For single-channel recordings, ltk– mouse fibroblast (LM) cells were used. They were plated to a confluence of 10–20% on sterile glass coverslips. These cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) and with DNA ratios of 1:3:1.5:1 of KCNQ1, KCNQ1-S209F, KCNQ1-S27D, or KCNQ1-S27D/S92D:KCNE1:AKAP-9:GFP. For some experiments, KCNE1-GFP was used to reduce the number of transfected plasmids. The cells were grown in MEM with 10% FBS (Gibco) and incubated in air/5% CO2 at 37°C. All recordings were done at room temperature, 24–48 h post-transfection.  For total internal reflection fluorescence (TIRF) microscopy, Chinese hamster ovary (CHO) cells were grown in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Thermo Fisher Scientific). Cells were cultured at 37°C in an air/5% CO2incubator. 1 d before transfection, cells were rinsed with 1× PBS (Thermo Fisher Scientific), lifted via 3-min exposure to trypsin-EDTA (Thermo Fisher Scientific), and replated on 35-mm2 glass-bottom microdishes (Ibidi). The cells were transiently transfected using Lipofectamine 2000 according to the manufacturer’s protocol. KCNE1-mCherry was coexpressed with KCNQ1-GFP and AKAP-9 in a 4:1:1 ratio. Cells were imaged 24–48 h after transfection.  2.2.4 Patch-clamp electrophysiology Methods for single-channel recordings were previously published (Werry et al., 2013; Eldstrom et al., 2015). Whole-cell recordings were performed, and patch electrodes were 53  fabricated as previously described (Murray et al., 2016). Data were collected and analyzed using Axopatch hardware and pCLAMP 10.5 software (Molecular Devices).  2.2.5 Microscopy KCNQ1 and KCNE1 subunits were expressed in CHO cells and were visualized at ∼27°C with TIRF microscopy as described previously (Boycott et al., 2013). Cells were imaged for 15 or 30 min, with 200 µM 8-CPT-cAMP added to the bath after 5 or 15 min, respectively.  2.2.6 Solutions For single-channel recordings, the solution in the bath contained 135 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM Hepes and was adjusted to pH 7.4 with KOH. The solution in the patch pipette contained 6 mM NaCl, 129 mM MES, 1 mM MgCl2, 10 mM Hepes, 5 mM KCl, and 1 mM CaCl2 and was adjusted to pH 7.4 with NaOH.   For whole-cell recordings, the solution in the bath contained 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.8 mM NaAcetate, and 10 mM Hepes, pH adjusted to 7.4 with NaOH. The solution in the pipette contained 130 mM KCl, 5 mM EGTA, 1 mM MgCl2, 4 mM Na2-ATP, 0.1 mM GTP, and 10 mM Hepes, pH adjusted to 7.2 with KOH. For TIRF microscopy, the external solution contained 135 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2.5 mM Na-pyruvate, 1 mM Na2HPO4, 20 mM D-glucose, and 10 mM Hepes, pH adjusted to 7.4 with NaOH.  54  2.2.7 Data analysis For TIRF microscopy imaging, overall fluorescence intensity of the cells was measured using Xcellence software (Olympus). Fluorescence of specific regions of interest (ROIs) was quantified using ImageJ with the Time Series Analyzer v3 plugin (National Institutes of Health).  G-V relations were acquired from normalized tail current amplitudes. Fitting the data from each cell with a Boltzmann sigmoidal function allowed us to obtain the V1/2 values. Data analysis was done using Prism 7 (GraphPad Software). Single-channel current records were acquired at 2 kHz and digitized using Digidata 1440A hardware (Molecular Devices). For presentation and for all-points histograms (unless otherwise stated), data were filtered at 200 Hz. To compile events lists, data were filtered at 500 Hz and idealized using the Single-Channel Search function in Clampfit. Cursors were set between capacity transients at the beginning and end of the test pulse. Levels were set to 0, 0.145, 0.22, 0.33, 0.5, and 0.75 pA, and the “update level automatically” function was disabled. The program automatically detects each event and is then added to the events worksheet from which idealized amplitudes can be extracted. The final mean amplitudes of events at each sublevel are returned by the program (see legend to Fig. 2.5). Only events longer than 1.5 ms in duration were used in the analysis. A short section of current idealization shown in Appendix A, Figure 8B.  55  2.2.8 Statistics Results reported here are expressed as mean ± SE and represent data from at least three independent experiments. The median value and 95% confidence intervals are also reported for first-latency data. To compare control and cAMP-treated cells, nonparametric Mann–Whitney test was used (GraphPad Software). The statistically significant threshold was set at 0.05 or less. Gaussian fits and single-channel analysis were performed using Clampfit. Nonlinear regressions were used on G-V relations using the Boltzmann sigmoidal function in Prism 7.  2.2.9 Supplemental material Appendix A, Figures 1 and 2 show KCNQ1 + KCNE1 single-channel data before and after cAMP. Appendix A, Figure 3 shows V1/2 changes in macropatches before and after cAMP of EQ and S27D + KCNE1. Appendix A, Figures 4 and 5 show a detailed subconductance and kinetic analysis of S27D + KCNE1. Appendix A, Figure 6 shows whole-cell data of S209F + KCNE1 before and after cAMP. Appendix A, Figure 7 shows further analysis of the TIRF data focusing on ROIs and how those change after cAMP addition. Appendix A, Figure 8 shows a snapshot of the idealization process used for subconductance analysis in Figure 2.5 and Appendix A, Figure 4.  56  2.3 Results 2.3.1 The 8-CPT-cAMP–induced increase in IKs current is seen at room temperature.  It is not possible to resolve the kinetics of single-channel IKs currents at 37°C because of the rapid shifts between subconductance levels and the need to keep noise levels extremely low. Thus, experiments were done at room temperature (20–22°C). To confirm that the expected modulation of IKs as a result of PKA phosphorylation was still present at these temperatures in our hands, we first used whole-cell recording to look at the effect of 8-CPT-cAMP on macroscopic IKs currents.  KCNQ1 was transiently transfected with KCNE1 and Yotiao into a mouse fibroblast cell line, and whole-cell currents were recorded (Fig. 2.2). Representative currents during a 4-s pulse to 60 mV before (control) and after 200 µM 8-CPT-cAMP (Fig. 2.2A) show that IKs increased significantly even at room temperature. The time course of the increase in peak current is illustrated in the diary plot (Fig. 2.2B). There was an initial decrease in current as the 8-CPT-cAMP was added to the bath, and then a delayed onset as it permeated across the membrane, followed by a progressive increase in current amplitude.  57   Figure 2.2: Increase in whole-cell IKs current at room temperature after 8-CPT-cAMP addition.  (A) Representative traces of KCNQ1 (Q1) + KCNE1 (E1) and Yotiao before (control) and after 8-CPT-cAMP (200 µM) + OA (0.2 µM). Cells were held at −80 mV and then pulsed to 60 mV for 4 s before being pulsed to −40 mV for 2 s. (B) Diary plot of the peak outward current during a 4-s pulse to 60 mV over time (same protocol as in A). Bar indicates the addition of 200 µM 8-CPT-cAMP/0.2 µM OA to the bath. (C) I-V current traces and their peak amplitude plotted against voltage (n = 1) before and after 8-CPT-cAMP. Cells were held at −90 mV and pulsed from −80 mV to 100 mV in 10-mV steps for 4 s. Tail currents were recorded at −40 mV for 900 ms. Representative traces are shown from every other voltage. (D) G-V curves before (V1/2 = 28.2 ± 5.4 mV) and after (V1/2 = 10.5 ± 2.6 mV) 8-CPT-cAMP; n = 4. Error bars show ±SE.  The characteristic sigmoidal shape of IKs current activation is shown in the representative traces in Figure 2.2. Apart from the increased current size apparent in the current–voltage (I-V) relationship (Fig. 2.2C), the mean G-V curve (Fig. 2.2D), obtained from analysis of 58  the tail currents, hyperpolarized after the addition of 8-CPT-cAMP (V1/2 changed from 28 to 10.5 mV; Fig. 2.2D). Saturation of current activation occurred at less-depolarized voltages in the presence of 8-CPT-cAMP compared with control (Fig. 2.2D). This indicates that a major action of 8-CPT-cAMP phosphorylation is to increase the macroscopic channel Po at more negative membrane potentials. These results also show that, at room temperature, we are still able to see the classic effect of 8-CPT-cAMP on the IKs current.  2.3.2 Response of IKs to cAMP in cell-attached recordings In cell-attached recordings, an EQ tandem construct was used to force a 4:4 KCNQ1:KCNE1 IKs stoichiometry and ensure that the channel complex was fully saturated with KCNE1. This construct has been previously characterized by Murray et al. (2016); in the present experiments, single-channels were found to be modulated by 8-CPT-cAMP in the same manner as KCNQ1 + KCNE1 expressed separately (Appendix A, Figs. 1 and 2). Cell-attached macropatch recordings were made from multiple channels under the pipette tip. 8-CPT-cAMP addition caused a hyperpolarizing shift in the V1/2 of activation for EQ and KCNQ1 + KCNE1 (Fig. 2.3A and Appendix A, Fig. 2A), also seen at the whole-cell level with KCNQ1 + KCNE1 (Fig. 2.2D). This shift increased with longer exposure to 8-CPT-cAMP, with a mean hyperpolarizing shift of −25 ± 8.5 mV in the V1/2 of activation (Appendix A, Fig. 3A). Deactivation appears to also be affected by the addition of 8-CPT-cAMP, as the macropatch tail currents decay more slowly (Fig. 2.3A, insets). Of six macropatches for EQ and KCNQ1 + KCNE1, two showed an increase in the amplitude of cell-attached macropatch currents. 59   Figure 2.3: 8-CPT-cAMP hyperpolarizes EQ activation and can increase the number of active channels in cell-attached patches.  (A) G-V curves from a single macropatch before (control, circles, V1/2 = 16.4 mV) and after 5-min (squares, #1, V1/2 = 5 mV), 8-min (triangles, #2, V1/2 = 0.5 mV), and 14-min (upside-down triangles, #3, V1/2 = −8 mV) after 200 µM 8-CPT-cAMP/0.2 µM OA exposure. Insets show macropatch I-V currents (control and 8-CPT-cAMP). Cells were held at −60 mV and pulsed from −70 mV to 80 mV in 10-mV steps for 4 s, then to −40 mV for 900 ms. Every other sweep is shown. (B) Sample single-channel sweeps from an IKs-expressing cell-attached patch in control, after adding 8-CPT-cAMP, and then in 50 µM chromanol 293B. Bottom, ensemble average current before and after 8-CPT-cAMP and after adding chromanol 293B. Control is the mean of 100 sweeps, 8-CPT-cAMP is the mean of 79 sweeps, and chromanol 293B is the mean of 38 sweeps. (C) Raw all-point amplitude histograms of active sweeps in control (left, 21 active sweeps) and when exposed to 8-CPT-cAMP (right, 47 active sweeps).  2.3.3 IKs single-channel kinetics in the presence of 8-CPT-cAMP 8-CPT-cAMP stimulation affected single-channel properties of IKs in a number of ways, as illustrated in Figures 2.3 and 2.4. Characteristically, in control, IKs single-channel 60  currents have a long first latency, and once they appear, channels flicker rapidly in bursts with infrequent longer closings (Figs. 2.3B and 2.4A). After 200-Hz filtering, single-channel openings peak at less than 0.5 pA with only one active channel present. The all-points histogram of amplitude events from 21 active of 100 sweeps peaked at 0.38 pA with evidence of only a single-channel present despite tens of thousands of opening events (Fig. 2.3C, left). Similar results were seen in 100 control sweeps from a second patch (single sweep histogram shown in Fig. 2.4B). However, after the administration of 8-CPT-cAMP, an increase in the current amplitude was observed from just under 0.5 pA to 1.5 pA (Fig. 2.3B) and up to 2.5 pA in Figure 2.4B (bottom). An additional event peak was seen in the histogram of the 47 active 8-CPT-cAMP sweeps at 0.75 pA, and the range of the histogram included events at 1.5–2.0 pA, suggesting that there were now two to three channels active in the patch (Fig. 2.3C, right). Data from the patch shown in Figure 2.4 suggest that there were now approximately six channels active after addition of 8-CPT-cAMP (Fig. 2.4B, bottom). In the presence of the selective blocker chromanol 293B, this increased current was almost abolished, suggesting that it arose from the presence of more active IKs channels. In the bottom panel of Figure 2.3B, ensemble averages are shown of the current in all 100 control, 79 8-CPT-cAMP, and 38 chromanol 293B sweeps. The mean current was much greater when 8-CPT-cAMP was present and least when chromanol 293B was added. Overall, in 20 initially single-channel EQ or KCNQ1 + KCNE1 patches in control, there was an increase in the apparent number of active channels in nine exposed to 8-CPT-cAMP. 61   Figure 2.4: Figure 2.4. 8-CPT-cAMP increases the ensemble average current and shortens the first latency in EQ.  (A) Single-channel sweeps of EQ coexpressed with Yotiao in control (top) and when exposed to 200 µM 8-CPT-cAMP/0.2 µM OA (bottom). (B) Raw all-points histogram of an example active control sweep (top) and an active sweep during cAMP exposure (bottom). (C) Ensemble average currents from a single patch: 93 sweeps in control, 17 active and 76 blank (black line), and 93 sweeps in 8-CPT-cAMP/OA, 39 active and 54 blank (red line). (D) Cumulative latency histogram. Mean first latency for the 43 active control sweeps of 367 was 1.61 ± 0.13 s. In 200 µM 8-CPT-cAMP/0.2 µM OA, mean first latency for the 69 active sweeps of 345 was 1.06 ± 0.11 s; P = 0.0005 (see Table 2.1). Sweeps without activity were given a first latency >4 s; arrows indicate sweep total in each case. Note split-scale ordinate.  The amplitude of single-channel openings appeared unchanged in the presence of 8-CPT-cAMP. For EQ, the mean single-channel conductance in control was 2.9 pS and unchanged after cAMP. For KCNQ1 + KCNE1, the mean single-channel conductance in 62  control was 2.7 pS and 2.8 pS after 8-CPT-cAMP. These values are close to the single-channel conductance of 3.2 pS reported by Werry et al. (2013). Ensemble average (93 sweeps) currents at 60 mV from a single-channel patch before and after cAMP are shown in Figure 2.4C. There is an increase in peak ensemble current from 0.04 to 0.12 pA in the presence of 8-CPT-cAMP, which corresponds to an increase in Po from 0.12 to 0.27 in this patch and agrees quite well with the ∼2.5-fold increase in whole-cell current at 60 mV shown in Figure 2.2C.  A consistent increase in the number of active sweeps occurred in the presence of 8-CPT-cAMP and is clearly seen in the latency histogram (Fig. 2.4D). In control, 43 of 367 sweeps were active (black line), whereas in 8-CPT-cAMP, 69 sweeps of 345 were active (red line). The mean first latency of EQ IKs currents was measured from patches in which only a single-channel was found to be present, even after exposure to 8-CPT-cAMP, and was 1.61 ± 0.13 s in control. It was significantly shortened upon 8-CPT-cAMP addition to 1.06 ± 0.11 s (Fig. 2.4D and Table 2.1; P = 0.0005, n = 3). These numbers correlate well with the first latency of KCNQ1 and KCNE1 expressed separately (Appendix A, Fig. 1C; and Table 2.1) and favorably with changes in the activation time course of EQ macropatches. The time to half-activation at 60 mV was reduced from 1.04 to 0.65 s (n = 4) with 8-CPT-cAMP, a 37% reduction, compared with the 34.2% decrease in first latency for EQ after 8-CPT-cAMP addition (Table 2.1).63   Table 2.1: First latency data of EQ, S209F, S27D, S27D/S92D, and KCNQ1 (Q1) + KCNE1 (E1) before and after 200µM 8-CPT-cAMP/0.2 µM OA  * = Mann-Whitney test  Control 8-CPT-cAMP/OA  Construct Mean First Latency (s) ± SE 95% confidence interval of the mean (lower-upper) Median (s) # Active Sweeps # Total Sweeps Mean First Latency (s) ± SE 95% confidence interval of the mean (lower-upper) Median (s) # Active Sweeps # Total Sweeps P-value* n (cells) EQ 1.61 ± 0.13 1.35 - 1.87 1.57 43 367 1.06 ± 0.11 0.84 - 1.27 0.85 69 345 0.0005 3 S209F + E1 0.16 ± 0.05 0.07 - 0.25 0.07 67 128 0.18 ± 0.06 0.06 - 0.30 0.04 85 220 0.0208 2 S27D + E1 1.81 ± 0.13 1.55 - 2.08 1.60 57 302 1.44 ± 0.11 1.22 - 1.66 1.10 67 335 0.0320 3 Q1 + E1 1.32 ± 0.13 1.06 - 1.57 0.90 68 278 0.79 ± 0.08 0.63 - 0.95 0.50 104 309 0.0002 3 S27D/S92D + E1 1.62 ± 0.08 1.46 - 1.78 1.40 189 467 1.43 ± 0.13 1.18 - 1.69 1.26 60 119 0.332 3-5 64  2.3.4 Changes in substate occupancy in the presence of 8-CPT-cAMP are primarily VSD activation effects In patches with no increase in the number of active channels, we analyzed the occupancy of the subconductance levels of the single-channel present in control and after 8-CPT-cAMP addition. During the idealization process (Materials and methods and Appendix A, Fig. 8), we assumed that 8-CPT-cAMP did not change the subconductance amplitude levels reached in control, just the occupancy of these levels. The computer-defined event amplitudes usually clustered closely around these preset levels. The results of this analysis on sweeps from two different EQ patches are shown in Figure 2.5, 26 active sweeps before, and 25 sweeps after the addition of 8-CPT-cAMP. The results illustrate the occupancy of five defined EQ IKs substate levels (0.145–0.75 pA). In Figure 2.5A, the raw all-points histograms for control (blue) and 8-CPT-cAMP (red) are overlaid. The initial peak, or level 0, is the closed state (0 pA). It can already be clearly seen that the area of the envelope to the right of the 0 pA peak, corresponding to channel openings, is greater after exposure to 8-CPT-cAMP (red) than control (blue). 65   Figure 2.5: Subconductance analysis of EQ before and after 8-CPT-cAMP/OA.  (A) Raw all-points amplitude histogram of 26 active control sweeps (blue) and 25 active sweeps after 200 µM 8-CPT-cAMP/OA from two different EQ single patches. (B) Five initial thresholds were used for the idealization: 0.145, 0.22, 0.33, 0.5, and 0.75 pA. For the control histogram, the final idealized levels were 0.14 ± 0.05, 0.24 ± 0.06, 0.34 ± 0.07, 0.51 ± 0.08, and 0.74 ± 0.10. In the presence of 8-CPT-cAMP/OA, the final idealized levels for cAMP were 0.13 ± 0.05, 0.23 ± 0.05, 0.34 ± 0.06, 0.49 ± 0.07, and 0.70 ± 0.08. (C) Total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and number of events at each threshold before and after 8-CPT-cAMP. The data were filtered at 0.5 kHz, and the bin width used was 0.01 pA. Only events longer than 1.5 ms were included.  The idealized histograms of the subconductance analysis for control (blue) and 8-CPT-cAMP (red) show the grouping of opening events into the five subconductance levels (Fig. 2.5B). We have already established that the 0.29- and 0.44-pA sublevels when data are 66  filtered at 0.2 kHz are the most frequently occupied upper open levels (Werry et al., 2013). The present data were filtered at 0.5 kHz, and in Figure 2.5 (B and C), the 0.33- and 0.5-pA levels are the most frequently visited upper open levels, accounting for 2.6% and 3.1% of the total dwell time, respectively. In the presence of 8-CPT-cAMP, there is a clear shift of occupancy away from the closed state to the higher subconductance opening levels. For example, 2.6% of events occurred at the 0.33-pA sublevel in control, but after 8-CPT-cAMP addition, this almost doubled to 4.5%. The 0.5-pA level also saw an increase in the number and doubling in the proportion of events from 3.1 to 7.6%, and the 0.75-pA sublevel increased from 0.8 to 1.7%. The first latencies to opening of each of the six sublevels before and after 8-CPT-cAMP also showed a trend to shorter times to reach each of the levels in the presence of 8-CPT-cAMP, although it was not statistically significant. Similar results were seen in two other EQ or KCNQ1 + KCNE1 patches, indicating that phosphorylation of IKs leads to more frequent high-subconductance-state occupancy, consistent with the increased current levels observed at the microscopic and macroscopic levels.  An analysis of EQ burst kinetics in controls and in the presence of 8-CPT-cAMP is shown in Figure 2.6. Bursts of channel openings such as those shown in Figure 2.4 and Appendix A, Figure 1 are largely outside the VSD-driven activation pathway (Werry et al., 2013) and allow us to examine the effects of 8-CPT-cAMP on pore kinetics of EQ. After idealization of 26 control and 25 8-CPT-cAMP 4-s sweeps, we found two resolvable closed states for EQ with similar time constants to those we reported previously in Werry et al. (2013). In the presence of cAMP, there is no change in the mean closed time for 67  the vast majority of events. For the longer closed time, there was an increase, but there are few events for this fit (Fig. 2.6A). Interestingly, the closed dwell times of EQ in 8-CPT-cAMP are very close to those reported for S209F + KCNE1 in Eldstrom et al. (2015). A probability plot of the closed dwell time distribution shows clearly how the burst closing kinetics are unaffected by 8-CPT-cAMP (Fig. 2.6B). Peaks of the closed dwell time distribution at ∼2 and 10 ms are very similar in both control and 8-CPT-cAMP.     68   Figure 2.6: EQ closed dwell times and burst analysis.  (A) Closed dwell time distributions for EQ from 26 sweeps before and 25 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA, filtered at 500 Hz. Data were fitted with the sum of two exponential functions. τ1: 1.07 ± 0.04 ms (area under the curve [AUC 606.85 ± 19.05) and τ2: 5.07 ± 0.20 ms (AUC 359.10 ± 18.75) in control. After 8-CPT-cAMP/OA, τ1: 1.62 ± 0.05 ms (AUC 613.43 ± 14.06) and τ2: 8.19 ± 0.73 ms (AUC 117.49 ± 14.00). Bin width was 1 ms. (B) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (C) Probability distribution of burst durations in control (black) and after 8-CPT-cAMP/OA (red), from the data used in A. Event histograms were fitted with the sum of two exponential functions. In control τ1: 1.58 ± 0.05 ms (AUC 1662.64) and τ2: 17.26 ± 2.56 ms (AUC 136.94 ± 17.69). In 8-CPT-cAMP, τ1: 1.99 ± 0.06 ms (AUC 1073.46 ± 15.72) and τ2: 22.36 ± 3.31 ms (AUC 95.62 ± 12.35). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in this analysis.  The table in Figure 2.5C shows a measure of the mean dwell times at each sublevel; they were relatively unchanged in the presence of 8-CPT-cAMP, with some dwell times increasing and some decreasing. The data give an indication of the time the channel 69  spent bursting at a particular sublevel before moving to a higher or lower subconductance level. A more formal analysis of overall open burst durations was performed using a burst termination criterion of pore closing for >2 ms. The event histograms were fitted as the sum of two decay exponentials, the faster of which accounted for >90% of bursts and had time constants of 2.9 ± 0.07 ms in control and 2.5 ± 0.09 ms in 8-CPT-cAMP. The burst duration probability functions are shown together in Figure 2.6 and appear unaffected by cAMP addition (Fig. 2.6C). In fact, there is a small but significant decrease in the number of bursts at intermediate dwell times in the presence of 8-CPT-CAMP. Overall, these data suggest that 8-CPT-cAMP has only minor effects, both positive and negative, on channel pore kinetics.  2.3.5 8-CPT-cAMP effects on IKs mutants described as having fixed and activated VSDs E160R/R237E (E1R/R4E) KCNQ1, a charge reversal mutant suggested to lock the voltage sensors in an upward position and stabilize the position of the VSD (Wu et al., 2010a; Zaydman et al., 2014), was used to further test the action of 8-CPT-cAMP by isolating VSD-induced changes in the channel complex and allowing a direct test on pore kinetics. In controls, when E1R/R4E is coexpressed with KCNE1 (Fig. 2.7, A–C, left), at 40 mV channels opened without any latency and burst throughout 4-s sweeps, as well as during repolarization to −40 and −80 mV. The ensemble average current (Fig. 2.7A, middle) was time-independent compared with WT IKs (Fig. 2.3B) and unaffected by 8-CPT-cAMP, but still sensitive to chromanol 293B (Fig. 2.7A, bottom).  70   Figure 2.7: The mutant E1R/R4E + KCNE1 channel is unaffected by 8-CPT-cAMP.  (A) Top, single-channel sweep of KCNQ1 E1R/R4E + KCNE1 expressed with Yotiao before and after 200 µM 8-CPT-cAMP/0.2 µM OA. Middle, ensemble averages of 10 sweeps pulsed to 40 mV before and after 8-CPT-cAMP. Bottom, mean currents after addition of 50 µM chromanol 293B. (B) All-points histograms from 10 sweeps before and after 8-CPT-cAMP. (C) Gaussian fits of the histograms shown in B and the amplitudes of individual fitted peaks as determined with Clampfit.  All-points histograms reveal a distribution of openings with the major event peak at ∼0.35 pA at this potential. The mean single-channel conductance was 3.2 pS and was unchanged in the presence of 8-CPT-cAMP (n = 5). These values may be compared with a conductance of 2.9 pS in EQ channels before and after exposure to cAMP (see Fig. 71  2.4 legend). The histograms show that the effect of cAMP in the same single-channel patch was to decrease the closed-state occupancy without significantly changing numbers of events at the higher subconductance occupancy levels shown in Figure 2.7C at ∼0.55 pA. As a result, there was little effect on the ensemble current (Fig. 2.7A, middle). These changes are in contrast to the shortened latency to opening and occupancy of higher sublevels seen in EQ channels (Figs. 2.4 and 2.5) and increased EQ and KCNQ1 + KCNE1 ensemble currents (Fig. 2.4C and Appendix A, Fig. 2B) during exposure to 8-CPT-cAMP. Similar results have been observed in four other E1R/R4E patches.  KCNQ1 S209F is a gain-of-function mutation found in the S3 domain of the VSD (Eldstrom et al., 2010) and is like E1R/R4E in that it has a saturated Po over a wide potential range. S209F + KCNE1 has been previously characterized as having a much higher open probability than that of WT IKs, (Po = 0.6 compared with 0.15 in WT after 4 s at 60 mV; Werry et al., 2013; Eldstrom et al., 2015), with dominant residency in higher subconducting open states. At room temperature, whole-cell experiments on S209F revealed that it possesses almost voltage-independent gating when expressed with KCNE1 (Appendix A, Fig. 6A). Currents are fairly time independent, and the Po shows only a small decrease negative to −50 mV (Appendix A, Fig. 6B). No effect of 8-CPT-cAMP on G-V relationships or current amplitude was seen at the whole-cell level (Appendix A, Fig. 6, B and C).  72  Because this mutant already has activated and equilibrated VSD gating, it can provide a good test of cAMP on pore kinetics at positive potentials. Data in Figure 2.8A show single sweeps of S209F + KCNE1 at 60 mV. From these representative traces, it can be seen that the channel opens with little latency but still sometimes occupies lower sublevels before reaching higher subconductance states. The single-channel currents for S209F before and after 8-CPT-cAMP appear to be very similar and show that the time the channel is open is greatly increased compared with that of EQ (Figs. 2.3B and 2.4A), but perhaps less than E1R/R4E, as judged by the relative number of closed events in the all-points histograms (Fig. 2.8B). 73   Figure 2.8: 8-CPT-cAMP has a small kinetic effect on the high Po mutant KCNQ1 S209F coexpressed with E1.  (A) S209F+E1 and Yotiao. Representative single-channel sweeps are shown in control (top) and during exposure to 200 µM 8-CPT-cAMP/0.2 µM OA (bottom). (B) All-points amplitude histograms in control (top, 21 active sweeps) and during 8-CPT-cAMP exposure (bottom, 27 active sweeps). (C) Ensemble average currents from a single patch; 70 control sweeps (50 active and 20 blank) and 70 sweeps in 8-CPT-cAMP/OA (55 active and 15 blank). Control and 8-CPT-cAMP/OA averages peaked at 0.25 pA after 4 s at 60 mV. (D) Cumulative latency histogram. Mean first latency for the 67 active control sweeps of 128 was 0.16 ± 0.05 s. With 200 µM 8-CPT-cAMP/0.2 µM OA, mean first latency for the 85 active sweeps of 220 was 0.18 ± 0.06 s; P = 0.021 (see Table 2.1). Sweeps without activity were given a first latency >4 s.  The active peaks of the raw histograms higher than 0.4 pA enclose a much larger number of events than the closed events peak (compare with EQ or WT KCNQ1 + KCNE1; Fig. 2.3C and Appendix A, Fig. 1B). There is not much difference in the numbers of closed 74  events before and after cAMP, but there is a shift of open events from lower to higher open subconductance levels in S209F in the presence of cAMP and an increase in the amplitude corresponding to the event peak of the histogram. Before addition of 8-CPT-cAMP, the event peak amplitude was 0.38 pA, and after, 0.42 pA (Fig. 2.8B). As in whole-cell data, there was no change in ensemble average single-channel currents at 60 mV (Fig. 2.8C) and 0 mV (not depicted) after exposure to 8-CPT-cAMP, with a calculated Po of 0.71 at 60 mV (control) and 0.74 (8-CPT-cAMP; Fig. 2.8C). S209F first latency was markedly reduced compared with that of WT KCNQ1 + KCNE1. In S209F patches in control, first latency was 0.16 ± 0.05 s, and upon 8-CPT-cAMP addition, actually lengthened to 0.18 ± 0.056 s, although the median value declined (Fig. 2.8D; and Table 2.1). There was also no increase in the number of active sweeps and no recruitment of new or silent channels upon 8-CPT-cAMP administration, such as that seen with EQ or WT KCNQ1 + KCNE1 (Figs. 2.3B and 2.4B and Appendix A, Fig. 2C).  Both E1R/R4E and S209F are mutations described as having fixed and activated VSDs, and our data indicate that their overall gating is relatively unaffected by 8-CPT-cAMP, which suggests that the actions of 8-CPT-cAMP on the channel complex are mediated principally via the voltage sensor domains.  2.3.6 Effect of 8-CPT-cAMP on the pseudo-phosphorylated KCNQ1 mutants S27D and S27D-S92D The Ser27 residue in KCNQ1 is a phosphorylation site known to be important in the response of IKs to β-AR stimulation (Fig. 2.1) (Kurokawa et al., 2003), and S27D is a 75  phosphomimetic mutant that recapitulates most of the effects seen with phosphorylation of IKs. However, the S27D + KCNE1 macropatch data still showed a hyperpolarizing shift in the V1/2 of activation after 8-CPT-cAMP addition, as shown in Figure 2.9A (−15.4 mV). The mean hyperpolarization in S27D macropatches in the presence of 8-CPT-cAMP shifted from 13.1 to −1.1 mV (−14.3 mV, n = 3; Appendix A, Fig. 3B), compared with a hyperpolarization from 27.0 to 2.1 mV (−24.9 mV) for EQ (n = 4; Appendix A, Fig. 3A). Deactivation also appeared to be slowed when 8-CPT-cAMP was added (Fig. 2.9A, insets). The single-channel recordings are shown in Figure 2.9B, and there were more subtle changes in the kinetics with 8-CPT-cAMP than seen in EQ channels. The all-points histograms showed a relative reduction of closed-state occupancy and a small shift of open events to higher subconductance amplitudes (Fig. 2.9C). The amplitude of the single-channel events peak was unchanged though, as all-point histograms of 11 active control and 11 active 8-CPT-cAMP sweeps were fitted with an event peak at 0.365 pA in control and 0.361 pA with 8-CPT-cAMP/OA (Fig. 2.9C). As might be expected from the all-points histograms, there was little change in ensemble average currents at 60 mV (Fig. 2.9D) in S27D after exposure to 8-CPT-cAMP, with a calculated Po of ∼0.33 in control and 8-CPT-cAMP. 76   Figure 2.9: Effects of 8-CPT-cAMP on first latency of KCNQ1 S27D + KCNE1 and Yotiao.  (A) G-V curves from macropatches in control and after exposure to 200 µM 8-CPT-cAMP/0.2 µM OA. Graph shows mean of n = 2 in each case from a single macropatch in control (circles, V1/2 = 10.5 mV) and cAMP (squares, V1/2 = −4.9 mV). Insets show macropatch I-V currents in control and with 8-CPT-cAMP. Every other sweep is shown. Cells were held at −60 mV then pulsed from −80 mV to 80 mV in 10-mV steps for 4 s. They were then pulsed to −40 mV for 900 ms. (B) Single-channel sweeps of KCNQ1 S27D + KCNE1 and Yotiao in control (top) and exposed to cAMP (bottom). (C) All-point amplitude histograms of active control sweeps (left, 11 sweeps) and active sweeps during cAMP exposure (right, 11 sweeps) were fitted with event peaks in control of 0.37 pA and in 8-CPT-cAMP of 0.36 pA. (D) Ensemble average currents from a single patch; 31 control sweeps (14 active) and 31 sweeps in 8-CPT-cAMP/OA (15 active). Control and 8-CPT-cAMP/OA averages peaked at 0.12 pA after 4 s at 60 mV. (E) Cumulative latency histogram. Mean first latency for the 57 active control sweeps of 302 was 1.81 ± 0.13 s. With 200 µM 8-CPT-cAMP/0.2 µM OA, mean first latency for the 67 active sweeps of 335 was 1.44 ± 0.1 s; P = 0.032 (see Table 2.1). Sweeps without activity were given a first latency >4 s. Note split-scale ordinate. Bin width of the histogram was 0.01 pA. 77  The detailed changes in subconductance occupancy were analyzed after data idealization, and results are shown in Appendix A, Figures 4 and 5. It is of note that the closed dwell time in S27D as a proportion of the total in control (78%) is very similar to that seen in EQ after exposure to 8-CPT-cAMP (78.8%; Fig. 2.5C). However, there is a reduction of closed-state occupancy and a shift of subconductance occupancy to all the higher sublevels after cAMP/OA exposure. The first latency was also still significantly shortened by the addition of 8-CPT-cAMP (Fig. 2.9E), from 1.81 ± 0.13 to 1.44 ± 0.11 s (Table 2.1; P = 0.032), and overall, these changes after cAMP are very similar to those seen in EQ and KCNQ1 + KCNE1 channels but are attenuated as expected in this mutant. There were few changes in the burst kinetics in the S27D mutant after exposure to 8-CPT-cAMP (Appendix A, Fig. 5).  Ser92 is also a potential phosphorylation site in the N terminus of KCNQ1 (Lopes et al., 2007) that has been shown to increase IKs current (Lundby et al., 2013), and thus we were prompted to test the effects of 8-CPT-cAMP/OA on the double-mutant S27D/S92D-KCNQ1 channel (Fig. 2.10). Surprisingly, the G-V relationship was slightly depolarized from the WT IKs relationship, rather than hyperpolarized, but there was no further change upon exposure to 8-CPT-cAMP. There was no change in peak whole-cell currents with cAMP, and no change in single-channel peak event amplitudes or latency kinetics, although we did not perform a subconductance analysis (Fig. 2.10, C and D). The mean first latency in control was 1.62 ± 0.08 s and was not significantly different in the presence of 8-CPT-cAMP, at 1.43 ± 0.13 s (Table 2.1; P = 0.332). These results suggest that almost 78  all of the kinetic effects of 8-CPT-CAMP on the IKs current are mediated via phosphorylation of these two residues, Ser27 and Ser92.  Figure 2.10: Effects of 8-CPT-cAMP on currents in KCNQ1 S27D/S92D + KCNE1 and Yotiao.  (A) G-V curves from whole-cell measurements in control (V1/2 = 43 ± 6.9 mV) and after exposure to 200 µM 8-CPT-cAMP/0.2 µM OA (V1/2 = 47.5 ± 8.6 mV, n = 8 cells), compared with WT IKs (V1/2 = 25.1 ± 2.5 mV). Error bars show ±SE. Insets show I-V currents in control and with 8-CPT-cAMP. Every other sweep is shown. Cells were held at −90 mV and pulsed from −80 mV to 100 mV in 10-mV steps for 4 s. Tail currents were recorded at −40 mV for 900 ms. (B) Diary plot of the peak outward current during a 4-s pulse to 60 mV over time. Bar indicates the addition of 200 µM 8-CPT-cAMP/0.2 µM OA to the bath. (C) Sample single-channel sweeps of S27D/S92D + KCNE1 (E1) before (left) and after (right panel) 8-CPT-cAMP. (D) Raw all-point amplitude histograms of 21 active S27D/S92D sweeps before (left) and after (right) 8-CPT-cAMP/OA.  79  2.3.7 Effect of 8-CPT-cAMP on the surface expression of WT IKs To investigate whether the recruitment of WT IKs producing channel complexes in the single-channel patches and whether an increased current density at the whole-cell level could be in part because of an increase in channels present at the cell surface, total internal reflection fluorescence (TIRF) microscopy was used to allow visualization of channels within 80 nm of the surface of the cell and monitor channel trafficking (Schwarzer et al., 2013; Yamamura et al., 2015).  CHO cells transfected with WT KCNQ1-GFP and WT KCNE1-mCherry were recorded for a period of 15–30 min, and 200 µM 8-CPT-cAMP was added at 5 min. TIRF images of KCNQ1-GFP and KCNE1-mCherry at 0 and 15 min, respectively, are shown in Figure 2.11 (A and C); 8-CPT-cAMP was added at 5 min. After measuring the fluorescence intensity of the whole cell, there does not appear to be an increase in the overall surface expression of KCNQ1-GFP or KCNE1-mCherry after the addition of 8-CPT-cAMP (Fig. 2.11, B and D). This indicates that addition of cAMP does not increase the net trafficking of IKs to the cell surface. 80   Figure 2.11 200 µM 8-CPT-cAMP does not alter overall surface expression of KCNQ1 and KCNE1 subunits in CHO cells.  (A) TIRF images of KCNQ1-GFP before (left) and after (right) addition of 200 µM 8-CPT-cAMP. Images were taken at 0 and 15 min, respectively. (B) Diary plot of change in relative fluorescence units (RFUs) of KCNQ1-GFP with 200 µM 8-CPT-cAMP added at 5 min (n = 7). (C) TIRF images of KCNE1-mCherry before (left) and after (right) addition of 200 µM 8-CPT-cAMP. Images were taken at 0 and 15 min, respectively. (D) Diary plot of change in relative fluorescence intensity of KCNE1-mCherry with 200 µM 8-CPT-cAMP added at 5 min (n = 7). Error bars in B and D show ±SE.  It was clear from observation of the KCNQ1-GFP fluorescence signals over the time frame of 15–30 min that the channels are highly mobile in both the presence and absence of 200 µM 8-CPT-cAMP (Appendix A, Fig. 7A). Some ROIs did show a decrease or an increase (Appendix A, Figs. 7, A and B) in fluorescence intensity with cAMP exposure, indicating that channels had internalized or been inserted at these locations during the experiment. In Appendix A, Figure 7A, a cell with three chosen ROIs is shown before (0 81  or 15 min) and after (30 min) addition of 8-CPT-cAMP. ROI 1 shows a decline in fluorescence intensity immediately after addition of 8-CPT-cAMP (Appendix A, Fig. 7B, left). Before addition of 8-CPT-cAMP, the fluorescence at 15 min was 385 a.u.; after addition of 8-CPT-cAMP, at 30 min, it had dropped to 354 a.u. (Appendix A, Fig. 7B, left). In ROI 2, the number of KCNQ1-GFP channels increased shortly after 8-CPT-cAMP was added (Appendix A, Fig. 7B, middle). The fluorescence intensity went from a stable baseline at 215 a.u. to 230 a.u. (Appendix A, Fig. 7B, middle). Finally, the largest increase is seen in ROI 3, in which the fluorescence intensity increases from 270 to 350 a.u. after addition of 8-CPT-cAMP (Appendix A, Fig. 7B, right). However, when analysis was expanded to 54–61 ROIs from the border region of the cell and 52–64 ROIs from the cell center of 11 cells before and after 2-, 5-, and 10-min exposure to 8-CPT-cAMP (Appendix A, Fig. 7C), the mean fluorescence intensities of ROIs did not change significantly from those in the center to those in the border over the 10 min analyzed, in control conditions (Appendix A, Fig. 7C, left) or during exposure to 8-CPT-cAMP (Appendix A, Fig. 7C, right). This supports the conclusion that addition of cAMP does not lead to any organized redistribution of the channel or increase in net trafficking to the surface.  2.4 Discussion The mechanism behind the increase in IKs current after cAMP administration has not been fully defined. It is well understood at the whole-cell level that there is a hyperpolarization of the G-V relationship and that there must be effects on the channel complex Po, but these have not been studied before at the single-channel level. The underlying mechanisms for the cAMP-induced increase in current were an increased likelihood of 82  channels to open at all during 4-s depolarizations, the ability of the channels to open sooner during a depolarizing test pulse than in control, and to occupy higher subconducting states more frequently. The studies with the E1R/R4E and S209F KCNQ1 mutants indicated that these dominant effects of cAMP were mediated preferentially via effects on voltage sensor domain kinetics rather than changes in pore kinetics. Our experiments also revealed that the effects of 8-CPT-cAMP on IKs single-channel kinetics could largely be mimicked by Asp substitution at Ser27 and Ser92.  We have proposed that there may be an increase in the number of IKs producing channel complexes at the cell surface that could increase overall current. Indeed, in cell-attached single-channel and macropatch recordings, we could often see an increase in the number of active channels after the addition of the cAMP analog. However, our TIRF results showed that there is no net increase in the number of channels present and active on the surface of cells, and although the data do support a constant redistribution of channels across the cell surface, this was not significantly changed by cAMP administration.  2.4.1 Single-channel studies of IKs The fusion construct EQ is composed of one KCNE1 and one KCNQ1 subunit attached together by a linker as characterized in Murray et al., (2016). The response to 8-CPT-cAMP was tested on independently expressed KCNQ1 and KCNE1 subunits, and they responded similarly to the tandem EQ construct (Appendix A, Figs. 1 and 2). Half the time, both constructs also showed an increase in the number of channels in the patch after cAMP addition. Single-channel recordings of both EQ and KCNQ1 + KCNE1 show 83  the characteristic long latencies to the initial opening of the channel, and their properties are similar to those previously reported for these constructs when AKAP-9 was not transfected (Murray et al., 2016). However, once the cAMP analog was added, this first latency was significantly decreased (Fig. 2.4D; and Table 2.1) and the channel opened more quickly upon depolarization (Fig. 2.2C). The number of active sweeps also increased by ∼70% upon 8-CPT-cAMP stimulation (Fig. 2.4D).  IKs has a number of discernible subconducting levels (Appendix A, Fig. 8) that are transitioned through rapidly to produce its characteristic flickering single-channel behavior. Because β-adrenergic stimulation increases the amount of current, our hypothesis was that in the presence of 8-CPT-cAMP, occupancy of the higher sublevels would be favored. The time spent at each of these subconducting states was different before and after PKA activation, and in the presence of 8-CPT-cAMP, more time was spent at the higher sublevels without any change in subconductance amplitudes (Fig. 2.5). We also did not see the appearance of new, larger sublevels in the presence of cAMP (Figs. 2.3, 2.4, and 2.5). The shift of occupancy toward the higher subconducting levels mediated by PKA was accompanied by a 10% decrease in the time spent closed, and together they translate into the well-known increase in macroscopic channel current seen during cAMP administration or β-adrenergic activation. In contrast to the marked effects on latency and subconductance state occupancy, the effects on pore kinetics were minor, with channel closed times and burst kinetics remaining relatively unaffected by 8-CPT-cAMP (Figs. 2.5 B and 2.6). 84  The molecular mechanisms behind the PKA-dependent changes in IKs current kinetics that we have described are not fully understood. However, it has been suggested that the distance between the KCNQ1 N and C termini shortens once the channel opens (Haitin et al., 2009). Residue Ser27 is located in the N terminus, and PKA is bound to a complex in the C terminus. Shortening the distance between the N and C termini is thought to be necessary for this phosphorylation to occur (Haitin et al., 2009). How this affects IKs gating is not understood (Liin et al., 2015), but in the present experiments it appears that most of the effects on the single-channels can be explained through actions on the VSD.  2.4.2 Effects of cAMP on the E1R/R4E and S209F channels with augmented VSD function The E1R/R4E mutant is suggested to possess voltage-independent gating, as the KCNQ1 voltage sensors are suggested to be locked in an upward position (Wu et al., 2010a; Zaydman et al., 2014). The S209F KCNQ1 mutant is a VSD gain-of-function mutation that opens at highly hyperpolarized potentials compared with WT, but with much slower or absent deactivation at −80 mV. The increase in Po of S209F compared with WT has been previously reported by Werry et al. (2013), as has a much slower rate of deactivation. These properties are similar to those described for other KCNQ1 gain-of-function mutations (Restier et al., 2008). These mutants allowed us to separate the potential actions of cAMP on pore kinetics from any effects on the VSD, which would already be maximally activated in these mutants. As anticipated, we did not observe a decrease in the number of silent 4-s sweeps, significant decreases in the first latency, or increases in E1R/R4E or S209F channel current amplitudes after 8-CPT-cAMP addition 85  (Figs. 2.7 and 2.8). Interestingly, we did still see minor subconductance effects of cAMP on S209F (which presumably retains some ability to show VSD gating), but not on E1R/R4E. It is worth noting that in neither of these mutant channels did we observe an increased appearance of channels in cell-attached patches exposed to cAMP. Perhaps this is caused by trafficking deficiencies between these mutant channels and WT (Eldstrom et al., 2015).  2.4.3 Effects of cAMP on phosphomimetic mutants Although the main target of phosphorylation for KCNQ1 is Ser27, other phosphorylation sites are also important. Mutating Ser27 from a serine to aspartic acid using site-directed mutagenesis recreates the effects of phosphorylation, but to a lesser degree (Kurokawa et al., 2003). As described by Kurokawa et al. (2003), S27D + KCNE1 current densities were twofold higher than WT, and S27D had a slower rate of deactivation when cAMP was present. The V1/2 of activation (23.2 ± 4.2 mV in S27D) was hyperpolarized compared with WT (34 mV), but less than with cAMP (V1/2 = 4.1 ± 4.3 mV), suggesting that this mutation does not completely recapitulate the hyperpolarizing shift of the V1/2 seen in WT.  This is in agreement with our findings. The macropatch data (Appendix A, Fig. 3) show that S27D channels have a more negative V1/2 of activation than WT, and that there is still an effect, albeit attenuated, of 8-CPT-cAMP on them. The first latency of S27D is similar to EQ before the addition of cAMP, although the total closed time of S27D + KCNE1 in control is similar to that of EQ plus cAMP. Once 8-CPT-cAMP was added, there was a significant shortening of the first latency (Fig. 2.9E and Table 2.1), but this was 86  proportionally far less (20%) than that observed for either EQ (34%) or KCNQ1 + KCNE1 expressed separately (39%). Therefore, this phosphomimetic residue is responsible in part for the shortening of first latency seen when IKs is exposed to 8-CPT-cAMP, but an additional effect remains. This construct also shows a recruitment of channels upon 8-CPT-cAMP (unpublished data) similar to that of EQ and KCNQ1 + KCNE1.  The S27D result suggests that there might be other relevant phosphorylation sites in KCNQ1, and Ser92 is another potential N-terminal site (Lopes et al., 2007) that has been shown to increase IKs current when phosphorylated (Lundby et al., 2013). In the double serine to aspartic acid mutant, S27D/S92D-KCNQ1 channel, changes in macroscopic current amplitude or in the G-V relationship were no longer observed (Fig. 2.10). At the single-channel level, no significant changes were seen in latency or single-channel conductance, and no obvious differences were seen in subconductance distribution in the raw event histograms. Subconductance analysis of KCNQ1 S27D + KCNE1 patches showed that before 8-CPT-cAMP, this construct occupied the higher open sublevels, much like EQ and KCNQ1 + KCNE1 after cAMP (Appendix A, Fig. 4). This suggests that phosphorylation of Ser27 is largely responsible for changes in channel Po once activated. However, when 8-CPT-cAMP is present, the time the channel spends closed is further reduced, so other sites like Ser92 may be involved in this response, as with the shift in V1/2 in macropatches, and the shortening of the first latency that was seen with S27D.  Other than phosphorylation sites in the N terminus of KCNQ1, Yotiao itself is phosphorylated by PKA at a residue in its N terminus, Ser43. Mutation of this serine 87  residue to alanine prevents phosphorylation from occurring and reduces the response to cAMP (Chen et al., 2005). KCNE1, although not a substrate for PKA phosphorylation, is also required for the effect of phosphorylation to occur. KCNQ1 can still be phosphorylated if KCNE1 is not present, but the functional response to phosphorylation is not apparent (Kurokawa et al., 2003). This highlights the importance of KCNE1 in sympathetic regulation of IKs activity. The KCNE1 C-terminal domain has been suggested to be partly responsible for the IKs response to phosphorylation. KCNE1-D76N is a LQT-5 mutation in the C terminus that does not respond to cAMP (Kurokawa et al., 2003), and there are a number of other LQT-5 mutations that occur in this region (Splawski et al., 1997; Bianchi et al., 1999).  2.4.4 Trafficking and expression of IKs TIRF experiments were done to assess whether the increase in current after cAMP addition was at least in part the result of an increase in trafficking of the channels to the cell membrane. This, however, was not the case, as there appeared to be no overall change in expression levels of KCNQ1 and KCNE1 at the surface of the cell after cAMP exposure (Fig. 2.11). However, there were clearly areas of local rearrangement of channels (Appendix A, Fig. 7). There are regions from which the channels seem to exit, and areas where there is increased channel density, often where channels are moving toward the edges of the cells. This phenomenon did not seem to be dependent on cAMP, but rather the result of normal turnover of the channel in heterologous cells. This might explain, however, why during single-channel recordings after adding cAMP we 88  sometimes see an increase in the number of channels (Figs. 2.3 and 2.4), or even a decrease/complete loss of channel activity (data not depicted).  Others have also shown no increase in KCNQ1 movement to the cell surface upon isoproterenol exposure in COS-7 cells (Wang et al., 2013), although PKA activation is known to alter the rate of trafficking in several channels, such as the cystic fibrosis transmembrane conductance regulator and voltage-gated sodium and potassium channels (Levin et al., 1995; Lehrich et al., 1998; Zhou et al., 2000). Kv1.1 channels are affected by PKA activation by two different mechanisms; one where an increase in channel synthesis occurs in response and the second where there is a redistribution of already synthesized channels to the membrane (Levin et al., 1995). It is also worth mentioning that other AKAPs, such as D-AKAP2, have been found to interact with Rab4 and Rab11 proteins and allow for protein recycling regulation (Eggers et al., 2009). In rats, cardiac sodium channels have been shown to increase in number at the surface, not directly through PKA modulation, but via the G protein stimulatory α subunit (Lu et al., 1999). The remodeling effect may not be a direct effect on IKs but an effect on cytoskeleton remodeling that consequently causes relocalization of IKs producing channel subunits.  2.5 Conclusion The increase in IKs current after addition of cAMP is caused by phosphorylation of the VSD at Ser27and other sites, which moves the VSD into more activated states and allows the channels to open more often, more quickly, and to higher sublevels. The direct effects 89  on pore opening and closing are less significant. Our results also suggest that Ser27 and Ser92 are important phosphorylated residues in this response, but that others may also be of importance. The overall membrane surface expression of IKs is unchanged by cAMP stimulation, but local rearrangement may occur.     90  Chapter 3: Single-Channel Kinetic Analysis of the cAMP Effect on IKs Mutants, S209F and S27D/S92D. 3.1 Introduction The IKs potassium current is involved in terminating the plateau phase of the cardiac action potential and becomes more important as sympathetic activity increases the heart rate (Terrenoire et al., 2005). Under sympathetic stimulation, the IKs current is enhanced in a cAMP-dependent fashion, leading to faster action potential repolarization, which shortens the action potential and allows adequate time for ventricular refilling (Stengl et al., 2003; Jost et al., 2005; Silva and Rudy, 2005; Terrenoire et al., 2005). Mutations in the channel can give rise to long and short QT syndrome as well as familial atrial fibrillation (Moss et al., 1991; Chen et al., 2003c; Bellocq et al., 2004).  A homotetramer of peptides encoded by the KCNQ1 gene form the channel and are modulated by KCNE1 accessory subunits to produce the IKs current. When KCNE1 subunits are a part of the channel complex, the current is converted from one that activates rapidly and inactivates, to one with very slow activation, deactivation kinetics and no inactivation (Barhanin et al., 1996; Sanguinetti et al., 1996). The number of these accessory subunits in the channel complex can vary between 1 to 4 units per channel (Murray et al., 2016), which allows the channel to be modulated and perhaps regulated by the number of KCNE1s in the complex. Two residues in the N-terminus are phosphorylated by cAMP, S27 and S92 (Marx et al., 2002; Lopes et al., 2007; Lundby et al., 2013) and under sympathetic stimulation, the N-terminus of KCNQ1 becomes 91  phosphorylated by PKA, which is part of a macromolecular complex bound to the C-terminal domain of KCNQ1 (Haitin et al., 2009).  Exogenous 8-CPT-cAMP decreases the voltage threshold for activation and increases the magnitude of IKs, but only when the KCNE1 subunit and Yotiao are present (Marx et al., 2002; Dilly et al., 2004; Terrenoire et al., 2005; Kurokawa et al., 2009; Li et al., 2012; Thompson et al., 2017) (See Chapter 2). Single-channel recording has revealed the detailed changes in channel kinetics in the presence of 8-CPT-cAMP. We showed that the increase in current observed upon 8-CPT-cAMP addition is caused by channels opening more quickly, more often and to higher open sublevels, and suggested that these effects were caused by increased activation of the voltage sensor domains (VSDs) as shown in Chapter 2 (Thompson et al., 2017). Mutant KCNQ1 channels with enhanced or fully activated VS were used to characterize this effect.  In this addendum, we have further analyzed the single-channel kinetics of two KCNQ1 mutants used in the original paper. One is S209F, a high Po mutant with enhanced gating, where 8-CPT-cAMP has some effect on the subconductance occupancy. The other is a double phosphomimetic mutant (S27D/S92D), which appeared to show no response to 8-CPT-cAMP, but that has kinetic properties more similar to WT than the single phosphomimetic mutant, S27D. 92  3.2 Methods 3.2.1 Reagents To activate PKA, 200 μM of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT-cAMP) (Sigma-Aldrich), a membrane-permeable cAMP analog, was used. To sustain the cAMP analog, OA (EMD Millipore) was used at 0.2 µM concentration.  3.2.2 Molecular biology Constructs used were as previously described in Chapter 2.  3.2.3 Cell culture and transfections For single-channel recording, ltk− mouse fibroblast cells were used. They were grown in MEM (Thermo Fisher Scientific) with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific) added. Cells were kept at 37°C in a humid atmosphere containing 5% CO2. Constructs were overexpressed by transient transfection using Lipofectamine2000 (Thermo Fisher Scientific). KCNQ1 S209F or S27D/S92D, KCNE1-GFP and AKAP-9 (required cofactor for phosphorylation of KCNQ1) were co-expressed in a 1:3:1 ratio. All recordings were performed 24–48 hr after transfection at room temperature.  93  3.2.4 Patch-clamp electrophysiology Single-channel recording methodology was performed as previously described in Werry et al., 2013 and Chapter 2. Data were obtained and analyzed using Axopatch hardware and pCLAMP 10.5 software (Molecular Devices).  3.2.5 Solutions The solutions used for both single-channel recordings were as described in Chapter 2.  3.2.6 Data analysis Data analysis was performed using Prism 7 (GraphPad Software). Single-channel data were acquired at 2 kHz and digitized using Digidata 1440A hardware (Molecular Devices). All recordings used for subconductance analysis were filtered at 500 Hz. Only events over 1.5 ms in duration were included in the analysis. One-way ANOVAs using Sidak’s multiple comparisons test were performed using Prism 7 (GraphPad Software).  3.2.7 Statistics Results shown here are mean values ± SE (Prism 7, GraphPad Software).  3.3 Results and discussion 3.3.1 Kinetic analysis of 8-CPT-cAMP on an enhanced gating mutant, S209F S209F is a kinetic gain-of-function mutation, which can be described as having a preferentially activated VSD stabilizing the open state. When complexed with KCNE1, it has a Po between 0.6 and 0.7 (Werry et al., 2013; Thompson et al., 2017) (See Chapter 94  2), which is about 4x that of WT KCNQ1 + KCNE1 (0.15–0.2) (Werry et al., 2013; Eldstrom et al., 2015; Murray et al., 2016). In our previous paper and in Chapter 2 (Thompson et al., 2017), at the single-channel level, there was no change in the Po (control 0.71 and 0.74 in 8-CPT-cAMP at + 60 mV (Thompson et al., 2017) (See Chapter 2)), but there was a reduction in the number of closed events and some changes in subconductance levels from the all-point histogram distribution in the presence of 8-CPT-cAMP.  To further investigate S209F changes in subconductance in response to 8-CPT-cAMP, analysis of 20 S209F + KCNE1 sweeps before and after 8-CPT-cAMP was carried out and shows changes in the substate occupancy levels in the presence of 8-CPT-cAMP (Figure 3.1). The closed dwell time percentage in control is ~ 44%, which is considerably less than EQ even in the presence of 8-CPT-cAMP (Control 88% and 8-CPT-cAMP 79%, Thompson et al., 2017, also see Chapter 2). This closed dwell time drops to ~ 21% in the presence of 8-CPT-cAMP. The mean closed dwell time duration are also significantly shorter in the presence of 8-CPT-cAMP (p-value: < 0.0001). The two highest sublevels 0.5 and 0.75 pA both increased in total dwell time by ~ 18 and 9% respectively (Fig. 3.1C), showing a shift in occupancy to higher sublevels. There are no significant changes in the mean dwell times at any of the open levels (p-value: > 0.999) (Fig. 3.1C). 95   Figure 3.1: Subconductance analysis of KCNQ1 S209F + KCNE1 before and after 8-CPT-cAMP/Okadaic acid (OA).  (A) Raw all-points histograms display the distribution of amplitudes from 20 active control sweeps (blue) and 20 sweeps in the presence of 200 µM 8-CPT-cAMP and 0.2 µM OA (red) from one representative cell were pulsed to + 60 mV for 4 s from a holding potential of −80 mV and filtered at 500 Hz. (B) Five initial thresholds used for idealization were 0.145, 0.22, 0.33, 0.5 and 0.75 pA and are shown in the table headers. The final idealization levels for control histograms were 0.13 ± 0.03, 0.23 ± 0.03, 0.35 ± 0.03, 0.50 ± 0.05 and 0.70 ± 0.10. In the presence of 8-CPT-cAMP/OA the levels were, 0.13 ± 0.03, 0.23 ± 0.03, 0.35 ± 0.03, 0.51 ± 0.05, 0.71 ± 0.07. (C) Total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP. These data were filtered at 0.5 kHz, and the bin width used was 0.01 pA. Only events longer than 1.5 ms were included. One-way ANOVA was used to compare control and 8-CPT-cAMP mean dwell times of each sublevel. The mean dwell times between control and 8-CPT-cAMP were significantly different (p-value < 0.0001). The p-values for all other sublevels were > 0.9998.  96  The S209 residue is buried in the hydrophobic core in the open state. The mutation converts a polar uncharged residue into a bulky hydrophobic one, which is thought to stabilize the open state and destabilize the closed state, resulting in gain-of-function activity (Eldstrom et al., 2010). This mutant can presumably gate normally as the voltage sensor is not “locked” unlike the E160R/R237E (E1R/R4E) mutant, which is locked in an activated state and appears to be unaffected by 8-CPT-cAMP (Haitin et al., 2009). While S209F has been described as “fully” activated, 8-CPT-cAMP does appear to further stabilize the open state by increasing events at higher subconducting states (Fig. 3.1). A reduction to ~ 20% in closed time in the presence of 8-CPT-cAMP may also suggest a destabilization of the closed state (Fig. 3.1C).  The closed dwell time constants between control and 8-CPT-cAMP remain relatively unaffected by cAMP (control τ1: 1.11 ± 0.04, τ2: 8.61 ± 0.55 and 8-CPT-cAMP τ1: 1.17 ± 0.06, τ2: 8.66 ± 3.96 ms) (Fig. 3.2A)) and are similar to the closed times of EQ in the presence of 8-CPT-cAMP (Thompson et al., 2017) (See Chapter 2). The probability distribution of the closed dwell times shows that the closed times are not affected by 8-CPT-cAMP (Fig. 3.2B). 97   Figure 3.2: KCNQ1 S209F + KCNE1 closed dwell times and burst analysis.  (A) Closed dwell time distributions for S209F + KCNE1 from 20 sweeps before and 20 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA from one representative cell. Data were fitted with the sum of two exponential functions. τ1: 1.11 ± 0.04 ms (area under the curve, AUC, 309 ± 5.5) and τ2: 8.61 ± 0.55 ms (AUC 75.4 ± 5.0 in control. After 8-CPT-cAMP/OA, τ1: 1.17 ± 0.06 ms (AUC 110 ± 3.9) and τ2: 8.66 ± 3.96 ms (AUC 14.2 ± 3.0). Bin width was 1 ms. (B) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (C) Probability distributions of burst durations based on open dwell time histograms, control (black) and after 8-CPT-cAMP/OA (red). Dwell-time histograms were fitted with the sum of three exponential functions in control τ1: 1.70 ± 0.09 ms (AUC 334 ± 10.7), τ2: 13.87 ± 2.41 ms (AUC 140 ± 16.7) and τ3: 44.38 ± 12.75 ms (AUC 39.5 ± 21.6). In 8-CPT-cAMP only the sum of two exponential functions could be fit, τ1: 2.62 ± 0.29 ms (AUC 88.2 ± 4.5) and τ2: 41.35 ± 3.36 ms (AUC 51.5 ± 2.7). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in this analysis.  Data in Figure 3.2C show the probability distribution plot of burst durations before and after 8-CPT-cAMP. There is a small decrease in the number of short and intermediate 98  bursts (Fig. 2C). The open dwell times in control could be fit with three-time constants (τ1: 1.70 ± 0.09, τ2: 13.87 ± 2.41, τ3: 44.38 ± 12.75 ms), however the second time constant was absent in the presence of 8-CPT-cAMP, as only two-time constants could be fit (τ1: 2.62 ± 0.29 and τ2: 41.35 ± 3.36 ms). 8-CPT-cAMP may be having a small effect on the pore as well as the VSD. However, the fits for these 8-CPT-cAMP data may reflect the presence of fewer events. Comparing the EQ burst probability distribution plots from Chapter 2 to the S209F + KCNE1 plots, S209F has a wider range of burst durations, whereas EQ has the majority of its events taking place at the shorter burst durations.  S209F + KCNE1 is still able to respond to 8-CPT-cAMP at the single-channel level, with a reduction in the total closed dwell time and an increase in the time spent at the higher subconducting levels. This suggests that even though this channel has enhanced activation, it can be still further increased by cAMP.  3.3.2 Single-channel analysis of KCNQ1 double-phosphomimetic mutant S27D/S92D The Ser27 residue in the N-terminus of KCNQ1 is an important phosphorylation site during adrenergic stimulation (Marx et al., 2002), which can be pseudo-phosphorylated by mutating the serine to aspartic acid, S27D (Kurokawa et al., 2003). This mutation produces currents of greater magnitude and with a more hyperpolarized V1/2 (Kurokawa et al., 2003), but can be further phosphorylated, which suggests that there are other potential significant phosphorylation residues (Thompson et al., 2017) (See Chapter 2). Ser92 has been shown by others to be phosphorylated (Lundby et al., 2013), so we 99  investigated the importance of this residue by using a double phosphomimetic mutant (S27D/S92D) and tested its response to 8-CPT-cAMP.  At the whole-cell level, there was no change in current size or V1/2 of activation in response to 8-CPT-cAMP. At the single-channel level, there was also no change in first latency or any significant change in the all-points histograms (Figs. 3.3A & 2.10C) (Thompson et al., 2017). We carried out further investigation by analyzing subconductance occupancy (Figure 3.3). The idealized histograms in Figure 3.3B show no significant change in the substate occupancy rates in the presence of 8-CPT-cAMP and no significant change in the time spent at each subconducting level between control and 8-CPT-cAMP (Fig. 3.3C) (p-value: > 0.1). 100   Figure 3.3: Subconductance analysis of KCNQ1 S27D/S92D + KCNE1 before and after 8-CPT-cAMP/OA.  (A) Raw all-points histograms display the distribution of amplitudes from 20 active control sweeps (blue) and 20 sweeps in the presence of 200 µM 8-CPT-cAMP and 0.2 µM OA (red) (20 sweeps before and after 8-CPT-cAMP from one representative cell). (B) Five initial thresholds used for idealization were 0.145, 0.22, 0.33, 0.5 and 0.75 pA and are shown in the table headers. The final idealization levels for control histograms were 0.12 ± 0.03, 0.23 ± 0.03, 0.34 ± 0.04, 0.50 ± 0.05 and 0.71 ± 0.07. In the presence of 8-CPT-cAMP/OA the levels were, 0.11 ± 0.03, 0.23 ± 0.03, 0.34 ± 0.04, 0.51 ± 0.05, 0.74 ± 0.10. (C) Total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP. These data were filtered at 0.5 kHz, and the bin width used was 0.01 pA. Only events longer than 1.5 ms were included. A one-way ANOVA was used to compare control and 8-CPT-cAMP mean dwell times of each sublevel. None of the 8-CPT-cAMP mean dwell times were significantly different from control (p-value between closed sublevels was 0.1355, all other sublevels were > 0.9999). 101  With respect to the closed and open dwell times (Fig. 3.4), two resolvable closed states were found for both control and 8-CPT-cAMP events. The faster time constant in control and cAMP was similar, 1.56 ± 0.06 and 1.24 ± 0.14 ms respectively (Fig. 3.4A). The second time constant was slower in control 7.18 ± 1.03 compared to 3.44 ± 0.89 ms in 8-CPT-cAMP (Fig. 3.4A). However, this could be due to a smaller number of events in the control fit. When the closed dwell times are plotted as a probability distribution (Fig. 3.4B), there is no clear effect of 8-CPT-cAMP on the closed dwell times. Finally, 8-CPT-cAMP does not appear to affect the open dwell time distribution (Fig. 3.4C).  102   Figure 3.4: KCNQ1 S27D/S92D + KCNE1 closed dwell times and burst analysis.  (A). Closed dwell time distributions for S27D/S92D + KCNE1 from 20 sweeps before and 20 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA (20 sweeps before and after 8-CPT-cAMP from one representative cell). Data were fitted with the sum of two exponential functions. τ1: 1.56 ± 0.06 ms (AUC 547 ± 20.3) and τ2: 7.18 ± 1.03 ms (AUC 158 ± 16.4) in control. After 8-CPT-cAMP/OA, τ1: 1.24 ± 0.14 ms (AUC 616 ± 132.3) and τ2: 3.44 ± 0.89 ms (AUC 416 ± 97.6). Bin width was 1 ms. (B) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (C) Probability distributions of burst durations based on open dwell time histograms, control (black) and after 8-CPT-cAMP/OA (red). Dwell-time histograms were fitted with the sum of three exponential functions in control τ1: 1.11 ± 0.12 ms (AUC 464 ± 56.8), τ2: 4.50 ± 0.88 ms (AUC 469 ± 56.9) and τ3: 11.10 ± 2.99 ms (AUC 126 ± 92.4). In 8-CPT-cAMP only the sum of two exponential functions could be fit, τ1: 1.59 ± 0.06 ms (AUC 792 ± 28.3) and τ2: 7.82 ± 1.52 ms (AUC 229 ± 20.1). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in this analysis.  In Xenopus oocytes, the KCNQ1 S27D/S92D mutant appears to have a more hyperpolarized V1/2 compared to WT (Li et al., 2011b), which is what one might expect of 103  a phosphomimetic mutant. However, in mammalian cells this construct produced a V1/2 that was more depolarized than WT (KCNQ1 S27D/S92D + KCNE1 V1/2: 47.5 ± 8.6 mV and KCNQ1 + KCNE1 V1/2: 25.1 ± 2.5 mV (Thompson et al., 2017) (See Chapter 2). This was surprising, and interestingly from the single-channel analysis (Fig. 3.4), we can see that this mutant does not quite behave like WT either. There is no visible main active opening amplitude peak of ~ 0.45 pA that is usually observed in WT channels (Fig. 3.3A), although the first latency of the channel was similar to that of EQ (1.62 ± 0.08 s and 1.61 ± 0.13 s respectively) (Thompson et al., 2017) (See Chapter 2). While this double pseudo-phosphorylation mutant does not appear to be affected kinetically by 8-CPT-cAMP, this mutant still showed altered single-channel kinetics compared to WT.  3.4 Conclusion Subconductance analysis allows us to better understand the kinetic changes that 8-CPT-cAMP causes at a single-channel level. Even though S209F has highly enhanced gating, 8-CPT-cAMP is able to stabilize the channel open state further. The double pseudo phosphorylated mutant, S27D/S92D does not respond to 8-CPT-cAMP.  104  Chapter 4: The IKs Current Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry 4.1 Introduction Normal cardiac action potential repolarization and changes of duration in response to inotropic and dromotropic modulation depend on the integrated function of potassium (K) and sodium (Na) channels, most importantly, IKr, IKs, and Late INa (Silva and Rudy, 2005). IKs has been identified in the heart of several mammalian species (Sanguinetti and Jurkiewicz, 1990; Salata et al., 1996), where outward potassium currents through voltage-gated potassium (Kv) channels contribute to the initiation of repolarization and the subsequent termination of the action potential. Functional loss of IKs and IKr due to inherited mutation(s) or acquired conditions or late INa gain of function account for ∼95% of genetically identified long QT interval syndrome (LQTS) (Ackerman et al., 2011), which may cause ventricular arrhythmias and sudden death in children and adults, as well as ∼30% of unexplained Sudden Infant Death Syndrome (Tester and Ackerman, 2009).  Although IKs is relevant at normal heart rates, its major role appears to be in shortening the action potential duration and the QT interval at elevated heart rates (Hund and Rudy, 2004) via two main mechanisms. Firstly, although the activation kinetics of IKs are relatively slow compared to the duration of a single action potential, the slowed deactivation kinetics are thought to allow accumulation of IKs in the open state during repetitive high-frequency activity. Such accumulation of open channels allows greater outward potassium efflux, and this is referred to as a “repolarizing reserve,” which in turn allows the physiological abbreviation of the cardiac action potential, and 105  therefore systole, at high heart rates (Stengl et al., 2006). Secondly, β-adrenergic stimulation results in the phosphorylation of KCNQ1 channels and increases IKs by causing a hyperpolarizing shift in the voltage dependence of activation, accelerating activation and slowing deactivation kinetics, both of which contribute to action potential shortening to accommodate rapid heart rates (Terrenoire et al., 2005). As a result, individuals with LQTS type I (deficiency of IKs) tend to suffer arrhythmic events during exercise or at times of high emotion when their heart rates and/or sympathetic drive to the heart is elevated.  The IKs current in the heart is composed of a homotetramer of peptides encoded by the KCNQ1 gene. This tetramer forms the potassium-selective pore and has a voltage-sensitive domain and multiple regulatory regions, including two that are phosphorylated by PKA via cAMP activation. An accessory peptide encoded by the gene KCNE1 is typically coassembled with KCNQ1 and greatly modifies the ion channel kinetics (Takumi et al., 1988; Barhanin et al., 1996). It slows the rate of activation and deactivation and prevents inactivation of the channel (Sanguinetti et al., 1996). The number of accessory peptides that coassemble with the channel has been the topic of much debate over the years. Some groups believe that the stoichiometry of the channel exists in a strict 2:4 (KCNE1:KCNQ1) ratio (Wang and Goldstein, 1995; Chen et al., 2003a; Kang et al., 2008; Morin and Kobertz, 2008; Plant et al., 2014), whereas others propose that the number of KCNE1 subunits may vary from one to four units per channel (Cui et al., 1994; Wang et al., 1998; Morokuma et al., 2008; Nakajo et al., 2010; Zheng et al., 2010; Strutz-Seebohm et al., 2011; Wang et al., 2011; Yu et al., 2013; Murray et al., 2016). Our lab has previously 106  published a study in favor of a variable stoichiometry (Murray et al., 2016). However, the physiological stoichiometry in cardiomyocytes is still unknown. A study by Yu et al., (2013) investigated the composition in cardiomyocytes using ML277, a KCNQ1 activator that reduces in efficacy as the number of KCNE1 subunits increases (Yu et al., 2013). The cultured human cardiomyocytes’ response to ML277 suggested that there were unsaturated channel complexes present that did not have a full complement of KCNE1 (Yu et al., 2013). Another study using human embryonic-stem-cell-derived cardiomyocytes produced biophysical characteristics consistent with an unsaturated channel complex. However, when the expression level of KCNE1 was increased, they saw a change in the activation kinetics implying that more KCNE1 was now present in the channel complex (Wang et al., 2011). This suggests that the stoichiometry is dependent on the level of KCNE1 expression, which may provide another means to regulate IKs activity in the heart.  Despite the importance of IKs, the biophysics of its adrenergic regulation have not been fully investigated. We, as well as others, have previously shown that exogenous cAMP shifts the voltage dependence of activation to more negative potentials and increases the magnitude of IKs when the KCNE1 subunit is present (Marx et al., 2002; Dilly et al., 2004; Terrenoire et al., 2005; Thompson et al., 2017) as seen in Chapter 2. Using single-channel recording techniques to investigate changes in the IKs current kinetics in the presence of 8-4-chlorophenylthio (CPT)-cAMP, we showed that the increase in IKs current seen with 8-CPT-cAMP addition is caused by channels that open faster, more often, and to higher-conducting open levels. We suggested that these effects were mediated through 107  voltage-sensor activation (Thompson et al., 2017) (See Chapter 2). However, our studies were done under transfection conditions designed to ensure a fully saturated complex of KCNQ1 and KCNE1 (four KCNQ1 to four KCNE1 subunits). The experiments in the current study use both whole-cell and single-channel recording techniques to examine the effect of varying the ratio of KCNE1 to KCNQ1 on the response to cAMP using previously validated (Murray et al., 2016) fusion constructs that fix the ratio of KCNE1 to KCNQ1 in the channel complex at different stoichiometries. Our major findings are that the IKs response to 8-CPT-cAMP is graded depending on the number of KCNE1 subunits present within the complex and that both KCNE1-dependent and KCNE1-independent components of the IKs response to 8-CPT-cAMP exist. Some of these findings were more obvious at the single-channel level.  4.2 Methods 4.2.1 Reagents 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt (8-CPT-cAMP) (Sigma-Aldrich, St. Louis, MO), a membrane-permeable cAMP analog, was used at 200 μM to activate protein kinase A (PKA). OA at 0.2 μM (EMD Millipore, Burlington, MA) was used to inhibit protein phosphatase 1 to sustain the response to the cAMP analog.  4.2.2 Molecular biology Both KCNQ1 and KCNE1 were purchased from OriGene (Rockville, MD). The fusion constructs (EQ, EQQ, and EQQQQ) were made as described in Murray et al., (2016). Yotiao (AKAP-9) was a gift from Dr. R. Kass of Columbia University (New York, NY). 108   4.2.3 Cell culture and transfections For both single-channel and whole-cell recordings, ltk− mouse fibroblast and tsA201 transformed human embryonic kidney 293 cells were used. Both were grown in Minimum Eagle Medium (Thermo Fisher Scientific, Waltham, MA) with 100 U/mL penicillin, 10% fetal bovine serum, and 100 μg/mL streptomycin (Thermo Fisher Scientific) added. Cells were kept at 37°C in a humid atmosphere containing 5% CO2. The cells were exposed for 1 min to trypsin/EDTA to lift the cells and replated on 25 mm2 glass coverslips one day before transfection. Constructs were overexpressed by transient transfection using Lipofectamine 2000 (Thermo Fisher Scientific). EQQQQ, EQQ, or EQ, Yotiao (required cofactor for phosphorylation of KCNQ1), and GFP (green fluorescent protein) were coexpressed in a 1.5:1.5:1 ratio. All recordings were performed 24–48 h after transfection at room temperature (Werry et al., 2013; Eldstrom et al., 2015; Murray et al., 2016; Thompson et al., 2017).  4.2.4 Patch-clamp electrophysiology Cells attached to a glass coverslip were placed in a chamber containing control bath solution. Cells selected for both whole-cell and single-channel recordings were GFP-fluorescent. Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA) and a Digidata 1440A digitizer (Molecular Devices). The software used was pClamp 10.5 (Molecular Devices) (Murray et al., 2016; Thompson et al., 2017).  109  Whole-cell recording patch electrodes were made from borosilicate glass (World Precision Instruments, Sarasota, FL) and were pulled into electrodes using a linear multistage puller (Sutter Instruments, Novato, CA). Glass pipettes were then fire polished, leading to pipette resistances between 1 and 3 MΩ. Currents were filtered at 5 kHz and sampled at 10 kHz (Murray et al., 2016).  Single-channel electrodes were made of a thick-walled borosilicate glass (Sutter Instruments), fire polished and coated with Sylgard (Dow Corning, Midland, MI). Electrodes had resistances between ∼40 and 60 MΩ. Acquired data were passed through a low-pass filter at 2 kHz (−3 dB, four-pole Bessel filter) (Werry et al., 2013; Eldstrom et al., 2015; Murray et al., 2016; Thompson et al., 2017).  4.2.5 Solutions For single-channel recordings, the bath solution contained 135 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM Hepes and was adjusted to pH 7.4 with KOH. The pipette solution contained 6 mM NaCl, 129 mM MES, 1 mM MgCl2, 10 mM Hepes, 5 mM KCl, and 50 μM CaCl2 and was adjusted to pH 7.4 with NaOH (Murray et al., 2016; Thompson et al., 2017).  For whole-cell recordings, the bath solution contained 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.8 mM NaAcetate, and 10 mM Hepes, pH adjusted to 7.4 with NaOH. The pipette solution contained 130 mM KCl, 5 mM EGTA, 1 mM MgCl2, 4 mM Na2-ATP, 0.1 mM GTP, 110  and 10 mM Hepes, pH adjusted to 7.2 with KOH (Murray et al., 2016; Thompson et al., 2017).  4.2.6 Data analysis Data analysis was performed using Prism 7 (GraphPad Software, La Jolla, CA). Using normalized tail current amplitudes, G-V relations were obtained. The data were fitted for each cell using a Boltzmann sigmoidal function:  GV = Gmax/(1 + exp [−(V − V1/2)/k]), where Gmax is max conductance, V membrane voltage, V1/2 the half-activation voltage and k the slope factor.  All single-channel data shown—sweeps and all-points histograms—were filtered at 200 Hz unless otherwise stated, and analysis of channel dwell times at different sublevels (Fig. 4.5) was done using data filtered at 500 Hz (Thompson et al., 2017).  The signal/noise ratio of single-channel data is an obstacle in the analysis, particularly for IKs, and filtering after acquisition is required to reduce the noise. 500 Hz filtering provides a better signal/noise ratio while keeping the amount of filtering to a minimum (Appendix B, Fig. 1). At this level of filtering, the dead time is 0.36 ms (0.54 × 0.66, rise time of the filter), and this means some brief events, both openings and closings, may be missed, thereby leading to an overestimation of the dwell times.  111  Sublevel analysis was performed in Clampfit using the single-channel search function. Sublevels are detected across many different filter frequencies, and therefore it seems unlikely that they are an artifact of filtering (Appendix B, Fig. 2). Levels were set to 0, 0.145, 0.22, 0.33, 0.5, and 0.75 pA following the 3/2 rule (Pollard et al., 1994). The “update level automatically” function was disabled. This program detects each event, amplitude level, and length of the event. Each event is then added to a spreadsheet, from which data for the idealized amplitude histograms and dwell time analysis are extracted. An example of the idealization process is shown in Appendix A, Figure 8 (Thompson et al., 2017). Because of the rise time of the system at 500 Hz, which was calculated to be 0.66 (0.332/0.5 kHz), only events over 1.5 ms (two times the rise time) in duration were included in the analysis (Werry et al., 2013; Thompson et al., 2017).  4.2.7 Statistics Results shown here are mean values ± SE and are averages of at least three independent experiments. Both the median and the 95% confidence intervals are also shown for the first latency data. A nonparametric Mann-Whitney test (Prism 7; GraphPad Software) was used to compare first latencies of both control and 8-CPT-cAMP-treated groups. Unpaired t-tests were used to compare V1/2 of control and 8-CPT-cAMP/OA groups as not all experiments were paired. Significant differences between means are considered as p-values < 0.05.  112  4.3 Results 4.3.1 The effect of cAMP on IKs in whole-cell recordings In whole-cell recordings (Fig. 4.1), exogenous 8-CPT-cAMP had no effect on currents in cells transfected with KCNQ1 and Yotiao. Data show no overall change in the KCNQ1 current amplitude or the waveform of whole-cell currents after 8-CPT-cAMP addition (Fig. 4.1, A–C) and no shift of the normalized conductance to hyperpolarizing potentials (Fig. 4.1D). KCNQ1 displayed an average V1/2 in control of −22.0 mV and in the presence of 8-CPT-cAMP of −22.7 mV (Fig. 4.11D; Table 4.1). However, during whole-cell recordings from cells transfected with both KCNQ1 + KCNE1 (transfected DNA ratio, 3:1 KCNE1:KCNQ1), 8-CPT-cAMP caused an increase in peak current and a large hyperpolarizing shift of ∼−18 mV in the G-V relationship, as we have previously shown in Chapter 2, Table 2.1. 113   Figure 4.1: KCNQ1 does not respond to 8-CPT-cAMP in whole-cell recordings.  Currents were recorded at room temperature. (A) Representative traces for KCNQ1 alone with Yotiao before (control, black line) and after 200 μM 8-CPT-cAMP/0.2 μM OA (gray line), pulsed to +60 mV for 2 s and then −40 mV for 900 ms. (B) A diary plot of the peak current of a 2 s +60 mV pulse over time. The addition of 200 μM 8-CPT-cAMP and 0.2 μM okadaic acid (OA) is marked by a solid bar. (C) Representative traces are shown from every other voltage starting from −80 to +60 mV for both control (top panel) and in the presence of 200 μM 8-CPT-cAMP and 0.2 μM OA (bottom panel). (D) G-V curves recorded before (black circles) and after 8-CPT-cAMP (gray squares). Cells were held at −90 mV and pulsed from −80 to +60 mV in 10-mV steps for 2 s. Tail currents were recorded at −40 mV for 900 ms. Control V1/2: −22.0 mV ± 1.8 SE, n = 5 and 8-CPT-cAMP V1/2: −22.7 mV ± 1.8 SE, n = 4, unpaired t-test p-value: 0.8038. 114   Table 4.1: V1/2 of Activation Before and After 8-CPT-cAMP/OA  Control 8-CPT-cAMP/OA  Unpaired T test Construct V1/2 k-factor n V1/2 k-factor n ∆ in V1/2 p-value KCNQ1 -22.0 ± 1.8 10.8 ± 0.3 5 -22.7 ± 1.8 11.5 ± 0.2 4 -0.7 ± 2.6 0.8038 EQQQQ 6.3 ± 2.3 16.1 ± 1.2 4 -3.2 ± 3.0 13.7 ± 1.2 4 -9.5 ± 3.7 0.0435 EQQ 10.4 ± 2.2 16.1 ± 1.6 8 -2.7 ± 1.2 12.3 ± 0.4 4 -13.1 ± 3.3 0.0024 EQ 26.7 ± 2.5 18.9 ± 1.0 6 4.2 ± 6.0 16.0 ± 1.9 4 -22.5 ± 5.7 0.0042 KCNQ1 + KCNE1 * 28.2 ± 5.4 21.3 ± 2.9 4 10.5 ± 2.6 18.5 ± 0.8 4 -17.7 ± 6.0 0.0261   V1/2 of activation was obtained from -40 mV tail portion of activation protocols (-80 mV holding potential, pulsed to -90 up to +100 mV in 10 mV steps for 4 s, then -40 mV). P-values compare control and the 200 µM 8-CPT-cAMP and 0.2 µM okadaic acid (OA) group using an unpaired t-test. * indicates data taken from Thompson et al., (2017) and Chapter 2.      115  Cells transfected with an EQ linked construct, which forces a 4:4 KCNE1:KCNQ1 stoichiometry (Appendix B, Fig. 3), show shifts in current magnitude (Appendix B, Fig. 3, A and B) and V1/2 that were similar to those observed when cells were transfected with KCNQ1 and KCNE1 separately. EQ had an average control V1/2 of 26.7 mV that was hyperpolarized to 4.2 mV in the presence of 8-CPT-cAMP (Appendix B, Fig. 3). 8-CPT-cAMP displayed a significant average shift of ∼−23 mV (p-value: 0.0042, Table 4.1).  When the stoichiometric ratio of KCNE1:KCNQ1 was reduced to 2:4 using an EQQ construct, 8-CPT-cAMP caused only small changes in the overall waveform (Fig. 4.2A) and a small increase in peak current at +60 mV (Fig. 4.2B) but not as profound an increase as when fully saturated with KCNE1 (Appendix B, Fig. 3). The waveform of both the control and 8-CPT-cAMP traces in Figure 4.2C suggests from the tail currents that saturation of the current upon activation is occurring at more negative potentials in the presence of cAMP. This steepening and hyperpolarizing shift of the G-V relationship is shown in Figure 4.2D, which was obtained from tail current analysis. The average V1/2 in EQQ was 10.4 mV, and the V1/2 in the presence of 8-CPT-cAMP was −2.7 mV (p-value = 0.0024). The shift in normalized conductance (∼−13 mV) is smaller than that seen with EQ (∼−23 mV) (Table 4.1). 116   Figure 4.2: EQQ V1/2 is hyperpolarized in response to 8-CPT-cAMP addition.  Currents were recorded at room temperature. (A) Representative traces for EQQ with Yotiao before (control, black line) and after 200 μM 8-CPT-cAMP/0.2 μM OA (gray line), pulsed to +60 mV for 2 s and then −40 mV for 900 ms. (B) A diary plot of the peak current of a 2 s +60 mV pulse over time. The addition of 200 μM 8-CPT-cAMP/0.2 μM OA is marked by a solid bar. (C) Representative traces are shown from every other voltage starting from −80 to +100 mV for both control (top panel) and in the presence of 200 μM 8-CPT-cAMP and 0.2 μM OA (bottom panel). (D) G-V curves recorded before (black circles) and after 8-CPT-cAMP/OA (gray squares). Cells were held at −90 mV and pulsed in 10-mV steps for 4 s from −80 to +100 mV in control and +60 mV in 8-CPT-cAMP/OA. Tail currents were recorded at −40 mV for 900 ms. Control V1/2: 10.4 mV ± 2.2 SE, n = 8 and 8-CPT-cAMP V1/2: −2.7 mV ± 1.2 SE, n = 4, unpaired t-test p-value: 0.0024. ΔV1/2: −13.1 mV ± 3.3 SE.  117  Having shown a reduction in the response to cAMP of the IKs current with a half-saturated complement of KCNE1 (EQQ 2:4) compared to a full complement of KCNE1 (EQ 4:4), it was of interest to examine the response of an IKs complex with only one KCNE1 (EQQQQ 1:4) to cAMP. EQQQQ was transfected along with Yotiao similarly to EQ and EQQ. Representative +60 mV traces (Fig. 4.3A) and a diary plot (Fig. 4.3B) of EQQQQ do not show increases in the current amplitude over time with exposure to cAMP. The waveforms of both control and 8-CPT-cAMP exposed cells do not appear to be dissimilar to each other (Fig. 4.3C). Upon further analysis of the tail currents before and after 8-CPT-cAMP, a significant hyperpolarization of the V1/2 of activation was observed. The average V1/2 in control cells was 6.3 mV and the V1/2 of 8-CPT-cAMP/OA cells was −3.2 mV (p-value = 0.0435, Fig. 4.3D; Table 4.1).  118   Figure 4.3: EQQQQ V1/2 is slightly hyperpolarized in response to 8-CPT-cAMP addition.  Currents were recorded at room temperature. (A) Representative traces for EQQQQ with Yotiao-Ro before (control, black line) and after 200 μM 8-CPT-cAMP/0.2 μM OA (gray line), pulsed to +60 mV for 4 s and then −40 mV for 900 ms. (B) A diary plot of the peak current of a 4 s +60 mV pulse over time. The addition of 200 μM 8-CPT-cAMP/0.2 μM OA is marked by a solid bar. (C) Representative traces are shown from every other voltage starting from −80 to +90 mV for both control (top panel) and in the presence of 200 μM 8-CPT-cAMP and 0.2 μM OA (bottom panel). (D) G-V curves recorded before (black circles) and after 8-CPT-cAMP/OA (gray triangles). Cells were held at −90 mV and pulsed from −80 to +90 mV in 10 mV steps for 4 s. Tail currents were recorded at −40 mV for 900 ms. Control V1/2: 6.3 mV ± 2.3 SE, n = 4 and 8-CPT-cAMP V1/2: −3.2 mV ± 3.0 SE, n = 4, unpaired t-test p-value: 0.0435. ΔV1/2: −9.5 mV ± 3.7 SE.  119  4.3.2 The effect of cAMP on IKs single-channel openings Data in Figures 4.4, 4.5, and 4.6 demonstrate the effects of 8-CPT-cAMP on the single-channel properties of EQQ and EQQQQ. Single-channel recordings in control solutions have been reported previously (Murray et al., 2016). IKs characteristically has a long first latency to opening after depolarization and, once open, exhibits bursts of rapid brief openings. EQ, which has 4 KCNE1 subunits, exhibits the longest first latency and more openings to high amplitudes (∼0.5 pA) as shown in Chapter 2 (Murray et al., 2016; Thompson et al., 2017). When the number of KCNE1 subunits within the IKs complex is reduced to a 2:4 ratio of KCNE1:KCNQ1, these properties change. The characteristic fast-flickering opening behavior is still intact, but the channel opens more quickly and visits the higher-amplitude levels less often (Figs. 4.4A and 4.5A). EQQQQ has the shortest first latency and the smallest amplitudes, never reaching the higher subconducting states (Fig. 4.6).  120   Figure 4.4: 8-CPT-cAMP shortens the first latency of EQQ single-channels.  Currents were recorded at room temperature. (A) Representative 4 s cell-attached single-channel recordings of control (left panel) and 8-CPT-cAMP/OA (right panel). Data were filtered at 200 Hz. (B) All-points event histograms of 10 active sweeps in control (left panel) and 8-CPT-cAMP/OA-treated EQQ (right panel). (C) A cumulative latency histogram showing that the mean first latency for the 107 active control sweeps (black line and arrow) of 259 was 0.90 ± 0.07 s. In 200 μM 8-CPT-cAMP/0.2 μM OA (gray line and arrow), mean first latency for the 129 active sweeps of 378 was 0.71 ± 0.06 s; p = 0.0032 (see Table 4.2). Sweeps without activity were given a first latency >4 s; arrows indicate sweep total in each case. Note split-scale ordinate. 121   Figure 4.5: Subconductance dwell time analysis of EQQ before and after 8-CPT-cAMP/OA.  (A) Raw all-points histograms of 10 active control sweeps (black line) from an EQQ patch and after 200 μM 8-CPT-cAMP/0.2 μM OA (gray dashed line). (B) Idealized histograms of 14 active control sweeps (black line) and 14 8-CPT-cAMP sweeps (gray dashed line). The idealization process used five different thresholds: 0.145, 0.22, 0.33, 0.5, and 0.75 pA. (C) Data in the table show total and average dwell times (ms) for each of the thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP. The bin width is 0.01 pA. Only events longer than 1.5 ms were used. Data were filtered at 500 Hz. 122   Figure 4.6: 8-CPT-cAMP shortens the first latency of EQQQQ single-channels.  Currents were recorded at room temperature. (A) Representative 4 s single-channel sweeps of control (left panel) and 8-CPT-cAMP/OA (right panel). Data were filtered at 200 Hz. (B) All-point event histograms of 10 active sweeps in control (left panel) and 8-CPT-cAMP/OA (right panel). Inset panels show the enlarged foot of each histogram. (C) A cumulative latency histogram showing that the mean first latency for the 50 active control sweeps (black line and arrow) of 171 was 0.94 ± 0.09 s. In 200 μM 8-CPT-cAMP/0.2 μM OA (gray line and arrow), mean first latency for the 62 active sweeps of 314 was 0.56 ± 0.07 s; p-value = <0.0001 (see Table 4.2). Sweeps without activity were given a first latency >4 s; arrows indicate sweep total in each case. Note split-scale ordinate.   In Figure 4.4A, representative EQQ single-channel traces are shown for both control (left panel) and 8-CPT-cAMP (right panel) environments. The all-points histograms in Figure 4.4B display the number of events recorded in each 0.01 pA amplitude bin. In 123  control sweeps, the peak of the main Gaussian distribution was at 0.17 pA, and in 8-CPT-cAMP, the peak was at 0.18 pA. There did not appear to be any great change in the distribution of events like that seen previously with the 4:4 stoichiometries of EQ and KCNQ1 + KCNE1 in Chapter 2 (Thompson et al., 2017). There was a subtle decrease in the smaller subconductance events (0.08 pA) and an increase in events around 0.2 pA.  Although there was no significant change in amplitude, the channel opened significantly more quickly in the presence of 8-CPT-cAMP, which is also illustrated in the representative sweeps in Figure 4.4A. This was quantified as a significant reduction in the time to first opening of the channel in the presence of 8-CPT-cAMP (Fig. 4.4C). The average first latencies of 107 active control sweeps was 0.90 s, whereas the average first latency of 129 active sweeps in 8-CPT-cAMP was 0.71 s (Fig. 4.4C; Table 4.2, p-value = 0.0032).         124  Table 4.2: First Latency Data for KCNQ1 (Q1) + KCNE1 (E1), EQ, EQQ, and EQQQQ, Before and After 200 μM 8-CPT-cAMP/0.2 μM OA   Control 8-CPT-cAMP/OA  Construct Mean First Latency (s) ± SE 95% Confidence Interval Of The Mean (lower-upper) Median (s) # Active Sweeps # Total Sweeps Mean First Latency (s) ± SE 95% Confidence Interval Of The Mean (lower-upper) Median (s) # Active Sweeps # Total Sweeps p-value n (cells) *Q1 + E1 1.32 ± 0.13 1.06 - 1.57 0.90 68 278 0.79 ± 0.08 0.63 - 0.95 0.50 104 309 0.0002 3 *EQ 1.61 ± 0.13 1.35 - 1.87 1.57 43 367 1.06 ± 0.11 0.84 - 1.27 0.85 69 345 0.0005 3 EQQ 0.90 ± 0.07 0.76 - 1.0 0.64 107 259 0.71 ± 0.06 0.58 – 0.84 0.46 129 378 0.0032 4-5 EQQQQ 0.94 ± 0.09 0.76 – 1.1 0.76 50 171 0.58 ± 0.07 0.41 – 0.70 0.38 62 314 <0.0001 4-5  First latencies were obtained from 4 s sweeps pulsed to +60 mV. Only active sweeps used for average. P-values compare control and 8-CPT-cAMP/OA group using the Mann-Whitney test. * indicates data taken from Chapter 2 (Thompson et al., 2017).      125  The number of active sweeps did not increase in the presence of 8-CPT-cAMP. In control, there were 107 active sweeps out of 259 (∼41% active), and in 8-CPT-cAMP, there were 129/378 (∼34% active) (Fig. 4.4C; Table 4.2). This is unlike the saturated 4:4 complex, EQ, which showed a large increase in the number of active sweeps from 43/367 to 69/345 as shown in Chapter 2 and (Thompson et al., 2017).  An analysis of the dwell times in five identifiable open states of EQQ channels before and in the presence of 8-CPT-cAMP is shown in Figure 4.5. These averaged data were obtained from 14 active sweeps in each case and show only small differences induced by the presence of cAMP. The all-points and idealized histograms clearly diverge at the higher open levels (Fig. 4.5), and this corresponds to an increased occupancy of the 0.5 and 0.75 pA levels (Fig. 4.5C). At the lower levels, no differences were observed, and data are remarkably concordant before and after 8-CPT-cAMP.  EQQQQ single-channel responses to 8-CPT-cAMP are described in Figure 4.6. Representative traces before (left panel) and after 8-CPT-cAMP (right panel) are shown in Figure 4.6A. The EQQQQ single-channel open amplitudes are very small in the presence of only one KCNE1 subunit—so small that the main opening peak is hard to distinguish from the closed peak (Fig. 4.6B). The all-points histograms do show a small decrease in the number of closed events and a small increase in the number of active events with 8-CPT-cAMP (Fig. 4.6B), but unlike EQQ constructs, there was no increase in range of amplitudes visited between control (Fig. 4.6B, left panel inset) and 8-CPT-cAMP (Fig. 4.6B, right panel inset). In both control and 8-CPT-cAMP, EQQQQ opening 126  events larger than 0.2 pA were very rarely seen. However, like EQQ, EQ, and KCNQ1 + KCNE1, EQQQQ channel first latency to opening was reduced upon 8-CPT-cAMP exposure (Fig. 4.6C). The average first latency in 50 active control sweeps was 0.94 s compared to 0.56 s in 62 active sweeps in the presence of 8-CPT-cAMP (Fig. 4.6C; Table 4.2, p < 0.0001). As for EQQ, the number of active sweeps after 8-CPT-cAMP did not increase (50/171 in control and 62/314 in 8-CPT-cAMP, Table 4.2).  Ensemble averages of EQ, EQQ, and EQQQQ single-channel sweeps are shown in Appendix B, Figure 4A. EQ, EQQ, and EQQQQ all show faster activation kinetics in the presence of 8-CPT-cAMP compared with control, which is consistent with a shortening of the first latency for all three constructs. Activation of whole-cell currents also appears to be faster in EQ, EQQ, and EQQQQ in the presence of 8-CPT-cAMP, as shown by the representative sweeps in Appendix B, Figure 4B.  4.4 Discussion The functional effect of phosphorylation of KCNQ1 in response to β-adrenergic stimulation has long been thought to require the KCNE1 subunit to be present. Under sympathetic stimulation, the S27 and possibly S92 residues in the N-terminal region of the KCNQ1 subunit become phosphorylated (Marx et al., 2002; Kurokawa et al., 2003; Lopes et al., 2007; Lundby et al., 2013). However, phosphorylation is not enough to produce a functional effect, as KCNE1 is also a requirement (Kurokawa et al., 2003). In Figure 4.1, data show that there was no change in the V1/2 or increase in current when KCNQ1 alone was exposed to 8-CPT-cAMP, in agreement with previous 127  studies that have also shown no functional response in the absence of KCNE1 despite KCNQ1 still being phosphorylated (Kurokawa et al., 2003). There are several LQT missense mutations in the KCNE1 C-terminal region that have an impaired β-adrenergic response, such as D76N, and deletions within the KCNE1 C-terminus (Takumi et al., 1991; Sesti and Goldstein, 1998; Kurokawa et al., 2003; Chen et al., 2009; Kurokawa et al., 2009; Dvir et al., 2014) have similarly led to the conclusion that the KCNE1 C-terminus is important in the sympathetic response of IKs to cAMP.  The present experiments confirm the importance of the KCNE1 subunit in the response of IKs to 8-CPT-cAMP. As stated above, the KCNQ1 tetramer alone is unresponsive to cAMP exposure (Fig. 4.1). It has also previously been shown that a 2:4 KCNE1:KCNQ1 stoichiometry showed a 34% reduction in the current amplitude response to cAMP in Chinese hamster ovary cells compared with a 4:4 KCNE1:KCNQ1 IKs construct or independently transfected KCNQ1 + KCNE1 (Kurokawa et al., 2003). This result is comparable with some of the findings of this study. In data from whole-cell experiments, exposure to 8-CPT-cAMP also produced a ∼35% increase in current at +60 mV in the EQQ (2:4 KCNE1:KCNQ1) construct, although this was often not sustained (Fig. 4.2B). This increase is rather less than that seen in the EQ construct (Appendix B, Fig. 3). In our experiments, the increased current in the EQQ construct was associated with a steepening of the G-V relationship (Fig. 4.2D; Table 4.1) and a −13 mV hyperpolarization of the V1/2. This may be compared with a −23 mV hyperpolarization in the EQ construct and −18 mV in independently expressed KCNQ1 and KCNE1 (Table 4.1). 128  It appears that KCNE1 subunits depolarize the V1/2 of activation of KCNQ1 in a graded manner so that the more KCNE1 subunits in the complex, the more depolarized the V1/2 (Appendix B, Fig. 5B). Phosphorylation by PKA antagonizes this shift, and the magnitude of the hyperpolarizing response to cAMP is reduced as the number of KCNE1 subunits is reduced. This seems to imply that phosphorylation is directly acting to reverse the depolarizing action of KCNE1 subunits on the V1/2. However, in extremely rare longer-lasting cells of both EQ and EQQQQ, 8-CPT-cAMP hyperpolarized the V1/2 of activation to ∼−10 mV, which suggests that perhaps all the constructs are able to converge around this value, upon phosphorylation. An example of this effect in EQ is shown in Appendix B, Figure 6, in which an initial V1/2 of activation of +36 mV shifts to −11 mV as time progresses. KCNQ1 alone has a V1/2 of activation of −22 mV, and a shift that negative was not seen in any of our KCNE1-containing constructs after cAMP (Appendix B, Fig. 5B). Such long time-course data are experimentally difficult to obtain, but we can speculate that the hyperpolarizing limit of the cAMP response may be at ∼−10 mV and might explain the apparent convergence of the first latencies between the different constructs (Appendix B, Fig. 5A; Table 4.2). From the cumulated data, a linear relationship exists between the V1/2-values of EQ, EQQ, and EQQQQ in the absence and presence of 8-CPT-cAMP (Appendix B, Fig. 5A). But this linear relationship does not extend to KCNQ1 alone, as a gap of around 10 mV is seen between the KCNQ1 V1/2 and the most hyperpolarized V1/2 seen in response to cAMP in the presence of KCNE1 (Appendix B, Fig. 5B). Given the discussion above, this suggests that although cAMP appears to reverse the depolarizing action of KCNE1 subunits, the two phenomena are not directly related. 129  Experiments with the EQQQQ construct, in which only one KCNE1 subunit is present in the IKs complex, did not show a significant effect on the current amplitude at the whole-cell level (Fig. 4.3, A and B) but did show a small but significant hyperpolarizing shift in the V1/2 of activation. As the number of KCNE1 subunits present in the IKs complex decreases, the amplitude response and the hyperpolarizing shift of the activation V1/2 is reduced. These data all support the idea that the degree of response to cAMP is graded to the number of KCNE1 accessory subunits present (although a 3:4 KCNE1:KCNQ1 construct was not tested) and that at least one KCNE1 subunit is required to observe the effects of cAMP (Appendix B, Fig. 5).  Many of the whole-cell findings were mirrored in the single-channel data. One of the important characteristics of the IKs current complex is its low open probability. However, EQ and KCNQ1 + KCNE1 expressed separately become more active in response to 8-CPT-cAMP and show an increase in the number of active sweeps per patch (See Chapter 2) (Thompson et al., 2017). This was not seen with either EQQ (Fig. 4.4C) or EQQQQ (Fig. 4.6C). In control EQQ, ∼41% of sweeps showed IKs activity, but in the presence of 8-CPT-cAMP, this decreased to ∼34% (Fig. 4.4C; Table 4.2). EQQQQ showed a similar reduction in active sweeps from ∼29% in control to ∼20% in 8-CPT-cAMP (Fig. 4.5C; Table 4.2). As previously shown in Chapter 2 and (Murray et al., 2016; Thompson et al., 2017), EQ (4:4) is able to visit many different subconductance levels with the main peak around 0.45 pA, as also seen when KCNQ1 and KCNE1 are expressed independently. However, EQQ and EQQQQ both no longer consistently reach the main open amplitude, with EQQ rarely having events at 0.5 pA and EQQQQ not at 130  all. The occupancy of the higher amplitudes was increased in EQQ in the presence of 8-CPT-cAMP but only reached 2.6% of the total dwell time, and there was no effect for EQQQQ. However, there was an increase in the number of events for both EQQ and EQQQQ due to the shorter first latencies (Figs. 4.4 and 4.5B).  Interestingly, the response of the first latency to channel opening to 8-CPT-cAMP was not affected by subunit composition in these experiments. The first latency was reduced by cAMP in EQ by 35% and in KCNQ1 and KCNE1 expressed separately by 41% (Table 4.2), compared with 22% in EQQ and 39% in EQQQQ (Figs. 4.4C and 4.6C). The robust reduction in first latency seen in the single-channel data from the EQQ and EQQQQ constructs suggests that phosphorylation may have both KCNE1-dependent and independent actions on the gating of the IKs producing channel complexes. Given the similar reduction in first latency across the constructs, this effect could be an all-or-none response to phosphorylation by PKA. KCNQ1 alone may also be affected, but we are not able to reliably determine its first latency. When the IKs channel first opens, it characteristically does so to very small amplitudes, representing the lowest subconductance levels (Werry et al., 2013), as also clearly seen in Figure 4.4A. It is possible that the modulation by 8-CPT-cAMP of the earliest steps in channel activation that lead to the first subconductance level openings are not as dependent on the presence of KCNE1 as openings to the higher subconductance levels. This finding is consistent with the idea that KCNE1 itself does not affect the rate of voltage-sensor activation in the KCNQ1 subunit (for review, see Liin et al., (2015)).  131  4.4.1 Mechanisms for the KCNE1-dependent action of 8-CPT-cAMP Previous studies have shown that the C-termini of KCNE1 and KCNQ1 interact with each other to cause a conformational change in the KCNQ1 subunit that brings the N- and C-termini closer together as the channel opens (Zheng et al., 2010). This step has been thought to be crucial for the phosphorylation of serine residues in the N-terminus of KCNQ1. However, these residues have been shown to still be phosphorylated when KCNE1 is not present or when the KCNE1 C-terminus is truncated (Δ109–129 (Dvir et al., 2014)). This suggests that both the N- and C-termini can come close enough for phosphorylation to occur without KCNE1. When the KCNE1 C-terminus is truncated, not present, or has one of a few known point mutations, there is no longer an effect of this phosphorylation (Kurokawa et al., 2003; Haitin et al., 2009; Kurokawa et al., 2009; Dvir et al., 2014). The interaction between the C-termini of KCNQ1 and KCNE1 may not be facilitating phosphorylation but rather allowing another conformational change to occur that allows the KCNE1 subunit to modulate the interaction between the voltage-sensor domains and the pore domains of the channel complex, perhaps by changing the affinity of KCNQ1 to phosphatidylinositol 4,5-bisphosphate (PIP2) (Zaydman et al., 2014), among other mechanisms (Cui, 2016).  KCNE1 D76N is an LQT mutation that does not respond to cAMP, has much faster deactivation, and causes a ∼35 mV depolarization in the V1/2 of activation (Chen et al., 2009). Both the KCNE1 C-terminus and KCNQ1 can still associate with each other, and phosphorylation of KCNQ1 is still observed, but there is no functional response to phosphorylation by PKA. The movement of the KCNE1 and KCNQ1 C-termini coming 132  closer together as seen in the wild type is no longer observed with this mutant (Chen et al., 2009). Chen et al. suggested that the change from an acidic to a polar, uncharged R-group may result in an KCNE1 C-terminus that cannot adequately slow deactivation or stabilize the open state of the channel (Chen et al., 2009).  An NMR study (Kang et al., 2008) of the distal KCNE1 C-terminus (residues 106–129) suggests that it is quite disordered and flexible. Shortening of the KCNE1 C-terminus (Δ69–77) was found to increase the binding of both KCNE1 and KCNQ1 C-termini compared to the wild type. Molecular modeling has suggested that the KCNE1 C-terminus is able to fold back toward the membrane, which could allow it to interact with the S4-S5 linker and/or the pore (Kang et al., 2008). Using a cysteine scanning method, Lvov et al. postulated that the KCNE1 C-terminus could interact with the S6 activation gate and/or the S4-S5 linker of KCNQ1 (Lvov et al., 2010). This flexible tail may allow KCNE1 the freedom/ability to stabilize IKs channel open states or destabilize closed states. If KCNE1 is able to cause a conformational change in response to phosphorylation of KCNQ1, then this may explain the graded response to 8-CPT-cAMP as additional KCNE1 subunits are present. What is clear is that voltage sensor to pore coupling in KCNQ1 is strongly regulated by the presence of KCNE1 (Cui, 2016), and thus, changes in KCNQ1 after phosphorylation require KCNE1 to be present to be transduced into higher occupancy of subconductance states later in the channel-activation process.  133  4.4.2 IKs in the heart In normal human cardiac myocytes, the IKs current is clearly important because of the high number of LQTS mutations in KCNQ1 (and KCNE1), which have a phenotype, but as yet, even the physiological composition of the channel complex remains uncertain. One study recorded a current-voltage relationship for IKs from human left ventricular myocytes that appeared to have a V1/2 of activation of ∼5 mV (Virág et al., 2001). Importantly, for our study, these data were collected from cells exposed to forskolin, which was required to increase the amplitude of IKs to enable the study. EQQ and EQQQQ exposed to 8-CPT-cAMP have V1/2-values of activation of ∼−3 mV, and EQ is ∼5 mV at room temperature (Table 4.1) but could be as hyperpolarized as −10 mV (Appendix B, Fig. 3). An increase in recording temperature leads to an additional ∼10 mV shift in the hyperpolarizing direction (Eldstrom et al., 2010). Together, these data suggest that the stoichiometry in the human left ventricle could well include a 4:4 ratio of KCNQ1:KCNE1.  4.5 Conclusions At least one KCNE1 subunit appears to be needed to observe a functional response to 8-CPT-cAMP. At the whole-cell level, with only one KCNE1 subunit, the EQQQQ channel shows smaller changes in the V1/2 of activation in response to PKA phosphorylation compared with the fully saturated EQ complex. The actions of 8-CPT-cAMP are graded depending on the number of KCNE1s present, and this graded effect indicates that complexes with fewer KCNE1s may show a blunted sympathetic response. At the single-channel level, changes in subconductance occupancy and channel activity mirror the whole-cell effects of reduced numbers of KCNE1 in 134  the IKs complex. However, changes in the latency to first opening of IKs, induced by cAMP, were not affected by the number of KCNE1 subunits present, which has highlighted the possibility of KCNE1-dependent and KCNE1-independent actions of 8-CPT-cAMP on the gating of IKs channel complex.     135  Chapter 5: Discussion 5.1 Summary of thesis findings The increase in IKs current seen under b-adrenergic stimulation is important at high heart rates for its role in the repolarization reserve and termination of the cardiac action potential. The response of IKs to PKA phosphorylation had been characterized from the macroscopic level, but the work in this thesis was planned to investigate the microscopic changes and the mechanisms behind the current increase seen at the whole-cell level. Previous work had established that upon b-adrenergic stimulation, the current amplitude of IKs increases, activation is faster, deactivation is slowed, and the V1/2 of activation is hyperpolarized, which results in a shortening in the APD (Dilly et al., 2004; Terrenoire et al., 2005). An immunoprecipitation study conducted by Nicolas et al., (2008) in COS-7 cells using a VSV tagged KCNQ1-KCNE1 fusion protein, found no increase in the expression of IKs complexes at the cell membrane when cAMP was present (Nicolas et al., 2008). In a study by Andersen et al. (2015), in Madin-Darby canine kidney cells stably expressing GFP tagged KCNQ1 channels, confocal microscopy was used to show that PKA phosphorylation increased the expression of KCNQ1 subunits and inhibition of PKA induces endocytosis of KCNQ1 (Andersen et al., 2015). Neither looked at the expression of both KCNQ1 and KCNE1 subunits expressed separately.   The experiments conducted in Chapter 2 were designed to investigate how the IKs channel complex responds to PKA phosphorylation at the microscopic level. Using single-channel recording and TIRF microscopy in CHO cells, we were able to elucidate what happens mechanistically to the channel that results in an increase in both current size 136  and conductance, as well its increased activation. We found using TIRF microscopy that there was no change in the cell surface expression of either the KCNQ1 or KCNE1 subunits following 8-CPT-cAMP addition, although there were dynamic movements of channel complexes to and from different regions of the imaged cells. This result shows that the increase in IKs is not a result of an overall increase in the number of channels at the cell surface, which has been shown to be the case in other channels and transporters (Gonin et al., 2001).   Our single-channel recordings demonstrated that 8-CPT-cAMP has many effects on the channel complex. The first finding is that the channel displays a reduced first latency to opening, which results in the channels opening more quickly than WT control channels. This shows a channel that activates faster, which reflects what has been shown previously at the macroscopic level (Terrenoire et al., 2005). The second finding at the single channel level was that the Po of the channel increased by almost 2.5 times in the presence of 8-CPT-cAMP and the number of active sweeps increased by ~70%. It had been previously thought that the Po of the channel was increased, as the rate of deactivation is slower in the presence of cAMP and the channel is more likely to stay open. The third finding of our single-channel recordings was that in the presence of 8-CPT-cAMP, the channel occupies the higher subconducting states more often than in control. The total dwell times of the two largest subconducting states, doubled in the presence of 8-CPT-cAMP. This shift in subconducting activity had not been seen before. EQ channels also spent less time in the closed state, which shows the channel’s shift in the presence of 8-CPT-cAMP towards its activated state.  137  Further analysis of the single-channel behaviour showed that the burst kinetics of IKs were not affected, which suggests that the pore is largely unaffected by 8-CPT-cAMP. However, through enhanced gating mutations, particularly E160R/ R237E, we found that the effects of 8-CPT-cAMP seen are most likely a result of enhanced voltage sensor activation. These changes in the channels’ kinetics in response to PKA phosphorylation results in a larger, faster activating current, observed at the macroscopic level.   Chapter 2 also investigated the residues at which PKA phosphorylation occurs. The most well described phosphorylation site is Ser27(Marx et al., 2002; Lopes et al., 2007; Zheng et al., 2010; Lundby et al., 2013). When Ser27 is mutated to an alanine (A), the large increase in current size is no longer seen upon cAMP addition. Mutating the serine to aspartic acid (D), recapitulates some of the effects that cAMP has on the channel, but not all (Kurokawa et al., 2003). We used this phosphomimetic mutant, KCNQ1 S27D, and found that it was indeed still sensitive to 8-CPT-cAMP. Similar to previous whole-cell recordings, our macropatch data showed an initial V1/2 of activation which was more hyperpolarized than the EQ control, but 8-CPT-cAMP could still further hyperpolarize the V1/2 of S27D. From our single-channel recordings, we saw that this mutant had an increased Po similar to EQ + 8-CPT-cAMP, as well a similar subconductance occupancy shift towards the higher conducting levels. However, the first latency of the channel was unchanged from EQ control and did become shorter when 8-CPT-cAMP was added. This suggests that Ser27 may be responsible for the increase in Po seen upon cAMP addition and at least partially responsible for the increased activity at higher subconducting levels. 138  Another putative phosphorylation site is Ser92, which has been shown to be phosphorylated in IKs channel complex. When Ser92 is mutated to aspartic acid together with S27D, a current which is no longer responsive to 8-CPT-cAMP is produced (Lopes et al., 2007). We also found KCNQ1 S27D/S92D to be non-responsive to 8-CPT-cAMP. No changes in first latency, amplitude, subconducting states, or V1/2 of activation were seen. The double mutant did, however, have many more active sweeps than EQ control and was also more active than EQ + 8-CPT-cAMP. The first latency was not changed from control and was unaffected by cAMP.   In Chapter 3, we further investigated the effects of 8-CPT-cAMP on S27D/S92D by performing a subconductance analysis. Previous studies had shown no effect of cAMP on this channel. We wanted to see if 8-CPT-cAMP did have any effect on the open and closed dwell times. We found that there were no observable changes in the number of events or total time spent at any sublevel. KCNQ1 S27D/S92D + KCNE1 showed no kinetic changes in response to cAMP, suggesting that both of these residues are phosphorylated. However, this double mutant does not recapitulate the kinetic effect seen upon PKA phosphorylation of WT channels. In Chapter 3, we also analyzed further the kinetic behavior of another KCNQ1 mutant, S209F, which produces a channel with enhanced gating and a high Po. During our analysis in Chapter 2, we noted that this current did still respond to 8-CPT-cAMP, and we wanted to see the extent of this response by performing subconductance analysis. We found that 8-CPT-cAMP increased activity and total dwell time at the higher subconducting states but did not affect the burst kinetics 139  of the channel. 8-CPT-cAMP further enhanced voltage-sensor activation in this enhanced gating channel.  Chapter 4 investigated the effect that varying the number of KCNE1 subunits has on the channel complex’s response to cAMP. Previously Kurokawa et al., (2003) had shown that KCNE1 was required for a functional effect of PKA phosphorylation to be seen (Kurokawa et al., 2003). They also showed, using a fusion construct of one KCNE1 subunit fused to two KCNQ1 subunits to produce a 2:4 stoichiometry similar to our EQQ, that the current increase in response to cAMP was still present but to a much smaller extent (0.5-fold increase as opposed to 2.5-fold increase seen with WT). Their EQQ did have different kinetic properties to ours. We confirmed that KCNQ1 alone did not respond to 8-CPT-cAMP and that whole-cell recordings also showed that, as the number of KCNE1 subunits is reduced, a gradual hyperpolarization of the V1/2 of activation is seen. At the single-cell level, a similar graded effect was seen in response to cAMP concerning subconductance occupancy and channel activity; the fewer KCNE1 subunits, the smaller the response. The EQQ (2:4) channel complex showed a small shift in subconducting activity with a reduction in activity in the small subconducting states moving to higher sublevels. There was however no increase in the number of active sweeps seen in EQQ after 8-CPT-cAMP, but EQQ is more active than the EQ control in general. However, while there was an E1-dependent effect of phosphorylation, the first latency to opening for all KCNQ1-KCNE1 complexes were reduced in a non-graded manner, suggesting that this effect was not dependent on the number of KCNE1 subunits present.   140  5.2 Different pharmacological sensitivity of IKs when PKA phosphorylated Loss of function mutations of KCNQ1 and KCNE1 can lead to LQT syndrome, type 1 and 5 respectively. As a result, the identification and development of IKs activators has been of interest. However, the pharmacological sensitivity of the IKs activators is in many cases quite dependent on the number of KCNE1 subunits in the channel complex. cAMP activates the IKs current as a result of phosphorylation of PKA as shown in Chapter 2.  However, as shown in Chapter 4, cAMP has a graded effect on the channel and is dependent on the number of KCNE1 subunits. The greatest effect is seen when more KCNE1s are present in the complex. Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), shows a similar effect to cAMP. Mefenamic acid has no effect on KCNQ1 alone, but does stabilize the channel in the open state, increase the IKs current and slow deactivation (Busch et al., 1994; Busch et al., 1997; Toyoda et al., 2006). This increase in potency of the drug may be as a result of a conformational change that occurs when KCNE1 is bound, which reveals a novel binding site or increases the affinity of the channel for the drug.   The stoichiometry of the channel can affect its pharmacological sensitivity. A similar suggestion has been made for the channel’s phosphorylated state. Blockers of IKs, chromanol-293B and quinidine, have both shown a reduced inhibitory effect on PKA phosphorylated channels compared to WT (Yang et al., 2003). It has been suggested that this is possibly due to conformational changes that occur during phosphorylation, which may affect the binding sites of these drugs or reduce their affinity. This suggests that phosphorylated channels, similar to IKs channel with different stoichiometries, can have 141  different pharmacological sensitivities and that the different phosphorylated states should be considered when novel drugs or the efficacy of an existing drug is being investigated.  5.3 Species variation of IKs in the heart The experiments conducted in Chapter 4 show that the stoichiometry of the IKs channel complex is important for how the channel responds to PKA phosphorylation. IKs channel complexes that are not fully saturated with KCNE1 may produce a blunted response to PKA phosphorylation. If this is the case, PKA phosphorylation may provide an indication of the in vivo stoichiometry. It is well-known that the expression levels of KCNQ1 and KCNE1 varies between species, but it is also thought that the in vivo stoichiometry may also vary between species.   Mice and rabbits have been shown to typically express lower levels of IKs compared to guinea pigs which are known to express more IKs (Lu et al., 2001; Zicha et al., 2003). In guinea pig ventricular myocytes, under basal conditions, inhibition of IKs greatly prolonged the cardiac action potential, which shows that even without b-adrenergic stimulation IKs contributes a significant amount to the IK in guinea pigs (Lu et al., 2001; Volders et al., 2003). However, in other species such as canines, humans and rabbits, the role of IKs in the heart at the basal level is considered negligible as the inhibition of IKs only prolongs cardiac action potential when the IKs current is under b-adrenergic stimulation.   The stoichiometry of IKs will most likely vary based on the expression levels of the two subunits. We know that expression levels vary between species; mice and rabbits 142  typically have low KCNE1 expression levels and guinea pigs express more (Lu et al., 2001; Zicha et al., 2003). As shown in Chapter 4 and in (Murray et al., 2016) tandem KCNQ1-KCNE1 constructs with fixed stoichiometries have suggested that the V1/2 of activation is depolarized as the number of KCNE1 subunits is increased in the channel complex. The V1/2 of activation for IKs differs substantially between guinea pig and rabbit. At a recording temperature of 35°C, guinea pig ventricular myocytes have a more depolarized V1/2 of ~18 mV compared with ~-1mV, which is seen in rabbit ventricular myocytes (Lu et al., 2001). This suggests that these species may have different stoichiometries, with the guinea pig most likely having more KCNE1 than that of the rabbit. This is of course speculative, and more studies need to be carried out to fully elucidate species specific stoichiometry.  The results from Chapter 4, suggest that if there is stoichiometric difference between these species, there may be a difference in how IKs responds to PKA phosphorylation in different species. A number of studies have shown that b-adrenergic stimulation of IKs results in an increase in IKs current in canines, humans, rabbits, guinea pigs, and mice (Lu et al., 2001; Virág et al., 2001; Obreztchikova et al., 2003; Volders et al., 2003). Most studies report an increase in the activation kinetics of the current as well as a slower rate of deactivation. However, for a number of these studies an accurate V1/2 could not be determined as the G-V relationships do not saturate.  143  5.4 Variation in stoichiometry of IKs in the diseased heart Expression levels of KCNQ1 and KCNE1 have been shown to fluctuate in different disease states. Several animal studies of heart failure have found that the IKs current is downregulated (Ramakers et al., 2003; Aflaki et al., 2014; Hegyi et al., 2018). Interestingly, in patients with congestive heart failure, KCNE1 total RNA levels were significantly elevated (Watanabe et al., 2007). However, total RNA levels do not necessarily predict protein expression levels and as mentioned above, protein expression of KCNE1 is reduced in animal models (Aflaki et al., 2014). During heart failure, the sympathetic nervous system activity increases to maintain cardiac output by increasing the number of catecholamines in circulation, such as norepinephrine. Initially, this is beneficial and helps stimulate the heart to contract. However, chronic exposure to catecholamines causes b-adrenergic receptors to become dysfunctional; b1 receptors are downregulated by ~50% at the cell surface and both b1 and b2 receptors can become uncoupled from G proteins (de Lucia et al., 2018). Sustained b-adrenergic stimulation has been shown to reduce IKs current in guinea pigs by ~60% (Aflaki et al., 2014). In a rabbit model of heart failure, under basal conditions IKs is upregulated by calcium/CaM to compensate for the reduction in IK1 current. However, when under b-adrenergic stimulation, IKs is much less responsive and the repolarization reserve is severely reduced. IKs upregulation by calcium/CaM is independent of PKA phosphorylation (Hegyi et al., 2018). The decrease in b1 receptors may be a reason for this reduced response to PKA, but it could also be as a result of a change in the channel’s stoichiometry. If KCNE1 144  protein expression is reduced in heart failure, channel complexes with less KCNE1 subunits may be produced, which may have a blunted response to PKA phosphorylation.   5.5 Enhanced PIP2 affinity in PKA phosphorylated channels The KCNE1 C-terminus is key to the functional response of IKs to PKA phosphorylation. Mutations in, as well as truncation of KCNE1’s C-terminus removes the channel’s response to cAMP. The C-termini of both KCNQ1 and KCNE1 interact with one another, which results in a conformational change in the channel complex (Zheng et al., 2010). This conformational change may allow KCNE1 subunits the ability to modulate the interaction between the VSD and the pore (Lvov et al., 2010). One mechanism by which KCNE1 may be modulating this interaction between the VSD and pore is by altering the channel’s affinity for PIP2, which is required for the coupling of the VSD to the PD (Zaydman et al., 2013). When PIP2 is unbound from the channel, the voltage sensor can uncouple from the pore and as a result can be in its activated state, but the pore remains closed (Sun and MacKinnon, 2017); PIP2 is required for channel pore opening. Similar to PKA phosphorylation, PIP2 sensitivity is significantly affected by KCNE1. IKs is ~100-fold more sensitive to PIP2 than KCNQ1 alone (Li et al., 2011a). KCNE1’s C-terminus is suggested to be key in regard to the channel’s sensitivity to PIP2. PIP2 has several putative binding sites in KCNQ1; S2-S3 linker, S4-S5 linker and the KCNQ1 C-terminus, where groups of basic residues are found. Both PIP2 modulation and PKA regulation of the IKs current are critically dependent on the interaction between KCNQ1 and KCNE1 subunits and the C-terminus of KCNE1 (Dvir et al., 2014). 145  These two regulatory signaling pathways have been proposed to undergo crosstalk between each other. KCNQ1 phosphorylation by PKA has been suggested to modulate the channel’s affinity to PIP2. Lopes et al. (2007) found that IKs currents which were exposed to PKA phosphorylation prior to having the cell’s PIP2 levels depleted, experienced less current inhibition than in non-phosphorylated control cells. Consequently, an increase in the amount of current inhibition was seen when a PKA inhibitor was administered prior to PIP2 depletion. This was seen in both the Xenopus laevis oocytes and guinea pig isolated ventricular myocyte systems (Lopes et al., 2007). The interface at which the KCNQ1 and KCNE1 C-termini interact houses a number of LQT mutations which results in reduced PIP2 binding and reduced upregulation of IKs caused by PKA phosphorylation. Of particular interest are two LQT mutations, KCNQ1 R366Q and R555C, which are found in the proximal and distal KCNQ1 C-terminus respectively (Matavel et al., 2010). Both of these mutations were more sensitive to changes in PIP2 levels in the cell, which suggests that PIP2 binds to these channels with less affinity than to WT channels. PIP2 may dissociate from the channel more quickly and only partially bind making these mutants more sensitive to changes in PIP2 levels. However, these mutant channels were significantly more sensitive and produced a larger response to PKA phosphorylation than WT channels.   The presence of KCNE1 drastically increases the sensitivity of the channel to PIP2 and stabilizes the channel in the open state. As shown in Chapter 4, KCNE1 also increases the functional response to PKA phosphorylation; the more KCNE1 subunits, the greater the shift towards the open state. If the channel’s affinity to PIP2 is further strengthened by 146  the channel’s phosphorylated configuration, then perhaps the increase in Po and shift in subconducting behavior seen in response to PKA stimulation is a result of increased affinity to PIP2. Enhanced voltage sensor activation, which is seen in response to PKA could be mediated at least in part by PIP2 further enhancing the coupling between the VSD and the PD. The crosstalk between PKA and PIP2 could be responsible for the KCNE1-dependent effects of PKA.  5.6 Limitations As mentioned in the Chapter 1, the signal-to-noise ratio is a problem for single-channel analysis. This is particularly the case for IKs, whose single-channel current amplitude is small at ~0.45 pA under our recording conditions. This requires that, before acquisition, as many sources of noise are eliminated or reduced as much as possible to improve the signal-to-noise ratio and reduce the amount of post-acquisition filtering required to improve the resolution of the recording. We want to limit the amount of filtering required as filtering data results in a loss of brief events, which can result in an overestimation of the dwell times. The balance between the resolution of the recording and the amount of filtering required is important as noise can also create false events or disrupt real events.  Tethered constructs are a useful tool to investigate heteromeric channels and produce channels of fixed stoichiometries. The work in this thesis required the use of three concatemer constructs; EQ, EQQ, and EQQQQ, where the subunits are tethered together and assemble to produce channels with defined stoichiometries. While this technique is very useful and has been widely used, it does rely on the concatemers assembling 147  correctly, which for some constructs may not occur and more than one type of channel assembly can be seen. For EQ, EQQ, and EQQQQ, electrophysiological studies suggest that this is not the case and that the concatemers assemble to produce the desired stoichiometry as the conductance-voltage relationship (G-V curve) for these constructs are well fit with a single Boltzmann fit suggesting that there is a uniform population of channels for each construct. Pharmacological validation has also been used to determine the uniformity of the channel populations. WT IKs has a very low sensitivity to tetraethylammonium chloride (TEA+), however, the KCNQ1 V319Y mutation increases the current’s sensitivity to TEA+ (Kurokawa et al., 2001). In Westhoff et al. (2019), EQQ + E1 is unresponsive to TEA+, but when a TEA+ binding site is introduced (V319Y) into the first Q1 subunit of the construct, the current is sensitive to TEA+. The dose response curve was fit with a Hill slope which resulted in Hill coefficient of 1.3 suggesting that there is only one population of channels (Westhoff et al., 2019). If the channels were able to assemble in more than one configuration, which would most likely result in channel assemblies with differing numbers of TEA+ binding sites, a dose response curve with multiple phases would be produced.  In this study, the IKs single channels were recorded from cells in a heterologous expression system rather than cardiomyocytes. Heterologous expression systems allow for the desired currents to be expressed and studied but they may not express all the necessary components to recreate regulatory mechanisms in the cardiomyocyte which may be important for channel function/regulation. However, preforming single-channel recording on IKs channels in cardiomyocytes is very challenging and hard to obtain. 148  5.7 Future directions Our experiments suggest that PKA phosphorylation increases the current amplitude through phosphorylation of Ser27 and Ser92, which causes a conformational change resulting in enhanced voltage sensor activation. The mechanisms involved in this conformational shift have not been fully elucidated. To further identify and characterize key regions and specific interactions that are required for this conformational change, single-channel studies of LQT mutations with an impaired PKA response may be beneficial. A recent review by Policarova et al. (2019) stated that ~ <3% of known KCNQ1 mutations have had their PKA sensitivity tested for. Identifying and characterizing some of these mutations may lead to important information concerning how these KCNE1-dependent and independent effects are produced. As we have shown in Chapter 4, depending on the channel’s stoichiometry, a varied response to PKA can be seen. It would be very interesting to perform single-channel recording experiments similar to those done in Chapter 1, in cardiomyocytes where the channel complexes are in their ‘natural’ configuration. It would also be interesting to investigate the potential crosstalk between PKA regulation of the IKs channel complex and PIP2. This could possibly be done by using wortmannin, which inhibits PIP2 synthesis, on IKs single-channel recordings that have either been exposed to 8-CPT-cAMP prior to the experiment or not exposed.   5.8 Conclusion The work produced in this thesis was performed to better understand the mechanisms underlying the kinetic changes in the IKs channel complex, triggered by sympathetic stimulation, that result in an increased current required for the initiation and completion of 149  cardiac repolarization. From our single-channels recordings of IKs, we can see that PKA phosphorylation increases the channel’s open probability, which results in IKs becoming more active. 8-CPT-cAMP also causes the channel to open more quickly upon depolarization and once open more activity is seen at the higher subconductance states. All of these effects result in an increase in current passed by the channel and reflects what is seen at the macroscopic level. The hyperpolarizing shift in the V1/2 of activation caused by 8-CPT-cAMP is reflected by the reduced first latency to opening. We suggested that the effect of PKA phosphorylation is through enhanced voltage sensor activation. Many of the effects seen with 8-CPT-cAMP were dependent on the stoichiometry of KCNE1 in the channel complex with the largest effect of 8-CPT-cAMP seen in channel complexes saturated with KCNE1 subunits. 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With 200 µM 8-CPT-cAMP/0.2 µM OA, mean first latency for the 104 active sweeps of 309 was 0.79 ± 0.08 s; P = 0.0002 (see Table 2.1). Sweeps without activity were given a first latency >4 s. Note split-scale ordinate.  181   Appendix A, Figure 2: 8-CPT-cAMP increases the number of active channels and causes a hyperpolarizing shift of the V1/2 with Q1+E1.  (A) G-V curves from a macropatch before and after 8-CPT-cAMP exposure. Control (circles, V1/2 = 21.6 mV), 8-CPT-cAMP (squares, V1/2 = −23.9 mV). Inset shows macropatch currents in control and with 8-CPT-cAMP. Cells were held at −80 mV and pulsed from −60 mV to 80 mV in 10-mV steps (every other trace is shown). Tails were recorded at −40 mV for 900 ms. (B) Sample single-channel sweep from IKs plus Yotiao-expressing patch (control) and after adding 8-CPT-cAMP/OA. Bottom, ensemble average of 24 sweeps before and after 8-CPT-cAMP/OA addition. (C) All-points histograms of active sweeps in control (left, 24 sweeps) and when exposed to 8-CPT-cAMP (right, 24 sweeps). 182   Appendix A, Figure 3: Effects of cAMP on V1/2 of activation in macropatches.  (A) V1/2 of activation for EQ macropatches before (circles, mean V1/2 = 27 ± 3.82 mV) and after (squares, mean V1/2 = 2.07 ± 2.03 mV) 200 µM 8-CPT-cAMP/0.2 µM OA, n = 4. (B) V1/2 of activation for Q1 S27D+E1 macropatches before (circles, mean V1/2 = 13.1 ± 4.7 mV) and after (squares, mean V1/2 = −1.13 ± 8.24 mV) 200 µM 8-CPT-cAMP/0.2 µM OA, n = 3. 183   Appendix A, Figure 4: Subconductance analysis of S27D+E1 before and after 8-CPT-cAMP/OA.  (A) Raw all-points histograms of 13 control sweeps (blue) from an S27D+E1 patch and after 200 µM 8-CPT-cAMP/0.2 µM OA (red). (B) Idealized histograms of 13 control sweeps (blue) and 13 8-CPT-cAMP sweeps (red). Five thresholds were used for the idealization process: 0.145, 0.22, 0.33, 0.5, and 0.75 pA. Right, also 13 sweeps, but in the presence of 8-CPT-cAMP/OA. (C) The table shows total and mean dwell times (milliseconds) for each of the different thresholds, the percentage of time spent at each level, and the number of events at each threshold before and after 8-CPT-cAMP addition. The bin width is 0.01 pA. Only events longer than 1.5 ms were used. Data were filtered at 500 Hz. 184   Appendix A, Figure 5: S27D+E1 closed dwell times and burst analysis.  (A) Closed dwell time distributions for S27D+E1 from 13 sweeps before and 13 sweeps after 200 µM 8-CPT-cAMP/0.2 µM OA, filtered at 500 Hz. The data were fitted with the sum of two exponential functions. τ1: 0.82 ± 0.04 ms (AUC 236.61 ± 7.16) and τ2: 5.85 ± 0.34 ms (AUC 105.79 ± 6.69) in control. After 8-CPT-cAMP/OA, τ1: 1.14 ± 0.05 ms (AUC 258.48 ± 7.48) and τ2: 7.00 ± 0.59 ms (AUC 71.52 ± 7.18). Bin width was 1 ms. (B) Probability distribution of closed time durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. (C) Probability distribution of burst durations in control (black) and after 8-CPT-cAMP/OA (red), from data in A. Event histograms were fitted with the sum of two exponential functions. In control τ1: 1.26 ± 0.04 ms (AUC 663.79 ± 7.28) and τ2: 15.55 ± 2.53 ms (AUC 43.65 ± 5.87). In 8-CPT-cAMP, τ1: 1.23 ± 0.05 ms (AUC 452.64 ± 7.03) and τ2: 21.04 ± 2.89 ms (AUC 47.582 ± 4.95). Bin width was 2 ms. Only events longer than 1.5 ms in duration were used in this analysis. 185   Appendix A, Figure 6: Whole-cell currents from S209F+E1 and Yotiao are unaffected by 8-CPT-cAMP.  (A) Currents from S209F+E1 before (left) and after (right) 200 µM 8-CPT-cAMP + 0.2 µM OA. The holding potential was −80 mV, and the cell was pulsed for 1 s from −80 to 80 mV in 10-mV steps. Every other current record is shown. The tail current was recorded at −40 mV for 1 s. (B) S209F+E1 G-V relationship plotted using the tail currents, data from a single cell. G-V data for WT IKs is also included (V1/2 = 25.1 ± 2.5 mV, n = 11). (C) Diary plot of the peak S209F+E1 current at 60 mV before and after the addition of 200 µM 8-CPT-cAMP + 0.2 µM OA. Black line denotes the time at which 8-CPT-cAMP was present in the bath. 186   Appendix A, Figure 7: KCNQ1 channels are highly mobile in CHO cells.  (A) TIRF images of KCNQ1-GFP control at 0 min (left), 15 min (middle), and 15 min after addition of 200 µM 8-CPT-cAMP at 30 min (right). (B) Diary plots representing change in fluorescence of ROI 1 (left), ROI 2 (middle), and ROI 3 (right) over time. 200 µM 8-CPT-cAMP was added at 15 min. (C) Percentage change from baseline in different ROIs after control solution (left) and 8-CPT-cAMP (right) was added to the bath. Black lines overlaying points show means ± SE of the distributions. The cell is divided into many ROIs and grouped into either the border (red) or center (blue) of the cell at three different time points: 2, 5, and 10 min after either 8-CPT-cAMP or control vehicle was added. The number of ROIs in each group and time point is shown. Above the figure are the number of ROIs in which a significant change in fluorescence was seen, relative to the total number of ROIs observed. 187   Appendix A, Figure 8: Example of idealization of a single-channel current trace in Clampfit 10.5.  (A) Scaled version of a KCNQ1 + KCNE1 single-channel recording filtered at 1000 Hz, highlighting two >2-ms subconductance events (arrows). (B) Section of an EQ single-channel current record during a step to 60 mV over a period of 95 ms. The data were filtered at 500 Hz after acquisition at 2 kHz (red line). Dashed lines indicate the five conductance levels (0.145–0.75 pA) as well as the closed level. The idealized events are color coded by level (closed is blue, level 1 is red, level 2 is green, level 3 is maroon, level 4 is purple, and level 5 is tan). Two subconductance events are highlighted as having durations of >4 ms (identified by arrows), a level 1 and a level 3 event.     188  Appendix B: Supplementary Figures for Chapter 4.  Appendix B, Figure 1: Different filter frequency effects on single-channel records and all-points histograms.  (A) Two (left and right panels) +60 mV 4 s EQQ sweeps at different filter frequencies, 2 kHz filter at acquisition, further filtered at 1000, 500 and 200 Hz. Arrows point to potential sublevel events at 2 kHz, clarified by further filtering. (B) All-points histograms for 14 EQQ sweeps filtered at 1000, 500, and 200 Hz.  189   Appendix B, Figure 2: All-points histograms of EQQ at different filter frequencies. 14 active EQQ +60 mV 4 s sweeps acquired at 2 kHz, and then filtered at 1000 (top), 500 (middle), and 200 Hz (bottom). Gaussian fits of each all-points histogram are shown, and the amplitude of each main open peak was determined by Clampfit. 190   Appendix B, Figure 3: EQ V1/2 hyperpolarizes in response to 8-CPT-cAMP addition.  Currents were recorded at room temperature. (A) Representative traces for EQ with Yotiao before (control, black line) and after 200µM 8-CPT-cAMP/0.2 µM OA (gray line). Pulsed to +60 mV for 2 s and then to -40 mV for 900 ms. (B) Diary plot of the peak current of a 4 s +60 mV pulse over time. The addition of 200 µM 8-CPT-cAMP/0.2 µM OA is marked by a solid bar. (C) Representative traces are shown from every other voltage (Δ 20 mV) starting from -80 mV to +100 mV for both control (top panel) and in the presence of 200 µM 8-CPT-cAMP/0.2 µM OA (bottom panel). D. G-V curves recorded before (black circles) and after 8-CPT-cAMP/OA (gray triangles). Cells were held at −90 mV and pulsed from −80 mV to +100 mV in 10 mV steps for 4 s. Tail currents were recorded at −40 mV for 900 ms. Control V1/2: 26.7 mV ± 2.5 SE, n=6 and 8-CPT-cAMP V1/2: 4.2 mV ± 6.0 SE, n=4, unpaired t-test p-value: 0.0042. ∆ in V1/2 -22.5 mV ± 5.7 SE. 191   Appendix B, Figure 4: Activation is faster in the presence of 8-CPT-cAMP for EQ, EQQ and EQQQQ.  (A) Ensemble control (black) and 8-CPT-cAMP (red) averages of 10 active single-channel 4 s sweeps of EQ (top), EQQ (middle), and EQQQQ (bottom). (B) Representative whole cell current traces at +60 mV for 4 s. Control sweeps are shown in black and 8-CPT-cAMP sweeps in red.  192   Appendix B, Figure 5: Graded contribution of KCNE1 to PKA modulation of IKs.  (A) The average V1/2 of activation in control (top panel) and in the presence of 200 µM 8-CPT-cAMP/0.2 µM OA (bottom panel) is plotted against the number of E1 subunits present in the channel complex. A linear regression line was fit through the E1 containing constructs. When x=0 the y-intercept in control was -1.85 and -6.7 in the presence of 8-CPT-cAMP. (B) Individual V1/2s of activation per cell for each construct in control and in the presence of 8-CPT-cAMP/OA. The averages for these data are shown in Table 4.1 and used in panel A.  193   Appendix B, Figure 6: EQ V1/2 over time can become progressively more hyperpolarized.  Currents were recorded at room temperature. (A) Representative traces from every other activation voltage are shown (Δ 20 mV) starting from -80 mV to +80 mV except in the bottom panel where only up to +60 mV is shown. All panels shown are in the presence of 200 µM 8-CPT-cAMP and 0.2 µM OA. These activations were taken at different exposure times. The top activation is after 8 minutes of 8-CPT-cAMP/OA exposure, the second after 27 minutes, the third after 36 minutes, and the final activation after 54 minutes. (B) G-V curves were recorded at different exposure times to 8-CPT-cAMP ranging from 8 to 71 min. A progressive hyperpolarization of the V1/2 of activation from +36.85 mV to -11.16 mV is displayed below the G-V curves alongside the slope values (k) for each activation, which range from 20.4 to 15.2 mV. Cells were held at -90 mV and pulsed from -80 mV up to +110 mV in 10 mV steps for 4 s. Tail currents were recorded at −40 mV for 900 ms.   

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