Medicine, Faculty of
Medicine, Department of
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UBCV
Chan, Magnus
Doctor of Philosophy - PhD
University of British Columbia
The cardiac delayed rectifier potassium current, IKs, formed by the co-assembly of KCNQ1 and KCNE1 subunits, plays a critical role in cardiac repolarization, acting as a reserve to adapt to higher heart rates and compensating when other repolarization currents are impaired, thereby preventing arrhythmias. Loss of function mutations in these subunits impair cardiac repolarization and are associated with inherited long QT syndrome, increasing the risk of developing a polymorphic ventricular tachycardia known as Torsades de Pointes, which can result in syncope and sudden death. Therapeutic interventions targeting the IKs channel directly to reverse the malignant effects of long QT syndrome hold significant potential but have historically been challenging due to the previously unknown binding sites for IKs activators. In this thesis, I delved into the binding mechanisms of two well-known IKs activators, mefenamic acid and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and successfully elucidated their binding sites using electrophysiological and structural biology techniques. These activators potentiate the IKs current by inducing and stabilizing a conformational change in an otherwise hidden binding pocket within the S1/KCNE1/pore complex, slowing channel deactivation. Furthermore, I uncovered a dual mechanism of action for mefenamic acid on the IKs channel. At high concentrations (300 µM), mefenamic acid inhibits wild-type IKs current amplitude while preserving the delay in channel deactivation. Moreover, KCNE1 and KCNQ1 mutations in the previously elucidated binding pocket unmasked the inhibitory actions at lower drug concentrations. I propose that a lower affinity, secondary binding site near the primary binding site is responsible for inhibition, and specific mutations reveal the site. Finally, I demonstrated the graded stoichiometric effects of mefenamic acid on the wild-type IKs channel complex. The K41C mutation in KCNE1, which disrupts the primary binding site, abolishes the effects of mefenamic acid. My findings further quantified this graded loss of effect, showing that the response to mefenamic acid diminishes as the proportion of K41C-mutated subunits increases. Using mefenamic acid as an archetype for designing therapeutically useful IKs agonists, results from this thesis provide critical insights into the molecular mechanisms of IKs modulation and offers future directions for developing targeted therapies.
MULTIPLE EFFECTS OF KCNQ1 ACTIVATORS ON THE DELAYED CARDIAC RECTIFIER POTASSIUM CHANNEL MOLECULAR COMPLEX by Magnus Chan B.Sc., The University of British Columbia, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2025 © Magnus Chan, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Multiple Effects of KCNQ1 Activators On The Delayed Cardiac Rectifier Potassium Channel Molecular Complex submitted by Magnus Chan in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine Examining Committee: Dr. David Fedida, Professor, Department of Anesthesiology, Pharmacology & Therapeutics, UBC Supervisor Dr. Michael J. A. Walker, Emeritus Professor, Department of Anesthesiology, Pharmacology & Therapeutics, UBC Supervisory Committee Member Dr. Eric Accili, Associate Professor, Department of Cellular & Physiological Sciences, UBC Supervisory Committee Member Dr. Ismail Laher, Professor, Department of Anesthesiology, Pharmacology & Therapeutics, UBC University Examiner Dr. Edwin Moore, Professor, Department of Cellular & Physiological Sciences, UBC University Examiner iii Abstract The cardiac delayed rectifier potassium current, IKs, formed by the co-assembly of KCNQ1 and KCNE1 subunits, plays a critical role in cardiac repolarization, acting as a reserve to adapt to higher heart rates and compensating when other repolarization currents are impaired, thereby preventing arrhythmias. Loss of function mutations in these subunits impair cardiac repolarization and are associated with inherited long QT syndrome, increasing the risk of developing a polymorphic ventricular tachycardia known as Torsades de Pointes, which can result in syncope and sudden death. Therapeutic interventions targeting the IKs channel directly to reverse the malignant effects of long QT syndrome hold significant potential but have historically been challenging due to the previously unknown binding sites for IKs activators. In this thesis, I delved into the binding mechanisms of two well-known IKs activators, mefenamic acid and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and successfully elucidated their binding sites using electrophysiological and structural biology techniques. These activators potentiate the IKs current by inducing and stabilizing a conformational change in an otherwise hidden binding pocket within the S1/KCNE1/pore complex, slowing channel deactivation. Furthermore, I uncovered a dual mechanism of action for mefenamic acid on the IKs channel. At high concentrations (300 µM), mefenamic acid inhibits wild-type IKs current amplitude while preserving the delay in channel deactivation. Moreover, KCNE1 and KCNQ1 mutations in the previously elucidated binding pocket unmasked the inhibitory actions at lower drug concentrations. I propose that a lower affinity, secondary binding site near the primary binding site is responsible for inhibition, and specific mutations reveal the site. iv Finally, I demonstrated the graded stoichiometric effects of mefenamic acid on the wild-type IKs channel complex. The K41C mutation in KCNE1, which disrupts the primary binding site, abolishes the effects of mefenamic acid. My findings further quantified this graded loss of effect, showing that the response to mefenamic acid diminishes as the proportion of K41C-mutated subunits increases. Using mefenamic acid as an archetype for designing therapeutically useful IKs agonists, results from this thesis provide critical insights into the molecular mechanisms of IKs modulation and offers future directions for developing targeted therapies. v Lay Summary A healthy heart maintains a steady rhythm through coordinated contraction and relaxation, regulated by ion channels—proteins that control ion movement. One such ion channel, called IKs, is a potassium channel composed of two proteins: the pore forming protein and an accessory subunit. Mutations that disrupt IKs function can cause irregular heartbeats, increasing the risk of stroke or sudden cardiac death. This research explores how IKs activators can restore less effective IKs function. I first investigated how mefenamic acid and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), two IKs activators, bind to IKs to enhance its current flow. Next, I determined that mefenamic acid can also paradoxically block the ion flow of the channel concomitant to its activating effects. Finally, I determined how many accessory subunits are needed for mefenamic acid to work effectively. These findings provide valuable insights into how IKs activators function, which may help in developing better treatments for heart rhythm disorders. vi Preface The work presented in this thesis was primarily conducted in Dr. David Fedida’s laboratory at the University of British Columbia (Vancouver Campus) in collaboration with Dr. Marc Pourrier and Dr. Jodene Eldstrom. Dr. Harutyan Sahakyan and Dr. Vitya Vardanyan at the Institute of Molecular Biology in Yerevan, Armenia made significant contributions. This research was further supported by Dr. Ying Dou, Dr. Daniel Sastre and Dr. Yundi Wang. Generative AI was not used for any aspects of the work. Chapter 2: A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes I was involved in the study's conceptualization, acquisition of cell recordings, data interpretation, figure production, and manuscript review and editing. Dr. Harutyan Sahakyan was involved in the study’s conceptualization, methodology, acquisition and interpretation of structural biology data, and manuscript writing and reviewing. Dr. Jodene Eldstrom was involved in the study’s conceptualization, methodology, supervision, acquisition of cell recordings, data analysis and manuscript writing and review. Dr. Yundi Wang was involved in the study’s conceptualization and acquisition of cell recordings. Dr. Daniel Sastre and Dr. Ying Dou were involved in the acquisition of cell recordings. Dr. Marc Pourrier was involved in the study’s methodology, supervision, data analysis, and manuscript writing and editing. Dr. Vitya Vardanyan was involved in the study’s conceptualization and supervision, structural biology data acquisition and analysis, and manuscript revision and editing. Dr. David Fedida was involved in the study’s conceptualization, methodology and supervision, acquisition of cell recordings, data analysis, and manuscript writing. vii A version of Chapter 2 has been published: Chan, M., Sahakyan, H., Eldstrom, J., Sastre, D., Wang, Y., Dou, Y., Pourrier, M., Vardanyan, V., & Fedida, D. (2023). A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes. elife, 12:RP87038 https://doi.org/10.7554/eLife.87038 Chapter 3: Dual effects of mefenamic acid on the IKs molecular complex I was involved in the study’s conceptualization and methodology, acquisition of cell recordings, data analysis and manuscript writing, reviewing and editing. Dr. Marc Pourrier was involved in the study’s conceptualization, methodology and supervision, and manuscript writing, reviewing and editing. Dr. Jodene Eldstrom was involved in the study’s conceptualization, manuscript review, and editing. Dr. Harutyan Sahakyan was involved in data analysis, manuscript review and editing. Dr. Vitya Vardanyan was involved in manuscript review and editing. Dr. David Fedida was involved in the study’s conceptualization, supervision, and manuscript revision and editing. A version of Chapter 3 has been published: Chan, M., Pourrier, M., Eldstrom, J., Sahakyan, H., Vardanyan, V., & Fedida, D. (2024). Dual effects of mefenamic acid on the IKs molecular complex. British Journal of Pharmacology, 1–15. https://doi.org/10.1111/bph.17389 viii Chapter 4: Graded activation of K41C-KCNQ1:KCNE1 channel complexes by mefenamic acid Dr. Yundi Wang and I were involved in the acquisition of cell recordings, data analysis, manuscript reviewing and editing. Dr. Harut Sahakyan was involved in the acquisition of structural biology data and manuscript reviewing and editing. Dr. Jodene Eldstrom was involved in the study’s conceptualization, methodology, supervision, data analysis, manuscript review and editing. Dr. Marc Pourrier was involved in the study’s supervision, manuscript writing, review, and editing. Dr. Vitya Vardanyan was involved in the study’s supervision, manuscript review, and editing. Dr. David Fedida was involved in the study’s conceptualization, methodology, supervision, data analysis, manuscript writing, review and editing. A version of Chapter 4 is anticipated to be submitted in Q2 2025. ix Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface .......................................................................................................................................... vii Table of Contents ......................................................................................................................... ix List of Tables ..............................................................................................................................xvv List of Figures ............................................................................................................................. xvi List of Multimedia Material .................................................................................................... xviii List of Abbreviations ............................................................................................................... xvix Acknowledgements ....................................................................................................................xxv Dedication ............................................................................................................................... xxviii Chapter 1: Introduction ................................................................................................................1 1.1 Voltage-gated potassium channels (VGKCs) ................................................................. 1 1.2 Voltage-gated K+ channels in cardiac health and disease ............................................... 2 1.3 The KCNQ1 macromolecular complex .......................................................................... 4 1.3.1 KCNQ1 ....................................................................................................................... 4 1.3.2 KCNE1 ........................................................................................................................ 7 1.3.3 Other KCNEs .............................................................................................................. 9 1.4 Pharmacological modulations of KCNQ1/KCNE1 Channels ...................................... 11 1.4.1 Activators of KCNQ1 ............................................................................................... 11 1.4.1.1 Stilbenes ............................................................................................................ 12 1.4.1.2 Mefenamic acid ................................................................................................. 12 x 1.4.1.3 ML277............................................................................................................... 14 1.4.1.4 Polyunsaturated fatty acids ............................................................................... 16 1.4.1.5 R-L3 .................................................................................................................. 19 1.4.1.6 Other IKs Activators .......................................................................................... 20 1.4.2 Activators of other KCNQ isoforms ......................................................................... 21 1.4.2.1 Retigabine ......................................................................................................... 21 1.4.2.2 Ztz240 ............................................................................................................... 22 1.4.2.3 ML213............................................................................................................... 23 1.4.2.4 Cannabidiol ....................................................................................................... 23 1.4.3 Blockers of KCNQ1 .................................................................................................. 24 1.4.3.1 Chromanol 293B ............................................................................................... 24 1.4.3.2 HMR1556 ......................................................................................................... 25 1.4.3.3 JNJ-303 ............................................................................................................. 25 1.4.3.4 UCL2077........................................................................................................... 26 1.5 Scope of the thesis ........................................................................................................ 27 Chapter 2: A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes .......................................29 2.1 Introduction ................................................................................................................... 29 2.2 Material and methods .................................................................................................... 31 2.2.1 Molecular docking and molecular dynamic simulations .......................................... 31 2.2.2 Reagents and solutions .............................................................................................. 34 2.2.3 Molecular biology, cell culture and whole cell patch clamp .................................... 34 2.2.4 Data analysis ............................................................................................................. 35 xi 2.3 Results ........................................................................................................................... 36 2.3.1 The mefenamic acid binding site on the KCNQ1/KCNE1 complex ........................ 36 2.3.2 Mefenamic acid binding site predicted by molecular modeling ............................... 39 2.3.3 Mutational impact on EQ current changes induced by mefenamic acid .................. 43 2.3.4 Augmented activation of EQ-L142C in the absence of mefenamic acid .................. 47 2.3.5 MD simulations of mutant ps-IKs channels exposed to mefenamic acid .................. 48 2.3.6 Mutational impact on IKs current changes induced by DIDS .................................... 52 2.4 Discussion ..................................................................................................................... 62 2.4.1 Mefenamic acid binding site in IKs ........................................................................... 63 2.4.2 Common binding site for IKs activators, mefenamic acid and DIDS ........................ 66 2.4.3 Proposed mechanism of action for mefenamic acid and DIDS ................................ 69 2.5 Conclusion .................................................................................................................... 72 Chapter 3: Dual effects of mefenamic acid on the IKs molecular complex .............................73 3.1 Introduction ................................................................................................................... 73 3.2 Methods......................................................................................................................... 74 3.2.1 Reagents and solutions .............................................................................................. 74 3.2.2 Cell culture and molecular biology ........................................................................... 75 3.2.3 Electrophysiology ..................................................................................................... 76 3.2.4 Trajectory clustering of mutant W323A, Y46C and WT channels via molecular dynamics simulation ............................................................................................................. 77 3.2.5 Data and analysis ...................................................................................................... 77 3.2.6 Nomenclature of targets and ligands ......................................................................... 80 3.3 Results ........................................................................................................................... 80 xii 3.3.1 Mefenamic acid has dual effects on WT IKs current in mammalian cells ................. 80 3.3.2 Mutations of residues in the mefenamic acid binding pocket unmask its inhibiting properties ............................................................................................................................... 81 3.3.3 Effects of mefenamic acid on E43C-E1Q1 and E1Q1-A300C G-V relationships and deactivation kinetics .............................................................................................................. 84 3.3.4 KCNE1 is essential for the inhibitory action of mefenamic acid in E1Q1-A300C .. 87 3.3.5 Removing the charge at position 43 of KCNE1 is insufficient to explain mefenamic acid’s inhibitory effect on E43C-E1Q1 ................................................................................ 89 3.3.6 The inhibitory action of mefenamic acid on E1-E43C is not dependent on the movement of the voltage-sensing domain (VSD) ................................................................. 91 3.4 Discussion ..................................................................................................................... 94 3.4.1 Inhibitory actions of mefenamic acid on the WT IKs channel complex .................... 94 3.4.2 Mutations enhance mefenamic acid’s inhibitory actions .......................................... 96 3.4.3 Mechanisms of the inhibitory effects of mefenamic acid ......................................... 98 3.4.4 The effects of mefenamic acid depend on the presence of KCNE1 ....................... 101 3.4.5 Relevance to drug design and therapeutic application ............................................ 102 Chapter 4: Graded activation of K41C-KCNQ1:KCNE1 channel complexes by mefenamic acid ..............................................................................................................................................104 4.1 Introduction ................................................................................................................. 104 4.2 Materials and methods ................................................................................................ 105 4.2.1 Reagents and solutions ............................................................................................ 105 4.2.2 Molecular biology, cell culture and whole cell patch clamp .................................. 106 4.2.3 Data analysis ........................................................................................................... 107 xiii 4.2.4 Modeling ................................................................................................................. 107 4.3 Results ......................................................................................................................... 108 4.3.1 Mefenamic acid actions are prevented by the KCNE1 mutation, K41C in a stoichiometric manner ......................................................................................................... 108 4.3.2 Varying the number of K41C-containing subunits in models of mefenamic acid activation of IKs ................................................................................................................... 115 4.4 Discussion ................................................................................................................... 118 4.4.1 Insights from K41C-KCNE1 on mefenamic acid action ........................................ 118 4.5 Conclusion .................................................................................................................. 121 Chapter 5: Discussion ................................................................................................................122 5.1 Summary of findings ................................................................................................... 122 5.2 Comparative analysis of the novel IKs activator binding activators’ binding sites and mechanism of action ............................................................................................................... 124 5.3 Comparative analysis of the novel IKs activator binding site with other potassium channel activators binding sites .............................................................................................. 128 5.4 Comparative analysis of the IKs activators and other potassium channel modulators 130 5.5 Graded activation of potassium channels by activators .............................................. 131 5.6 Therapeutic relevance of the novel IKs activator binding site ..................................... 132 5.7 Clinical relevance of the new findings of the novel IKs activator binding site ........... 135 5.8 Limitations .................................................................................................................. 136 5.9 Future directions ......................................................................................................... 138 5.10 Conclusions ................................................................................................................. 140 Bibliography ...............................................................................................................................141 xiv Appendix A: Supplementary Figures for Chaper 2 ...............................................................161 Appendix B: Supplementary Figures for Chaper 3 ................................................................173 xv List of Tables Table 2.1: Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of mefenamic acid for fully saturated IKs channel complexes ....................................... 38 Table 2.2: Mean frequency of H-bonds between MEF or DIDS and residues in ps- IKs during 500 ns MD simulations or until drug unbound .................................................................................... 42 Table 2.3: Mean V1/2 of activation (mV) and slope values (k-factor, mV) for WT EQ treated with 100 µM mefenamic acid, and untreated mutant EQ-L142C ......................................................... 48 Table 2.4: Average free interaction energies of MEF-bound ps-IKs complexes calculated according to MM/GBSA methods from three independent MD simulation runs using the AMBER force field ....................................................................................................................... 49 Table 2.5: Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of 100 µM DIDS for fully saturated IKs channel complexes ......................................... 54 Table 2.6: Average free interaction energies of DIDS-bound ps-IKs complexes calculated according to MM/PBSA and MM/GBSA methods from three independent MD simulation runs of 300 ns duration using the AMBER force field ......................................................................... 60 Table 2.7: Average free energy of mefenamic acid and DIDS-bound ps-IKs and mutant complexes ..................................................................................................................................... 65 Table 3.1: Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and in the presence of mefenamic acid .................................................................................................... 87 Table 4.1: V1/2 of activation and slope value (k) for Boltzmann fits in the absence and presence of mefenamic acid for fully saturated IKs channel complexes .................................................... 110 xvi List of Figures Figure 1.1 Cryo-EM structure of KCNQ1EM/CaM ......................................................................... 5 Figure 1.2 Current traces of Q1-WT (left) and KCNQ1/KCNE1 4:4 (EQ-WT) (right) in tsA201 transformed human embryonic kidney 293 cells ............................................................................ 9 Figure 1.3 Schematic illustration of the various regulatory accessory subunits associated with KCNQ1 channels .......................................................................................................................... 11 Figure 2.1 K41C-KCNE1 mutants prevent the agonist effect of mefenamic acid ....................... 37 Figure 2.2 MD prediction of mefenamic acid binding site in the ps-IKs model ............................ 40 Figure 2.3 Current waveform and G-V changes induced by mefenamic acid in binding site mutants .......................................................................................................................................... 45 Figure 2.4 K41C, Y46C, and W323A mutant impact on mefenamic acid binding energy and flexibility of external KCNE1 residues ......................................................................................... 51 Figure 2.5 Effect of DIDS on IKs .................................................................................................. 53 Figure 2.6 Ligand interaction and energy decomposition per amino acid for DIDS binding to ps-IKs .................................................................................................................................................. 56 Figure 2.7 Binding site mutants important for the action of DIDS .............................................. 59 Figure 2.8 Subtle differences in activator binding to Y46C- and A44C-ps-IKs mutant channels . 68 Figure 3.1 Current waveform changes and inhibitory effects of mefenamic acid (Mef) ............. 79 Figure 3.2 Concentration-dependent effects of mefenamic acid (Mef) on E43C-E1Q1 and E1Q1-A300C ........................................................................................................................................... 84 Figure 3.3 G–V and deactivation kinetic changes induced by mefenamic acid (Mef) in E43C-E1Q1 and E1Q1-A300C ............................................................................................................... 86 xvii Figure 3.4 Current waveform and G–V changes induced by mefenamic acid (Mef) on KCNQ1 alone .............................................................................................................................................. 89 Figure 3.5 Current waveform, current amplitude and G–V changes induced by mefenamic acid (Mef) on E43A-E1Q1, E43K-E1Q1 and E43D-E1Q1 ................................................................. 91 Figure 3.6 Effects of mefenamic acid (Mef) on E1Q1-R4 and E43C-E1Q1-R4 .......................... 93 Figure 3.7 Mefenamic acid (Mef) binding to ps-IKs-W323A ....................................................... 97 Figure 3.8 Mefenamic acid (Mef) binding to Y46C. Snapshots of mefenamic acid binding patterns over time to ps-IKs carrying the Y46C mutation ............................................................. 98 Figure 3.9 Structure showing region of interest for IKs block by mefenamic acid ..................... 100 Figure 4.1 K41C-KCNE1 mutants prevent the agonist effect of mefenamic acid ..................... 109 Figure 4.2 Stoichiometric prevention of mefenamic acid agonism by K41C-KCNE1 mutants . 113 Figure 4.3 Model of mefenamic acid action in K41C mutants ................................................... 117 Figure 5.1 Binding sites of PUFA and ML277 on the KCNQ1-KCNE1 molecular complex .... 126 xviii List of Multimedia Material Video 1 MD simulations at the molecular level of binding of mefenamic acid to ps-IKs, and K41C-, W323A-, Y46C- ps-IKs mutants Video 2 Mefenamic acid binding to K41C ps-IKs Video 3 Mefenamic acid binding to W323A ps-IKs Video 4 Mefenamic acid binding to Y46C ps-IKs Video 5 MD simulations at the molecular level of binding of DIDS to ps-IKs, and K41C-, W323A-, Y46C- ps-IKs mutants Video 6 DIDS binding to K41C ps-IKs Video 7 DIDS binding to W323A ps-IKs Video 8 DIDS binding to Y46C ps-IKs Video 9 Binding pocket fluctuations before and after exit of mefenamic acid Video 10 Mefenamic acid bound to ps-IKs-W323A xix List of Abbreviations ± – Plus/minus 4D – Four-dimensional AF – Atrial fibrillation AMBER – Assisted Model Building with Energy Refinement Å – Angstrom ANOVA – Analysis of variance cDNA – Complementary deoxyribonucleic acid CHARMM – Chemistry at Harvard Macromolecular Mechanics CHO – Chinese hamster ovary Cl- – Chloride ion Cos7 – CV-1 in Origin, carrying the SV40 genetic material CRRRR – Four independent voltage sensors in the closed resting states Cryo-EM – Cryogenic electron microscopy Cs+ – Cesium ion Δ – Change ΔG – Change in free energy DIDS – 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid EC50 – Half maximal effective concentration EGTA – Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid EQ – a linked wild-type KCNE1 and KCNQ1 cDNA which assembles as a 4:4 ratio of KCNE1 to KCNQ1 xx EQQ – A linked wild-type KCNE1 and KCNQ1 cDNA which assembles as a 2:4 ratio of KCNE1 to KCNQ1 EQQQQ – A linked wild-type KCNE1 and KCNQ1 cDNA which assembles as a 1:4 ratio of KCNE1 to KCNQ1 fs – Femtosecond G – Conductance G-V – Conductance-voltage relationship GBSA – Generalized Born surface area GFP – Green fluorescent protein Gmax – Maximum conductance GROMACS – Groningen Machine for Chemical Simulations H-bond – Hydrogen bond HEPES – 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid hiPSC-CM – Human-induced pluripotent stem cells - derived cardiomyocytes HMR1556 – (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide h or hr – Hour IC50 – Half maximal inhibitory concentration ICM – Internal Coordinate Mechanics IK – Delayed rectifier potassium current IKs – Slow delayed cardiac rectifier potassium current K – Potassium k – Slope factor xxi K+ – Potassium ion kcal – Kilocalorie KCl – Potassium chloride KCNE1 – Potassium voltage-gated channel subfamily E member 1 KCNQ1 – Potassium voltage-gated channel subfamily Q member 1 KCNQ1EM – A KCNQ1 construct encompassing amino acids 67–610 of frog (Xenopus laevis) KCNQ1, which shares 78% sequence identity with human KCNQ1 (Sun & MacKinnon, 2017) KcsA – K channel of streptomyces A Kd – Dissociation constant kHz – Kilohertz kIA – Intrinsic rates of forward voltage sensor transitions at 0 mV, between intermediate and activated states kJ – Kilojoules KOH – Potassium hydroxide kRI0 – Intrinsic rates of forward voltage sensor transitions at 0 mV, between resting and intermediate states Kv – Voltage-gated potassium channel L – Litre LM – Mouse fibroblast cells LQTS – Long QT syndrome LSD – Fishers Least Significant Difference MD – Molecular dynamics Mef – Mefenamic acid xxii MgCl2 – Magnesium chloride µg – Microgram µM – Micromolar ML277 – (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2-piperidinecarboxamide ml – Millilitre ml-1 – Per millilitre mM – Millimolar MM – Molecular mechanics MM/GBSA – Molecular mechanics/generalized Born surface area MM/PBSA – Molecular mechanics/Poisson-Boltzmann surface area mols – Moles MTSET – 2-(Trimethylammonium)ethyl methanethiosulfonate, bromide mV – Millivolts MΩ – Megaohms n – number N-terminal – Amino-terminus Na-acetate – Sodium acetate Na+ – Sodium ion Na2ATP – Adenosine 5′-triphosphate disodium salt hydrate NaCl – Sodium chloride NaOH – Sodium hydroxide nH – Hill coefficient xxiii nm2 – Square nanometer NPT – Constant temperature, constant pressure ns – Nanosecond NSAID – Nonsteroidal anti-inflammatory drug NVT – Constant temperature, constant volume OAAAA – Four independent voltage sensor in the fully activated open states P-helix – Pore helix PBSA – Poisson-Boltzmann surface area PIP2 – Phosphatidylinositol 4,5-biphosphate PME – Particle Mesh Ewald POPC – 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ps – Picosecond ps-IKs – Pseudo-KCNE1-KCNQ1 PUFAs – Polyunsaturated fatty acids R-L3 (or L-364,373) – 5-(2-fluorophenyl)-1,3-dihydro-3-(1H-indol-3-ylmethyl)-1- methyl-2H-1,4-benzodiazepin-2-one Rb+ – Rubidium ion RMSD – Root mean square deviation RMSF – Root mean square fluctuation s – seconds SD – Standard deviation of the mean SEM – Standard error of the mean SHAKE – Self-consistent Homonuclear Algebraic Key xxiv SITS – 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid SQTS – Short QT syndrome 𝜏deact – Deactivation time constant V1/2 – Voltage at half-maximal activation VS – Voltage-sensor VSD – Voltage-sensing domain WT – Wild type Amino Acids Three-letter One-letter Amino Acid Ala A Alanine Arg R Arginine Asn N Asparagine Asp D Aspartic acid Cys C Cysteine Glu E Glutamic acid 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 Behind every successful PhD candidate is a strong support system. Over the years, I have developed my organizational, interpersonal, critical thinking, project management, and written and oral communication skills. I am immensely grateful for all the opportunities I’ve had and would like to express my deepest gratitude to my mentors and peers who have supported me throughout this journey. This thesis is a reflection of the unwavering support I received from you all—I could not have done this without you. To my supervisor, Dr. David Fedida, thank you for being an exceptionally compassionate, responsible, and dedicated mentor. You took me in and gave me a chance, even when I had minimal experience with ion channels and neuroscience. You continuously sought out opportunities for me, whether through scholarships or conferences, helping me refine my communication skills and become a stronger candidate for my future career. You were always the one to take responsibility and push projects forward when they seemed to stall, and for that, I am truly grateful. Dr. Marc Pourrier, I am forever indebted to you. From the beginning, your guidance and unwavering support helped me navigate every challenge. You were always there to fix things—both in science and in life—offering advice, troubleshooting experiments, and ensuring that I never felt alone in this journey. No matter how busy you were, you always had time to help, to counsel, and to listen when things weren’t working. I will miss having someone who always has my back on every project, but even more, I will miss our coffee chats. Thank you for always going above and beyond. Dr. Jodene Eldstrom, you are the spark behind so many ideas. Your insights often made the difference between our papers being published in good journals versus very good ones. Thank you for keeping the lab running smoothly and ensuring that everything was always in place. xxvi To the one who ignited my passion for research and inspired the start of my PhD journey, Dr. Michael J.A. Walker, thank you for your guidance and support since my undergraduate years—both academically and personally. You not only believed in me but also helped me find the perfect supervisor. Your mentorship has always been rooted in genuine care for my best interests, and for that, I am truly grateful. Dr. Eric Accili, thank you for serving on my supervisory committee and for your insights and support, always accommodating my schedule with flexibility. Fariba Ataei, thank you for your assistance in molecular biology, your patience, and your willingness to help troubleshoot every error. Dr. Ying Dou, I appreciate your technical expertise in cell culture and your companionship throughout our electrophysiology projects. Dr. Efthimios Kyriakis, thank you for your friendship in the lab and for always offering honest advice and a listening ear. Dr. Daniel Sastre Martínez, working on projects with you was a joy, but even more so was our friendship. Thank you for your unwavering support and encouragement. Sophia Russo, thank you for your time, effort, and kindness—not only in supporting my PhD journey but also in always being there to help out whenever needed. Thank you to the Canadian Institutes of Health Research (CIHR) for their grant support for the CGS-M and CGS-D awards. Thank you to the University of British Columbia (UBC) for the multiple Faculty of Medicine Graduate Awards and the 4Y Doctoral Fellowship. My journey with all of you doesn’t end here. As I embark on my next chapter, I will always look back on my time in the lab and the support that shaped both my academic and personal growth. "I don’t ever say goodbye, I always just say, I’ll see you down the road. And I do." – Nomadland xxvii Dedication To my parents, thank you for your steadfast support and for believing in me from day one, always doing all you can to support me in every imaginable and unimaginable way. To Elaine, thank you for being the kind of supportive sister that every brother wishes they had. To Jocelyn, thank you for your loving partnership – always lifting me up when I’m down, cheering me on, and being the rock in my life. To Marc, thank you for going above and beyond in all that you do for me. To David F, thank you for your unparalleled guidance. You all kept my best interests at heart. 1 Chapter 1: Introduction 1.1 Voltage-gated potassium channels (VGKCs) Potassium (K+) channels are the largest and most diverse group of ion channels, encoded by about 70 to 80 different genes. Found in both excitable and non-excitable cells, they play critical roles in cellular signaling processes regulating neurotransmitter release, heart rate, neuronal excitability, insulin secretion, epithelial electrolyte transport, smooth muscle contraction, cell volume regulation and cell proliferation (Blaine & Ribera, 2001; Joseph et al., 2013; Murakoshi & Trimmer, 1999; Pardo et al., 1999; Preston et al., 2010; Yang & Zheng, 2014). Functionally, the K+ channels form four major families: (1) calcium-activated K+ channels (KCa); (2) inwardly rectifying K+ channels (Kir), which favor the inward flow of current; (3) tandem pore domain K+ channels (K2P), which constitutively open to maintain negative membrane potential; and (4) voltage-gated K+ channels (Kv), which activate predominantly in response to changes in transmembrane voltage. Voltage-gated K+ (Kv) channels are diverse and can be divided into 12 subfamilies, from Kv1 to Kv12. They share the same structure, forming tetramers from four accessory subunits arranged in a ring to create the transmembrane pore (Robbins, 2001; Yellen, 2002). Each subunit comprises six transmembrane segments (S1-S6) and a pore loop (P loop), with S1-S4 functioning as the voltage-sensing domain (VSD) and S5-P loop-S6 segments making up the pore domain to create the channel’s conduction pathway (Cui, 2016; Liin et al., 2015a; Nakajo & Kubo, 2015; Smith et al., 2007). Both the N-termini and C-termini are located on the intracellular side. Kv channel activation follows a three-step molecular process. (1) The voltage-sensing domain (VSD) moves from a resting to an activated conformation upon membrane depolarization (Bezanilla, 2000; 2 Bezanilla & Stefani, 1994; Jensen et al., 2012; Wu et al., 2010). (2) Electro-mechanical coupling (ECM) resulting from the rearrangement in interactions between the VSD and the pore, and (3) opening of the gate to conduct potassium ions. Compared to other voltage-gated ion channels, Kv channels share two universal traits: (1) selectivity for K+ ions over others like Na+ and Ca2+ (Hille, 2001; LeMasurier et al., 2001; Neyton & Miller, 1988; Yellen, 1984) and (2) their fast rate of ion conductance (Doyle et al., 1998; Hille, 2001; Hodgkin & Keynes, 1955). In KCNQ channels, recent structural studies show that ion permeation occurs as ions become partially dehydrated at the cavity constriction (Mkrtchyan et al., 2024). Voltage-gated potassium channels regulate cellular excitability by selectively allowing potassium ions to flow out of the cells in response to a change in membrane voltage. Their primary function is to repolarize the cell membrane, rapidly returning the cell to its resting state. Kv channels are expressed throughout the body and are linked to multiple diseases. 1.2 Voltage-gated K+ channels in cardiac health and disease The normal electrophysiologic activity of the heart is determined by the ordered propagation of an electrical impulse along the heart conduction system resulting from the generation of action potentials. The cardiac action potential morphology originates from the orchestrated opening and closing of multiple ion channels, each with its unique time- and voltage-dependent amplitudes (Pourrier & Fedida, 2020). In ventricular and atrial cells, the cardiac action potential is divided into five distinct phases. A propagating impulse leads to the activation of sodium (Na+) channels and Na+ influx, which results in action potential rapid depolarization (Phase 0). A transient and fast repolarization follows Phase 0 due to the combined inactivation of Na+ channels and K+ 3 efflux resulting from the activation of the transient outward K+ current (Ito) (Phase 1). This early, rapid repolarization phase controls the height and duration of the plateau phase (Phase 2), which relies on the fine balance of inward (Ca2+ and Na+) and outward (K+) currents. Phase 2 is critical in triggering calcium-induced calcium release, which is central to excitation-contraction coupling. As Ca2+ channels inactivate, outward K+ currents predominate, while the driving force for the K+ efflux through the voltage-gated K+ channels becomes high during the plateau. This results in the final repolarization phase (Phase 3), where the membrane potential returns toward diastolic levels (Phase 4). Although the action potential shape differs in various regions of the heart, Kv channels are typically responsible for the repolarization phase. Kv4.3 and Kv1.4, Kv1.5, Kv11.1 (or hERG), and Kv7.1 (or KCNQ1) are pore-forming subunits that underlie Ito, IKur (ultrarapid delayed rectifier K current), IKr (rapid delayed rectifier K current), and IKs (slow delayed rectifier K current), respectively. While IKur is an important repolarizing current in the atrium, it is absent in the ventricle (Amos et al., 1996; Burashnikov & Antzelevitch, 2009; Fedida et al., 1993; Wang et al., 1993; Wettwer et al., 2004; Yue et al., 1996). IKr and IKs are the two main repolarizing currents in atrial and ventricular cells and, thus, are crucial in regulating the action potential duration (APD) (Abbott et al., 1999; Barhanin et al., 1996; Cheng & Kodama, 2004; Sanguinetti et al., 1996; L. Wang et al., 1996). As a result, dysfunction of Kv channels can lead to major changes in cardiac repolarization potentially leading to life-threatening arrhythmias. Mutations in genes encoding hERG or KCNQ1 and some associated 𝛽 subunits are linked to inherited cardiac diseases. Whereas loss of function mutations are associated with long QT syndrome, gain of function mutations can lead to short QT syndrome and atrial fibrillation (Abraham et al., 2010; Bartos et al., 2014; Bellocq et al., 2004; Y. H. Chen 4 et al., 2003; Das et al., 2009; Hong et al., 2005; Lundby et al., 2007; Moreno et al., 2015; Wu et al., 2015). My work focused on IKs, which is formed by the co-assembly of the KCNQ1 pore subunits, with its 𝛽 subunit KCNE1, other KCNEs, and multiple accessory regulatory molecules such as phosphatidylinositol 4,5-biphosphate (PIP2), calmodulin and yotiao (Wang et al., 2020a). KCNQ1 belongs to the KCNQ family of voltage-gated potassium channels and is found in cardiomyocytes, the crypt cells of the small intestine and colon, the kidney, the pancreas and the inner ear (Abbott, 2014; Cui, 2016; Jespersen et al., 2005; Liin et al., 2015a; Nakajo & Kubo, 2015; Robbins, 2001). This channel complex is discussed in detail in Section 1.3. 1.3 The KCNQ1 macromolecular complex 1.3.1 KCNQ1 Like other Kv channels, KCNQ1 consists of six transmembrane segments, with S1-S4 forming the VSD and S5-S6 forming the PD, connected by the S4-S5 linker (Figure 1.1) (Labro et al., 2011; Ma et al., 2022; Willegems et al., 2022; Wu et al., 2021). In its domain swapped configuration, the VSD from one subunit interacts with the PD of a neighboring subunit rather than its own. KCNQ1 functions as part of a macromolecular complex with KCNE1 and other regulatory proteins, including calmodulin (CaM), phosphatidylinositol 4,5-biphosphate (PIP2), adenosine triphosphate (ATP), and protein kinase A (PKA) (Figure 1.1 and 1.3). CaM binds to the KCNQ1 C-terminus and a novel site in the S2-S3 loop to facilitate VSD-PD coupling (Sun & MacKinnon, 2017, 2020; Tobelaim et al., 2017). PIP2 stabilizes the open state and dilates the 5 cytoplasmic pore entrance by interacting with positively charged residues in S0, the S2-S3 loop, and the S4-S5 linker regions (Boulet et al., 2007; Seebohm et al., 2007; Sun & MacKinnon, 2017, 2020). ATP enhances channel activation and increases current amplitude, while PKA-dependent phosphorylation increases current amplitude and slows deactivation in the presence of KCNE1 (Li et al., 2013; Lopes et al., 2007; Marx et al., 2002; Thompson et al., 2017). Figure 1.1 Cryo-EM structure of KCNQ1EM/CaM. Left: Top view of the KCNQ1EM/CaM complex with S1-S6 labeled. Right: Model of one subunit. C = C-terminus of KCNQ1, CaM = calmodulin, HA HB HC = cytosolic HA, HB and HC helices of KCNQ1, N = N-terminus of KCNQ1, S = segment. Extracted from Sun and MacKinnon 2017. Upon depolarization, KCNQ1 generates a fast-activating current (Figure 1.2) (Hou et al., 2017; Pusch et al., 1998; Sanguinetti et al., 1996; Tristani-Firouzi & Sanguinetti, 1998). Activation is voltage-dependent, and channels open to allow ions to flow. Like other Kv channels, such as the Shaker channel, KCNQ1 undergoes a two-step activation in which the voltage-sensing domain (VSD) first transitions from the resting state to an intermediate state before reaching the fully activated state (Barro-Soria et al., 2014; Hou et al., 2017; Hou et al., 2020; Osteen et al., 2010; Silva et al., 2009; Taylor et al., 2020; Wu et al., 2010; Zaydman et al., 2014). When the membrane is depolarized, the positively charged S4 segment moves toward the extracellular space, inducing a conformational change in the VSD, i.e., VSD activation followed by pore 6 opening. When S4 moves into the intermediate state, the S4-S5 linker interacts with the bottom of the S6 within the same subunit, enabling channel opening at the IO (Intermediate-Open) state (Sun & MacKinnon, 2017, 2020). When S4 transitions to the fully activated state, the S4-S5 linker interacts with the S5 and S6 of the neighboring subunit, opening the pore at the AO (Activated-Open) state. However, unlike most Kv channels, which have a non-conducting intermediate state and only conduct current when fully activated, KCNQ1 can conduct current in both the IO and AO states (Barro-Soria et al., 2014; Cui, 2016; Osteen et al., 2010; Taylor et al., 2020; Wu et al., 2010; Zaydman et al., 2014). In contrast, other KCNQ channels, such as KCNQ2 and KCNQ3, activate in a single step and conduct current exclusively in the AO state (Yang et al., 2022). Beyond the differences in conductance, these states also differ in their modulation by auxiliary KCNE subunits, pharmacological properties, and regulation by cell-signaling pathways (Hou et al., 2019; Kurokawa et al., 2003; Kurokawa et al., 2004; Lin et al., 2021; Marx et al., 2002; Taylor et al., 2020; Zaydman et al., 2014). Notably, while both the IO and AO states conduct current in KCNQ1, the VSD-pore coupling in the AO state results in a lower open probability than in the IO, so that the transitions from IO to AO give rise to an inactivation-like behavior (Cui, 2016; Hou et al., 2017; Hou et al., 2020; Hou et al., 2019; Taylor et al., 2020; Wu et al., 2021; Zaydman et al., 2014). Inactivation is typically seen as a characteristic “hook” in the tail current i.e., the initial increase of current, which reflects recovery from inactivation, followed by a decay as the channels deactivate (Figure 1.2) (Hou et al., 2017; Pusch et al., 1998; Sanguinetti et al., 1996). The half-activation voltage (V1/2)—the voltage at which 50% of the channels are activated—is 7 approximately -10 to -20 mV, with a slope factor (k, voltage sensitivity) of around 12 mV per e-fold increase in conductance (Meisel et al., 2018; Westhoff et al., 2019). KCNQ1 channel properties are modulated by multiple regulatory subunits which are discussed in the following sections. 1.3.2 KCNE1 KCNE1 is a member of the KCNE family of auxiliary subunits, all of which contain a single transmembrane domain and modify the properties of KCNQ1 channels in diverse tissues (Liin et al., 2015a). In the heart, KCNE1 interacts with KCNQ1 to underlie IKs (Barhanin et al., 1996; Bendahhou et al., 2005; Sanguinetti et al., 1996). When co-expressed with KCNE1, the characteristics of the KCNQ1 channel are significantly altered (Figure 1.2). Compared to KCNQ1 alone, the co-assembled KCNQ1/KCNE1 complex (1) shifts the half-activation voltage to more positive potentials, (2) slows activation by approximately 1000-fold, (3) decelerates deactivation kinetics, (4) increases the overall macroscopic current amplitude, and (5) eliminates the inactivation observed in KCNQ1-only channels (Hou et al., 2017; Meisel et al., 2018; Murray et al., 2016; Tristani-Firouzi & Sanguinetti, 1998). This resulting current morphology closely resembles the IKs current found in cardiac cells (Wang et al., 2012; Y. H. Wang et al., 2011). The slow activation of this KCNQ1/KCNE1 current is particularly important, as it plays a key role in regulating the APD and the QT interval in the heart (Song & Lee, 2022). Single-channel studies reveal that the slow activation of IKs results in part from single channel showing very long latencies to first opening to a first sub-conductance level. Full opening is further delayed as channels occupy partially open sub-conductance states before reaching the fully open state (Werry et al., 2013; Yang & Sigworth, 1998). In addition, KCNE1 modifies the ion selectivity of 8 KCNQ1 channels, as demonstrated by the significantly lower Rb⁺/K⁺ permeability ratio observed in KCNQ1/KCNE1 channel complexes compared to KCNQ1 channels alone (Pusch et al., 2000; Zaydman et al., 2014). The KCNQ1:KCNE1 ratio is of great interest because it critically modulates channel gating, which is highly dependent on KCNE1 expression levels (Cui et al., 1994; Wang et al., 1998; Yu et al., 2013). Studies using link constructs to force the stoichiometric ratios have resulted in functional channels, supporting the potential for assembly in different stoichiometries (Murray et al., 2016; Nakajo et al., 2010). Various studies have shown that the stoichiometric ratio between the KCNQ1 channel subunit and the KCNE1 accessory subunit may vary from 1:4 to 4:4. Biochemical studies like subunit counting and fluorescence photobleaching, have suggested a fixed ratio of 2:4 (H. Chen et al., 2003; Morin & Kobertz, 2008; Plant et al., 2014) while others suggested that this ratio could go up to 4:4 (Nakajo et al., 2010). Compared to the fully saturated 4:4 KCNQ1:KCNE1 ratio (EQ-WT), as the stoichiometric ratio of KCNQ1/KCNE1 decreases to 4:2 or 4:1, the channel's properties gradually revert to those observed in KCNQ1 alone. At a 4:2 ratio, the effects of KCNE1 on KCNQ1 are about half that at the 4:4 ratio (e.g., half-activation voltage is approximately midway between that of the 4:4 and 4:0 (KCNQ1-alone ratios)). At 4:1, the properties shift even closer to the KCNQ1-alone state, with the effects of KCNE1 on KCNQ1 reduced to about half of those observed at the 4:2 ratio (Hou et al., 2017; Murray et al., 2016). In humans, the 4:2 stoichiometry of KCNQ1:KCNE1 is supported by experiments in human myocytes, which examined the channel’s kinetics, and in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), where the drug sensitivity to ML277, a KCNQ1 activator, 9 effectively potentiates the channel at concentrations consistent with this stoichiometry (Bosch et al., 1998; Virág et al., 2001; Yu et al., 2013). Figure 1.2 Current traces of Q1-WT (left) and KCNQ1/KCNE1 4:4 (EQ-WT) (right) in tsA201 transformed human embryonic kidney 293 cells. A 4 s protocol was used with pulses from –80 mV to +100 mV, followed by a repolarization step to -40 mV for 2 s (left) and 800 ms (right). Holding potential and interpulse interval were -80 mV and 15 s, respectively. Data extracted from Chan et al., 2023. 1.3.3 Other KCNEs In addition to KCNE1, other KCNE subunits (KCNE2-KCNE5) are expressed in the heart to various levels and modulate KCNQ1 activity (Bendahhou et al., 2005; Lundquist et al., 2005). KCNE2 and KCNE3 both render KCNQ1 constitutively active at physiologically relevant voltages; however KCNE2 reduces current amplitude and slows channel kinetics, while KCNE3 maintains or increases current amplitude through its interaction with the voltage sensor domain (Barro-Soria et al., 2015; Bendahhou et al., 2005; Choi & Abbott, 2010; Jiang et al., 2009; Nakajo & Kubo, 2007; Panaghie & Abbott, 2007; Schroeder et al., 2000; Sun & MacKinnon, 2020; Tinel et al., 2000; Toyoda et al., 2006; Wu et al., 2006). In contrast, KCNE4 act as an KCNQ1 KCNQ1/KCNE1 4:4 0.5 nA 0.5 nA 500 ms 500 ms 10 inhibitor of KCNQ1. KCNE4 stabilizes the closed state by significantly shifting the voltage-dependence of activation to more positive potentials, a mechanism that can suppress constitutive activation caused by certain KCNQ1 mutations (Bendahhou et al., 2005; Manderfield et al., 2009; Manderfield & George, 2008). Recent work indicates that KCNE3 and KCNE4 are uniquely upregulated at arrhythmia sites within scarred myocardium following myocardial ischemia, suggesting an important role in IKs-associated repolarization heterogeneity (Kelemen et al., 2022). KCNE5 has a similar inhibitory role, shifting KCNQ1 activation beyond physiological voltages, thereby minimizing its contribution under physiological conditions (Angelo et al., 2002; Bendahhou et al., 2005; Piccini et al., 1999). However, KCNE5 mutations have been linked to cardiac arrhythmias, including Brugada syndrome and atrial fibrillation, likely due to its altered regulation of the transient outward current or KCNQ1/KCNE1 complex, respectively (Liin et al., 2018; Lundquist et al., 2005; Ohno et al., 2011; Ravn et al., 2008). 11 Figure 1.3 Schematic illustration of the various regulatory accessory subunits associated with KCNQ1 channels. α = alpha protein, ATP = adenosine triphosphate, β = beta protein, CaM = calmodulin, cAMP = cyclic adenosine monophosphate, γ = gamma protein, GDP = guanosine diphosphate, GTP = guanosine triphosphate, PKA = protein kinase A, P = phosphate, PIP2 = phosphatidylinositol 4,5-bisphosphate, TM = transmembrane, Created in https://BioRender.com 1.4 Pharmacological modulations of KCNQ1/KCNE1 channels Currently, a few potentiators have been identified for both the KCNQ1/KCNE1 channel and the KCNQ1 channel alone. The mechanisms and effects of these agents vary, with some acting only in the presence of KCNE1 while others acting only in its absence. The discovery and usage of KCNQ1/KCNE1 activators have allowed us to study the mechanisms of this channel in-depth. Identifying their binding sites and effects has laid the groundwork for developing small modulators as potential therapeutic solutions to address KCNQ1/KCNE1 channel deficiencies. 12 1.4.1 Activators of KCNQ1 1.4.1.1 Stilbenes Stilbenes, such as 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS) were one of the earliest groups identified as activators of the KCNQ1/KCNE1 channel complex (Abitbol et al., 1999; Busch et al., 1994b). Stilbenes have significant and multi-faceted potentiation effects on KCNQ1/KCNE1 channels expressed in Xenopus oocytes and iPSC cardiomyocytes (Abitbol et al., 1999; Bollmann et al., 2020). These effects include increasing current amplitude, slowing deactivation kinetics, and hyperpolarizing the voltage dependence of activation. In contrast, the effect of DIDS on the KCNQ1 channel alone is more muted, with only a hyperpolarizing shift in the voltage dependence of activation observed. These findings suggest that KCNE1 plays a key role in modulating the effects of stilbenes on the KCNQ1/KCNE1 channel complex. While identifying the binding site, DIDS was found ineffective when injected intracellularly into Xenopus oocytes, suggesting that it acts extracellularly rather than intracellularly (Abitbol et al., 1999). Residues at positions 39-43 located extracellularly in KCNE1 were found to be important for DIDS potentiating effects. This supported the idea that DIDS acts extracellularly and highlighted KCNE1 as a crucial modulator of DIDS effects on the KCNQ1/KCNE1 channel complex. Successful rescue of the LQT5 phenotype associated with the KCNE1 mutation D75N by DIDS, revealed the therapeutic potential of IKs activators that bind to this region (Abitbol et al., 1999). 1.4.1.2 Mefenamic acid The fenamate mefenamic acid has been shown to potentiate KCNQ1/KCNE1 currents similar to DIDS (Abitbol et al., 1999). Like DIDS, mefenamic acid increases current amplitude, slows 13 deactivation kinetics, and hyperpolarizes the voltage dependence of activation (Abitbol et al., 1999; Busch et al., 1994b; Wang et al., 2020b). Wang et al. demonstrated that mefenamic acid’s potentiation is due to its ability to decrease the voltage sensitivity of the KCNQ1/KCNE1 channel and shift gating kinetics toward more negative potentials, i.e., enhanced channel activation gating in the presence of mefenamic acid (Wang et al., 2020b). These findings are further supported by single-channel experiments from the same study, which showed an increase in the open probability of the KCNQ1/KCNE1 channel complex in the presence of mefenamic acid (Wang et al., 2020b). Identical to DIDS, KCNE1 is a strong modulator of mefenamic acid’s effect. Wang et al. discovered that mefenamic acid's effect is KCNQ1:KCNE1 stoichiometry-dependent, with the greatest effect seen in channels with a 4:4 KCNQ1/KCNE1 ratio (Wang et al., 2020b). The effect weakened as the number of KCNE1 subunits decreased, with no significant effect observed when KCNE1 was absent. Mutation of K41 in KCNE1, a residue found to be part of the mefenamic acid binding, completely abolished mefenamic acid's potentiation effect, further highlighting the role of KCNE1 in modulating mefenamic acid's effects on the KCNQ1/KCNE1 channel complex (Wang et al., 2020b). Cross-linking studies have highlighted this extracellular region of KCNE1 as particularly interesting due to its interaction with the S1 and S6 transmembrane domains of KCNQ1. These regions of KCNQ1 likely play a significant role in the effects that mefenamic acid exerts on the KCNQ1/KCNE1 channel (Chung et al., 2009; Xu et al., 2008). In terms of binding, Abitbol et al. found that mefenamic acid also acts extracellularly and mefenamic acid could rescue the effects of the LQT5-associated D76N mutation (Abitbol et al., 1999). 14 1.4.1.3 ML277 ML277 first identified through a high-throughput screen, is a potent activator of the KCNQ1 channel with an EC50 of 260 nM (Mattmann et al., 2012). It is more than 100x selective to KCNQ1 over other KCNQs and cardiac ion channels such as hERG, Nav1.5 and Cav1.2 (Yu et al., 2013). The selectivity is driven by specific residues that make up the ML277 binding pocket in KCNQ1, which are not conserved in other KCNQ subunits. Several key residues identified as critical for ML277 binding in KCNQ1 are substituted by bulkier or smaller residues in other subunits, altering their binding properties (Willegems et al., 2022). Low docking scores from MD simulations for KCNQ2 and KCNQ4 further confirm this selectivity (Willegems et al., 2022). ML277 shows a strong preference for channels composed of KCNQ1 only and loses its potentiating effects as the number of KCNE1 subunits increases within the channel complex (Xiong et al., 2008; Yu et al., 2013). ML277 potentiates the KCNQ1 current by increasing the current amplitude and slowing deactivation kinetics (Eldstrom et al., 2021; Willegems et al., 2022; Xu et al., 2015; Yu et al., 2013). It was proposed that the hyperpolarization of the voltage-dependence of KCNQ1 activation and removal of voltage-dependent inactivation contributed to the increase in current amplitude (Eldstrom et al., 2021; Willegems et al., 2022; Xu et al., 2015; Yu et al., 2013), although others have indicated that ML277 instead induces a right shift in the GV relationship of KCNQ1, reflective of ML277’s enhanced occupancy of the AO state (Chen et al., 2022; Hou et al., 2019; Ma et al., 2022). Mechanistically, these effects are considered to result from enhanced VSD-pore coupling (Chen et al., 2022; Ma et al., 2022; Willegems et al., 2022). ML277 enhances the electro-mechanical coupling (EMC), which stabilizes the AO state 15 and reduces inactivation, increasing the open probability of the channel and thus increasing the macroscopic current (Eldstrom et al., 2021; Hou et al., 2019; Ma et al., 2022). An initial attempt to determine key domains and residues critical for drug effects using site-directed mutagenesis and voltage clamp studies suggested that ML277 binds to a site in a hydrophobic pocket in the KCNQ1 S5-S6 pore domain with minimal impact on the voltage sensor movement (Chen et al., 2022). An MD simulation study proposed that the ML277 binding site is either at the boundary of the S2-S3 loop and the S4-S5 linker of KCNQ1 or between the S5-S6 helices, with the latter confirmed by another experimental and docking study (Hou et al., 2020; Xu et al., 2015). Cryo-EM structures of Xenopus and human KCNQ1 with ML277 offered a high resolution of the binding site. While the structures capture the channels in different states (closed xKCNQ1 vs. open hKCNQ1), the two studies show that the drug resides in a hydrophobic pocket (termed as hydrophobic “elbow” by Hou et al., 2020) defined by the S4-S5 linker helix, S5 and S6 transmembrane segments from one subunit and the S5’ and S6’ helices from an adjacent subunit (Willegems et al., 2022). The binding of ML277 to the S4-S5 linker enhances the electro-mechanical coupling by lowering the energy barrier to enter the channel's AO state (Willegems et al., 2022 ). The enhancement of ECM pulls the N-terminus of the linker upward to induce the opening of the activation gate via a mechanism described as a “hand-and-elbow gating mechanism” (Hou et al., 2019; Ma et al., 2022). The hand-like C-terminus of the S4-S5 linker interacts with the pore in the VSD intermediate state, while the “elbow” links the S4 segment to the S4-S5 linker and engages with the S5 and S6 domains of the neighboring subunit to facilitate pore opening as the VSD transitions to the fully activated state (Hou et al., 2020). 16 Physiologically, ML277 has been shown to shorten the APD in hiPSC-CMs and guinea pig ventricles, presumably due to its enhancement of KCNQ1/KCNE1 channel current (Xu et al., 2015; Yu et al., 2013). This further suggests that IKs in cardiomyocytes may not be fully saturated with KCNE1 (4:4 ratio) and highlights ML277's potential as an archetype for developing therapeutic agents in the human heart. In addition, ML277 has been reported to have cardioprotective effects in acute coronary syndromes (Brennan et al., 2023). In cardiac models, ML277 modulates action potential duration, enhancing contractile recovery and cell survival rate while reducing infarct size in the coronary ligation model (Brennan et al., 2023). 1.4.1.4 Polyunsaturated fatty acids Polyunsaturated fatty acids (PUFAs) are amphipathic compounds with a long hydrocarbon tail with ≥2 double bonds and a charged hydrophilic head group (Benatti et al., 2004). PUFAs are believed to exhibit antiarrhythmic properties (Endo & Arita, 2016; Kang & Leaf, 2000), initially through the inhibition of Na+ and Ca2+ channels and, more recently, by activating the KCNQ1 channel (Liin et al., 2015b). PUFAs (1) increase the current amplitude of the channel by increasing the maximum K+ conductance and (2) hyperpolarize the voltage dependence of activation (Bohannon et al., 2020a; Liin et al., 2015b; Liin et al., 2016; Liin et al., 2018). PUFAs exert their effects on KCNQ1/KCNE1 through two distinct binding sites (Bohannon et al., 2020a; Bohannon et al., 2020b; Golluscio et al., 2024; Larsson et al., 2020; Liin et al., 2015b; Liin et al., 2018 ; Yazdi et al., 2021). The first site, located in the VSD, involves the interaction of the negatively charged PUFA head group with positively charged residues on the S4 segment 17 of KCNQ1 (R228 in KCNQ1 and R231 in the KCNQ1/KCNE1 complex). The subtle difference in the residues in which PUFAs bind in KCNQ1 and KCNQ1/KCNE1 channels arises from the distinct opening states of KCNQ1. Unlike KCNQ1, the KCNQ1/KCNE1 complex only opens after the second S4 transition (R231), and PUFA can, therefore, not exert its effects on R228, the residue responsible for the first S4 transition (Liin et al., 2018; Yazdi et al., 2021). PUFAs’ interaction with KCNQ1 stabilizes the S4 segment in its upward position, hyperpolarizing the voltage dependence of activation (Bohannon et al., 2020b; Liin et al., 2015b; Liin et al., 2016; Liin et al., 2018). The second site is in the pore domain, where PUFAs bind to residues K326 and D301. This binding stabilizes the selectivity filter in its conductive state; a mechanism elucidated through recent cryo-EM structures (Yazdi et al., 2021). These structures reveal that the interactions critical for stabilizing the selectivity filter are present only in structures with activated VSD, not those with a resting VSD. Thus, the ability of PUFAs to potentiate the KCNQ1/KCNE1 channel lies in its ability to stabilize the channel pore in its conductive state. Specifically, PUFA binding stabilizes interactions involving the aromatic ring cuff (W304, W305 and P320), which are critical for maintaining the conductive conformation of the selectivity filter – a feature relatively conserved among K+ channels (Doyle et al., 1998; Kurata & Fedida, 2006; Larsson & Elinder, 2000). During the rare transition from the non-conducting to the conducting state, the proline retreats from its position near the two tryptophan and flips outwards, enabling this conformation change. These findings support the idea that PUFA binding to the crevice between K326 and D301 – accessible only in the activated VSD state – stabilizes the usually unstable network of interactions responsible for maintaining the conductive form of the selectivity filter (Golluscio et al., 2024; Mandala & MacKinnon, 2023). 18 The key to the mechanism of action of PUFAs lies in its negatively charged head, which is central to the lipoelectric hypothesis (Elinder & Liin, 2017). This hypothesis suggests that the PUFA molecule integrates into the phospholipid bilayer through its hydrocarbon tail while its negatively charged head interacts to modulate the activity of KCNQ1 and KCNQ1/KCNE1 channels (Bohannon et al., 2020b; Borjesson & Elinder, 2008). PUFAs exhibit partial KCNE1 dependence, with certain analogs, e.g., docosahexaenoic acid (DHA), being less effective in the presence of KCNE1 (Liin et al., 2015b). The potentiation ability of PUFAs rest on their negatively charged head group interacting with arginine residues. However, KCNE1 reduces the local pH near the PUFA head group, leading to its protonation. This protonation renders PUFAs ineffective on KCNQ1/KCNE1 complexes. Since the deprotonated, negatively charged PUFA head is crucial for its effects on KCNQ1/KCNE1 channels, it is essential to maintain this charge, especially at physiological pH (7.5), for potential therapeutic applications. While the PUFA DHA is effective on KCNQ1 channels, the presence of KCNE1 in the KCNQ1/KCNE1 channel complex induces a conformation change. This brings the acidic residue (E290) closer to the PUFA binding site, leading to the protonation of the PUFA head and a loss of its effects (Larsson et al., 2018). Consequently, PUFA analogs capable of remaining negatively charged at physiological pH, such as N-arachidonoyl taurine (N-AT) and DHA-glycine, and most recently, linoleoyl taurine (Lin-taurine) are currently being explored for potential therapeutic use in modulating KCNQ1/KCNE1 channels (Bohannon et al., 2020a; Bohannon et al., 2020b; Liin et al., 2015b; Liin et al., 2016; Liin et al., 2018). 19 In more physiologically relevant settings like isolated guinea pig hearts, negatively charged PUFA analogs can reduce pharmacologically prolonged ventricular action potentials and QT intervals (Liin et al., 2015b). 1.4.1.5 R-L3 R-L3 or L-364,373 is a benzodiazepine that potentiates IKs and shortens the APD in guinea pig cardiac myocytes, and suppresses early afterdepolarizations (EAD) in ventricular myocytes isolated from rabbit hearts (Salata et al., 1998; Xu et al., 2002). In KCNQ1, the drug increases current amplitude, slows deactivation kinetics, and hyperpolarizes the voltage dependence of activation (Chen et al., 2022). Mutagenesis studies propose that R-L3 shares the hydrophobic pocket in the S5-S6 pore domain of KCNQ1 with ML277 (Chen et al., 2022). However, their mechanisms of action differ slightly, as R-L3 exerts a dual effect on both the pore and the VSD. It remains unclear whether the effects of R-L3 on the VSD occur via direct interaction or as an allosteric effect resulting from disturbances at the pore. The aromatic residues (Y267, F339, and F340) and G269 in the KCNQ1 pore domain are critical for the binding of R-L3, as revealed by mutagenesis studies (Chen et al., 2022). While these residues are similarly essential for ML277 binding, R-L3 exhibits unique behavior. Mutations in these residues abolish the ability of R-L3 to increase current amplitude but do not eliminate its effect on slowing deactivation kinetics. This suggests that R-L3 also modulates the VSD (Chen et al., 2022). Fluorescence experiments confirm this hypothesis by demonstrating slowed fluorescence deactivation kinetics, indicative of the influence of R-L3 on voltage sensor movements. Moreover, computational modeling reveals that the effects of R-L3 on KCNQ1 20 channels can be recapitulated fully by incorporating a decreased deactivation rate of the voltage sensor transition into the same model used to simulate the action of ML277 (Chen et al., 2022). These findings support the hypothesis of a shared binding site while demonstrating the additional ability of R-L3 to influence the VSD. Additionally, it is likely that the binding of R-L3 to KCNQ1 overlaps, at least partially, with KCNE1. Like ML277, R-L3 exerts its strongest effects on KCNQ1-only channels, with its efficacy diminishing as the stoichiometric ratio of KCNQ1:KCNE1 approaches 4:4 (Xiong et al., 2008; Yu et al., 2013). This may result from competitive interactions between R-L3 and KCNE1 at overlapping binding sites. Crosslinking studies have demonstrated that KCNE1 interacts with the S6 segment of KCNQ1 (Chung et al., 2009). Furthermore, residues such as S338, F339, and F340 on KCNQ1, which are critical for the action of R-L3, have also been found to interact with KCNE subunits (Melman et al., 2004; Panaghie & Abbott, 2006). 1.4.1.6 Other IKs activators Other IKs activators that have been identified but less extensively studied include hexachlorophene (HCP) and phenylboronic acid (PBA). HCP is the active ingredient in pHisoHex, a topical anti-infective drug (Jungermann, 1968). HCP has been shown to potentiate the KCNQ1/KCNE1 channel by increasing current amplitude, slowing deactivation, and hyperpolarizing the voltage dependence of activation (Zheng et al., 2012). These potentiation effects reduce APD in pig cardiomyocytes. However, the precise binding mechanism remains unknown. PBA is a boronic acid shown to hyperpolarize the voltage dependence of activation of KCNQ1/KCNE1 channels (Mruk & Kobertz, 2009). Experimental evidence suggests that PBA 21 may also target the permeation pathway of KCNQ1 channels. However, the binding mechanism remains unknown. 1.4.2 Activators of other KCNQ isoforms 1.4.2.1 Retigabine Retigabine (Ezogabine) is an antiepileptic drug that was used as an adjunctive treatment for refractory partial-onset seizures in adults from its approval in 2011 till its discontinuation in 2017 due to poor target selectivity and resultingly serious side effects. Retigabine promotes KCNQ2 channel activation by slowing deactivation and hyperpolarizing the voltage dependence of activation while having little effect on the maximum current amplitude (Kanyo et al., 2020; Li et al., 2021; Main et al., 2000; Vicente-Baz et al., 2016; Wickenden et al., 2000). Cryo-EM studies have identified the binding site of retigabine at the inter-subunit interface of the pore domain within a hydrophobic pocket formed by S5, pore helix, and S6 from the neighboring subunit, where it binds to KCNQ2 primarily via hydrogen bonds (Li et al., 2021). The residues lining the retigabine-binding pocket are highly conserved across the KCNQ family, except for KCNQ1, which accounts for the high selectivity of retigabine for other KCNQ channels over KCNQ1 (Li et al., 2021). The binding of retigabine leads to conformational changes in the pore domain. When the pore domain is viewed from the extracellular side, there is a clockwise rotation of the upper halves of S5, the pore helix, and S6, which may couple the conformational changes of the lower activation gate when it opens. Moreover, retigabine binding increases the mobility of the VSD, likely lowering the energy required for the VSD to transition from the resting to the activated state (Li et al., 2021). 22 1.4.2.2 Ztz240 Ztz240 is a KCNQ activator first identified using high throughput screening parameters in 2010 (Gao et al., 2010). Ztz240 is most selective for KCNQ4 and KCNQ5, followed by KCNQ2, while exhibiting low selectivity for KCNQ1 and KCNQ3 due to their low sequence similarity in the binding pocket of ztz240 (Gao et al., 2010; Li et al., 2021). In KCNQ2, KCNQ4 and KCNQ5, ztz240 increases current amplitude and hyperpolarizes the voltage dependence of activation, with a larger shift in KCNQ4 and KCNQ5. Ztz240 also eliminates the two-phase deactivation in KCNQ4 and KCNQ5, but this effect is not observed in KCNQ2, where two-phase deactivation remains (Gao et al., 2010). Cryo-EM studies show that ztz240 sits at the gating charge transfer centre – where a large quantity of electric charge (gating charge) is transferred across the membrane voltage difference when a sensor switches from off to on – to form mainly hydrophobic interactions with S3 and S4 (Li et al., 2021; Tao et al., 2010). Upon binding, ztz240 induces local conformational changes in the VSD. The S2, S3, and a significant part of S1 shift to create a deeper binding pocket for ztz240. Then, S4 rotates closer to the pore domain, strengthening hydrophobic interactions with the S5 of the neighboring subunit. This interaction helps maintain S4 in its 'up' conformation, thereby stabilizing VSD in its activated state (Li et al., 2021). 1.4.2.3 ML213 ML213 was originally identified as a selective KCNQ2 and KCNQ4 activator through high-throughput screening in 2011(Yu et al., 2011). However, ML213 was later found also to activate KCNQ3 and KCNQ5 (Kim et al., 2015; Provence et al., 2018). ML213 increases the current 23 amplitude, slows the channel deactivation, and hyperpolarizes the voltage dependence of activation (Jepps et al., 2014; Kanyo et al., 2020; Kim et al., 2015; Yu et al., 2011; Zheng et al., 2021). Cryo-EM studies reveal that ML213 targets the pore, and its binding site is enclosed by four walls formed by hydrophobic residues from two adjacent subunits, (1) S5, (2) S6, (3) the S4-S5 linker, and (4) S6 from the neighboring subunit (Zheng et al., 2021). The activation mechanism of ML213 is similar to that of retigabine (Kanyo et al., 2020; Vicente-Baz et al., 2016; Zheng et al., 2021). 1.4.2.4 Cannabidiol Cannabidiol (CBD), one of the main active compounds in cannabis, is a clinically effective antiepileptic drug. CBD directly potentiates KCNQ2 and KCNQ3 channels by increasing the current amplitude and hyperpolarizing the voltage dependence of activation (Ma et al., 2023; Pisanti et al., 2017; Zheng et al., 2021). Cryo-EM studies show that two CBD molecules bind simultaneously to KCNQ2 at a hydrophobic cleft formed by the first subunit, S5, the pore helix, S6 from the second subunit, and S1 from the third subunit, primarily via hydrophobic interactions (Ma et al., 2023). One CBD molecule occupies the deep, classical fenestration pocket of voltage-gated ion channels, lined by S6 from the first subunit and S5 and S6 from the second subunit. The other CBD molecule localizes in a shallower, upper pocket formed by S5, and the pore helix from the second subunit, along with S1 from the third subunit, covering the upper part of the first CBD molecule (Ma et al., 2023). The binding of CBD molecules stabilizes the PIP2-bound state by pushing the VSD away, ensuring a sufficiently large hydrophobic pocket between the VSD and PD for PIP2, which is known to enhance EMC. The first CBD molecule stabilizes approximately 20% of the total channels in the open conformation, while the second 24 CBD molecule further reinforces this stabilization, increasing the proportion of channels in the open conformation (Ma et al., 2023). 1.4.3 Blockers of KCNQ1 1.4.3.1 Chromanol 293B Chromanol 293B is a highly selective and specific blocker of the KCNQ1/KCNE1 channel (IC50 = 1.8 µM) (Bosch et al., 1998; Bosch et al., 2003; Busch et al., 1996; Gerlach et al., 2001; Lerche et al., 2007; Lerche et al., 2000). Chromanol 293B blocks the current amplitude of the channel but keeps the voltage dependence of activation and channel kinetics unaffected (Lerche et al., 2007; Lerche et al., 2000; Seebohm et al., 2001; Yang et al., 2000). KCNQ1/KCNE1 channels expressed in Xenopus oocytes and IKs current recorded from guinea pig cardiomyocytes are blocked by chromanol 293B. Based on its ability to prolong APD, chromanol 293B has been suggested to function as a Class III antiarrhythmic drug. This property is particularly useful in the treatment of tachyarrhythmias, where chromanol 293B can help to prolong the otherwise shortened APD and increase the refractory period (Bosch et al., 1998). As proposed by multiple studies, the chromanol 293B binding site is located in the central cavity of KCNQ1 (Decher et al., 2004; Lerche et al., 2007; Seebohm et al., 2003). Binding to KCNQ1 occurs via hydrophobic interactions with specific residues, I337 and F340, to stabilize the ligand-receptor-K+ ion complex (Lerche et al., 2007). The result is a plug in the ion permeation pathway. Mutation of these residues, along with T312, has been shown to impair the blocking effect of Chromanol 293B. This is likely due to T312’s involvement of the selectivity filter in forming the K+ binding site. After the initial binding of chromanol 293B to the channel, it is 25 stabilized by electrostatic interactions between its oxygen atoms and a potassium ion in the inner vestibule, effectively blocking further K+ ion permeation (Lerche et al., 2007). 1.4.3.2 HMR1556 HMR1556, a derivative of chromanol 293B, is a more potent KCNQ1/KCNE1 channel blocker compared to chromanol 293B (HMR1556 IC50 = 10.5 nM vs Chromanol 293B IC50 = 1.8 µM in canine ventricular myocytes) due to its higher binding affinity (Thomas et al., 2003). The mechanism of action of HMR1556 is thought to be similar to that of chromanol 293B, but the details are not elucidated. 1.4.3.3 JNJ-303 JNJ-303, developed by Janssen Pharmaceutical, is an IKs blocker (IC50 = 12.6µM). Studies using hiPSC models found that JNJ-303 decreases current amplitude and prolongs the APD (Bjork et al., 2017). Unfortunately, its torsadogenic effects have limited its potential as a therapeutic drug. JNJ-303 caused marked QT prolongation in anesthetized guinea-pig models and even more extreme QT prolongation (>600 ms) in anesthetized canine models, triggering pulse-dependent Torsades de Pointes events spontaneously, and led to the unexpected deaths of two animals during pharmacology and pharmacokinetics studies (Towart et al., 2009). These findings suggest that JNJ-303 may also have off-target effects, affecting other ion channels (Gallacher et al., 2007; Towart et al., 2009). 26 1.4.3.4 UCL2077 Triphenylmethylaminomethyl pyridine, also known as UCL2077, was developed to inhibit small-conductance Ca²⁺-activated K⁺ channels (SK channels), which are believed to contribute to the slow afterhyperpolarization (sAHP) in hippocampal neurons (Shah et al., 2006). With the knowledge that UCL2077 blocks K+ channels, Soh et al. tested its effects on mammalian cells expressing KCNQ1 channels and found that it selectively inhibits KCNQ1 channels compared to other KCNQ subtypes (Soh & Tzingounis, 2010). UCL2077 has been demonstrated to be an open-channel blocker (Barro-Soria et al., 2014; Soh & Tzingounis, 2010). It has been proposed that, when KCNQ1/KCNE1 channels open, UCL2077 enters the pore, binds to the S6 segment, and stabilizes the gate in the closed state, inhibiting the second S4 movement to prevent it from reopening. This finding is supported by fluorescence data showing that UCL2077 eliminates the second component of the fluorescence signal, corresponding to the second S4 movement necessary for channel opening (Barro-Soria et al., 2014). Furthermore, UCL2077 has proven helpful in experiments distinguishing the contributions of voltage sensor and the pore to channel currents. It helped demonstrate that KCNE1 divides the voltage sensor movement in KCNQ1/KCNE1 channels in two distinct steps – an initial intermediate state followed by full activation – before the pore opens (Peng et al., 2017). 27 1.5 Scope of the thesis The introduction highlights the powerful impact of IKs activators on the IKs current and, consequently, their immense therapeutic potential. Thus, understanding the binding sites of IKs activators and their effects across the full concentration spectrum is crucial for guiding future drug development efforts in identifying clinically useful IKs agonists. IKs activators such as ML277 and PUFAs have been studied extensively. Our lab, along with another, previously published a cryo-EM structure of the ML277-bound KCNQ1 channel (Ma et al., 2022; Willegems et al., 2022). Significant work has been done to characterize the binding site and mechanism of PUFAs on the IKs complex. However, despite mefenamic acid being one of the few activators that enhance a fully saturated KCNQ1/KCNE1 complex, mefenamic acid and DIDS have not been studied in detail since 1999, when Abitbol and colleagues determined they act extracellularly (Abitbol et al., 1999). Previous studies used site-directed mutagenesis and electrophysiology to identify key residues involved in mefenamic acid binding to the IKs complex. A few years ago, our lab demonstrated that mefenamic acid enhances IKs activity in a dose- and stoichiometry-dependent manner (Wang et al., 2020b). Nevertheless, significant gaps remain in understanding how mefenamic acid and DIDS bind to the IKs complex and exert their effects. For instance, the precise binding site had not yet been fully mapped – which is exactly what we set out to accomplish. In Chapter 2, by integrating results from molecular dynamics (MD) simulations and extensive electrophysiology experiments, I not only contributed to the successful characterization of an occult and flexible binding pocket at the S1/KCNE1/pore interface, where mefenamic acid and DIDS bind, but also how they modulate the interactions between KCNQ1 and KCNE1. 28 During these experiments, I observed that while mefenamic acid potentiated IKs at moderate concentrations (~100 µM), higher concentrations paradoxically reduced current amplitude. However, key potentiating effects – such as slowed deactivation kinetics and instantaneous current onset – were preserved. In Chapter 3, I sought to identify the underlying mechanism behind this blocking effect. My results suggest that high concentrations of mefenamic acid induces an allosteric conformational change that occludes the pore, driving the channel into a non-conducting state. Finally, Chapter 4 builds on previous work (Wang et al., 2020b) by investigating how many subunits are required to support the activating action of the mefenamic acid. I contributed to examining the impact of incorporating the K41C-KCNE1 mutation into individual subunits, which destabilizes local drug binding. Our findings demonstrate that the potency of mefenamic acid progressively decreases as the number of mutant K41C-KCNE1 subunits increases, supporting a graded, stoichiometric activation model. 29 Chapter 2: A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes 2.1 Introduction Potassium ion (K+) channel activators are important compounds in human health, as partial or complete loss of function of many K+ channels may lead to inherited or acquired diseases that have significant morbidity and mortality. The delayed cardiac rectifier potassium current, IKs, plays an important role, especially at high heart rates, in the physiological shortening of the cardiac action potential (Sanguinetti et al., 1996). Unsurprisingly, mutations in both KCNQ1 (α-subunit) and KCNE1 (β-subunit), which when paired together give rise to the IKs current (Barhanin et al., 1996; Bendahhou et al., 2005; Sanguinetti et al., 1996), have been implicated in cardiac arrhythmia syndromes such as long QT syndrome (LQTS) and atrial fibrillation (Y. H. Chen et al., 2003; Eldstrom & Fedida, 2011; Jervell & Lange-Nielsen, 1957; Olesen et al., 2014; Q. Wang et al., 1996). With nearly all mutations seen in LQTS patients identified as loss-of-function and 50% of those loss-of-function mutations identified in the KCNQ1 subunit (Ackerman et al., 2011; Hedley et al., 2009), enhancing and activating IKs currents has long been suggested as a promising therapeutic approach for treating LQTS. It is a curiosity of IKs channels that the known activators fall into two general groupings: those that work best on the α-subunit, KCNQ1, alone, including zinc pyrithione, L-364,373, and one of the most studied activators, ML277 (Eldstrom et al., 2021; Mattmann et al., 2012; Xu et al., 2015; Yu et al., 2013); and those that work best in the presence of the auxiliary β-subunit, KCNE1, compounds like mefenamic 30 acid and DIDS (4,4'-diisothiocyano-2,2'- stilbene disulfonic acid) (Abitbol et al., 1999; Wang et al., 2020b). Using cryo-EM, we recently visualized the binding of ML277 deep in the central core of KCNQ1 channels in a pocket lined inferiorly by the S4-S5 linker, laterally by the S5 and S6 helices of two separate subunits, and above by pore domain residues (Willegems et al., 2022). The location of the binding pocket and its structural inter-relationships help to explain the underlying mechanism of action of ML277, its specificity for KCNQ1, and the lack of efficacy due to steric hindrance in the presence of a β-subunit. However, it is known neither where activators of IKs bind that require the presence of the β-subunit, such as phenylboronic acid (Mruk & Kobertz, 2009), hexachlorophene, stilbenes such as DIDS and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), and diclofenac acid derivatives such as mefenamic acid (Abitbol et al., 1999; Wang et al., 2020b; Zheng et al., 2012), nor how they mediate their activator action. We have some clues for DIDS and mefenamic acid that their binding sites are not in the central channel core, as is the case for ML277, but depend on KCNE1 β-subunit residues at the extracellular surface of the channel, residues 39–43 in KCNE1 for DIDS (Abitbol et al., 1999) and K41 for mefenamic acid (Wang et al., 2020b). Our recent cysteine scanning data revealed that although other extracellular KCNE1 residues in the same region to varying degrees impacted the effect of mefenamic acid, only the K41C mutation completely abolished mefenamic acid effect up to a concentration of 1 mM (Wang et al., 2020b). Previous cross-linking studies have identified key interactions between this extracellular region of KCNE1 and the S1 and S6 transmembrane segments of KCNQ1 (Chung et al., 2009; Xu et al., 2008), 31 suggesting that residues in either of these regions could also provide important clues to explain mefenamic acid’s mechanism of action. To further explore the dependence of residues in KCNE1 and those in adjacent KCNQ1 sites on the binding of mefenamic acid to IKs, we first examined the role of K41C in preventing the drug effect. Docking in combination with molecular dynamics (MD) simulations of mefenamic acid binding to IKs followed by mutagenesis were used to map out critical KCNE1 and KCNQ1 residues. Further, we expanded on the idea that the stilbene, DIDS (Abitbol et al., 1999), which is structurally quite different from mefenamic acid, shares a common binding site. Our results showed that both compounds bind in the same general region formed by elements of the pore and S6 domains of KCNQ1 and the near extracellular region of KCNE1 but depend on different critical residues for their binding stability. Exposure of the channel complex to either compound induces subtle structural changes that subsequently stabilize the conformation of the S1/outer pore/S6 of KCNQ1 and slow the IKs deactivation gating kinetics. The results suggest the existence of a common drug-induced binding site and a mechanism of action for small molecule IKs activators that is distinct from that of specific compounds that activate KCNQ1 alone. 2.2 Material and Methods 2.2.1 Molecular docking and molecular dynamic simulations A model of the IKs channel complex – termed pseudo-KCNE1-KCNQ1 (ps-IKs) – was created based on the cryogenic electron microscopy (cryo-EM) structure of KCNQ1-KCNE3 (PDB ID: 6v01; (Sun & MacKinnon, 2020). In this structure, the extracellular residues of KCNE3, R53-Y58, were substituted with homologous KCNE1 residues D39-A44. Conformational sampling 32 was then performed for substituted residues and the lowest free energy conformations were selected for subsequent docking experiments applying a four-dimensional (4D) docking approach which accounts for the flexibility of the receptor site configuration (Bottegoni et al., 2009). After docking, a receptor ensemble with multiple conformations of the putative binding site region was created via another round of conformational sampling for the external part of ps-KCNE1 and its KCNQ1 neighborhood (8 Å cut-off distance) and generated conformations were used for a new docking iteration. A ligand-channel conformation with the lowest free energy was chosen from the final docking iterations. The docking and conformational sampling as well as substitution of amino acids were performed with ICM-pro 3.8 software (Neves et al., 2012). A schematic representation of the general workflow for ps-IKs model construction and drug docking can be found in Figure A.1. The coordinates of mefenamic acid-bound ps-IKs channel complexes with the lowest free energy were then used for two sets of MD simulations with AMBER and CHARMM force fields (see below). Considering the complexity of the binding site located on the periphery of the pore, ps-KCNE1 and VSD, docking and subsequent MD simulations were performed for only one binding site (Figure 2.2D). To keep the voltage-sensing domain (VSD) in its activated state conformation, we restrained the PIP2 molecules in their cryo-EM positions with a force constant of 1000 kJ/mol/nm2 during MD simulations. Three 300 ns duration simulations were performed in a water environment with AMBER20 using a ff14SB force field for protein and GAFF/AM1-BCC scheme for the ligand parameterization and calculation of the atomic point charges (Case et al., 2005; Jakalian et al., 33 2002; Maier et al., 2015). The complex was solvated with TIP3P water models and Na+/Cl- at 100 mM concentration. The system was minimized and equilibrated in the NVT and NPT ensembles for 10 ns, gradually releasing spatial restraints from the backbone and sidechains. During the last 5 ns only backbone atoms were restrained with a force constant of 50 kJ/mol/nm2. The same procedure was used for equilibration of mutant complexes after introduction of mutations using ICM-pro 3.8 software. We used the Langevin thermostat with a collision frequency of 2 ps-1, a reference temperature of 310 K, and Monte Carlo barostat with reference pressure at 1 bar (Oliver et al., 1997; Wu et al., 2016). The long-range electrostatic interactions with a cut-off at 10 Å were treated with the Particle Mesh Ewald (PME) algorithm. Bonds involving only hydrogens were constrained with the SHAKE algorithm and a 2 fs integration time step was used. Five 500 ns duration simulations were performed using a CHARMM36m force field and ps-IKs complex inserted into a POPC membrane. The same MD parameters described in our previous study were used for this set of simulations (Willegems et al., 2022). Trajectories from MD simulations were clustered with TTclust based on ligand and its binding site RMSD. The elbow method was used to find the optimum number of clusters. From each cluster, a centroid with the lowest RMSD to all other ligand conformations in the cluster was selected as a representative structure. GROMACS-2021.4 was used for RMSD, RMSF, and H-bond calculation. RMSD was calculated from the initial position of the ligand after the least squares fit alignment of the protein backbone. For RMSF calculations only backbone and C-beta atoms were used. 34 Trajectories from MD simulations with a CHARMM36m force field are available at https://doi.org/10.5281/zenodo.8226585. The 2D diagrams and other molecular visualizations were generated by ICM-pro and VMD software. The MM/PBSA and MM/GBSA methods were used to calculate the free energy of binding (Miller et al., 2012). We collected 1000 snapshots with equal intervals (every 0.3 ns) if ligands did not leave the binding site during 300 ns. 2.2.2 Reagents and solutions DIDS (Sigma-Aldrich, Mississauga, ON, Canada) was prepared as a 50 mM stock solution dissolved in 100% dimethyl sulfoxide. Stock DIDS solution was diluted in control whole-cell bath solution to obtain a final DIDS concentration of 100 µM which was perfused onto mammalian cells for whole-cell experiments. All other reagents and solutions were prepared as described (Wang et al., 2020b). Mefenamic acid (Tocris Bioscience, Oakville, ON, Canada) was used to activate IKs at concentrations of 100 μM and 1 mM. 2.2.3 Molecular biology, cell culture and whole cell patch clamp tsA201 transformed human embryonic kidney 293 cells were purchased directly from Sigma-Aldrich. After culturing, they were plated for whole-cell experiments and subsequently transfected with Lipofectamine2000 (Murray et al., 2016; Wang et al., 2020b; Westhoff et al., 2019). All mutations were first generated using site-directed mutagenesis and Pfu turbo, then sequence confirmed. Whole-cell experiments were conducted 24-48 hr post transfection. For wild type (WT) EQ and mutant x-EQ-Y (where ‘x’ denotes a KCNE1 mutation and ‘y’ denotes a KCNQ1 mutation; for example, K41C-EQ, EQ-W323A), cells were transfected with a linked KCNE1 and KCNQ1 cDNA (2-3 µg was used) which assembles as a fully saturated 4:4 ratio of 35 KCNE1 to KCNQ1. All constructs were also co-transfected with 0.8 µg of GFP to allow transfected cells to be identified. Data were obtained using an Axopatch 200B amplifier, Digidata 1440 A digitizer and pCLAMP 11 software (Molecular Devices, LLC, San Jose, CA). 2.2.4 Data analysis Conductance-voltage (G-V) relationships were obtained from the normalized peak of the initial tail current (G/Gmax) and plotted against the corresponding voltage. G-V plots were fitted with a Boltzmann sigmoid equation to obtain the voltage at half-maximal activation (V1/2) and slope (k) values (Tables 2.1, 2.3 and 2.5). For each EQ mutant, the change in activation V1/2 (∆V1/2=V1/2 in the presence of drug-V1/2 control) was also determined (Figures 2.3E and 2.7D). In some cases, the foot of the G-V curve did not reach 0 (essentially in the presence of drug) and as a result, the V1/2 was read directly from the normalized plots at the voltage point where the curve crossed the 0.5 value on the y coordinate. Slowing of tail current decay was used as a measure of mefenamic acid and DIDS impact on WT and mutated EQ channel complexes. Specifically, the peak to end difference currents were calculated by subtracting the minimum amplitude of the deactivating current from the peak amplitude of the deactivating current. The difference current in mefenamic acid or DIDS was normalized to the maximum control (in the absence of drug) difference current and subtracted from 1.0 to obtain the normalized response (Figures 2.3D and 2.7C). Data files collected during this study and used in the preparation of results and figures are available at https://doi.org/10.5281/zenodo.8226585. GraphPad Prism 9 (GraphPad Software, San Diego, CA) was used to analyze all the data. Where 36 applicable, unpaired t-test or one-way ANOVA followed by the Fisher’s least significant difference (LSD) test was used to determine statistical significance. In all figures ****, ***, **, * denotes a significance where p<0.0001, p<0.001, p<0.01 and p<0.05, respectively. All data in the figures and tables are shown as mean ± SD or SEM. Bar graphs showing mean ∆V1/2 (Figures 2.3E and 2.7D) were generated by calculating changes in V1/2 induced by drug treatment vs. control in separate cells. ∆V1/2 values reported in the Results were calculated from the mean V1/2 values shown in Tables 2.1 and 2.3. 2.3 Results 2.3.1 The mefenamic acid binding site on the KCNQ1/KCNE1 complex Exposure of wild type IKs complexes (4:4 ratio; WT EQ) to 100 μM mefenamic acid transforms the slowly activating IKs current into one with an almost linear waveform and completely inhibits tail current decay at -40 mV (Figure 1A; (Abitbol et al., 1999; Unsöld et al., 2000; Wang et al., 2020b). G-V relations obtained from peak initial tail currents show that 100 μM mefenamic acid hyperpolarizes the G-V (ΔV1/2 = -105.7 mV, Figure 2.1B and C) and decreases the slope (control k=19.4 mV, Mef k=41.3 mV, Table 2.1). We previously showed that introduction of a cysteine mutation at residue K41 in all four KCNE1 subunits (4:4 ratio of mutant K41C-KCNE1 to KCNQ1; K41C-EQ) itself had only a minor effect on the G-V, but prevented changes to currents and the G-V relationship on exposure to 100 μM or 1 mM mefenamic acid (Figure 2.1B and C and Table 2.1; (Wang et al., 2020b). The data suggest that residue K41 in KCNE1 is critical to the action of mefenamic acid, but give no information why K41 is so important, and prompted us to question how this residue and other adjacent residues in KCNE1, and those nearby on KCNQ1 acted together to form a binding pocket for mefenamic acid. 37 Figure 2.1 K41C-KCNE1 mutants prevent the agonist effect of mefenamic acid. (A) Current traces of WT EQ (top) and K41C-EQ (bottom) in the presence of 100 µM mefenamic acid (Mef). A 4 s protocol was used with pulses from -150 mV or higher to +100 mV, in 10 mV steps, followed by a repolarization step to -40 mV for 1 s. Holding potential and interpulse interval were -80 mV and 15 s, respectively. (B) G-V plots obtained from WT EQ tail currents (triangles) and K41C-EQ (circles) in the absence (control: black) and presence of Mef (100 µM: grey; 1 mM: light blue). Boltzmann fits were: WT EQ control (n = 6): V1/2 = 25.4 mV, k = 19.4 mV; WT EQ 100 μM Mef (n = 3): V1/2 = -80.3 mV, k = 41.3 mV; K41C-EQ control (n = 4): V1/2 = 15.2 mV, k = 18.4 mV; K41C-EQ 100 μM Mef (n = 4): V1/2 = 11.4 mV, k = 19.4 mV; and K41C-EQ 1 mM Mef (n = 3): V1/2 = 16.7 mV, k = 19.8 mV. Error bars shown are SEM. (C) Summary plot of V1/2 change (ΔV1/2) for WT EQ in the presence of 100 µM mefenamic acid, WT EQ vs K41C-EQ in control and K41C-EQ in the presence of 100 µM and 1 mM mefenamic acid. Data are shown as mean ± SEM and unpaired t-test was used. **** denotes a significant ΔV1/2 compared to control where p<0.0001. 38 Table 2.1 Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of mefenamic acid for fully saturated IKs channel complexes. A statistical difference in V1/2 compared to control is shown as p-value, determined using an unpaired t-test. NS denotes not significant. Values are shown ± SEM. * For K41C-EQ, the concentration of mefenamic acid used was either 100 µM (upper row values) or 1 mM (lower row values). For all other constructs, 100 µM mefenamic acid was used. ** An estimation of the activation V1/2 was calculated from a right shifted, non-saturating GV curve. Control 100 µM or 1 mM mefenamic acid * p-value V1/2 (mV) k-factor n V1/2 (mV) k-factor n WT EQ 25.4 ± 2.4 19.4 ± 1.2 6 -80.3 ± 4.1 41.3 ± 8.4 3 <0.0001 K41C-EQ 15.2 ± 1.1 18.4 ± 1.7 4 11.4 ± 1.0 19.4 ± 0.8 4 <0.05 16.7 ± 2.0 19.8 ± 1.4 3 NS L42C-EQ 68.9 ± 1.5 21.5 ± 3.7 3 31.8 ± 0.4 14.8 ± 4.27 3 <0.01 E43C-EQ 70.8 ± 1.8 27.0 ± 2.8 4 46.4 ± 2.1 29.1 ± 1.7 4 <0.01 A44C-EQ 4.1 ± 1.8 17.6 ±1.4 4 -5.6 ± 2.7 18.3 ± 2.0 4 <0.05 Y46A-EQ** 76.4 ± 1.5 57.3 ± 1.6 3 -29.8 ± 4.1 18.9 ± 3.1 3 <0.05 EQ-W323A 47.8 ± 2.7 23.7 ± 2.2 4 33.7 ± 1.2 28.5 ± 3.1 3 <0.05 EQ-W323C 54.0 ± 2.1 22.2 ± 2.1 4 27.5 ± 3.3 25.6 ± 1.8 4 <0.05 EQ-V324A 34.4 ± 2.3 20.7 ± 1.1 6 15.5 ± 2.3 27.9 ± 1.6 5 <0.05 EQ-V324W 41.0 ± 2.4 18.4 ± 0.3 4 27.3 ±6.0 25.5 ± 1.3 4 NS EQ-Q147C 63.9 ± 5.8 25.4 ± 2.0 4 26.3 ± 5.7 32.7 ± 1.7 4 <0.05 EQ-Y148C 36.8 ± 0.3 20.3 ± 0.4 4 17.5 ± 4.0 36.7 ± 3.7 4 <0.05 39 2.3.2 Mefenamic acid binding site predicted by molecular modeling Initially, to visualize potential drug binding sites and understand how mutation of KCNE1 and KCNQ1 residues might prevent drug action, in-silico experiments of drug docking with subsequent MD simulations were performed on a model of IKs channels. The IKs model was constructed based on the recent cryo-EM structure of KCNQ1-KCNE3, which is thought to represent the activated-open state of the channel complex (Sun & MacKinnon, 2020). Taking into consideration the sequence similarity of KCNE1 and KCNE3 subunits in their transmembrane segments, it has been suggested that the main interface of these subunits with KCNQ1 is preserved in this region. Our initial data indicated that the extracellular residues of KCNE1 are involved in the action of IKs activators, so we constructed a model where external KCNE3 residues R53-Y58 were substituted with homologous KCNE1 residues, D39-A44 (Figure 2.2A). The resulting 4:4 IKs channel complex was termed pseudo-KCNE1-KCNQ1, ps-IKs, and Figure 2.2B shows the essential elements of the ps-IKs subunits which form the extracellular interface of KCNQ1 and KCNE and served as a basis for drug docking and MD simulations. Details of the docking procedure are described in the Materials and methods section and schematically summarized in Figure A.1. 40 Figure 2.2 MD prediction of mefenamic acid binding site in the ps-IKs model. (A) Pseudo-KCNE1 (ps-KCNE1) used to predict Mef binding site. Extracellular residues of KCNE1 (top), ps-KCNE1 (middle) and KCNE3 (bottom). Below, cartoon topology of the single transmembrane ps-KCNE1 β-subunit and the six transmembrane KCNQ1 α-subunit. S1-S4 transmembrane segments form the voltage sensor domain and S5-S6 form the pore domain. (B) Binding pose of Mef (yellow) in external region of the ps-IKs channel complex obtained with docking (side view). Pore domain residues are blue, ps-KCNE1 subunit red, and the VSD of a neighbouring subunit is in yellow. (C) Ligand interaction map of Mef with ps-IKs from molecular docking. Size of residue ellipse is proportional to the strength of the contact. The distance between the residue label and ligand represents proximity. Grey parabolas represent accessible surface for large areas. Light grey ellipses indicate residues in van der Waals contacts, light green ellipses are hydrophobic contacts, and light blue are H-bond acceptors. Red borders indicate KCNE1, yellow are KCNQ1 VSD, and blue are pore residues. Dashed lines indicate H-bonds. The 2D diagram was generated by ICM pro software with cut-off values for hydrophobic contacts of 4.5 Å and hydrogen bond strength of 0.8. Further details in Materials and Methods. (D) Mef binding pose observed in MD simulations in space-fill to highlight pocket formed by external S1 (yellow), S6 (blue) transmembrane domains of KCNQ1, and extracellular region of the ps-KCNE1 subunit (red). This binding conformation is the most frequent binding pose of Mef observed in ∼50 % of frames and corresponds to the blue-framed conformation in Figure A.3. 41 Briefly, conformational sampling was performed on ps-IKs residues D39-A44, and conformations showing the lowest free energy were selected for docking using a four-dimensional (4D) docking approach to find the best binding pose of the ligand. A conformation with the best docking score (Figure 2.2B) shows mefenamic acid binding to the pocket formed between extracellular KCNE1 residues, the external S6 transmembrane helix of one subunit, and the S1 transmembrane domain of the neighboring subunit and the pore turret of a third subunit. The estimated volume of the pocket in the mefenamic acid bound state is ~307 Å3 with a hydrophobicity value of ~0.65 kcal/mol (Figure 2.2D). The free energy of the mefenamic acid-ps-IKs interaction estimated by the MM/GBSA method from analysis of 300 ns MD simulations was –37.9±1.22 kcal/mol (Figure 2.4A, Table 2.4), while a similar value of –31.8±1.46 kcal/mol was calculated using the Poisson-Boltzmann surface area (MM/PBSA) model. The MM/GBSA and MM/PBSA data were used to identify ps-IKs residues contributing to the free energy of interaction with mefenamic acid for further analysis (Figure A.2), although it should be noted that root mean square deviation (RMSD) analysis of trajectories indicated that the binding was dynamic as there were other binding conformations possible (Figure A.3). Mefenamic acid in this complex was stabilized by two hydrogen bonds formed between the drug and ps-KCNE1 residues Y46 and E43 (Figure 2.2C). Analysis of MD simulations showed that these two H-bonds were the most stable, the Y46 H-bond to mefenamic acid had a mean frequency of 79.7%, and E43 22.5% of the drug bound time (Figure A.4; Table 2.2). 42 Donor Hydrogen Acceptor % Frames with H-Bonds SEM MEF MEF-N1 MEF-H1 GLU43-O 22.5 10.2 TYR46-N TYR46-HN MEF-O2 79.7 4.2 ILE47-N ILE47-HN MEF-O2 7.9 2.7 DIDS DIDS-O2 DDS357-H10 TRP323-NE1 12.9 2.1 DIDS-O1 DDS357-H9 GLU43-OE2 25.1 4.0 DIDS-O1 DDS357-H9 GLU43-OE1 25.2 4.6 ILE47N ILE47-HN DIDS-O5 22.0 3.2 TYR46-N TYR46-HN DIDS-O6 56.8 10.4 TYR46-N TYR46-HN DIDS-O5 7.1 1.8 TYR299N TYR299-HN DIDS-N2 11.9 0.6 SER298-OG SER298-HG1 DIDS-N2 26.0 10.6 The energy decomposition per amino acid using MM/GBSA and MM/PBSA methods revealed several residues in KCNQ1 and ps-KCNE1 with significant contributions to mefenamic acid coordination (Figure 2.2C, Figure A.2). As expected, these are the KCNE1 residues located at the external region of the auxiliary subunit – amino acids K41 to Y46. In addition, residues W323 and V324 located on the S6 helix as well as residues L142, Q147, and Y148 located on the S1 helix exhibited the lowest interaction free energy. We focused on functional validation of the KCNE1 residues K41, L42, E43, A44, and Y46, and KCNQ1 residues W323, V324, L142, Q147, and Y148 by mutation to cysteine, alanine and/or tryptophan, and examining the sensitivity of fully saturated EQ channel complexes to 100 μM mefenamic acid. Although A300, located in the turret region was identified as potentially important (but less so than KCNE residues and W323), we could not get adequate expression from A300C for functional analysis. Table 2.2 Mean frequency of H-bonds between MEF or DIDS and residues in ps- IKs during 500 ns MD simulations or until drug unbound. MEF357-N1 indicates the nitrogen of aminobenzoic acid; MEF357:O1 and O2 are the oxygens of the aminobenzoic acid; DDS357:O1-O6 are the oxygens of the sulfonic acids; DDS357:N1 and N2 are nitrogens of the isothiocyanates. n = 5 for each. 43 2.3.3 Mutational impact on EQ current changes induced by mefenamic acid The effect of mutations on the current waveform and tail current response to 100 μM mefenamic acid treatment was examined on IKs channels identified as: x-EQ-y where ‘x’ denotes a KCNE1 mutation and ‘y’ denotes a KCNQ1 mutation. In the absence of mefenamic acid (control), most mutations, with the exception of EQ-L142C (Figure A.6), produced slowly activating currents with rapid tail current decay (Figure 2.3A). In the presence of 100 μM mefenamic acid, the waveforms of WT EQ, S6 and pore mutations EQ-V324A, EQ-V324W, EQ-Q147C and EQ-Y148C were all transformed into ones with instantaneous current onset and slowed tail current decay (Figure 2.3A, Figure A.5). Only the EQ-W323A waveform and tail current were largely unaffected by 100 μM mefenamic acid (like K41C-EQ, Figure 2.1). The EQ-W323C and A44C-EQ current waveforms were also unchanged by 100 μM mefenamic acid, but their tail current decay was slowed (Figure 2.3A, Figure A.5). We interpreted this to mean that mefenamic acid binds to EQ-W323C and A44C-EQ mutant open channels and slows closing, but that the drug-channel complex is less stable and mefenamic acid unbinds during the interpulse interval, which relieves drug action between pulses. The summary of the normalized response of the different mutants to mefenamic acid is shown in Figure 2.3D. G-V plots were obtained from the tail current amplitudes in the absence and presence of 100 μM mefenamic acid, and unlike WT EQ (Figure 2.1), minimal change in the shape and position of the EQ-W323A G-V plot was seen after exposure to 100 μM mefenamic acid V1/2 shift of -14.1 mV compared with -105.7 mV seen in WT EQ (Figure 2.1, Figure 2.3B and E, and Table 2.1). For the W323C mutation, a less drastic decrease in size compared to alanine, the V1/2 shift seen with 100 μM mefenamic acid increased 44 (EQ-W323C ΔV1/2 = -26.5 mV), suggesting that the size of the W323 residue is important (Figure 2.3E and Table 2.1). However, when the neighboring V324 KCNQ1 residue was mutated to a smaller (alanine) or bulkier (tryptophan) residue they both showed the same response to mefenamic acid. Both the V324W and V324A mutations reduced V1/2 shifts caused by 100 μM mefenamic acid to between -14 to -19 mV (Figure 2.3E, Table 2.1, Figure A.5). 45 Figure 2.3 Current waveform and G-V changes induced by mefenamic acid in binding site mutants. (A) Current traces from WT EQ and key residue mutants in control (black) and 100 µM Mef (colors). (B) EQ-W323A and (C) EQ-Y148C current traces in control (top) and presence of 100 μM Mef (below). G-V plots in control (black) and presence of 100 μM Mef (colors). Boltzmann fits were: EQ-W323A control (n = 4): V1/2 = 47.8 mV, k = 23.7 mV; EQ-W323A Mef (n = 3): V1/2 = 33.7 mV, k = 28.5 mV; EQ-Y148C control (n = 4): V1/2 = 36.8 mV, k = 20.3 mV; and EQ-Y148C Mef (n = 4): V1/2 = 17.5 mV, k = 36.7 mV). Voltage steps were from -80 mV to +100 mV for 4 s, followed by a 1s repolarization to -40 mV. Interpulse interval was 15 s. Error bars shown are ± SEM. (D) Summary plot of the normalized response to 100 µM Mef (see Materials and Methods). Data are shown as mean +SEM and one-way ANOVA statistical test was used. **p<0.01 and *p<0.05 denote a significantly reduced response compared to WT EQ. N.S. denotes not significant. (E) Change in V1/2 (ΔV1/2) for WT EQ and each IKs mutant in control versus mefenamic acid. Data are shown as mean -SEM and unpaired t-test was used, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate significant change in V1/2 comparing control to the presence of drug. n-values for mutants in D and E are stated in Table 2.1. 46 Extracellular S1 residues identified in the MD simulations also proved to be important for mefenamic acid binding, although less so than W323 and K41 residues found in the KCNQ1 extracellular regions of the S6 segment and KCNE1, respectively. Compared to WT, EQ-Y148C reduced the V1/2 shift after exposure to 100 μM mefenamic acid (EQ-Y148C ΔV1/2 = -19.3 mV) and lessened the slope of the G-V relationship (Figure 2.3C). The Q147C mutant on the other hand, only partially prevented the V1/2 shift observed after mefenamic acid treatment (EQ-Q147C ΔV1/2 = -37.6 mV, Figure 2.3E). Other KCNE1 residues located at the N-terminal limit of the KCNE1 transmembrane segment (L42, E43, A44, Y46) were also investigated. Unlike with K41C, but similar to WT EQ, tail current decay was inhibited in the presence of 100 μM mefenamic acid in all of these mutants, reflected by the normalized response (Figure 2.3D, Figure A.5). In addition, a reduced but still significant shift in V1/2 was observed with 100 μM mefenamic acid compared to control in most mutants tested, except K41C. (K41C ΔV1/2 = -3.8 mV; L42C ΔV1/2 = -37.1 mV; E43C ΔV1/2 = -3.9 mV; A44C ΔV1/2 = -9.7 mV; Figure 2.3E). Representative current traces and G-V curves for all mutants are shown in Figure A.5. Consistent with previous literature (Gofman et al., 2012; Kuenze et al., 2020; Wang et al., 2012), Y46C-EQ in control conditions produced a current with faster activation and a complex GV curve which made analysis and an assignment of slope and V1/2 difficult, though a small left shift in the GV curve was visible (data not shown). In lieu of Y46C, G-V data from the Y46A-EQ mutant showed a potent effect of mefenamic acid, equivalent to that of WT with a ΔV1/2 = -106.2 mV 47 (Figure A.5D). These results suggest that mutation of residues further away from the N-terminus of KCNE1 than K41 has diminishing effects on the activator action of mefenamic acid. 2.3.4 Augmented activation of EQ-L142C in the absence of mefenamic acid Unlike other mutations, the control EQ-L142C current waveform displayed an almost instantaneous current onset, and tail currents showed no decay with our standard protocol (Figure A.6A). As the control EQ-L142C current waveform qualitatively resembled WT EQ currents in the presence of 100 µM mefenamic acid, G-V plots at different interpulse intervals were compared. At the standard interval of 15 s, the EQ-L142C G-V plot (blue) closely overlapped that of WT EQ in the presence of Mef (solid grey), and at a 30 s interpulse interval, the position of the WT EQ + Mef (grey open circles) and EQ-L142C plots (green) both depolarized significantly (Figure A.6B). It appeared that the L142C mutation augmented channel activation as much as 100 µM mefenamic acid on WT channels. In addition, mefenamic acid made the EQ-L142C G-V plot voltage independent, overlapping with the G-V obtained for EQ-L142C with a 7 s interpulse interval (pink and red respectively, Figure A.6B). The data indicate that EQ-L142C is still responsive to mefenamic acid but does so from a heightened state of activation (Table 2.3). 48 Table 2.3 Mean V1/2 of activation (mV) and slope values (k-factor, mV) for WT EQ treated with 100 µM mefenamic acid, and untreated mutant EQ-L142C. Interpulse interval used is as indicated. Values are shown ± SEM. An interpulse interval of 7 s created such a dramatic change in the shape of the EQ-L142C G-V plot (see Figure A.5) that a Boltzmann curve could not be fit and V1/2 and k-values are therefore not available. V1/2 k-factor n WT EQ + 100 µM mefenamic acid: Interpulse interval 15 s -80.3 ± 4.1 41.3 ± 8.4 3 WT EQ + 100 µM mefenamic acid: Interpulse interval 30 s 26.7 ± 10.5 66.6 ± 28 8 EQ-L142C control: Interpulse interval 15 s -80.3 ± 4.5 30.0 ± 2.9 6 EQ-L142C control: Interpulse interval 30 s -28.7 ± 19 62.5 ± 9.6 3 2.3.5 MD simulations of mutant ps-IKs channels exposed to mefenamic acid To explore the role played by critical residues, in-silico homologous mutations K41C, Y46C and W323A were introduced into the mefenamic acid-bound ps-IKs channel (Mef-ps-IKs) and the stability of the Mef-mutant complexes was assessed in MD simulations. Remarkably, mefenamic acid detached from mutant K41C and W323A channels within 120 ns in all three independent MD simulations with an AMBER force field (Table 2.4). In contrast, mefenamic acid remained bound during the entire simulation time to WT and Y46C-ps-IKs complexes (Table 2.4). The last mutant was tested as we could not determine the functional effect of mefenamic acid on this residue in electrophysiological experiments, but in the simulation, Y46C-ps-IKs behaved like Y46A from electrophysiological data (Figure 2.3, Figure A.5). Mefenamic acid unbound from A44C-ps-IKs channel complexes within 80, 100, and 110 ns during three different simulations, changing binding pose several times before doing so. 49 Table 2.4 Average free interaction energies of MEF-bound ps-IKs complexes calculated according to MM/GBSA methods from three independent MD simulation runs using the AMBER force field. Mean values are in kcal/mol, ± SD. For W323A and K41C mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. Note that unbinding occurred in K41C and W323A in all 3 simulations with 300 ns duration. Run Method ps-IKs W323A Y46C K41C I GBSA -39.3 ± 4.1 -13.0 ± 5.1 -31.2 ± 2.9 -10.5 ± 3.8 - Unbinding after ~75 ns - Unbinding after ~25 ns II GBSA -39.0 ± 3.3 -23.1 ± 3.1 -32.2 ± 3.8 -17.0 ± 2.6 - Unbinding after ~120 ns - Unbinding after ~70 ns III GBSA -35.5 ± 2.6 -22.6 ± 4.1 -28.8 ± 5.7 -17.2 ± 2.5 - Unbinding after ~85 ns - Unbinding after ~20 ns Similar results were obtained in MD simulations with the CHARMM force field where Mef-ps-IKs was embedded in the lipid membrane (Video 1), although in two of five 1250 ns long duration simulations we observed unbinding of Mef from WT ps-IKs complexes, which suggested that the lipid environment allowed a more dynamic interaction between Mef and IKs. In contrast, Mef left the binding site in all five 500 ns runs for W323A and K41C mutants (Figure A.7, Videos 2 and 3), and when the Y46C mutant was placed in a lipid environment, mefenamic acid left the binding site or changed its binding conformation in three of five 500 ns MD simulations (Video 4). A significantly reduced free interaction energy (ΔG) of ligand binding for K41C and W323A mutant Mef-ps-IKs complexes in 300 ns simulations was observed compared to WT Mef-ps-IKs (Figure 2.4A, Table 2.4), and the small but statistically significant change in free energy observed for the Y46C mutant complex confirms that the Y46 residue is not as important as K41 50 and W323 for mefenamic acid binding. It should be noted that the absolute values of ΔG or corresponding dissociation constants (Kd) calculated from MD simulations do not reflect apparent ΔG and Kd values determined from electrophysiological experiments or biochemical essays. The flexibility of the external ps-KCNE1 protein residues of the mutants W323A, K41C, and Y46C ps-IKs channels was also analyzed from 300 ns duration trajectories, by monitoring their average root mean square fluctuation (RMSF) during the last 100 ns of simulations after the detachment of mefenamic acid from the molecular complex or in the case of Y46C without introduction of the ligand. The RMSF values obtained from the mutant channels were then compared to that of WT ps-IKs channels where MD simulations of the same duration were conducted after removing the mefenamic acid molecule from the complex (Figure 2.4B). The results indicate that K41C and W323A mutations modestly increased the RMSF of D39-A44 ps-KCNE1 residues when compared to the WT ps-IKs channel complex without mefenamic acid bound (Figure 2.4C and D). The Y46C mutation seems to impact the mobility of the N-terminal portion of KCNE1 much less (Figure 2.4E), perhaps owing to its location further into the membrane. These results suggest that the side chains of K41 and W323 residues normally stabilize the conformation of the external region of KCNE1, so that mutation of these residues increases random motion and reduces the ability of drugs like mefenamic acid to remain bound at this site. 51 Figure 2.4 K41C, Y46C, and W323A mutant impact on mefenamic acid binding energy and flexibility of external KCNE1 residues. (A) Average free interaction energy of Mef-bound ps-IKs complexes calculated using MM/GBSA methods from three independent MD simulation runs. For K41C and W323A mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. * and *** denote significant differences in average free interaction energy compared to ps-IKs. Student`s unpaired t-test was used for comparison of groups. (B) Surface representation of ps-IKs after removal of Mef. The pore residues are in blue and the VSD of a neighbouring subunit is yellow. (C-E) Root mean square fluctuations (RMSF) of ps-KCNE residues (Å) in the ps-IKs complex during the last 100 ns of simulations. Three separate MD simulations shown for the ps-IKs channel without Mef (black lines) and three for K41C (C, red), W323A (D, blue) after ligand detachment, and Y46C in absence of ligand (E, purple). Dashed lines show average values of RMSF calculated from three simulations. ***p = 0.001; **p =>0.01 *p =>0.05 using an unpaired t-test. GROMACS software was used for RMSF analysis. 52 2.3.6 Mutational impact on IKs current changes induced by DIDS To establish whether the binding pocket for mefenamic acid can be generalized to other IKs activators, we also examined the binding of the structurally unrelated IKs activator, DIDS. Stilbenes such as DIDS (Figure 2.5A) also activate IKs (Abitbol et al., 1999; Bollmann et al., 2020), and given their molecular differences from fenamates, it is of interest to explore common structural and dynamic features of their binding to the IKs channel complex. 100 µM DIDS had no effect on endogenous currents in GFP-transfected tsA201 cells but treatment of WT EQ channels with 100 µM DIDS transformed the slowly activating waveform into one with faster onset (although not instantaneous like mefenamic acid) and inhibited tail current decay (Figure 2.5B). G-V plots were obtained from the tail amplitudes in the absence and presence of 100 µM DIDS (Figure 2.5C and D). The overall shape, slope, and V1/2 of the WT EQ G-V relationship changed with 100 µM DIDS in the direction of increased activation (ΔV1/2 = -46.6 mV; Table 2.5; control k=20.3 mV, DIDS k=25.3 mV). However, the effects on WT EQ were less pronounced with DIDS than mefenamic acid at the 100 µM concentration. 53 Figure 2.5 Effect of DIDS on IKs. (A) Molecular structure of mefenamic acid and DIDS. (B) WT EQ current in control (black) and exposed to 100 µM DIDS over time (grey). Pulses were from -80 to +60 mV every 15 s, and current traces are shown superimposed. Lower panel shows no effect on currents from GFP-only transfected cells exposed to 100 µM DIDS over time (grey). (C) Current traces from WT EQ in control and presence of 100 µM DIDS as indicated. Pulses were from -80 to +100 mV for 4s, with a 1 s repolarization to -40 mV. Interpulse interval was 15 s. (D) Corresponding G-V plot in control (black) and DIDS (grey) from data as shown in panel C. Boltzmann fits were: WT EQ control (n = 8): V1/2 = 30.5 mV, k = 20.3 mV; WT EQ in the presence of DIDS (n = 5): V1/2 = -16.1 mV, k = 25.3 mV. Error bars shown are ± SEM. 54 Table 2.5. Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of 100 µM DIDS for fully saturated IKs channel complexes. A statistical difference in V1/2 compared to control is shown as p-value determined using an unpaired t-test. NS denotes not significant. Values are shown ± SEM. * An estimation of the activation V1/2 was calculated from a right shifted, non-saturating GV curve. The ps-IKs construct and docking procedures used previously to explore the binding site of mefenamic acid were used as a basis for DIDS docking and subsequent MD simulations. We found that DIDS also bound to a location formed between extracellular KCNE1 residues, the external S6 transmembrane helix of one subunit, the S1 transmembrane domain of the neighboring subunit, and the pore turret of the opposite subunit (Figure 2.6A). In docking experiments, DIDS is stabilized by its hydrophobic and van der Waals contacts with KCNQ1 and KCNE1 subunits as well as by two hydrogen bonds formed between the drug and ps-KCNE1 residue L42 and KCNQ1 residue Q147 (Figure 2.6B). These hydrogen bonds however were not Control 100 µM DIDS p-value V1/2 k-factor n V1/2 k-factor n WT EQ 30.5 ± 4.3 20.3 ± 0.9 8 -16.1 ± 2.8 25.3 ± 1.9 5 <0.001 K41C-EQ 23.1 ± 3.0 20.2 ± 1.8 3 -1.6 ± 3.7 24.0 ± 3.6 3 <0.01 L42C-EQ 55.4 ± 3.3 19.9 ± 1.5 3 21.4 ±10.7 28.5 ± 2.6 4 <0.05 A44C-EQ 24.1 ± 1.7 29.6 ± 3.3 4 5.6 ± 2.4 17.6 ± 1.8 3 <0.05 *Y46A-EQ 75.2 ± 2.1 59.1 ± 5.3 5 25.8 ± 0.9 39.2 ± 1.4 5 <0.0001 EQ-Y148C 51.8±4.6 23.5±1.7 4 40.0 ± 3.1 26.3 ± 1.5 5 NS EQ-W323A 50.7 ± 3.6 23.9 ± 1.6 4 23.4 ± 4.9 24.1 ± 1.3 4 <0.05 55 stable during MD simulations and instead H-bonding occurs between E43, Y46, and I47 in KCNE1 as well as W323, S298 and Y299 in KCNQ1 (Figure A.9, Table 2.2). The energy decomposition per amino acid using MM/GBSA and MM/PBSA methods based on 300 ns simulations revealed several residues in KCNQ1 and ps-KCNE1 with significant contributions to DIDS coordination (Figure 2.6C). Although residues identified as critical to mefenamic acid binding and action also generally appear to be important for DIDS, especially W323, K41 was noticeably not as critical to DIDS coordination, since the ΔG values for this residue were significantly smaller compared to those of the Mef (see Figure A.2). 56 Figure 2.6 Ligand interaction and energy decomposition per amino acid for DIDS binding to ps-IKs. (A) Binding pose of DIDS (yellow) in external region of the ps-IKs channel complex obtained with molecular docking (side view). The residues of the pore domain are coloured in blue, ps-KCNE1 subunit in red, and the voltage-sensor domain of a neighbouring subunit is presented in yellow. (B) Ligand interaction map of DIDS with ps-IKs from molecular docking. Size of residue ellipse is proportional to the strength of the contact. Light grey indicates residues in van der Waals contacts, light green hydrophobic contacts, and light blue are hydrogen bond acceptors. Red borders indicate KCNE1 residues, yellow are KCNQ1 VSD residues, and blue are pore residues. Dashed lines indicate H-bonds. The distance between the residue label and ligand represents proximity. Grey parabolas represent accessible surface for large areas. The 2D diagram was generated by ICM pro software with a cut-off value for hydrophobic contacts 4.5 Å and hydrogen bond strength 0.8. (C) Energy decomposition per amino acid for DIDS binding to ps-IKs. Generalized Born Surface Area (MM/GBSA; orange) and Poisson-Boltzmann Surface Area (MM/PBSA; blue) methods were used to estimate the interaction free energy contribution of each residue in the DIDS-bound ps-IKs complex. The lowest interaction free energy for residues in ps-KCNE1 and selected KCNQ1 domains are shown as enlarged panels (n=3 for each point). Error bars indicate ±SD. 57 Similar to mefenamic acid, in AMBER force field simulations, DIDS stayed bound to WT ps-IKs, but detached from the W323A mutant during two of three runs of 300 ns duration, and from Y46C channels in one of three runs after 240, 239, and 122 ns of simulation, respectively (Table 2.6). Interestingly, DIDS did not unbind from K41C mutant ps-IKs channels, even after 300 ns of simulation, although the binding pose did change in one run. Calculations of free interaction energy shown in Table 2.6 using both MM/PBSA and MM/GBSA methods are of mean values before the detachment of the drug if that occurred. If DIDS detachment did not occur during 300 ns MD runs, the average free energy was calculated over the duration of the entire MD run for that construct. Only DIDS binding to W323A, and to Y46C in one simulation run, showed significantly lower free energy than DIDS binding to WT, using both methodologies. The small change in free energy and more stable binding of DIDS to K41C suggest that this residue is not as important as W323 or Y46 for DIDS binding. In longer 500 ns simulations using a CHARMM force field with the channel complexes embedded in lipid, trajectory analysis revealed clusters of DIDS poses in complex with the WT ps-IKs channel, which are much more closely related than those identified for mefenamic acid (Figure A.10, note the ordinate scale). Still, DIDS remained bound to WT ps-IKs, during all runs throughout 500 ns simulations (Figure A.11, Video 5), and to the K41C mutant in two runs (Video 6). Both W323A and Y46C mutants unbound during 500 ns simulations (all runs for Y46C, Figure A.11, Videos 7 and 8), as was seen with the AMBER force field. These model predictions were tested experimentally using fully saturated mutant IKs channel complexes, K41C-EQ, L42C-EQ, A44C-EQ, Y46A-EQ, EQ-W323A, EQ-L142C, and EQ-Y148C (Figure 2.7A, Figure A.12). Y46 was identified as an important residue for DIDS binding in MD simulations, but as before, the complexity of the GV curve and low functional 58 expression of the Y46C construct led us to substitute Y46A for this mutation in electrophysiology experiments (Figure 2.7 and A.12). 59 Figure 2.7 Binding site mutants important for the action of DIDS. (A) Current traces from WT EQ and key mutants in the absence (control; black) and presence (colors) of 100 µM DIDS. (B) Data and G-V plots in control (black) and DIDS (teal). Boltzmann fits were: K41C-EQ control (n = 3): V1/2 = 23.1 mV, k = 20.2 mV; K41C-EQ DIDS (n = 5): V1/2 = -1.6 mV, k = 24.0 mV. Voltage steps from a holding potential of -80 mV to +70 mV for 4 s, followed by repolarization to -40 mV for 1 s. Interpulse interval was 15 s. Error bars shown are ± SEM. All calibration bars denote 0.5 nA/0.5 s. (C) Summary plot of the normalized response to 100 µM DIDS. Data are shown as mean ± SEM and *p<0.05, **p<0.01, ***p<0.001 denote significant change in mutant versus WT currents (one-way ANOVA, see Materials and Methods). For calculation, see Materials and Methods. (D) Change in V1/2 (ΔV1/2) for WT EQ and each IKs mutant in control versus DIDS. Data are shown as mean ± SEM and unpaired t-test was used, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate significant change in V1/2 comparing control to the presence of drug. n-values for mutants in C and D are stated in Table 2.5. 60 Table 2.6 Average free interaction energies of DIDS-bound ps-IKs complexes calculated according to MM/PBSA and MM/GBSA methods from three independent MD simulation runs of 300 ns duration using the AMBER force field. Mean values are in kcal/mol, ± SD. For W323A and Y46C mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. Note that unbinding occurred in W323A in 2 of 3 simulations, and K41C did not unbind but the binding pose shifted. Run Method ps-IKs W323A Y46C K41C A44C Y148C I PBSA -32.3 ± 6.7 -25.7 ± 4.9 -35.5 ± 6.3 -33.5 ± 4.4 -29.2 ± 4.2 -34.1± 4.6 GBSA -35.5 ± 4.6 -30.2 ± 4.5 -42.7 ± 5.4 -41.5 ± 5.8 -36.8 ± 2.9 -40.7 ± 4.0 unbinding after 240 ns changed binding pose after ~140-150 ns II PBSA -32.6 ± 4.8 -25.3 ± 5.5 -25.7 ± 6.5 -36.4 ± 4.2 -29.0 ± 4.0 -29.3 ± 3.7 GBSA -36.0 ± 4.8 -30.0± 4.8 -32.4 ± 6.4 -31.6 ± 4.0 -34.0 ± 5.7 -31.8 ± 4.7 unbinding after ~100-132 ns III PBSA -29.2 ± 8.1 -27.1± 3.9 -38.3 ± 4.8 -35.1 ± 4.3 -31.2 ± 45.0 -35.7 ± 5.8 GBSA -34.0 ± 8.9 -35.1± 4.8 -42.5 ± 4.7 -30.8 ± 3.9 -35.6 ± 5.6 -40.3 ± 5.9 unbinding after ~240 ns K41C-EQ G-V plots were obtained from peak tail current amplitudes in the absence and presence (Figure 2.7B) of 100 µM DIDS. Unlike with mefenamic acid, but in agreement with the modeling, K41C only partially prevented the action of DIDS. Treatment of K41C-EQ with 100 µM DIDS hyperpolarized the V1/2 and changed the shape of the G-V relationship compared to 61 control (Figure 2.7B and D, Table 2.5). The current waveform of K41C-EQ activated more quickly with less sigmoidicity after treatment with DIDS, and tail current decay was slowed as indicated by the normalized response (Figure 2.7B and C). As K41C-EQ remained responsive to DIDS, and L42C responded similarly to K41C-EQ (for K41C-EQ, ΔV1/2 was -24.7 mV, and for L42C-EQ ΔV1/2 was -34 mV), we hypothesized that DIDS binding to IKs channel complexes may involve KCNE1 residues closer to the transmembrane region. In agreement with this idea, A44C showed little response to 100 µM DIDS both in terms of current waveform and tail current decay. The shift in V1/2 was also reduced compared to WT-EQ (A44C-EQ ΔV1/2 = -18.5 mV; WT EQ ΔV1/2 = -46.6mV, Figure 2.7C and D). In Y46A, the effect of DIDS on the current waveform was greatly reduced as was slowing of tail current decay (Figure 2.7A and C). We could only estimate an activation V1/2 for Y46A as the G-V curve was right shifted to very positive potentials and non-saturating. Nevertheless, hyperpolarization of the V1/2 (Figure 2.7D, Figure A.12) in the presence of DIDS was observed, suggesting that unstable and short-lived binding of the drug to the channel complex was sufficient to interfere with channel gating. The modeling studies (Figure 2.6) suggested that W323 remained a key residue for IKs activator sensitivity, and in agreement with this, EQ-W323A was only partially responsive to DIDS. In EQ-W323A, the tail current decay was affected but the slowly-activating current waveform was preserved in the presence of DIDS, supporting the idea that the drug dissociates from the channel complex between pulses (Figure 2.7A and C, Table 2.6). However, a significant shift in the V1/2 remained (ΔV1/2 = -27.3 mV; Figure 2.7D, Table 2.5). Comparable effects observed in EQ-W323A, A44C-EQ, and Y46A-EQ are consistent with the idea that the DIDS binding pocket involves the N-terminal end of S6 and the deeper extracellular surface of the KCNE1 62 transmembrane segment. In EQ-Y148C, the V1/2 and the shape of the G-V plot were not altered by 100 µM DIDS (Figure 2.7D, Table 2.5), and tail current decay was not markedly slowed (Figure 2.7A and C). This result suggests that unlike mefenamic acid, DIDS binding determinants extend to the S1 region of KCNQ1 and that Y148 is an important contributor. Notably, as with mefenamic acid, the L142C-EQ G-V plot became voltage-independent after treatment with 100 µM DIDS (data not shown) and so it proved hyper-responsive to both of the IKs activators, DIDS and mefenamic acid. 2.4 Discussion Mefenamic acid, a nonsteroidal anti-inflammatory drug (NSAID), and the structurally distinct stilbenes DIDS and SITS, among other compounds, have previously been identified by numerous groups to enhance IKs currents in various expression systems including canine and guinea-pig ventricular myocytes (Magyar et al., 2006; Toyoda et al., 2006) as well as heterologous expression systems such as Xenopus laevis oocytes, CHO, COS-7, tsA201 and LM cells (Abitbol et al., 1999; Busch et al., 1994a; Toyoda et al., 2006; Unsöld et al., 2000; Wang et al., 2020b). The extracellular region of KCNE1 between residues 39 and 44 was found important in mediating the effect of DIDS on IKs (Abitbol et al., 1999) and residue K41, located on the extracellular end of KCNE1, was found to be critical in mediating mefenamic acid’s activating effect on the fully saturated IKs channel complex (Wang et al., 2020b). Consistent with this idea, when all four WT KCNE1 subunits are replaced with mutant K41C-KCNE1, mefenamic acid up to a concentration of 1 mM is largely ineffective (Figure 2.1). Unlike in the WT complex, there is little change to the current waveform, slope or V1/2 of the G-V plot during activator exposure, 63 suggesting that all the drug binding site(s) on the channel complex are impaired, or that the mechanism of action is disabled, or that the mutation causes a combination of these actions. In the present study, we analysed these activator compound actions using molecular modeling approaches, and complementary mutational analysis. Our data describe the formation of a drug binding pocket between the immediate extracellular residues of KCNE1, S1 and pore residues of two KCNQ1 subunits, stabilized by the presence of structurally different activator compounds. The effect of mutations that negate the activator compound actions is to destabilize the binding pocket itself, reduce drug binding and limit activator residency time on the channel complex. 2.4.1 Mefenamic acid binding site in IKs Using data that suggested an extracellular binding site for mefenamic acid was at the KCNE1-channel interface, MD simulations were used to explore drug-channel interactions further. The drug binding pocket was defined as the extracellular space formed by KCNE1, the domain-- swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits (Figure 2.2; Video 1). Docking and molecular simulations identified a binding site made up of several residues across all of these elements (Figure 2.2 and Figure A.2, Table 2.7). Mutagenesis revealed that most mutations impacted at least one out of the three mefenamic acid effects: V1/2 shift, GV-plot slope change, and current waveform change related to slowed deactivation. However, only three of the mutants impacted all of the mefenamic acid actions (Figure 2.3). These were W323A in KCNQ1, as well as K41C, and A44C (to a lesser extent) in KCNE1, which define key components of the binding pocket for mefenamic acid. That W323 forms an important medial wall of the hydrophobic pocket to which mefenamic acid binds is suggested by data from mutants in which the size of the side chain at this location is 64 systematically made smaller to cysteine and then alanine, which results in diminished effectiveness of the drug, particularly with alanine. This mutation resulted in the detachment of mefenamic acid as well as lower interaction energy of drug-channel complexes during MD simulations (Figure 2.4A; Video 3, Figure A.7C) particularly loss of interaction strength with KCNE1 (Table 2.7). Likewise, MD simulations revealed detachment of mefenamic acid and reduced interaction energy values for K41C-Mef-IKs complexes (Video 2; Figure 4) again accompanied by loss of interaction strength with KCNE1 (Table 2.7). The drug also detached from the A44C complex, another mutant with a reduced response in terms of changes in waveform, V1/2 and slope. In contrast, while the free interaction energy of the mutant Y46C channel was significantly decreased in comparison to the WT channel complex (Figure 2.4A), detachment of mefenamic acid from the mutant channel during 300 ns MD simulations was not observed (Table 2.4). However, when placed in a lipid environment mefenamic only remained bound in three out of five 500 ns simulations with Y46C, suggesting a more dynamic interaction in this model (Figure A.7D, Video 4). Comparison of energies at specific residues shows a loss of interaction with Q147 and Y148 in S1 with this mutant and a small shift towards the turret at S298 and A300 (Figure 2.8A; Table 2.7, Figure 2.8—source data 1). While we were not able to obtain good electrophysiological data from Y46C, data from Y46A suggested that mefenamic acid still had potent actions on this mutant. Overall, the combination of binding studies and electrophysiological data indicate that Y46 was not as important for mefenamic acid binding and action as other KCNE1 N-terminal residues. Table 2.7 Average free energy of mefenamic acid and DIDS-bound ps-IKs and mutant complexes. Values are calculated according to the MM/GBSA method from three independent MD simulation runs using the AMBER force field and further broken down by residue and channel region. For K41C and W323C, calculations 65 correspond to the interval of simulations before detachment of the ligand from the complex. Values are in kcal/mol. Mutated residues are in bold italics. Residue ps-IKs MEF K41C MEF Y46C MEF W232A MEF ps-IKs DIDS A44C DIDS Y46C DIDS Y148C DIDS W323A DIDS Total -38.02 -16.39 -31.40 -20.31 -34.29 -35.40 -39.16 -37.60 -34.33 Drug -19.69 -8.79 -15.25 -11.65 -17.42 -17.52 -21.21 -18.18 -19.17 Channel -17.91 -6.52 -16.61 -7.55 -19.21 -18.40 -16.94 -19.21 -15.10 Ch+Drug -37.60 -15.31 -31.86 -19.20 -36.63 -35.92 -38.15 -37.39 -34.27 Difference -0.42 -1.08 0.46 -1.11 2.34 0.52 -1.01 -0.21 -0.06 K41 -1.548 -0.349 -1.411 -0.954 -0.639 -0.439 -1.148 -0.283 0.083 L42 -0.970 -0.895 -0.943 -1.193 -1.061 -0.798 -0.165 -0.320 -0.587 E43 -2.681 -0.472 -2.257 -0.309 -1.312 -0.660 -0.014 -2.264 -0.705 A44 -1.906 -0.886 -2.035 -1.102 -1.778 -2.062 -1.027 -1.061 -1.780 E1/E3 M45 -1.744 -0.163 -1.913 -0.064 -1.756 -2.154 -1.222 -2.810 -1.633 Y46 -2.391 -0.121 -2.702 -0.083 -1.985 -1.797 -1.245 -2.377 -1.603 I47 -0.771 -0.136 -0.412 -0.203 -2.036 -1.993 -1.004 -0.621 -1.528 L48 -0.010 -0.003 -0.025 0.005 -0.045 -0.100 -0.233 -0.081 -0.035 Sum -12.02 -3.03 -11.70 -3.90 -10.61 -10.00 -6.06 -9.82 -7.79 T322 -0.125 -0.047 -0.091 -0.968 -0.200 -0.213 -0.127 -0.152 -0.410 W323 -2.159 -0.736 -2.226 -0.932 -3.312 -3.395 -2.279 -3.566 -1.590 Pore V324 -0.644 -0.037 -0.123 -0.047 -0.172 -0.210 -0.111 -2.016 -0.063 G325 -0.038 -0.006 -0.006 0.017 -0.004 0.000 0.003 -0.048 0.032 K326 -0.185 -0.015 -0.024 0.008 0.038 0.058 0.091 0.000 0.118 T327 -0.130 -0.015 -0.020 0.027 -0.040 -0.022 -0.018 -1.298 -0.014 Sum -3.28 -0.86 -2.49 -1.89 -3.69 -3.78 -2.44 -7.08 -1.93 V141 -0.061 -0.014 -0.247 -0.006 -0.569 -0.411 -0.660 -0.076 -0.504 L142 -0.861 -0.434 -1.052 -0.267 -1.525 -0.822 -1.285 -0.460 -1.036 S143 -0.028 0.004 -0.014 -0.063 -0.008 -0.006 -0.104 0.026 -0.017 T144 -0.091 -0.006 -0.242 -0.526 -0.015 -0.122 -0.982 -0.002 -0.001 S1 I145 0.004 0.005 0.002 0.009 0.003 -0.329 -0.067 -0.009 -0.001 E146 0.000 0.048 0.056 0.114 0.028 -0.014 -1.601 0.113 0.013 Q147 -0.230 -0.365 0.006 -0.244 0.012 -0.143 -1.061 -0.974 -0.054 Y148 -0.565 -1.556 0.006 -0.128 -0.428 -0.336 0.002 -0.022 -0.223 Sum -1.83 -2.32 -1.49 -1.11 -2.50 -2.18 -5.76 -1.40 -1.82 G297 -0.024 -0.001 -0.010 -0.027 0.016 -0.027 -0.089 -0.001 -0.076 S298 -0.320 -0.163 -0.419 -0.427 -0.791 -0.848 -1.107 -0.216 -1.026 Turret Y299 -0.103 -0.046 -0.104 -0.052 -0.718 -0.598 -0.563 -0.167 -0.673 A300 -0.618 -0.183 -0.636 -0.431 -1.130 -1.112 -0.751 -0.652 -1.093 D301 0.311 0.075 0.248 0.299 0.231 0.172 -0.181 0.136 -0.683 66 2.4.2 Common binding site for IKs activators, mefenamic acid and DIDS Although it was suggested that DIDS and mefenamic acid have the same binding site (Abitbol et al., 1999), the extent of overlap was unknown. Initially, we examined the effect of DIDS on WT EQ. Consistent with previous studies (Abitbol et al., 1999; Bollmann et al., 2020) we confirmed that 100 µM DIDS enhanced WT EQ activity (Figure 2.5B) with a V1/2 shift of -46.6 mV and a decrease in the slope of the G-V curve (Table 2.5). As most IKs activators are dependent on the KCNQ1-KCNE1 stoichiometry, this more potent effect of DIDS seen in our study may be explained by the higher dose we used and our KCNE1-KCNQ1 linked channel constructs, which ensured fully KCNE1-saturated complexes. Furthermore, due to the large size and complex folds found on the surface of oocytes, higher drug concentrations than those used for cultured cells are often required in order to facilitate a similar effect in both expression systems (Kvist et al., 2011). All previous studies utilized Xenopus laevis oocytes, whereas in this study transiently transfected tsA201 cells were used. The results of in-silico experiments, including some binding properties and stability of some mutations, were only partially validated by electrophysiology data, which might be explained by the limitations of the applied methods. Nevertheless, docking analyses revealed that both mefenamic acid and DIDS bind to the same general extracellular inter-subunit interface with some differences in key residues revealed by the electrophysiology data. Mutation of Y148 in KCNQ1 to a cysteine (Y148C) or Y46 in KCNE1 to an alanine (Y46A) was found to reduce the effects of 100 µM DIDS, particularly in the case of Y148C (Figure 2.7). These data suggest that A302 -0.024 -0.003 -0.011 -0.007 -0.011 -0.020 0.009 -0.008 -0.015 Sum -0.78 -0.32 -0.93 -0.65 -2.40 -2.43 -2.68 -0.91 -3.57 67 DIDS resides deeper in the binding pocket formed by KCNE1 residues and the KCNQ1 pore/S6, and so was less dependent on the side chain of K41 to retain the activator on the channel complex (compare Videos 1 and 5). DIDS associated more strongly with the pore of the Y148C mutant, particularly with V324 and T327, and less across the ps-KCNE1, S1 and turret regions (Table 2.7). The Y46C mutation resulted in shifts away from ps-KCNE1 and towards the S1 domain (T144, E146 and Q147). In the case of A44C and DIDS, the changes were more subtle, with stronger interactions moving from central residues in KCNE1 (K41, L42, and E43) and S1 (V141 and L142; colored grey in Figure 2.8B, cf. Figure 2.8—source data 2) to more peripheral residues (I145, E146, and Q147) and lower in the KCNE TMD (A44 and M45; colored magenta in Figure 2.8B). 68 Figure 2.8 Subtle differences in activator binding to Y46C- and A44C-ps-IKs mutant channels. (A) Mefenamic acid bound to the ps-IKs channel. Residues that are part of the binding site are shown in stick format colored green, except those residues that were important in the WT channel that had reduced ΔG in Y46C (in grey). Residues that had slight increases in ΔG values in Y46C are shown in magenta (S298, A300). Mefenamic acid is in cyan. (B) DIDS bound to the ps-IKs channel. Residues that are part of the binding site are shown in stick format colored green except those residues that were important in the WT channel that had reduced ΔG in the A44C mutant (in grey). Residues that had slight increases in ΔG values in A44C are shown in magenta. DIDS is in cyan. W323 may be seen behind DIDS. Images were made with the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. Source data for Figure 2.8A https://cdn.elifesciences.org/articles/87038/elife-87038-fig8-data1-v1.zip. Source data for Figure 2.8B https://cdn.elifesciences.org/articles/87038/elife-87038-fig8-data2-v1.zip. 69 2.4.3 Proposed mechanism of action for mefenamic acid and DIDS Molecular modeling and docking revealed that mefenamic acid and DIDS induce conformational changes in the channel upon binding, to shape a binding pocket formed by residues from the external S1 domain, KCNE1 and the pore domain of IKs (Figure 2.2, Videos 1 and 5). This cryptic binding pocket is not detectable in the absence of the drug (Figure 2.4B), which suggests that it has been induced in a similar manner to previous reports of toxin interactions with KcsA-Kv1.3 that induce conformational changes in both the toxin and the channel structure to generate a high-affinity binding site (Lange et al., 2006; Zachariae et al., 2008). Indeed, analysis of the binding site before and after mefenamic acid unbinds shows the involved residues in the channel complex moving into the space vacated by mefenamic acid (Figure A.8, Video 9), a collapse of the pocket created by the drug-channel interactions. Given the high modeled binding energy of the channel-drug activated state complexes, ~ -39 kcal/mol for mefenamic acid and ~ -35 kcal/mol for DIDS, but the relatively low affinities, given the micromolar concentrations required experimentally, we can imagine a dynamic complex where mefenamic acid and DIDS bind/unbind from IKs at high frequency. Slowed deactivation may therefore be the result of these rapid binding/unbinding interactions slowing the dissociation of the S1/KCNE1/pore domain/drug complex by either providing steric hindrance to dissociation or by stabilizing the activated complex. MD simulations suggest the latter is most likely the case. Cross-linking studies have previously shown that placing cysteines at key locations in the KCNE1 N-terminus, the top of S1 and in S6 can lead to disulfide bond formation and slowing or elimination of deactivation (Chung et al., 2009) similar to what we observe when IKs is exposed to mefenamic acid and DIDS. Our data indicate that mutation of residue W323 to an alanine 70 would not only destabilize the external S1/KCNE1/pore domain interface (Figure 2.4D, Table 2.1) but also eliminate direct hydrophobic contacts which normally occur between the W323 side chain and mefenamic acid, thus facilitating drug dissociation (Figure 2.2C, Video 3). A reduced interaction with S1 could also conceivably curtail the ability of IKs activators to slow dissociation of the activated complex and restore faster deactivation rates such that there is no longer an enhanced step current in the presence of the drug, as seen in mutants K41C and A44C for mefenamic acid (Figures 2.1A and 2.3A) and A44C, Y46A, W323A, and Y148C for DIDS (Figure 2.7A). Similarly, electrostatic interactions of residue K41, which acts as a lid for the binding pocket for mefenamic acid (Video 2), could help stabilize the S1/KCNE1/pore complex by its contacts with drug, which in its turn links the static pore domain to the dynamic voltage-sensor. Mutation of residue K41 to a cysteine may prevent stable IKs activator binding to the channel by increasing the fluctuations of the external KCNE1 region (Figure 2.4C) and by reducing the contacts with the drug (Figure 2.2C). In simulations, mefenamic acid was seen dissociating from mutant K41C, A44C, and W323A channel complexes much more often compared to wild type IKs, and this explains why the electrophysiological effects of IKs activators were prevented by amino acid substitutions at these locations (Videos 2 and 3). Shifts in, and particularly reductions in ps-KCNE1 association for both drugs (Table 2.7) that lead to loss of efficacy suggest that it is the interactions with KCNE1 and S1 that are key to maintaining the activated state. This is similar to the proposal for tight interactions between S1 and KCNE3 in maintaining constitutive activity in KCNE3-associated channels (Kasuya & Nakajo, 2022). 71 In close proximity to this drug-binding pocket are known gain-of function mutations in S1, S140G and V141M, which have previously been reported to slow deactivation in the presence of KCNE1 (El Harchi et al., 2010; Peng et al., 2017). As their current waveforms are qualitatively similar to those seen after exposure of WT EQ to IKs activators, we propose that the same mechanism may underlie the effects of mefenamic acid, DIDS and these IKs S1 mutations. In addition, we find that mutation of the neighboring residue, L142, also produces current waveforms and G-V plots which mirror those seen when WT EQ is treated with 100 µM mefenamic acid (Figure A.6) or DIDS. In the cryo-EM structure of KCNQ1-KCNE3 (Sun & MacKinnon, 2020), residue L142 interacts with KCNE3, whereas V141 interacts with both KCNE3 and the pore domain. This suggests that both the V141M and L142C mutations could directly alter the interaction of the S1 domain with the pore and/or the position or movement of KCNE1. The co-evolved interface between the extracellular end of S1 and the pore domain is thought to be important for bracing the VSD, to allow efficient force transmission to the pore (Lee et al., 2009), but can also impact permeation, as the S140G and V141M mutations also enhance rubidium permeation through IKs complexes (Peng et al., 2017). The importance of this S1-pore coupling to channel function is supported by mutational analyses of S1 residues (Campbell et al., 2013; Y. H. Chen et al., 2003; Hong et al., 2005; Y. H. Wang et al., 2011) as well as the L142C mutation examined in this study (Figure A.6), which all display current waveforms with instantaneous onset. Incidentally, A300, a residue in the pore region of KCNQ1, which also interacts with both mefenamic acid and DIDS in MD simulations (Figure A.2; Figure 2.6), has also been implicated in the same gain-of-function cleft as S140G and V141M (Smith et al., 2007). The A300T mutant has a ~ -20 mV shift in V1/2 of activation and faster rates of 72 activation (Bianchi et al., 2000), once again showing how a mutation can mirror the effects of the two activators studied here. 2.5 Conclusion Binding of mefenamic acid and DIDS to the extracellular end of KCNE1 and the KCNQ1 S6 and S1 helices is facilitated by a number of key residues. Residue K41 acts as a ‘lid’ holding mefenamic acid in place, while residue W323 impacts the size of the binding pocket. Size reduction mutations of either residue destabilize mefenamic acid binding and ultimately lead to drug detachment from the channel complex. This explains why, when all four IKs subunits are mutated, as in the case of K41C-EQ, EQ-W323A, and A44C-EQ, little to no effect of the drug is seen. The larger drug, DIDS, interacts with many of the same residues but those deeper in the pocket appear more important than for mefenamic acid. Furthermore, the qualitative similarities between the S1 mutant channel, EQ-L142C and WT EQ in the presence of 100 µM mefenamic acid suggest that IKs activators most likely cause their effects by modulating interactions between the S1 helix, pore turret, KCNE1 and the S6 helix. Upon binding, both DIDS and mefenamic acid induce conformational changes in an occult binding pocket and stabilize the S1/KCNE1/pore complex, which ultimately slows deactivation. The results indicate that this extracellular inter-subunit interface forms a generalized binding site which different drugs can access and through which induce common effects on channel activation and deactivation. The presence of such a binding site and the variable nature of IKs complex composition may serve as starting points for future drug development projects targeted at discovering therapeutically-useful IKs agonists. 73 Chapter 3: Dual effects of mefenamic acid on the IKs molecular complex 3.1 Introduction In the heart, the pore-forming subunit KCNQ1 (Kv7.1) co-assembles with the accessory subunit KCNE1 and various accessory regulatory molecules to form the slow delayed cardiac rectifier K+ current (IKs) channel complex (Barhanin et al., 1996; Sanguinetti et al., 1996; Wang et al., 2020b; Willegems et al., 2022). IKs is active during phase 3 repolarisation of the action potential and is particularly important for its contribution to a ‘repolarisation reserve’ at elevated heart rates that shortens the action potential to maintain diastolic filling between heartbeats (Lundquist et al., 2005; Sanguinetti et al., 1996; Silva & Rudy, 2005; Wang et al., 2020b). Thus, it is not surprising that life-threatening arrhythmias including long and short QT syndromes (LQTS and SQTS) and familial atrial fibrillation (AF) have been associated with mutations in IKs (Y. H. Chen et al., 2003; Moss & Kass, 2005; Olesen et al., 2014; Peng et al., 2017; Steffensen et al., 2015; L. Wang et al., 1996). Most of these mutations lead to a loss of function so IKs activators have been proposed as a promising therapeutic strategy for dysfunctional channels (Abitbol et al., 1999; Xu et al., 2015). Activators show a wide spectrum of activity that often depends on the presence, nature and number of accessory subunits within the channel complex (Willegems et al., 2022; Xiong et al., 2008). In whole-cell patch clamp experiments, mefenamic acid potentiates IKs in both a dose-and stoichiometry-dependent manner by transforming the slow activating and deactivating current waveform into an almost instantaneous activating current with inhibition of tail current decay (Chan et al., 2023; Wang et al., 2020b). Single-channel and whole-cell studies suggested that mefenamic acid modulates gating kinetics and decreases voltage sensitivity of the KCNQ1/KCNE1 channel (Wang et al., 2020b). In our previous work, we determined that the 74 extracellular end of KCNE1, along with the KCNQ1 S1 and S6 helices, form a dynamic binding pocket for mefenamic acid, DIDS, and potentially other IKs activators (Chan et al., 2023). Molecular modelling and docking experiments revealed that activators stabilised the S1/KCNE1/pore complex by inducing a conformational change upon binding to shape a binding pocket which was not present in the absence of drug (Chan et al., 2023). Interestingly, in zebrafish, mefenamic acid inhibits KCNQ1/KCNE1 channels, perhaps because the interaction between KCNE1, KCNQ1 and mefenamic acid is different from that of the human channel due to a low sequence homology between the extracellular domain of human and zebrafish KCNE1 (Haverinen et al., 2022). In the present study, we explore a dual effect of mefenamic acid on KCNQ1/KCNE1 channels. We show that, in addition to its potentiator action, mefenamic acid can inhibit IKs. Single mutations in either KCNQ1 or KCNE1 subunits accentuate a concentration-dependent inhibition of IKs. The inhibiting activity of mefenamic acid may involve effects on the pore domain. This work highlights the importance of the IKs S1/KCNE1/pore structure and interactions in determining the nature of drug effects on the channel complex. 3.2 Methods 3.2.1 Reagents and solutions Except where indicated, all drugs and solutions were prepared using chemicals from Sigma-Aldrich (Mississauga, ON, Canada). For whole-cell experiments, the control bath solution contained 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.8 mM NaAcetate and 10 mM HEPES, pH 7.4 with NaOH. The pipette solution contained 130 mM KCl, 5 mM EGTA, 1 mM MgCl2, 4 mM 75 Na2-ATP, 0.1 mM GTP and 10 mM HEPES, pH 7.2 with KOH. Mefenamic acid was prepared as either a 5- or 50-mM stock solution dissolved in 100% dimethyl sulfoxide. The stock solution was then diluted with the control bath solution to achieve the desired concentrations (3, 10, 30, 100 and 300 μM) and then perfused into the recording chamber containing the mammalian cells. HMR1556 (Tocris Bioscience, Oakville, ON, Canada) was dissolved in 100% dimethyl sulfoxide to achieve a stock concentration of 1 mM which was then added to the control bath solution and superfused to the recording chamber to achieve the desired final concentration of 300 nM. Other reagents and solutions were prepared according to the materials and methods described previously (Wang et al., 2020b). 3.2.2 Cell culture and molecular biology For whole-cell experiments, tsA201 transformed human kidney 293 cells (ECACC Cat# 96121229, RRID:CVCL_2737) were sourced directly from Sigma-Aldrich (Mississauga, ON, Canada). Cells were cultured in a modified version of Eagle's medium, enriched with 10% fetal calf serum, 100 mg ml-1 each of penicillin and streptomycin, and 0.25 mg ml-1 of amphotericin. They were then prepared for whole-cell experiments following the methods outlined in prior studies (Wang et al., 2020b; Westhoff et al., 2019). Cells were plated for whole-cell studies and transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). Mutations were generated by site-directed mutagenesis (Chan et al., 2023) followed by sequencing confirmation. For WT EQ, Y46A-EQ and EQ-W323A, where for x-EQ-y, x denotes a KCNE1 mutation and y denotes a KCNQ1 mutation, and 3 μg of cDNA of the linked construct was transfected. In EQ, the stoichiometric ratio of KCNQ1:KCNE1 is set to 4:4. For E43C-KCNE1 + KCNQ1 (further referred to as E43C-E1Q1) and KCNE1 + A300C-KCNQ1 (further referred to 76 as E1Q1-A300C) constructs, KCNE1 and KCNQ1 cDNA were co-transfected in a 3:1 (1.5 μg:0.5 μg) ratio. In E43C-E1Q1 and E1Q1-A300C, linked constructs of the mutations were not used because of their low expression levels. For KCNQ1 alone constructs, 1.5 μg of cDNA were used. All constructs were co-transfected with 0.5 μg green fluorescent protein (GFP) for the identification of transfected cells except for E43A-KCNE1 and E43K-KCNE1, which were tagged with GFP (KCNE1-GFP). Previous studies have demonstrated that GFP-tagged KCNE1 does not impede the function of the KCNQ1 channel, as evidenced by the identical gating properties observed when KCNE1 is co-expressed with GFP (Murray et al., 2016). Whole-cell recordings were conducted 24 h post-transfection. 3.2.3 Electrophysiology Cells were continuously superfused at room temperature with the control bath solution. Current recordings were obtained using standard whole-cell patch clamp techniques. An Axopatch 200B amplifier, Digidata 1440A digitiser and pCLAMP 10 software (Molecular Devices, LLC, San Jose, CA) were used for all the experiments. Recording electrodes were first pulled from the thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL) using a horizontal electrode puller (Sutter Instrument, Novato, CA) and then fire-polished. Electrodes had resistances of 1.5-3 MΩ when filled with the pipette solution. Analog capacity compensation and 40-60% series resistance compensation were generally used during whole-cell measurements. Data were sampled at 20 kHz and filtered at 5 kHz during acquisition. Currents were recorded using a standard voltage protocol: Cells were held at -90 mV. Membrane potential was then stepped from -80 to +90 mV in 10 mV steps for 4 s. This was followed by a step to -40 mV for 2 s to record deactivating tail currents. 77 3.2.4 Trajectory clustering of mutant W323A, Y46C and WT channels via molecular dynamics simulation Clustering dendrograms based on root mean square deviation (RMSD) were generated from molecular dynamic simulations as described in Chan et al. 2023 (Chan et al., 2023) using methods previously described (Tubiana et al., 2018). 3.2.5 Data and analysis The manuscript complies with the British Journal of Pharmacology's (BJP) recommendations and requirements on experimental design and analysis (Curtis et al., 2022). In our experiments, we used the same cell population (tsA201) to determine the effects of mefenamic acid treatment on WT E1Q1 and various mutations. Given the homogeneity of the tsA cell line, randomisation cannot be applied. Similarly, the uniform properties of these cells, as well as the use of a single compound, rendered blinding unfeasible for a single experimenter. Therefore, no randomisation or blinding was applied. All data free from technical errors were included, with no exclusion criteria for outliers. Data are represented as mean ± standard error of the mean (SEM) unless otherwise specified. Tail current amplitudes in the presence of different drug concentrations were normalised to the tail current amplitude in control and used to generate concentration-response curves for changes in IKs current. Dose-response curves were fitted to the following equation: !!!"# = ""#($%&'[)] )+, to obtain the IC50 and Hill coefficients (nH), where IC50 is the concentration of inhibitor that gives a 78 response halfway between the lower and upper asymptotes of the dose-response curve. We set the upper limit (top constraint) to 1, indicating no effect. There was no constraint on the slope and the lower limit. Conductance–voltage (G-V) relationships were generated by plotting the normalised peak of the tail current against the corresponding voltage. The G-V relationships were fitted with a Boltzmann sigmoid equation to obtain the half-maximal activation voltage (V1/2) and the curves' slope (k-value). Activation V1/2 was calculated for each cell and averaged to obtain the mean V1/2 (Table 3.1). When the bottom of the G-V curve did not reach zero, as when the drug was present, the V1/2 was read directly from the voltage level where the G-V curve intersected the midpoint of the normalised current. Fractional tail current was calculated from the ratio of tail current amplitude in the presence of drug over tail current amplitude in control. Peak tail current amplitude was measured using 0 current as the baseline, denoted by the dotted line in figures representing current traces (e.g. Figure 3.1 A-D). HMR1556, a selective IKs inhibitor at 300 nM almost completely abolished the peak tail current in WT and mutant channels, validating the use of 0 current as the baseline (see Figure 3.1 C-F). In some cases, the ‘normalised response’ was used to quantify the extent of the slowing of tail current compared to control, as described in Chan et al. 2023 (Chan et al., 2023). Standard voltage protocols were used to examine the deactivation kinetics. Current traces were fitted at each voltage using a single-exponential model to determine the deactivation time constants (tdeact). GraphPad Prism 9 (GraphPad Software, San Diego, CA) and Microsoft Excel (Microsoft, Redmond, WA) were used for all data analysis. Statistical analysis was undertaken only for experiments where each group size was at least n = 5, where n represents independent samples. Datasets where n < 5 were not tested for significance. To calculate significance for two-group 79 comparisons (i.e. before and after drug treatment), the non-parametric (Mann-Whitney test) t test was used because normalisation generated a baseline value of 1 with no variance. For multiple group comparisons, the mixed-effects model followed by the Fisher's least significant difference (LSD) test was used to calculate statistical significance. The threshold for statistical significance for determining whether treated groups differ from control groups was set to P < 0.05. All data in the figures are shown as mean ± SEM. Figure 3.1 Current waveform changes and inhibitory effects of mefenamic acid (Mef). (A-D) Current waveforms for WT-EQ, E43C-E1Q1 and E1Q1-A300C. Control (black), Mef (grey, purple, red), wash-out (green), and 300 nM HMR1556 (brown). Cells were depolarised from -80 to +60 mV for 4 s, and stepped back to 40 mV for 2 s. Interpulse interval was 15 s. The dotted line denotes zero current. (E) Fractional tail current remaining after application of Mef (30 μM Mef: purple, n=7, red, n=12; 100 μM Mef: light grey, n=6, light purple, n=5, light red, n=5; 300 μM Mef: dark grey, n=5) and 300 nM HMR1556: brown. *P < 0.05 versus control. (F) Diary plot of complete E1Q1-A300C experiment showing the effects of Mef, wash-out and HMR1556 treatments on tail current amplitude. 80 3.2.6 Nomenclature of targets and ligands Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander et al., 2023). 3.3 Results 3.3.1 Mefenamic acid has dual effects on WT IKs current in mammalian cells Mefenamic acid is known to potentiate IKs currents (Abitbol et al., 1999; Wang et al., 2020b; Zheng et al., 2012), and this enhancement in the presence of 100 μM mefenamic acid is shown in Figure 3.1A. The slow sigmoidal activation of IKs is transformed into an almost square waveform because of the presence of a large instantaneous current (Figure 3.1A). In addition, the tail current decay seen in control is abolished in the presence of 100 μM mefenamic acid. The dramatic potentiation of current arises because of the combined effects of this drug concentration and the experimental protocol in which the cell is repeatedly pulsed to +60 mV for 4 s. Such a current facilitation would likely be pro-arrhythmic in vivo, but this protocol is used here to clearly separate the activating effects of the drug from its inhibiting action, which is the subject of the present study. At higher drug concentrations, activation remains almost instantaneous, and tail current decay is fully inhibited, but both step and tail current amplitudes decrease (300 μM, Figure 3.1B). Notably, this inhibition is not observed at 100 μM mefenamic acid, even when the activation pulse is considerably extended (Figure B.1). This current inhibition is reversed upon wash-out, with the current amplitude restored to the pre-drug level. These results suggest that mefenamic has a dual effect on the WT-EQ channels. It behaves as a relatively pure potentiator 81 at concentrations up to 100 μM, but this activating effect becomes outweighed by a strong inhibiting effect at higher concentrations (i.e., 300 μM). 3.3.2 Mutations of residues in the mefenamic acid binding pocket unmask its inhibiting properties We previously identified a binding pocket for mefenamic acid in the IKs channel complex, comprised of KCNE1 residues located at the extracellular membrane surface, residues in S1, S6, and in the turret region of KCNQ1 (Chan et al., 2023). In the present study, we explore more specifically the effects of mefenamic acid on two residues, KCNE1-E43 and KCNQ1-A300 (Figure 3.1). Zebrafish IKs is inhibited by mefenamic acid, and this was attributed to sequence divergence in KCNE1 at the extracellular transmembrane domain boundary. Whereas human KCNE1 has the sequence K41, L42 and E43, the zebrafish ortholog has the sequence H41, L42 and S43 (Haverinen et al., 2022). Current traces for E43C-E1Q1 and E1Q1-A300C in the absence and the presence of mefenamic acid are shown in Figure 3.1 C, D. Under control conditions, both E43C-E1Q1 and E1Q1-A300C display IKs-like current morphology (Figure 3.1 A, C, D). Exposure to 30 μM mefenamic acid markedly inhibits peak and tail current amplitudes in both mutants (Figure 3.1 C, D). While the current morphology in both cases is preserved, the maximum current amplitude at the end of the 4 s pulse is reduced when compared to control. Likewise, the peak tail current amplitude is reduced by 41% in E43C-E1Q1 and 38% in E1Q1-A300C in the presence of 30 μM mefenamic acid (Figure 3.1 C, D, E). Interestingly, slowing of the tail current decay is also observed in E43C-E1Q1, suggesting that the enhancing properties of mefenamic acid are partly maintained 82 in this mutant (Figures 3.1C and 3.2A). The slowing of deactivation is not as visible in E1Q1-A300C, where mefenamic acid inhibition dominates (Figures 3.1D and 3.2B). Current amplitude and morphology are restored close to control levels after drug wash-out, confirming that the inhibition was caused by mefenamic acid rather than being due to current run-down (Figure 3.1 C, D). In E43C-E1Q1, potentiation, as demonstrated by inhibition of tail current decay, precedes a decrease in current amplitude (Figure B.2A). Notably, the potentiating actions of mefenamic acid are not as prevalent in the E1Q1-A300C mutant (Figure B.2B) until after 90 s of washout, when the current amplitude has returned to near control levels, but the tail current decay has now slowed (Figure 3.1 D). As observed in WT EQ, the application of 300 nM HMR1556, a well-described IKs inhibitor, (Gögelein et al., 2000; Thomas et al., 2003) abolished the currents in both E43C-E1Q1 and E1Q1-A300C, confirming that the measured ionic current is flowing through KCNQ1/KCNE1 channels (Figure 3.1 C-E). This experiment also suggests that the binding of mefenamic acid in these mutants does not interfere with that of HMR1556. The diary plot of a complete experiment with E1Q1-A300C shown in Figure 3.1F illustrates the effects of mefenamic acid, wash-out and HMR1556 on tail current peak amplitude. Fractional tail current measured at 30 and 100 μM indicates that inhibition of E43C-E1Q1 and E1Q1-A300C is concentration dependent (Figure 3.1E). E43 has been shown to make direct contact with KCNQ1-W323 in S6 (Chung et al., 2009; Kuenze et al., 2020). The indole ring of W323 covers the side chain of residue Y46 in KCNE1, which is buried in between S6 and the P helix, and modulates pore conductance (Chung et al., 2009; Kuenze et al., 2020). Because of the proximity of W323 and Y46 to E43, we tested the possibility that mutations at Y46 and W323 may also unmask mefenamic acid inhibitory effects. Y46A-EQ and EQ-W323A were both inhibited by 100 μM mefenamic acid, and like E43C-E1Q1, slowing of tail current decay precedes a decrease 83 in current amplitude in both Y46A-EQ and EQ-W323A (Figure B.2C and D). The concentration-dependent inhibitory effects of mefenamic acid on E43C-E1Q1 and E1Q1-A300C tail current peak amplitude are shown in Figure 3.2. A decrease in current size was first detected in both mutants at a drug concentration of 3 μM, and this inhibition becomes greater with mefenamic acid concentrations up to 100 μM (Figure 3.2 A, B). While mefenamic acid is more potent on E43C-E1Q1 than on E1Q1-A300C, it is also less efficacious as inhibition is less complete (Figure 3.2 A, B) (E43C-E1Q1: fractional current remaining = 0.41; E1Q1-A300C: fractional current remaining = 0.29). Overall, these results suggest that in addition to its IKs enhancing activity, mefenamic acid possesses inhibitory effects which are mostly concealed in WT-EQ but are unmasked by mutations close to the mefenamic acid binding site. 84 Figure 3.2 Concentration-dependent effects of mefenamic acid (Mef) on E43C-E1Q1 and E1Q1-A300C. (A, B) Current traces and concentration–response curves for E43C-E1Q1 and E1Q1-A300C exposed to increasing concentration of Mef. Cells were depolarised from -80 to +60 mV for 4 s and stepped back to 40 mV for 2 s. Interpulse interval was 15 s. The dotted lines in the current traces denote zero current. The dotted line in the concentration-response curves is for reference only and denotes current remaining after 300 nM HMR1556, which fully suppresses IKs. Number of replicates for each concentration is indicated on the concentration–response curve below each mean data point. 3.3.3 Effects of mefenamic acid on E43C-E1Q1 and E1Q1-A300C G-V relationships and deactivation kinetics Mefenamic acid (100 μM) induces a large hyperpolarising shift in the WT-EQ G–V curve, which results in a very negative activation V1/2 (ΔV1/2 = -105.6 mV, (Chan et al., 2023)) as well as a 85 slowing of deactivation kinetics (Wang et al., 2020b). In E43C-E1Q1, mefenamic acid produces a hyperpolarising shift in the G-V curve (ΔV1/2 = -24.5 mV; Table 3.1, Figure 3.3A), as well as a slowing of deactivation kinetics compared to control (Figure 3.3C). In contrast, mefenamic acid causes a depolarising shift of the G-V curve in E1Q1-A300C resulting in a more positive activation V1/2 (ΔV1/2 = +35.2 mV) (Table 3.1 and Figure 3.3B) while slowing the deactivation kinetics (Figure 3.3D). These results confirm that mefenamic acid has a dual effect on E43C-E1Q1. It has both potentiating effects (inhibition of tail current decay, hyperpolarisation of the G-V curve) and inhibiting effects (decrease in step current and tail current amplitude). In contrast, mefenamic acid inhibiting effects in E1Q1-A300C are more prominent as current amplitude is reduced and the G-V relationship is depolarised although potentiating effects are still present as evidenced by slower deactivation kinetics. 86 Figure 3.3 G–V and deactivation kinetic changes induced by mefenamic acid (Mef) in E43C-E1Q1 and E1Q1-A300C. (A, B) Current traces of E43C-E1Q1 and E1Q1-A300C in control (top) and presence of 30 μM Mef (bottom). G-V plots in control (black) and 30 μM Mef (colours). Cells were held at -80 mV and depolarised from -80 to +100 mV for E43C-E1Q1 and +120 mV for E1Q1-A300C for 4 s, followed by a 2 s repolarisation to 40 mV. Interpulse interval was 15 s. Mean V1/2 of activation, slope and n values are reported in Table 3.1. (C, D) Deactivation time constants versus different membrane potentials for E43C-E1Q1 and E1Q1-A300C in the absence (black circles) and presence of 30 μM Mef (coloured circles). Number of replicates for each voltage is indicated next to each mean data point. Cells were held at -80 mV and depolarised to +60 mV for 4 s followed by 4 s repolarisation steps from 90 to 130 mV in 10 mV steps. Interpulse interval was 15 s. Representative tail currents (grey) fit with a single exponential function (blue curve) from 130 to 40 mV of E43C-E1Q1 and E1Q1-A300C in control (left) and presence of 30 μM Mef (right) after depolarising to +60 mV for 4 s. *P < 0.05 versus control. 87 Table 3.1: Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and in the presence of mefenamic acid. Hyphen means not tested. Data are shown as mean ± SEM with *p<0.05 vs. control. Control 30 µM Mef 100 µM Mef V1/2 (mV) k-factor (mV) n V1/2 (mV) k-factor (mV) n V1/2 (mV) k-factor (mV) n E43C- E1Q1 15.2 ± 6.5 25.7 ± 1.4 6 -9.3 ± 4.0* 19.9 ± 2.3 9 - - - E1Q1-A300C 28.7 ± 4.1 23.6 ± 0.9 14 63.9 ± 6.7* 32.0 ± 2.9 5 - - - E43A- E1Q1 -17.1 ± 3.0 10.0 ± 0.9 3 - - - -35.7 ± 3.6 15.4 ± 4.2 3 E43K- E1Q1 3.7 ± 2.8 13.8 ± 1.7 8 - - - -7.9 ± 4.4 16.9 ± 1.5 4 WT- Q1 -28.7 ± 1.2 11.2 ± 0.4 5 - - - -35.9 ± 0.7 18.3 ± 2.9 5 Q1-A300C -19.0 ± 2.0 9.5 ± 0.7 3 - - - -22.6 ± 3.5 13.4 ± 1.6 3 To compare the effects of mefenamic acid to those of a known IKs blocker (i.e., HMR1556), we applied 30 nM HMR1556 to E43C-E1Q1 (Figure B.3A). The results show a potent reduction of current amplitude with no changes to the voltage dependence of activation or deactivation kinetics of E43C-E1Q1 as evident in the overlap between the control and drug G-V relationships and deactivation time constant plots (Figure B.3B and C). 3.3.4 KCNE1 is essential for the inhibitory action of mefenamic acid in E1Q1-A300C To investigate the requirement for KCNE1 in the inhibitory action of mefenamic acid, we expressed homomeric KCNQ1-A300C and WT-KCNQ1 (WT-Q1) without the co-expression of KCNE1. Under control conditions, both channels show a rapidly activating current at +60 mV and a fast-deactivating current at -40 mV (Figure 3.4A, D, E). After application of 100 μM or 88 300 μM mefenamic acid, there is no significant change in the tail current amplitude and G–V relationships compared to control in either WT-Q1 or Q1-A300C (Figure 3.4A, B, D, E). In WT-Q1, tail current decay is somewhat slowed in the presence of 300 μM mefenamic acid, indicating a minor drug activation effect, but this is not observed in Q1-A300C (Figure 3.4A, C). Interestingly, the tail current hook typical of Q1 is eliminated by mefenamic acid, suggesting that mefenamic acid prevents or slows recovery from Q1 inactivation. These findings show that mefenamic acid does not inhibit Q1-alone channels, a direct contrast to the inhibitory effect seen in E1Q1-A300C and E43C-E1Q1. Thus, these results confirm that the presence of KCNE1 is necessary for the inhibiting effect of mefenamic acid. 89 Figure 3.4 Current waveform and G–V changes induced by mefenamic acid (Mef) on KCNQ1 alone. (A) Current traces from WT-Q1 and Q1-A300C in control (black), 100 μM Mef (red) and 300 μM Mef (dark grey). The voltage protocol is as described in Figure 3.1. The dotted line denotes zero current. (B) Fractional tail current remaining after application of 100 μM Mef (grey, n=6 and red, n=6) and 300 μM Mef (dark grey, n=6). *P < 0.05 versus control. (C) Summary plot of the normalised tail current decay response to 100 μM Mef (WT-Q1, n=7 and Q1-A300C, n=5) compared to control. (D, E) WT-Q1 and Q1-A300C current traces in control (top) and in the presence of 100 μM Mef (bottom). G–V plots in control (black) and 100 μM Mef (grey and red). Cells were held at -80 mV and depolarised from -80 mV to +100 mV in 10 mV steps for 4 s, followed by a 2 s repolarisation to 40 mV. Interpulse interval was 15 s. Mean V1/2 of activation, slope and n values are reported in Table 3.1. 3.3.5 Removing the charge at position 43 of KCNE1 is insufficient to explain mefenamic acid’s inhibitory effect on E43C-E1Q1 The glutamic acid residue at position 43 was substituted with the hydrophobic residue alanine (A), the positively charged residue lysine (K), or aspartic acid (D) which conserves the negative 90 charge but has a shorter side chain than glutamic acid (Figure 3.5). For E43A-E1Q1, the channel complex exhibits a current waveform like that of Q1 alone, with rapid activation at +60 mV and deactivation at 40 mV, including a hook indicative of recovery from inactivation also observed in WT-Q1 (Figures 3.4A and 3.5A). Upon exposure to 100 μM mefenamic acid, there is a hyperpolarising shift in the G–V relationship (Figure 3.5E and Table 3.1), and tail current decay is inhibited as evidenced by flattening of the tail current (Figure 3.5A). Interestingly, the hook disappears in the presence of mefenamic acid, suggesting that the drug prevents inactivation in this construct or slows the kinetics of recovery (Figure 3.5A). Notably, no inhibitory effect was observed as the peak tail current amplitude remains the same in the presence of mefenamic acid (Figure 3.5A, D). The E43K-E1Q1 current waveform resembles that of WT-EQ (Figures 3.1A and 3.5B). Post-treatment with 100 μM mefenamic acid, the current morphology of E43K-E1Q1 closely mirrors the potentiated response seen with WT-EQ. There is a slight hyperpolarising shift in the G–V curve (Figure 3.5F and Table 3.1), but there is no inhibition, as indicated by the tail current amplitude remaining constant relative to control (Figure 3.5D, F). In E43D-E1Q1, the current waveform exhibits a slow activation at +60 mV and a fast deactivation at 40 mV compared to WT-EQ (Figure 3.5C). In the presence of mefenamic acid, tail current amplitude is reduced (Figure 3.5C, D) and the tail current decay is slowed (Figure 3.5). These effects are very similar to those observed in E43C-E1Q1 with 100 μM mefenamic acid. Overall, the data show that inhibition of E43C-E1Q1 by mefenamic acid is not because of the removal of the negative charge, but rather the size of the side chain seems to be of significance. In addition, a negative charge at position 43 is not essential for the potentiation effect of mefenamic acid, as activation properties remain with either a hydrophobic (and shorter) or positively charged residue. 91 Figure 3.5 Current waveform, current amplitude and G–V changes induced by mefenamic acid (Mef) on E43A-E1Q1, E43K-E1Q1 and E43D-E1Q1. (A-C) Current waveform of E43A-E1Q1, E43K-E1Q1 and E43D-E1Q1 before (black) and after 100 μM Mef. (colours). The dotted line denotes zero current. (D) Fractional tail current remaining after application of 100 μM Mef (E43A-E1Q1, n=5, E43K-E1Q1, n=6, E43D-E1Q1, n=6). *P < 0.05 versus control. (E, F) E43A-E1Q1 and E43K-E1Q1 current traces in control (top) and in the presence of 100 μM Mef (bottom). G-V plots in control (black) and 100 μM Mef (colours). Cells were held at -80 mV and depolarised from -80 to +100 mV in 10 mV steps for 4 s, followed by a 2 s repolarisation to 40 mV. Interpulse interval was 15 s. Mean V1/2 of activation, slope and n values are reported in Table 3.1. 3.3.6 The inhibitory action of mefenamic acid on E1-E43C is not dependent on the movement of the voltage-sensing domain (VSD) To examine whether mefenamic acid's inhibitory effect on E43C-E1Q1 is the result of the modulation of VSD movement, we used the KCNQ1 (E160R/R237E) construct (referred to as R4), where the VSD of KCNQ1 is arrested in the fully activated position by charge reversal 92 mutations between S2 and S4 (Wu et al., 2010; Zaydman et al., 2014). By fixing the VSD domain, we can obviate any effects of mefenamic acid caused via VSD-induced changes in channel gating and isolate mefenamic acid effects on VSD-pore coupling and the pore itself. Data in Figure 3.6 show the result of experiments where R4 is co-expressed with either KCNE1 WT (E1Q1-R4) or KCNE1-E43C (E43C-E1Q1-R4) in the absence and the presence of mefenamic acid. In both constructs, the channel complexes exhibit an instantaneous current upon depolarisation to +60 mV with no measurable deactivation at 40 mV in control conditions (Figure 3.6A, B). In the presence of 30 μM mefenamic acid, there are no changes in the current morphology or tail current amplitude of E1Q1-R4, as indicated by the continued presence of the instantaneous current at +60 mV and the lack of deactivation at 40 mV. Increasing the concentration to 100 μM does not induce any effects either, as indicated by fractional tail currents (Figure 3.6 D). This finding is consistent with our previous work where we showed that mefenamic acid potentiates IKs by enhancing VSD activation, leading to more frequent channel openings, rather than affecting the pore dynamics (Wang et al., 2020b). In the presence of 300 μM mefenamic acid, E1Q1-R4 current morphology is unchanged, but tail current amplitude is reduced by 19% (Figure 3.6C, D). In the presence of 30 μM mefenamic acid, E43C-E1Q1-R4 remains constitutively open, as evident from the persistent instantaneous current, but the tail current amplitude is reduced by 38% compared to control (Figure 3.6D). In E43C-E1Q1-R4, 100 μM mefenamic acid does not inhibit the channel complex further (Figure 3.6D). Both channel complexes are inhibited by the pore blocker HMR1556 (300 nM), as current amplitude decreases to the level of endogenous currents in these cells, confirming that measured current flows through IKs (Figure 3.6A, B, D). Reduction in current amplitude in the presence of mefenamic acid is quantitatively similar in WT-EQ and E1Q1-R4 (Figures 3.1B, E and 3.6D) and in E43C-93 E1Q1 and E43C-E1Q1-R4 (Figures 3.2A and 3.6), which suggests that mefenamic acid's inhibitory action in WT-EQ and E43C-E1Q1 are primarily because of modulation of VSD-pore coupling and/or intrinsic pore kinetics. Figure 3.6 Effects of mefenamic acid (Mef) on E1Q1-R4 and E43C-E1Q1-R4. (A-C) Current waveform of E1Q1-R4 and E43C-E1Q1-R4 for control (black), 30 or 300 μM Mef (colours), 300 nM HMR1556 (brown) and wash-out (green). Voltage protocol is as described in Figure 3.1. The dotted line denotes zero current. (D) Fractional tail current remaining after application of 30 μM (E1Q1-R4, n=3, E43C-E1Q1-R4, n=5), 100 μM (E1Q1-R4, n=3, E43C-E1Q1-R4, n=6), 300 μM Mef (E1Q1-R4, n=6), or 300 nM HMR1556 (brown). *P < 0.05 versus control. 94 3.4 Discussion A hallmark of mefenamic acid's potentiating effect, and the primary reason that the drug is of interest, is the transformation, at sufficient drug concentration, of the slowly activating IKs current into one with an almost instantaneous current onset and slowed tail current (Chan et al., 2023; Wang et al., 2020b). This change in the current waveform is associated with a large shift in the voltage dependence of activation toward more negative potentials, resulting in a more negative activation V1/2. Activating effects have been well described in the literature (Busch et al., 1994a; Chan et al., 2023; Unsöld et al., 2000; Wang et al., 2020b). However, most studies only focus on a single concentration of mefenamic acid (100 μM), somewhat above the EC50 of 60 μM (Wang et al., 2020b). Although the present study confirms mefenamic acid's activating effect, we also reveal the previously undetected inhibition of the WT IKs channel complex by mefenamic acid at higher concentrations (300 μM). Inhibitory effects are mostly concealed in WT channels and were unrecognised in our prior study where we focused on current potentiation by mefenamic acid (Wang et al., 2020b). However, inhibition is unmasked by mutation of specific residues located at the extracellular intersubunit interface of KCNQ1 and KCNE1, where mefenamic acid was found to bind (Chan et al., 2023). Previous studies have reported the biphasic effects of the racemate benzodiazepine L-364,373 (R-L3) on IKs, which were because of the opposing actions of the two optical enantiomers (Corici et al., 2013; Salata et al., 1998). 3.4.1 Inhibitory actions of mefenamic acid on the WT IKs channel complex In the fully saturated IKs channel complex, inhibition is manifested by a decrease in macroscopic current amplitude of both step and tail currents. Interestingly, potentiating and inhibiting effects are not mutually exclusive as the current morphology observed in the presence of 300 μM 95 mefenamic acid maintains the potentiating effect phenotype (instantaneous current, inhibition of tail current decay) despite decreased current amplitude (Figure 3.1). Similar dual actions have been described before. Almokalant, a class III antiarrhythmic agent, was shown to have two effects on IK in rabbit ventricular myocytes where a hyperpolarising shift of the voltage dependence of activation (potentiating effects) was seen along with use-dependent block of the open channel (inhibitory effects) (Carmeliet, 1993). To explain its opposing effects, it was assumed that almokalant bound to two different binding sites. Similarly, Bay K8644, a calcium channel agonist, has dual effects as it shifts the G-V curve of the calcium channels to the left (potentiation) and also decreases current amplitude and contractility at depolarised holding potentials (Sanguinetti et al., 1986). The existence of two different binding sites for potassium channel openers with opposite effects has been suggested before, although modulation by regulatory molecules was not excluded (Teramoto et al., 2001; Tricarico et al., 2003). Models have been developed to explain the inhibition of ion channels or receptors by agonists at high concentrations by the existence of two different sites mediating opposite actions (Rovati & Nicosia, 1994). As the inhibition by mefenamic acid of WT IKs is only detected at higher concentrations, a model where two distinct binding sites coexisting is likely. The drug may bind mostly to the higher-affinity binding site at lower concentrations of mefenamic acid, potentiating IKs current. When higher concentrations of mefenamic acid are applied, it may initially bind to the higher affinity site, causing transient potentiation, and eventually bind to the lower affinity site, causing inhibition. This series of events is supported by our experimental approach using mutated channels where flattening of the tail current occurs before the decrease in current amplitude in our mutants (Figure B.2) and the persistence of slowed tail current decay during wash-out (Figure 3.1D). 96 3.4.2 Mutations enhance mefenamic acid’s inhibitory actions Investigation of the effects of mefenamic acid on mutated residues involved in its binding site demonstrates that the lower affinity inhibitory site in WT EQ can be turned into a higher affinity site in E43C, Y46A, A300C and W323A. In these mutants, IKs channels are inhibited by mefenamic acid with a higher potency than WT. However, in E43C-E1Q1, Y46A-EQ and EQ W323A, activating effects (hyperpolarised shift of the G-V curve and slowed deactivation) coexist with the decrease in current amplitude (Figures 3.1, 3.3 and Figure B.2; (Chan et al., 2023)), and potentiation is less apparent in the A300C phenotype (depolarised shift of the G-V and slowed deactivation but less so than in E43C), leaving the block relatively unopposed. This difference in gating properties between A300C and the other mutants may explain the stronger blocking efficacy of mefenamic acid for A300C, as evidenced by a more complete inhibition of the tail current amplitude on the concentration-response curve. These results indicate that the presence of a cysteine residue at position 300 in the nearby P helix of KCNQ1 can abolish most of the activating effects of mefenamic acid, turning a low-affinity site into a primary binding site for drug inhibitory action. In our previous paper, docking analysis showed that mefenamic acid was stabilised within the binding pocket by the formation of two hydrogen bonds between the drug and KCNE1 residues E43 and Y46 (Chan et al., 2023). Disruption of these hydrogen bonds by substitution of glutamic acid and tyrosine for cysteine or alanine residues may induce a change in drug-receptor stability and affinity, facilitating a change in drug orientation which ultimately favours binding to a different site, leading to channel inhibition. This change in drug orientation was observed in 97 molecular dynamics simulations (Chan et al., 2023). Cluster dendrograms generated from the MD simulation from our previous paper indicate that there are at least five major conformations adopted for each of the W323A and Y46C mutants highlighting the dynamic nature of the interaction (Figures B.4 and 5). The initial binding conformation is shown in Figures 3.7A and 3.8A, with the next largest cluster of the dendrogram in Figures 3.7B and 3.8B. Changes induced by the mutations may be explained by unmasking an alternate binding site for the drug with inhibitory actions. Figure 3.7 Mefenamic acid (Mef) binding to ps-IKs-W323A. Snapshots of mefenamic acid binding over time to ps-IKs carrying the W323A mutation (MD simulations from Chan et al. [2023]). (A) Initial position of bound mefenamic acid. (B) Over time, mefenamic acid changes position. E43, W323A, A300 and Y46 side chains are shown. The VSD is in yellow, the pore in blue and ps-KCNE1 in red. VSD, voltage-sensing domain. 98 Figure 3.8 Mefenamic acid (Mef) binding to Y46C. Snapshots of mefenamic acid binding patterns over time to ps-IKs carrying the Y46C mutation (MD simulations from Chan et al. [2023]). (A) Initial position of bound mefenamic acid. (B) Over time, mefenamic acid changes position. E43, W323, A300 and Y46C side chains are shown. The VSD is in yellow, the pore in blue and ps-KCNE1 in red. VSD, voltage-sensing domain. 3.4.3 Mechanisms of the inhibitory effects of mefenamic acid At the single channel level, potentiating effects of mefenamic acid are because of an increase in open probability rather than an increase in maximum channel conductance, suggesting enhanced channel activation gating in the presence of drug (Wang et al., 2020b). Consistent with this idea, modelling the effect of mefenamic acid on IKs determined that increasing voltage sensor forward rates for activation transitions could mostly explain the effects observed experimentally (Wang et al., 2020b). To investigate whether the voltage sensor was also involved in the inhibition mechanism, we used the R4 constructs (E1Q1-R4 and E43C-E1Q1-R4) in which the voltage sensing domain is locked in a fully activated state (Eldstrom et al., 2021; Zaydman et al., 2014). As the current amplitude decreases in the R4 mutants in the presence of mefenamic acid (Figure 99 3.6), and the relative degree of inhibition equals that seen in WT-EQ and E43C-E1Q1, our results imply that the primary mechanisms underlying the inhibiting effect of mefenamic acid in both WT-EQ and E43C-E1Q1 involve a reduction of VSD-pore coupling or effects on ion conduction through direct or allosteric effects on the pore domain, and are independent of VSD movement (Figure 3.6). It is clear that E43 plays a role in pore dynamics as the E43A mutant displays a hook in the tail current (Figure 3.5) indicative of a recovery from inactivation which is normally eliminated by the presence of KCNE1. The remaining residues are in or interact with the filter helix (Figure 3.9), and therefore, it is proposed that inhibitory effects of mefenamic acid on the pore are better explained by a model in which block of the IKs pore occurs via an allosteric mechanism rather than direct binding to occlude the pore as is proposed for HMR1556 (Lerche et al., 2000). 100 Figure 3.9 Structure showing region of interest for IKs block by mefenamic acid. Mefenamic acid is shown in stick (purple) bound to ps-IKs (structure from Chan et al. [2023]) showing residues (in yellow) that when mutated show block with mefenamic acid. Residues important for pore stability are in green. The ps-KCNE1/KCNE3 is in magenta. SF = selectivity filter. Recently, we have proposed a mechanism for the observed increase in IKs channel whole cell currents on exposure to polyunsaturated fatty acids (PUFAs) (Golluscio et al., 2024). The proposed mechanism starts with the idea that the IKs pore is unstable and contributes to the channel's low open probability (Werry et al., 2013). Polyunsaturated fatty acids (PUFAs), therefore, act by stabilisation of specific residue interactions within the selectivity filter of the channel to increase the number of active sweeps. These interactions may involve the aromatic ring cuff made up of W304 and W305, and analogous aromatic rings are implicated in the inactivation of other Kv channels (Larsson & Elinder, 2000). Interestingly, in one cluster of binding patterns from MD simulations of mefenamic acid with W323A (Figure 3.7), the drug 101 interacts with W304. It is therefore tempting to speculate that by interacting with this residue, mefenamic acid is displacing other interactions in this cuff to destabilise the pore, leading to a non-conductive state (Figure B.6). 3.4.4 The effects of mefenamic acid depend on the presence of KCNE1 Activators such as ML277 are most active on KCNQ1 with reduced activity in the presence of KCNE1 (Eldstrom et al., 2021; Mattmann et al., 2012; Willegems et al., 2022; Yu et al., 2013). Mefenamic acid, DIDS and unsaturated fatty acids are more potent on KCNQ1/KCNE1 channels (Wang et al., 2020b). Similarly, KCNE1 increases the sensitivity of KCNQ1 to classical pore blockers such as chromanol 293B and benzodiazepine (Busch et al., 1997; Lerche et al., 2007; Seebohm et al., 2003; Wang et al., 2020b). Although KCNE1 does not directly participate in the binding site of these inhibitors, it facilitates drug binding to the KCNQ1 pore subunit through allosteric modulation (Lerche et al., 2000). An allosteric inhibitory mechanism has also been described for KCNQ1 inhibition by adamantanes, involving fenestrations in the KCNQ1 channel that only form when the pore subunit interacts with KCNE1 (Wrobel et al., 2016). While our results confirm that mefenamic acid requires KCNE1 to display its activating effects on the channel complex, they show that its inhibiting effects also depend on the presence of KCNE1, highlighted by the lack of effects of mefenamic acid on WT-KCNQ1 and KCNQ1-A300C (Figure 3.4). While we lack any structures to confirm that KCNE1 makes direct contact with A300, certainly KCNE3 does (Sun & MacKinnon, 2020); therefore, it is expected that mutation to a cysteine residue would affect KCNE1 interaction or, indirectly, the pore conformation, which favours the binding of mefenamic to a new site to decrease ion conduction. Residues on the extracellular domain of KCNE1 are involved in the allosteric modulation of the backbone 102 conformation of the KCNQ1 selectivity filter (Xu et al., 2013). Specifically, a tyrosine residue at position 46 on KCNE1 applies steric pressure on the KCNQ1 pore domain to favour selectivity for K+ ions over bulkier Cs+ and Rb+ (Xu et al., 2013). E43 has been shown to make direct contact with W323 in S6. It is, therefore, possible that E43C, Y46A and Y46C in KCNE1 and W323A in KCNQ1 favour binding of mefenamic acid at the intersect of the extracellular domain of KCNE1, P-loop S6 regions of KCNQ1, either allosterically influencing the backbone conformation of the pore or exerting steric pressure on the KCNQ1 pore domain, resulting in reduced potassium flow and current inhibition. A300 is located at the interface between the voltage sensor domain, the pore domain and KCNE1 (Figure 3.9) (Chan et al., 2023). Proximity to the VSD may explain effects of mefenamic acid on gating mechanisms in A300C, differentiating it from E43C. 3.4.5 Relevance to drug design and therapeutic application Concentrations of 100 and 300 μM mefenamic acid are unlikely to be reached in a clinical setting (mean peak plasma concentrations of 82.9 μM have been reported; (Cryer & Feldman, 1998)), and the increase in instantaneous IKs current observed using our experimental protocol in the presence of mefenamic acid would therefore not reflect the action of mefenamic acid in a more physiological setting (i.e., rapid changes in membrane potential during a brief cardiac action potential). However, this study aims to shed light on how mefenamic acid and, incidentally, other IKs activators' effects on IKs channels may change based on the presence of disease mutations in KCNQ1 and KCNE1 subunits. In mutant channels studied in this work, inhibition of IKs can be seen at lower mefenamic acid concentrations (e.g. 40 μM). Of interest, KCNQ1-A300T is associated with a mild recessive form of the Romano-Ward long QT 103 syndrome (Bianchi et al., 2000; Priori et al., 1998). The A300T cellular phenotype shows a decrease in current amplitude, a hyperpolarised shift in the voltage dependence of activation, and faster activation kinetics (Bianchi et al., 2000; Priori et al., 1998). Thus, this work may highlight the importance of personalised in vitro drug testing before treating patients with diseases such as long QT syndrome (Blinova et al., 2017). In vitro clinical trials have received growing attention because it is now possible to test the effects of novel molecules on patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), which reproduce the patient's clinical phenotype at the cellular level (Pourrier & Fedida, 2020). Our results suggest that LQTS 1 mutations, notably the ones targeting residues located at the extracellular intersubunit interface of KCNQ1 and KCNE1 channel complexes, may impact drug response, turning a potential treatment for patients into a harmful one. 104 Chapter 4: Graded activation of K41C-KCNQ1:KCNE1 channel complexes by mefenamic acid 4.1 Introduction Varying numbers of one to four KCNE1 subunits have been shown to co-assemble with KCNQ1. While the exact stoichiometry of IKs remains unknown in humans, activators that are effective across different IKs stoichiometric ratios are of interest (Dvir et al., 2014; Murray et al., 2016; Nakajo et al., 2010; K. Wang et al., 2011; Wrobel et al., 2012). Most known activators of IKs such as ML277, zinc pyrithione and L-364,373 unfortunately are only effective on KCNQ1 alone, with limited efficacy on increasingly saturated IKs channel complexes (Gao et al., 2008; Magyar et al., 2006; Yu et al., 2013). Of interest, mefenamic acid has previously been identified by numerous groups to enhance IKs currents in primary cells such as canine and guinea-pig ventricular myocytes (Magyar et al., 2006; Toyoda et al., 2006) as well as in heterologous expression systems such as Xenopus laevis oocytes and CHO, COS-7, tsA201 and mouse LM(tk-) fibroblast cells (LM) (Abitbol et al., 1999; Busch et al., 1994b; Chan et al., 2024; Chan et al., 2023; Toyoda et al., 2006; Unsöld et al., 2000; Wang et al., 2020b). After treating IKs with 100 µM mefenamic acid, various amounts of instantaneous current and inhibition of tail current decay were reported. In our characterization of the effects of mefenamic acid on IKs, residue K41, located on the extracellular end of KCNE1, was found to be critical in mediating the activating effect of mefenamic acid on the fully saturated IKs channel complex (4:4 ratio of KCNE1 to KCNQ1, EQ) (Wang et al., 2020b). Cysteine scanning revealed that although other extracellular KCNE1 residues in the 105 same region to varying degrees impacted the effect of mefenamic acid, only the K41C mutation completely abolished mefenamic acid effects up to a concentration of 1 mM. Previous cross-linking studies have identified key interactions between this extracellular region of KCNE1 and the transmembrane segments of KCNQ1, S6 and S1, suggesting that residues in either the S6 and/or S1 region could also provide critical clues to explain the mechanism of action of mefenamic acid. Building on this, our recent study has identified a generalized activator binding pocket for mefenamic acid that is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits (Chan et al., 2023). Particularly K41 in KCNE1, but also other residues such as W323, in the adjacent KCNQ1 subunits appear critical for mefenamic acid binding. A remaining unresolved question is how many mefenamic acid binding sites need to be occupied to permit the activating action of the drug on KCNQ1-KCNE1 complexes, and how this affects VS transitions in the activation pathway. To address this, we have made fixed-stoichiometry IKs mutants containing K41C residues in different numbers of the four KCNE1 accessory subunits. We find that any reduction in the number of available mefenamic acid binding sites reduces the activating response to the drug, and the presence of K41C in more than one KCNE1 subunit further diminishes the response to 100 µM mefenamic acid. 4.2 Materials and Methods 4.2.1 Reagents and solutions Mefenamic acid (Tocris Bioscience, Oakville, ON, Canada) was used at concentrations of 10 μM, 30 μM, 100 μM, 300 μM, 500 μM and 1 mM. To block IKs, the specific IKs inhibitor 106 HMR1556 (Tocris Bioscience, Oakville, ON, Canada) was used at a concentration of 1 µM. All other reagents and solutions were prepared as previously described (Wang et al., 2020b). 4.2.2 Molecular biology, cell culture and whole cell patch clamp Transformed human embryonic kidney 293 cells (tsA201) were first cultured, then plated for whole-cell experiments and, finally, transfected with Lipofectamine2000 as previously described (Murray et al., 2016; Wang et al., 2020b; Westhoff et al., 2019). All mutations were first generated using site-directed mutagenesis and Pfu turbo then sequence confirmed. Whole-cell experiments were conducted 24-48 hr post transfection. For WT EQ and mutant x-EQ-Y (where “x” denotes a KCNE1 mutation and “y” denotes a KCNQ1 mutation; for example, K41C-EQ, cells were transfected with a linked KCNE1 and KCNQ1 cDNA (1.25 µg was used) which assembles as a fully saturated 4:4 ratio of KCNE1 to KCNQ1. For experiments where mutant K41C-KCNE1s were introduced into one (1:3 ratio of mutant to WT KCNE1), two (2:2 ratio of mutant to WT KCNE1) and three (3:1 ratio of mutant to WT KCNE1) out of the four β-subunits in a fully saturated IKs complex, cells were transfected with the following constructs: for the 1:3 ratio of mutant to WT KCNE1 IKs construct (K41C-EQQQQ + KCNE1), cells were co-transfected in an 9:2.25 µg ratio of WT KCNE1 to K41C-EQQQQ (a linked mutant K41C-KCNE1 and KCNQ1 cDNA which assembles as a 1:4 ratio of KCNE1 to KCNQ1); for the 1:1 ratio of mutant to WT KCNE1 IKs construct (EQQ + K41C-KCNE1), cells were co-transfected in an 8:2 µg ratio of mutant K41C-KCNE1 to EQQ (a linked WT KCNE1 and KCNQ1 cDNA which assembles as a 2:4 ratio of KCNE1 to KCNQ1); for the 3:1 ratio of mutant to WT KCNE1 IKs construct (EQQQQ + K41C-KCNE1), cells were co-transfected in an 9:2.25 µg ratio of mutant K41C-KCNE1 to EQQQQ (a linked WT KCNE1 and KCNQ1 cDNA which assembles 107 as a 1:4 ratio of KCNE1 to KCNQ1). All constructs were also co-transfected with 0.8 µg of GFP to allow transfected cells to be identified. Data were obtained using an Axopatch 200B amplifier, Digidata 1440A digitizer and pCLAMP 11 software. 4.2.3 Data analysis The normalized response to 100 µM mefenamic acid was calculated in the same manner as previously described (Wang et al., 2020b). Where applicable, one-way ANOVAs followed by the Bonferroni multiple comparison post-hoc test was used to determine statistical significance. In all figures ****, ***, **, * denote a significance where p<0.0001, p<0.0005, p<0.01 and p<0.05, respectively. All data in the figures and tables are shown as mean ± SE. Statistical analysis was done in Graphpad Prism 9. 4.2.4 Modeling IonChannelLab software (Santiago-Castillo et al., 2010) was used to model IKs in Figure 3.3, as we did previously (Westhoff et al., 2019) and in modeling the action of mefenamic acid on wild type (WT) IKs channels (Wang et al., 2020b). In this model KCNQ1:KCNE1 in a 4:4 stoichiometry assumes four VSs each undergo two activating transitions, to an intermediate and then activated conformation. Pore subconductance opening can occur as soon as each VS is fully activated, and thus pore opening does not require a concerted step after all four VSs have reached fully activated conformations. As in our prior study, to simulate the action of mefenamic acid on IKs currents, the intrinsic rates of forward VS transitions at 0 mV, between resting and intermediate states (kRI0) and between intermediate and activated states (kIA) were multiplied by the drug concentration (in µM), or log10 of the concentration, respectively (Wang et al., 2020b). 108 4.3 Results 4.3.1 Mefenamic acid actions are prevented by the KCNE1 mutation, K41C in a stoichiometric manner Consistent with our previous study and those of others (Abitbol et al., 1999; Unsöld et al., 2000; Wang et al., 2020b), treatment of fully saturated WT IKs complexes (4:4 ratio; WT EQ) with 100 µM mefenamic acid transformed the slowly activating IKs current and deactivating tail current into one with an almost linear waveform indicating instantaneous current onset and complete inhibition of tail current decay (Fig. 4.1A, top left). The corresponding G-V plot obtained from peak initial tail currents shows, in the presence of 100 µM mefenamic acid, a hyperpolarization (ΔV1/2 = -119.4 mV) and shape change (decreased slope) of the G-V relationship compared to control (control k-factor = 19.4 mV; Mef k-factor = 36.7 mV) (Fig. 4.1A, right, triangles, and Table 4.1). 109 -100 -50 0 50 1000.51.0Control 100 µM Mef -100 -50 0 50 1000.51.0-150 -100 -50 0 50 1000.51.0-100 -50 0 50 1000.51.0A G/Gmax Voltage (mV) Control 100 µM Mef 1 mM Mef B G/Gmax Voltage (mV) EQQQQ + K41C-KCNE1 G/Gmax Voltage (mV) G/Gmax Voltage (mV) D C EQQ + K41C-KCNE1 K41C-EQQQQ + KCNE1 Control 100 µM Mef Control 100 µM Mef + Mef K41C-EQ + Mef WT EQ Figure 4.1 K41C-KCNE1 mutants prevent the agonist effect of mefenamic acid. (A) WT EQ currents (top) and K41C-EQ (bottom) in 100 µM mefenamic acid (Mef). G-V plot from WT EQ tail currents (circles) and K41C-EQ (triangles) in the absence (control: black) and presence of Mef (100 µM: green; 1 mM: red) (right). Boltzmann fit parameters are in Table 4.1. Scale bars in all panels represent 0.5 nA and 0.5 s. (B-D) Current traces of EQQQQ + K41C-KCNE1 (B), EQQ + K41C-KCNE1 (C) and K41C-EQQQQ + KCNE1 (D) in control (top) and 100 µM Mef (bottom). G-V plots in control (black) and 100 μM Mef (colored). Boltzmann fits parameters are in Table 4.1. WT EQ, K41C-EQ, EQQQQ + K41C-KCNE1, EQQ + K41C-KCNE1 and K41C-EQQQQ + KCNE1 IKs constructs have a WT KCNE1:K41C-KCNE1 ratio of 4:0, 0:4, 1:3, 2:2 and 3:1, respectively. A 4 s protocol with pulses from -150 mV or higher to +100 mV followed by a repolarization step to -40 mV for 1 s was used. Holding potential and inter-pulse interval were -80 mV and 15 s, respectively. 110 Table 4.1 V1/2 of activation and slope value (k) for Boltzmann fits in the absence and presence of mefenamic acid for fully saturated IKs channel complexes. p-value indicates statistical difference between V1/2 in drug compared to control, determined using a One-way ANOVA and Bonferroni multiple comparisons test. NS denotes ‘not significant’. SEM is denoted by ±. * For K41C-EQ, the concentration of mefenamic acid used was either 100 µM (upper row values) or 1 mM (lower row values). For all other constructs 100 µM mefenamic acid was used. The introduction of a cysteine mutation at residue K41 in all four KCNE1 subunits (4:4 ratio of mutant K41C-KCNE1 to KCNQ1; K41C-EQ) abolished all changes to the G-V relationship and current waveform up to a concentration of 1 mM mefenamic acid. No significant shift in the V1/2 or changes to the slope of the G-V plot were seen after treatment with not only 100 µM but also 1 mM mefenamic acid (Fig. 4.1A, right, circles, and Table 4.1. Note the overlap of red circles and triangles). Additionally, the waveform of K41C-EQ remained sigmoidal at 100 µM (Fig. 4.1A, bottom left) and 1 mM mefenamic acid (data not shown) suggesting that the residue K41 in KCNE1 is critical to the action of mefenamic acid. Control 100 µM or 1 mM Mefenamic Acid * p-value V1/2 (mV) k (mV) n V1/2 (mV) k (mV) n Wildtype (WT) EQ 25.7 ± 2.6 19.4 ± 1.2 6 -93.8 ± 12.3 36.7 ± 14.2 5 <0.0001 EQQQQ + K41C-KCNE1 44.0 ± 2.2 27.2 ± 1.3 4 34.8 ± 2.6 30.4 ± 2.8 3 NS EQQ + K41C-KCNE1 50.3 ± 2.3 23.7 ± 1.6 4 45.3 ± 2.1 29.8 ± 1.4 3 NS K41C-EQQQQ + KCNE1 57.6 ± 1.5 27.6 ± 1.9 5 -2.4 ± 4.7 44.5 ± 5.8 4 <0.0001 K41C-EQ 17.0 ± 1.6 19.6 ± 1.6 7 11.7 ± 1.0 18.6 ± 0.6 3 NS 16.7 ± 2.0 19.8 ± 1.4 3 NS 111 Since the presence of four mutant K41C-KCNE1 subunits was shown to abolish the effect of mefenamic acid and a total of between one and four KCNE1 subunits are known to co-assemble with the KCNQ1 channel complex, we then examined whether the action of mefenamic acid was impacted by introducing different ratios of mutant K41C- and WT KCNE1 subunits into a fully saturated IKs channel complex. K41C and WT KCNE1 subunits were introduced in a ratio of 3:1 (EQQQQ, transfected with K41C-KCNE1), 2:2 (EQQ, transfected with K41C-KCNE1) and finally 1:3 (K41C-EQQQQ, transfected with KCNE1) K41C-KCNE1 to WT KCNE1 subunits (see Materials and Methods). In all three cases fully saturated IKs channel complexes will comprise a 4:4 ratio of KCNQ1:KCNE1). When three out of the four subunits were mutated (EQQQQ + K41C-KCNE1), no significant changes to the shape or V1/2 of the G-V relationship were seen after treatment with 100 µM mefenamic acid (Fig. 4.1B, right, and Table 4.1). Additionally, the EQQQQ + K41C-KCNE1 current waveform remained unaffected by the drug at this concentration. A sigmoidal waveform both in the absence (Fig. 4.1B, top left) and presence of 100 µM mefenamic acid (Fig. 4.1B, bottom left) was observed. The tail current decay also remained, unlike WT EQ (Fig. 4.1A, top left) where complete inhibition of tail current decay was observed after treatment with 100 µM mefenamic acid. Thus EQQQQ + K41C-KCNE1 constructs failed to display any of the three hallmarks of mefenamic acid (changes to current waveform, hyperpolarization of the V1/2 and shape change of the G-V plot) at a dose of 100 µM. However, at concentrations of mefenamic acid above 100 µM the sigmoidal EQQQQ + K41C-KCNE1 waveform was transformed into one with drastically faster onset and partially inhibited tail current decay (Fig. 4.2A). Mefenamic acid reached its maximum efficacy at 500 µM (Fig. 4.2A). At the highest concentration of 1 mM 112 mefenamic acid, the normalized response was approximately half that of WT EQ which indicates decreased efficacy and a heavily right-shifted dose-response relationship, but one that was still responsive to mefenamic acid (EQQQQ + K41C-KCNE1 EC50 = 1203 μM; WT EQ EC50 = 60 μM; Fig. 4.2C). 113 C !"#$ !%$ !&$ $100 µM Mef 1 mM Mef D ΔV1/2 (mV) EQQ + K41C-E1 WT K41C-EQQQQ + E1 K41C-EQ EQQQQ + K41C-E1 **** **** A B Control 100µM 1mM 300µM 500µM EQQQQ + K41C-KCNE1 + Mef !"!!"#$"!$ $! $!! $!!!Mefenamic Acid (µM) Normalized Response EQQQQ + K41C-KCNE1 WT EQ EQQ + K41C-KCNE1 K41C-EQQQQ + KCNE1 Control 100µM 1mM 300µM 500µM + 1 µM HMR1556 EQQ + K41C-KCNE1 + Mef Figure 4.2 Stoichiometric prevention of mefenamic acid agonism by K41C-KCNE1 mutants. (A, B) Currents (control: black) exposed to increasing concentrations of Mef (EQQQQ + K41C-KCNE1: purple; EQQ + K41C-KCNE1: blue), and in B, 1 µM HMR1556 (green). Complete inhibition by HMR1556 is indicated by the green arrow. Scale bars represent 0.5 nA and 0.5 s. (C) Mean log concentration-response curves for EQQQQ + K41C-KCNE1 (purple circles, n = 3-4 at each concentration; EC50 = 1203 [899, 1954; 95% CI] μM and nH = 0.95 [0.66, 1.3; 95% CI]); EQQ + K41C-KCNE1 (blue circles, n = 3-5 at each concentration; EC50 = 382 [323, 440; 95% CI] μM and nH = 1.9 [1.3, 2.8; 95% CI]); K41C-EQQQQ + KCNE1 (red circles, n = 3-5 at each concentration; EC50 = 30 [23, 39; 95% CI] μM and nH = 0.53 [0.47, 0.60; 95% CI]); and, WT EQ (black circles, n = 3-5 at each concentration: EC50 = 60 [38, 89; 95% CI] μM and nH = 0.49 [0.39, 0.60; 95% CI]). The WT EQ concentration-response curve was from (Wang et al., 2020). A 4 s protocol to +60 mV followed by a repolarization step to -40 mV for 1 s was used to obtain currents (see Methods for calculation of normalized response). Holding potential and interpulse interval were -80 mV and 15 s, respectively. (D) Summary plot of V1/2 change (Δ V1/2) in the presence of 100 µM mefenamic acid. **** denotes a significant Δ V1/2 compared to control where p<0.0001. Δ V1/2 were obtained from data in Figure 4.1. 114 The introduction of 2:2 K41C-KCNE1 to WT KCNE1 subunits (EQQ + K41C-KCNE1) produced the same results as EQQQQ + K41C-KCNE1 when treated with 100 µM mefenamic acid. The slope and V1/2 of the EQQ + K41C-KCNE1 G-V relationship remained almost the same after treatment with 100 µM mefenamic acid (Fig. 4.1C, right, and Table 4.1). A sigmoidal waveform with tail current decay was also seen both in the absence (Fig. 4.1C, top left) and presence of the drug (Fig. 4.1C, bottom left). EQQ + K41C-KCNE1 at increasingly higher concentrations of mefenamic acid also responded to the drug in a dose-dependent manner (Fig. 4.2B), and the response of EQQ + K41C-KCNE1 was greater than that of EQQQQ + K41C-KCNE1 (EQQQQ + K41C-KCNE1 EC50 = 1203 μM; EQQ + K41C-KCNE1 EC50 = 382 μM, Fig. 2C). At the highest concentration of mefenamic acid tested, 1 mM, instantaneous current onset and complete inhibition of tail current decay was seen with EQQ + K41C-KCNE1 (Fig. 4.2B). This mefenamic acid-enhanced EQQ + K41C-KCNE1 current was subsequently fully inhibited by 1 µM of the specific IKs inhibitor, HMR1556 (Fig. 4.2B, green trace). These results suggest that at least two WT KCNE1 subunits within the IKs complex are necessary to maintain maximal efficacy of mefenamic acid. Finally, the introduction of only one mutant subunit in a fully saturated channel complex (K41C-EQQQQ + KCNE1) was only able to partially prevent the three hallmarks of mefenamic acid at a dose of 100 µM. Although the V1/2 was hyperpolarized (ΔV1/2 = -59.9 mV) and the slope of the G-V curve was decreased for K41C-EQQQQ + KCNE1 in the presence of 100 µM mefenamic acid compared to control (control k-factor = 27.6 mV; Mef k-factor = 44.5 mV; Fig. 1D, right and Table 4.1), both of these effects were less than that of WT EQ (Fig. 4.1A right; triangles). Despite reducing changes in the G-V relationship, the presence of only one mutant K41C-115 KCNE1 subunit did not prevent changes to the current waveform. An instantaneous current onset with complete inhibition of tail current decay was seen after treatment of K41C-EQQQQ + KCNE1 with 100 µM mefenamic acid (Fig. 4.1D, bottom left). Furthermore, at all concentrations tested, the response of K41C-EQQQQ + KCNE1 to mefenamic acid was comparable to that of WT EQ (Fig. 4.2C). Overall, the results above indicate that the ability of K41C to prevent the activating effect of mefenamic acid is stoichiometrically graded. The fewer mutant K41C β-subunits present in the fully saturated IKs complex, the more effective mefenamic acid was in activating IKs. Additionally, the dramatic shift in V1/2 seen when WT EQ was treated with 100 µM mefenamic acid was found to be absent or reduced in a graded manner, when channel complexes contained four, three and two K41C-KCNE1 subunits (Fig. 4.2D). K41C-EQQQQ + KCNE1 still showed a significant shift in V1/2 suggesting that one mutant K41C-KCNE1 subunit in a saturated IKs channel complex is not enough to prevent the channel from being fully activated by 100 µM mefenamic acid. Importantly, at higher concentrations than 100 µM, all constructs except EQQQQ + K41C-KCNE1 which contained only one K41C subunit, could be activated to the level of WT in the presence of 1 mM mefenamic acid. 4.3.2 Varying the number of K41C-containing subunits in models of mefenamic acid activation of IKs In order to explore how inclusion of different numbers of K41C mutations in the KCNE subunits modifies drug action, a model of IKs channel activity that we have used previously was modified to allow the kinetics of individual VSs to simulate the WT channel response or K41C channel 116 insensitivity to mefenamic acid (Fig. 4.3). The model scheme is shown in Figure 4.3A, and further details, including the rates for all transitions may be found in the Materials and Methods in its original published derivation (Westhoff et al., 2019). Essentially, the model tracks the movement of four independent VS from closed resting states (CRRRR) to fully activated open states (OAAAA). A quantitative simulation of the WT response to mefenamic acid is obtained by speeding the forward rates of each of the two VS transitions (see Materials and Methods, (Wang et al., 2020b)), as shown in the current simulations with zero K41C-KCNE1 mutant subunits (Fig. 4.3B, K41C:0, i.e., WT) where 100 µM mefenamic acid induces an instantaneous current of about 50% of the peak activating current. WT and mutant K41C subunits were included in the model to simulate two extreme possibilities, as shown by the red-delineated areas on the model scheme, where WT VS transitions either dominate the early activation pathway of the channels (red solid outline area), and K41C mutant subunits underlie the later VS transitions (red dash outline area), or vice-versa. The results of the simulations in these two conditions are quite different. When WT subunits activate first, the effect of a single K41C subunit is to reduce the response to 100 µM mefenamic acid by 50%, and then to 10% or less with the addition of further mutant VS (Fig. 4.3B). On the other hand, delayed activation of WT subunits until after K41C subunit activation, blunts the effects of K41C mutants such that the response to mefenamic acid only starts to decline in the presence of three or more K41C-KCNE1 subunits (Fig. 4.3C). 117 Figure 4.3 Model of mefenamic acid action in K41C mutants. (A) Model of IKs. Closed-state transitions are grey, open-state are black. C-O transitions and their reverse as black/grey arrows. VS state subscripts: R = rest; I = intermediate-activated; and A is the activated state. Only open states with activated VS conduct current. Selected transition rates are shown, for other rates, see Methods. Red boxes divide VS movement into two early steps (solid) and two later steps (dashed). (B) Simulation of IKs currents at +80 mV in 100 µM Mef. Inset denotes number of K41C-KCNE1 subunits in a 4:4 complex, so K41C:0 = WT. WT subunits were added to model starting at the left of the scheme, replacing K41C subunits one at a time (red outline area in A). Dotted line represents zero current. (C) As for B, except that WT subunits were added from the right side of the scheme (dashed red outline). (D) Model Mef response (red curves) vs. number of K41C-KCNE1 subunits 118 compared with conductance (at 0 mV, Fig. 1) and 𝛥V1/2 (Fig. 2D) responses to Mef. Data normalized to WT response to 100 µM Mef. As WT VS have augmented kinetics in the presence of mefenamic acid, it is expected that they will activate before K41C subunits, so the first scenario described above with WT subunits activating first (area circumscribed by solid red line) is the most likely and fits the experimental data much better (Fig. 4.3D). This model response to mefenamic acid simulates well the changes in conductance at 0 mV (Fig. 4.1) and the ΔV1/2 (Fig. 4.2D) induced by 100 µM mefenamic acid in channel complexes that contain increasing numbers of K41C-KCNE1 subunits. 4.4 Discussion 4.4.1 Insights from K41C-KCNE1 on mefenamic acid action In a previous study we showed that mefenamic acid has little to no effect on KCNQ1 alone, which suggested that the drug required the presence of KCNE1 to either bind and/or facilitate its effect on the channel, and also that the effect was dependent on the stoichiometric ratio of KCNQ1:KCNE1 subunits (Wang et al., 2020b). The more saturated the IKs channel complex was, the greater the effect of mefenamic acid (1 KCNE1 subunit in the channel complex < 2 KCNE1 subunits < 4 KCNE1 subunits) intimating that the binding of each KCNE1 subunit to the channel in turn forms a binding site for the drug. A total of four identical mefenamic acid binding or regulatory pockets should be available on the IKs channel complex. Consistent with this idea, when all four WT KCNE1 subunits are replaced with mutant K41C-KCNE1 subunits, mefenamic acid up to a concentration of 1 mM is ineffective (Fig. 4.1A). No current waveform changes, slope changes to the G-V plot or V1/2 shifts were seen, suggesting that all the drug binding site(s) on the channel complex were impaired, or that the mechanism of action was 119 disabled. Further detailed studies of mefenamic acid binding to IKs channel complexes is the subject of a recent study from our laboratory, results of which support the conclusions above (Chan et al., 2023). When one to four K41C-KCNE1 subunits are replaced with WT KCNE1 subunits, the effect of 100 µM mefenamic acid is brought back in a stoichiometrically graded manner. Consistent with EQQ + K41C-KCNE1 (2:2 ratio of mutant to WT KCNE1 subunits) having one more WT binding site than EQQQQ + K41C-KCNE1 (3:1) and K41C-EQQQQ + KCNE1(1:3) having yet another additional WT binding site, the EC50 for EQQQQ + K41C-KCNE1 was found to be the greatest followed by that of EQQ + K41C-KCNE1 then finally K41C-EQQQQ + KCNE1 (EQQQQ + K41C-KCNE1 EC50 = 1203 μM; EQQ + K41C-KCNE1 EC50 = 382 μM; K41C-EQQQQ + KCNE1 EC50 = 30 μM) (Fig. 4.2C). A closer examination of results from channel complexes with different mutant to WT KCNE1 ratios reveals that K41C-EQ (Fig. 4.1A; circles), EQQQQ + K41C-KCNE1 (Fig. 4.1B) and EQQ + K41C-KCNE1 (Fig. 4.1C) all prevented the effects of 100 μM mefenamic acid. Only treatment of K41C-EQQQQ + KCNE1 with 100 μM mefenamic acid allowed instantaneous current onset, shape change to the G-V and a V1/2 shift (Fig. 4.1D, Table 4.1). This G-V plot shape change and V1/2 shift was still less than that seen after treatment of WT EQ with the same concentration of mefenamic acid, suggesting that four mefenamic acid molecules likely bind to and act on the WT EQ channel complex at the same time. The binding of four drug molecules is consistent with the presence of a total of four identical mefenamic acid binding pockets formed between KCNQ1 and KCNE1 (Chan et al., 2023). 120 In the case of K41C-EQQQQ + KCNE1 the Hill coefficient remains unchanged (nH = 0.53) from WT EQ (nH = 0.49, Fig. 4.2C), although one mutant K41C-KCNE1 is present in the channel complex, the three remaining WT binding sites are enough to overcome the effect of the mutation. Interestingly, the Hill coefficient of EQQ + K41C-KCNE1 (nH = 1.9) and EQQQQ + K41C-KCNE1 (nH = 0.95) dose-response curves are significantly different from WT EQ. The K41C mutation may alter residues in the binding pocket, which results in a different nH compared to the WT binding pocket (Cattoni et al., 2015). Since EQQ + K41C-KCNE1 and EQQQQ + K41C-KCNE1 have at least two K41C-KCNE1 subunits, combining the different nH values at each binding site may produce an overall value different from WT EQ. The steepness of the EQQ + K41C-KCNE1 and EQQQQ + K41C-KCNE1 concentration-response curves may also give insight into the mechanism of action of mefenamic acid. At lower concentrations, below 100 μM, mefenamic acid has little effect on these complexes. Since we now understand that the K41C mutation destabilizes drug binding (Chan et al., 2023), I hypothesize that below 100 μM, the unbinding rate may exceed the drug on rate, which is a function of concentration. However, once the mefenamic acid concentration is high enough, the destabilization induced by K41C is overcome, and the response to the drug then increases more steeply as a function of concentration. This mass action effect of the drug may also explain why, when sufficient drug is present, the efficacy of mefenamic acid is the same as WT in EQQ + K41C-KCNE1. Only the limiting solubility of the drug prevented us from testing this hypothesis in EQQQQ + K41C-KCNE1, and indeed in K41C-EQ as well. Simulation of the action of mefenamic acid on individual K41C subunits using the same model configurations that reproduced its characteristic drug action on WT IKs channels (Wang et al., 121 2020b) reproduced the new experimental data well. The model suggested that WT subunits, with accelerated VS activation kinetics in the presence of the drug outcompeted K41C-containing subunits in the activation pathway, so that later activating K41C subunits which determined the activation kinetics of the major channel open states were able to limit opening when one or more of them were present in the channel complex. 4.5 Conclusion The importance of residue K41 to the binding of mefenamic acid is further supported by the dependence of the mefenamic acid effect on the number of available binding sites. Two to four K41C containing KCNE1 subunits largely prevented the actions of mefenamic acid, reducing the maximum response by 50-100%. One K41C-KCNE1 subunit was found to be insufficient to fully prevent the effect of 100 µM mefenamic acid, which when compared to WT EQ in the presence of the drug suggested that four drug molecules can likely bind to the WT IKs channel complex at once. 122 Chapter 5: Discussion 5.1 Summary of findings The binding site for IKs activators, such as mefenamic acid and DIDS, has been the subject of multiple studies, but has never been mapped out in detail (Abitbol et al., 1999; Bollmann et al., 2020; Wang et al., 2020b). Abitbol et al. previously suggested that the binding site for mefenamic acid and DIDS lies between residues D39 and E43 in KCNE1, emphasizing residue 43 as critical for DIDS binding. Later, Wang et al. identified residue K41 on KCNE1 as the most critical for mefenamic acid binding through mutational analysis (Wang et al., 2020b). Most recently, our lab successfully mapped the precise binding sites of both drugs in the KCNQ1/KCNE1 channel complex by integrating results from MD simulations and whole-cell patch clamp experiments. These findings are presented in Chapter 2 and a recent publication (Chan et al., 2023). We show that the binding of mefenamic acid and DIDS is facilitated by several key residues on the extracellular end of KCNE1 and the S1 and S6 helices of KCNQ1. The binding of mefenamic acid and DIDS induces a conformational change that slows channel deactivation kinetics by stabilizing the S1/KCNE1/pore complex, with mefenamic acid acting as a bridge to stabilize interactions at the KCNE1/S1/pore interface in the activated conformation. These findings demonstrate that the extracellular inter-subunit interface can serve as a general binding site for enhancing channel activation and slowing channel deactivation, offering the potential to develop therapeutically useful IKs agonists. In some experiments described in Chapter 2, we observed that mefenamic acid exhibited dual effects on the wild-type KCNQ1/KCNE1 complex, as well as on the E43C-KCNE1 and A300C-KCNQ1 mutants. We explored these observations deeper in Chapter 3 (Chan et al., 2024). 123 Mefenamic acid slows the channel deactivation kinetics of the wild-type KCNQ1/KCNE1 channel complex at low concentrations, but at higher concentrations or in the presence of single mutations in the KCNQ1 or KCNE1 subunits, it inhibits the current amplitude. These dual actions disrupt traditional paradigms that classify ion channel modulators into rigid categories (activator vs. inhibitor). Instead, our findings establish that modulation depends on ligand concentration and structural interactions at the channel's extracellular side. Our findings suggest that the clearly distinguishable drug effects result from the coexistence of two distinct binding sites. At low concentrations, the drug binds to a high-affinity site to potentiate IKs, whereas at high concentrations, it shifts to a lower-affinity site that causes inhibition. In the A300C mutation, the lower-affinity site becomes the primary binding site as it abolishes most of the potentiating effects of mefenamic acid and leaves only inhibition. Mefenamic acid binding likely exerts allosteric effects on the pore backbone conformation or applies steric pressure on the pore domain, decreasing potassium current. As discussed in Chapter 3, these findings could have significant implications for therapeutic drug design in inherited diseases such as LQTS. In Chapter 4, I examined in detail the effects of mefenamic acid on the KCNQ1/KCNE1 complex carrying the KCNE1 K41C mutation, which destabilizes the drug’s binding. We showed that the loss of mefenamic acid’s efficacy occurs in a stoichiometrically graded manner: As the number of functional binding sites decreases due to the mutation, the drug's potency decreases. Inversely, the presence of multiple WT KCNE1 subunits partially compensates for the destabilizing effects of a K41C-mutated KCNE1 subunit, enabling some restoration of drug’s potency. This phenomenon was further explained by modeling data, complemented with experimental results, which demonstrated that WT subunits, with accelerated activation kinetics, 124 outcompete the slower-acting K41C mutant subunits during channel activation. As a result, when K41C subunits are present in the channel complex, they can influence the overall activation kinetics and limit channel opening. By bridging structural insights with functional outcomes, this binding site provides a foundation for developing more effective, and safe therapies, particularly for long QT type 1 patients. 5.2 Comparative analysis of the novel IKs activator binding site with other KCNQ1 channel activators’ binding sites and mechanism of action The four most commonly discussed KCNQ1 activators – (1) Mefenamic Acid, (2) DIDS, (3) PUFAs, and (4) ML277 – potentiate the KCNQ1 channel through distinct mechanisms of action. Despite their shared ability to potentiate channel activity, their binding sites and effects on the channel differ substantially. Mefenamic acid and DIDS share a general occult binding site at the extracellular KCNQ1-S1/KCNE1/pore interface, but with subtle differences (Abitbol et al., 1999; Busch et al., 1994b; Chan et al., 2023; Wang et al., 2020b). For mefenamic acid, K41 in KCNE1 acts as a “lid” for the binding pocket, while W323 in KCNQ1 dictates its size, holding the drug in place. In contrast, DIDS binds slightly deeper within the same pocket, relying on residues like Y148 and Y46, as shown by reduced DIDS effects in Y148C and Y46A mutants (Chan et al., 2023). Both drugs utilize interactions involving KCNE1 extracellular residues, the domain-swapped S1 helices, and the pore/turret regions of two additional KCNQ1 subunits to stabilize the binding pocket. Previous cross-linking studies have shown that the KCNE1 N-terminus, the top of S1, and S6 lie in close proximity 125 (Chung et al., 2009). Drug occupancy in this shared pocket likely helps maintain these segments in an activated conformation, coupling the static pore domain to the dynamic voltage sensor and thus stabilizing the activated complex, slowing deactivation (Chan et al., 2023). This slowed deactivation of the channel allows currents to summate between pulses. As current levels remain sustained even at hyperpolarized potentials, this results in a leftward shift in the channel’s V1/2 and renders activation effectively voltage-independent at physiological voltages. Consequently, the current waveform is altered, and the current persists even at longer pulse-to-pulse intervals (e.g., 15, 20 and 30 seconds) (Chan et al., 2023; Wang et al., 2020b). The mefenamic acid/DIDS mechanism contrasts with that of PUFAs on KCNQ1/KCNE1 channel complex, which bind to two sites to confer their two distinct effects. PUFA binding to arginine residues on the S4 segment keeps the voltage sensor in the activated “up” position, hindering deactivation. Although the resultant hyperpolarizing shift in voltage dependence is not as significant as with mefenamic acid (KCNQ1/KCNE1 Mef: ΔV1/2 = -105.7 mV; PUFAs (DHA-Gly, N-AT) ΔV1/2 = ~-26.0 mV), the qualitative effect is similar (Chan et al., 2023; Liin et al., 2015b; Wang et al., 2020b). The more substantial shift induced by mefenamic acid may result from stabilizing the VSD in a more upward position. Under standard voltage pulse protocols, PUFAs do not significantly modify the current waveform or increase instantaneous current like mefenamic acid. Despite limited data on the time-dependent effects of PUFAs on KCNQ1/KCNE1, they are unlikely to alter the current waveform or potentiate summation as dramatically as mefenamic acid at short inter-pulse intervals, given their smaller shift in the voltage dependence of activation. The KCNQ1 activator ML277 does not bind to the VSD but instead interacts with the S4-S5 linker to enhance VSD-pore coupling (Hou et al., 2019). As a 126 result, ML277 does not alter voltage sensor activation and causes only a minimal shift in the voltage dependence of activation compared to mefenamic acid or PUFAs (Hou et al., 2019; Ma et al., 2022). In some cases, ML277 has been shown to increase the occupancy of KCNQ1 in the AO state, slightly depolarizing the G-V. Figure 5.1 Binding sites of PUFA and ML277 on the KCNQ1-KCNE1 molecular complex. (A) Top view of KCNQ1 with PUFA binding sites: Site I, at the voltage sensor domain (VSD); and site II, at the pore domain. Extracted from Golluscio et al., 2024. (B) Top view of xKCNQ1-CaM-ML277 from the extracellular side is shown with ML277 (orange) bound in its hydrophobic pocket. Extracted from Willegems et al., 2022. In addition, PUFAs differ from mefenamic acid and DIDS in their ability to increase both the open probability (Po) and the maximum channel conductance, leading to a significant rise in current amplitude. PUFAs act on the selectivity filter to increase the current amplitude. They bind to a crevice between residues K326 and D301 in the selectivity filter, helping it remain in the conductive conformation (Golluscio et al., 2024; Liin et al., 2015b). In contrast, mefenamic acid and DIDS’ stabilization of the activated complex only increases the channel’s Po by enhancing activation gating, which likely contributes to the slowed deactivation and persistent currents between pulses (Wang et al., 2020b). They do not, however, increase the channel 127 conductance and, therefore, do not increase the current amplitude. ML277 enhances current primarily by removing inactivation via enhancing VSD-pore coupling (Eldstrom et al., 2021). Single-channel studies suggest that, in the presence of ML277, KCNQ1 behaves similarly to KCNQ1 channels locked in the IO state – it’s primary state for current conduction – while less frequently transitioning to the lower-conducting AO state (Eldstrom et al., 2021). Since mefenamic acid and DIDS are only effective in the KCNQ1/KCNE1 complex and KCNE1 itself reduces inactivation, this suggests that mefenamic acid does not exhibit the inactivation-removing effect seen with ML277. In the human heart, KCNQ1, together with KCNE1, forms the channel complex underlying the IKs current. As a result, the effects or modulation of drugs on the KCNQ1/KCNE1 complex are of critical importance. The effects of these activators differ in their reliance on KCNE1. Mefenamic acid and DIDS require KCNE1 for their effect, as KCNE1 constitutes part of the binding site. Supporting this, Wang et al., 2020 demonstrated that a reduction in the number of KCNE1 subunits linked to KCNQ1 correspondingly diminishes the potentiation effect of these activators (Wang et al., 2020b). In contrast, ML277’s drug-potentiating effect increases as the number of KCNE1 subunits decreases, likely due to the location in which ML277 binds to KNCQ1. ML277's effect is most pronounced when KCNQ1 is expressed alone, and its efficacy diminishes with increased KCNE1 subunits, as KCNE1 conformational changes hinder its binding, decreasing the number of available binding sites (Hou et al., 2019; Xu et al., 2015; Yu et al., 2013). In the latest resolved cryo-EM structures, although only the structure of KCNQ1-KCNE3 – a paralog of KCNE1 – has been determined, KCNE3 does not occupy the binding site of ML277 (Ma et al., 2022; Sun & MacKinnon, 2020; Willegems et al., 2022). This suggests that 128 KCNE1 is also unlikely to occupy this site. Based on the cryo-EM structures of KCNQ1-CaM-KCNE3 and KCNQ1-CaM, KCNE3 induces a ~2-3Å shift in both the S5 and S6 helices, narrowing the binding pocket (Sun & MacKinnon, 2020). The structural alignment of KCNQ1-CaM-KCNE3 and human KCNQ1-CaMML277 reveals that the side chain of F335 on S6 sterically clashes with ML277, which explains the reduced efficacy of ML277 (Ma et al., 2022). Structural studies of ML277 binding to Xenopus KCNQ1 indicate that ML277 induces a displacement of the L256 side chain (Willegems et al., 2022). In the presence of KCNE3, this displacement is sterically hindered due to clashes with T71 on KCNE3 and F270 in KCNQ1, interfering with ML277’s binding and efficacy. PUFAs exhibit partial KCNE1 dependence, with certain analogs e.g., DHA being less effective in the presence of KCNE1 (Liin et al., 2015b). The potentiation ability of PUFAs rests on their negatively charged head group interacting with arginine residues. However, KCNE1 reduces the local pH near the PUFA head group, causing the PUFA to become protonated. This protonation renders PUFAs ineffective on KCNQ1/KCNE1 complexes. Other PUFA analogs, such as N-AT (N-arachidonoyl taurine), DHA-Gly and linoleic glycine (Lin-Gly) have a lower pKa and can thus preserve their negatively charged head groups to modulate the KCNQ1/KCNE1 complex (Golluscio et al., 2024; Liin et al., 2015b; Liin et al., 2018). Moreover, the number of KCNE1s in the KCNQ1/KCNE1 complex has no bearing on the type of effect of the magnitude of PUFA on the complex. 5.3 Comparative analysis of the novel IKs activator binding site with other potassium channel activators binding sites Our results provide the first detailed mapping of the binding site for IKs activators on the IKs complex. While this represents a novel discovery, the binding sites of other activators, such as 129 retigabine for the voltage-gated potassium channel Kv7.2, have already been resolved, allowing for insightful comparisons (Li et al., 2021; Wuttke et al., 2005). Despite their shared ability to shift the voltage dependence of activation toward more negative potentials, stabilizing the open state of the channel and enhancing K+ current, distinct differences emerge in their mechanisms. Unlike other K+ channel activators, the binding mechanism of mefenamic acid to the KCNQ1/KCNE1 complex is less common as it requires the presence of the beta subunit KCNE1; without it, the drug cannot bind effectively. In contrast, retigabine, a KCNQ2 activator, does not require a beta subunit. Instead, retigabine binds to a pre-existing intracellular hydrophobic pocket in the pore domain between the cytoplasmic S5 and S6 segments in Kv7.2, involving the critical tryptophan residue 236 near the channel gate that forms a direct hydrogen bond with retigabine (Li et al., 2021; Wuttke et al., 2005). Its effects, however, are elicited via allosteric modulation - binding to the pore domain but acting on the voltage sensor domain (Li et al., 2021). The binding site is rigid, specific and requires no structural adaption. This mechanism contrasts with mefenamic acid’s binding on IKs, which occurs through an induced-fit mechanism at a binding site that is not detectable when the drug is not present. However, this induced-fit binding pocket offers greater flexibility, allowing accommodation of other structurally distinct IKs activators such as DIDS. Moreover, this binding pocket enables mefenamic acid to exert its effects more directly. Instead of crossing the lipid bilayer and binding intracellularly, it binds to an extracellular interface, stabilizing the complex in its activated state (Abitbol et al., 1999; Chan et al., 2023; Wang et al., 2020b). Additionally, retigabine’s mechanism of action is more typical of those found in other channels, such as Bay K8644 for Cav1.1, which binds to the pore domain, and HC-070 for the transient 130 receptor potential channel 5 (TRPC5), which binds to a hydrophobic pocket formed by the S5, pore helix of one subunit, and S6 of an adjacent subunit (Sanguinetti et al., 1986; Song et al., 2021; Zhao et al., 2019). 5.4 Comparative analysis of the IKs activators and other potassium channel modulators In our patch clamp experiments, 100 µM of mefenamic acid was observed to potentiate IKs. However, inhibitory effects emerge at higher concentrations. While the discovery of such a dual mechanism for mefenamic acid is noteworthy, it is not unprecedented in potassium channel pharmacology. A similar dual effect has been reported with almokalant, a class III antiarrhythmic drug that primarily targets the delayed rectifier K+ current, IK (Carmeliet, 1993). Almokalant hyperpolarizes the voltage-dependence of activation to enhance IK but simultaneously blocks open IK channels in a use-dependent manner. It was suggested that the drug binds to two different binding sites or that simultaneous binding to IKr and IKs complexes may produce opposing effects. This dual mechanism can pose a risk to patients undergoing physical exercise or experiencing emotional stress. At elevated heart rates, the duration of channel opening increases, and the cumulative inhibition of IKr + IKs may become excessive and lead to early afterdepolarizations and arrhythmias. Cromakalim and 2H-1,4-benzoxazine derivatives interact with KATP channels in an intracellular ATP concentration-dependent manner (Tricarico et al., 2003). In the presence of ATP, these compounds act as activators, increasing KATP currents, while in its absence, they function as inhibitors, reducing KATP currents. This dual effect suggests the compounds may interact with two receptor sites on the channel, mediating opposing actions. Nonetheless, a single binding site 131 explanation is plausible, with intracellular ATP concentration modulating the drug’s interaction with the channel (Tricarico et al., 2003). By contrast, other drugs with dual effects like the KATP channel opener ZD6169 is suggested to interact with two distinct target proteins rather than binding to two different sites on the KATP channel itself (Teramoto et al., 2001). ZD6169’s effects are concentration-dependent, and its dual effects are complementary rather than opposing. At low concentrations, the drug activates KATP channels for smooth muscle relaxation. At higher concentrations, it inhibits L-type calcium channels, further reducing smooth muscle contraction. Mefenamic acid’s dual effects are unique because they simultaneously oppose each other. I hypothesize that this may be due to the suggested binding site for inhibition being located in close proximity to the pore, making it very susceptible to changes in the immediate vicinity, and that activation or block rests on one residue. In the case of A300C on KCNQ1, the single mutation almost completely abolished mefenamic acid’s potentiation effect and allows it to block the current. 5.5 Graded activation of potassium channels by activators In Chapter 2, we investigated the impact of disabling binding sites through mutations in a stoichiometric manner. In Chapter 4, we discovered that reducing the number of functional binding sites by introducing mutations, i.e., K41C, resulted in a progressive, graded loss in the drug’s effects. This stoichiometric-dependent, progressive loss in effect is very similar to what has been suggested for the KCNQ1 activator ML277. Intriguingly, as the stoichiometric number 132 of KCNE1 subunits increases, the efficacy of ML277 decreases. This phenomenon has been attributed to ML277 binding at the interface where KCNE1 interacts with KCNQ1. As the number of KCNE1 subunits increase, competition at these binding interfaces may be reducing ML 277’s effects (Chan et al., 2012; Hou et al., 2019; Nakajo et al., 2010; Peng et al., 2017; Ramasubramanian & Rudy, 2018; Strutz-Seebohm et al., 2011; Xu & Rudy, 2018; Yu et al., 2013). By contrast, the stoichiometric and graded mechanism of mefenamic acid and ML277 differ from sulfonylurea’s action on another potassium channel complex, KATP. KATP channels comprise the inward rectifying K+ channel Kir6.x and sulfonylurea receptor (SUR) subunits in a 4:4 stoichiometric ratio (Drain, 2013; Proks et al., 2002). Sulfonylureas, used in the treatment of type 2 diabetes, act by binding to SUR1 subunits, inducing channel closure, which depolarizes the cell membrane and stimulates insulin secretion to lower blood sugar levels. Two theories have emerged: An “all-or-nothing” model, where sulfonylurea must bind to all four subunits to induce channel closure, or a model where binding to just one subunit is sufficient to close the channel. Currently, no evidence supports a stoichiometric, graded response in between these two models (Dörschner et al., 1999; Russ et al., 1999). 5.6 Therapeutic relevance of the novel IKs activator binding site Mefenamic acid’s enhancement of the KCNQ1/KCNE1 channel complex in vitro is well-documented (Abitbol et al., 1999; Chan et al., 2024; Chan et al., 2023; Jing et al., 2014; Wang et al., 2020b). However, the binding site of IKs activators like mefenamic acid and DIDS on the KCNQ1/KCNE1 complex was only recently identified (Chan et al., 2023). This discovery holds 133 significant implications for developing IKs-targeted drugs. The identified binding site accommodates diverse IKs activators through an induced-fit mechanism. Upon binding, the activator locks the channel complex in its activated state, slowing deactivation and hyperpolarizing the voltage dependence of activation in a rate-dependent fashion. The extent of hyperpolarization increases with decreasing interpulse intervals; a 7-second interpulse interval results in the greatest hyperpolarization, while a 30-second interval results in the least. In other words, shorter interpulse intervals correspond to more pronounced hyperpolarization of voltage dependence of activation (Wang et al., 2020b). Likely resulting from incomplete deactivation between pulses. In vivo, this mechanism will likely promote current summation at high heart rates as channels stay open longer, enhancing IKs' role as a repolarization reserve in the heart. This recent discovery will propel drug developments, opening new avenues for treatments targeting arrhythmogenic disorders such as LQTS. Earlier studies demonstrated the therapeutic potential of IKs activators (Abitbol et al., 1999; Wang et al., 2020b). Identifying a shared structural determinant for fenamates (e.g., mefenamic acid) and stilbenes (e.g., DIDS) enables the rational design of activators targeting this site. The IKs activator, PUFAs, are non-selective and bind to multiple ion channels (Bai et al., 2015; Elinder & Liin, 2017; Long et al., 2005; Long et al., 2007; Xiao et al., 2001) (Farag et al., 2016). As a result, their effects are less specific in correcting potassium current deficiencies and have a higher likelihood of off-target interactions. In contrast, ML277 binds only to the central core binding pocket of KCNQ1 and is only accessible when KCNQ1 is either alone or in unsaturated KCNQ1:KCNE1 complexes. Functionally, ML277 has been shown to decrease the APD in 134 hiPSC-CMs; it also enhances contractile recovery, and improves cell survival, offering protection against ischemia in both cell and whole-heart models (Brennan et al., 2023; Yu et al., 2013). These findings validate its therapeutic potential in vivo. The therapeutic potential of mefenamic acid, on the other hand, remains unproven, as it has not been tested in vivo. However, its key advantage is its ability to accommodate KCNQ1:KCNE1 complexes in any ratio, which aligns with the physiological composition of IKs channels in the human body (Wang et al., 2020b). This binding site offers an alternative approach to existing LQTS treatments - by directly targeting IKs. In cases of LQTS1 caused by IKs loss-of-function mutations, potentiating the IKs current at this site can restore repolarization currents and mitigate arrhythmic risk with minimal impact on other cardiac currents. This targeted approach reduces off-target effects compared to treatments that address insufficient IKs by slowing the heart rate. Using MD simulations in our studies, we gained a deeper understanding of binding dynamics, specifically how certain residues in the binding pocket stabilize mefenamic acid and the extent of their contributions to drug effect. MD simulations identified key residues critical for mefenamic acid binding, including K41, A44, and Y46 in KCNE1, as well as W323 and Y148 in KCNQ1 (Chan et al., 2023). These computational predictions were then validated through mutagenesis and electrophysiological studies, confirming their role in ligand coordination and IKs activation. This approach can guide the design of IKs activators by optimizing binding affinity and specificity for this novel binding site. With the binding pocket identified and its functional relevance confirmed, this model now supports targeted drug development for IKs modulation. 135 This novel binding site adds to the growing list of identified IKs activators binding sites. Each site has its own significance, offering additional targets for drug development and expanding therapeutic options. 5.7 Clinical relevance of the new findings of the novel IKs activator binding site Despite the potentiation effect of mefenamic acid observed in experimental studies, it has not been reported to be proarrhythmic in clinical settings. As an NSAID, it is currently prescribed for the short-term relief of mild to moderate pain associated with conditions such as headaches, menstrual cramps and post-operative pain (Haward, 1966; Hirata et al., 2007; Potter et al., 2021; Vaidya et al., 1974). At its current therapeutic dosage as an NSAID, the peak plasma concentration of mefenamic acid is 82.9 µM, which is significantly lower than the concentration required to block wild-type KCNQ1/KCNE1 channels (Cryer & Feldman, 1998). However, in laboratory-generated mutations such as E43C in KCNE1 and A300C in KCNQ1, the IC50 drops to ~30 µM from >300 µM (exact IC50 not determined for wild-type EQ) – well within the range of clinically relevant concentrations. While the A300C mutation is not currently associated with disease, sequence analysis has linked the A300T mutation in KCNQ1 to Romano-Ward syndrome. Disease-related mutations could affect drug potency and efficacy, potentially exacerbating the blocking effects of mefenamic acid and leading to adverse outcomes for patients if the drug’s blocking effect predominates. Adding to the complexity, patients with chronic heart failures exhibit elevated KCNE1 levels, which may enhance the effects of IKs activators like mefenamic acid more than anticipated if their condition warrants such treatment (Watanabe et al., 2007). This excess of KCNE1 can lead 136 to saturation of the IKs complex, thereby amplifying the efficacy of mefenamic acid which is most potent when the complex is fully saturated (Lundquist et al., 2005; Wang et al., 2020b; Watanabe et al., 2007). Furthermore, the variable expression of accessory subunits like KCNE1 across different tissues adds a layer of uncertainty regarding the magnitude of the drug’s effect (Lundquist et al., 2005). These findings emphasize the importance of genetic screening and mutation-specific drug development to mitigate the risks of unintended pharmacological effects. Moreover, these findings reinforce the need for rigorous drug concentration testing and in-vitro drug testing to ensure safety and efficacy across diverse patient populations. Personalized medicine, an emerging practice that tailors treatments based on an individual’s genetic profile, could be particularly effective in developing drug regimens like mefenamic acid. 5.8 Limitations One reason mefenamic acid's potentiating effects on IKs in clinical settings remain unconfirmed is the various stoichiometry and nature of accessory subunits associating with KCNQ1 (KCNE1-5) in the heart. While all KCNE subunits are present in cardiac tissue, their expression at the protein level remains unclear due to challenges in quantifying the precise amounts of these subunits (Bendahhou et al., 2005; Franco et al., 2001; Lundquist et al., 2005; Radicke et al., 2006; Soma et al., 2011). However, at the mRNA level, KCNE1 is known to have the highest expression levels, followed by KCNE4, KCNE5~KCNE3, and KCNE2, in descending order (Bendahhou et al., 2005; Lundquist et al., 2005). Although mRNA levels provide a proxy for protein expression, the correlation between mRNA and protein levels is uncertain (Bendahhou et al., 2005; Franco et al., 2001; Lundquist et al., 2005). The stoichiometric ratio significantly impacts the efficacy of mefenamic acid, as the number of available binding sites on the 137 KCNQ1/KCNE1 interface depends on this ratio (Chan et al., 2023). For example, its effect is halved in a 2:4 KCNQ1:KCNE1 ratio compared to a 4:4 ratio (Wang et al., 2020b), complicating dosage optimization and limiting its practical application due to the lack of stoichiometric clarity. Additionally, KCNQ1 interacts with other accessory subunits such as KCNE2-5 (Bendahhou et al., 2005; Lundquist et al., 2005; Radicke et al., 2006; Soma et al., 2011). KCNE2, for instance, negatively modulates IKs current amplitude, which may counteract the potentiating effects of mefenamic acid. This was demonstrated by Toyoda et al., where the efficacy of mefenamic acid was reduced in KCNQ1/KCNE1/KCNE2 complexes (Toyoda et al., 2006). Furthermore, these studies were conducted in human-transformed kidney cells transfected with KCNQ1/KCNE1 DNA under controlled laboratory settings. Despite their mammalian origins, they are not replicas of human cardiac cells. These cells are engineered to express the desired ion channels for research purposes but lack all the components necessary to replicate the regulatory mechanisms crucial for ion channel function and regulation in cardiomyocytes. In addition, other KCNQ1 accessory subunits (e.g., KCNE2) are not transfected in these kidney cells during experiments, with implications detailed below. Thus, further investigation is required to understand the effects of mefenamic acid on these KCNQ1 complexes in more human heart-relevant models. For a few of the mutations used in Chan et al., 2024 (e.g., E43C on KCNE1 or A300C on KCNQ1), we attempted to saturate KCNQ1 with KCNE1 by co-transfecting KCNE1 DNA at a 3:1 ratio. Despite these efforts, other factors, such as expression levels and subunit interactions, 138 can influence the final stoichiometric ratio, which may vary between cells, leading to inconsistent results. Moreover, the resultant ratio may not accurately reflect the true stoichiometry in cardiac tissue and the implications are discussed below. To fully understand how mefenamic acid affects these mutated channels, further experiments in native environments are warranted. In Chan et al., 2023, I observed that high concentrations of mefenamic acid inhibited current amplitude. This finding led me to propose a blocking mechanism in Chan et al., 2024, which is compelling and well-supported by molecular dynamics and electrophysiological data. However, more detailed structural studies are necessary to further elucidate and validate this mechanism. 5.9 Future directions In vitro studies suggest that mefenamic acid has therapeutic potential for treating patients with LQTS. However, its current clinical use remains limited to its role as an NSAID. Further laboratory experiments and subsequent clinical trials are necessary to evaluate the viability of mefenamic acid as a therapy for LQTS. It is important to note that many drugs are known to inhibit IKr (Kannankeril et al., 2010), which could result in more harm than benefit for patients with LQTS and can lead to drug-induced arrhythmias. Additionally, positive experimental results may not always translate into the desired clinical outcomes and, therefore, will require more rigorous validation. Before conducting further experiments in more physiologically relevant environments, running voltage-clamp protocols that mimic cardiac action potentials at various frequencies (1 Hz – 4 Hz) 139 on cells expressing IKs channels would be useful to investigate how the drug affects the channels’ summation properties. The key focus is determining whether mefenamic acid can restore the loss of summation in IKs caused by loss-of-function mutations associated with LQTS. If mefenamic acid can reverse the attenuated summation and re-establish the hallmark frequency-dependent behavior of IKs, it could offer significant therapeutic benefits to patients with LQT1. This is particularly relevant as arrhythmias are more likely to be triggered at high heart rates, i.e., during exercise or high emotional stress. The next logical step to build on these promising findings would be to conduct experiments with mefenamic acid in more physiologically relevant settings, such as hi-PSC-CMs which mimic the native physiological environment, allowing for studying how other regulatory mechanisms respond to the potentiation of IKs. The synergistic application of electrophysiology and structural biology has proven to be a powerful approach, yielding comprehensive insights that a single method could not achieve. By combining these techniques, I effectively linked structural data with functional outcomes. Looking ahead, advancements in cryogenic electron microscopy (cryo-EM) present opportunities for more sophisticated studies of drug-target interactions, potentially accelerating the translation of these findings into therapies at the bedside. The ideal IKs channel activator would be highly selective for the IKs channel complex while maintaining efficacy across various genetic mutations affecting the channel. The activator should function in a concentration-dependent manner, allowing fine-tuning of its effects while 140 maintaining non-opposing actions at all clinical concentrations. Additionally, the drug should preserve the channel’s voltage-dependent activation, ensuring it is not constitutively open across all physiologically relevant voltages and disrupts cardiac electrophysiology. Furthermore, the activator should maintain the IKs frequency-dependent properties if lost, enabling larger IKs currents at higher stimulation frequencies to protect against arrhythmias during exercise and stress. From a toxicological perspective, the activator should have a wide therapeutic window and minimal off-target effects, i.e., IKr or INa. 5.10 Conclusions The results and discussion points all suggest the need to search for an ideal channel activator, and I certainly made some progress toward that goal in this thesis. I have understood better the binding site for classic IKs activators, including its location, binding mechanism, and concentration-dependent effects. Furthermore, identifying the binding site has revealed the drug's graded, stoichiometric effects, which we successfully validated. The newly characterized mefenamic acid binding site presents promising therapeutic potential, warranting further exploration and development to optimize drug specificity. The search for the ideal IKs channel activator continues. 141 Bibliography Abbott, G. W. (2014). Biology of the KCNQ1 Potassium Channel. New Journal of Science, 1-26. Abbott, G. W., Sesti, F., Splawski, I., Buck, M. 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Free energy for residues located in ps-KCNE1, pore domain and voltage-sensor domain of KCNQ1, that stand above background, are shown as enlarged panels. Error bars represent ± SD. 1000 frames for each simulation (n=3) were analyzed. 163 Figure A.3: Clustering of MD trajectories based on mefenamic acid conformations. (A) The clustering dendrogram based on Mef RMSD illustrates the similarity of Mef conformations in MD trajectories. (B) Centroids from each cluster with the lowest RMSD compared to all other conformations in the cluster. The color frame around the panels shows to which cluster the depicted conformation belongs. 164 Figure A.4: H-bonds formed between mefenamic acid and protein residues in its binding site during MD simulations. MEF-N1 indicates the nitrogen of aminobenzoic acid; MEF:O1 and O2 are the oxygens of the aminobenzoic acid. Red lines indicate at which time point and between which atoms H-bond were formed. 165 Figure A.5: Current waveform and G-V changes induced by mefenamic acid for all binding site mutants. (A)-(H) current traces in control (above) and presence of 100 μM Mef (below) and G-V plots for binding site mutants, as indicated. Cells were held at –80 mV, then pulsed from –90 mV to +100 mV for 4 s followed by –40 mV for 2 s. Error bars shown are ± SEM. Refer to Table 2.1 for Boltzmann fit values. 166 Figure A.6: Augmented activation of EQ-L142C in the absence of mefenamic acid. (A) EQ-L142C current traces in control solutions. Voltage steps were from –130 mV or higher in 10 mV steps to +100 mV for 4 s, followed by a repolarization step to –40 mV for 1 s. Holding potential was –80 mV. Interpulse interval was as indicated above, 15 s, 30 s and 7 s. (B) G-V plot of WT EQ in control (interpulse interval 15 s: black) and in the presence of 100 µM Mef (interpulse interval 15 s: grey closed circle; 30 s: grey open circle) and EQ-L142C in control solutions (interpulse interval 7 s: red; 15 s: blue; 30 s: green). Boltzmann fits were: WT EQ 15 s control (n=6): V1/2 = 25.4 mV, k=19.4 mV; WT EQ 15 s Mef (n=3): V1/2 = -80.3 mV, k=41.3 mV; WT EQ 30 s Mef (n=8); V1/2 = 26.7 mV, k=66.6 mV; EQ-L142C 15 s control (n=6) V1/2 = -80.3 mV, k=30.0 mV; EQ-L142C 30 s control (n=3) V1/2 = -28.7 mV, k=62.5. Due to the dramatic change in G-V plot shape, Boltzmann fits were not possible for EQ-L142C with an interpulse interval of 7 s. All mutations are forced saturated IKs channel complexes. Error bars shown are ± SEM. 167 Figure A.7: Root-mean-square deviations (RMSD) of mefenamic acid from its initial position during MD simulations. (A) ps-IKs, (B) K41C, (C) W323A, and (D) Y46C-IKs mutants in the presence of mefenamic acid. Each color represents an individual simulation run, five in each case. Note that time scale in (A) is 2.5 x extended compared with (B–D). 168 Figure A.8: Result of loss of mefenamic acid from the binding site. The binding pocket viewed from above (left) and the side (right) during a 1250 ns MD simulation before drug detachment (grey superimposed structure) and after (red superimposed structure). Mefenamic acid is represented as licorice and colored green. Snapshots for superimposition were collected every 10 ns. Visible from the snapshots is that when the drug leaves the binding site (after 500 ns, structures colored red), the N-terminal residues of ps-KCNE1, as well as W323 and other residues that form the pocket, shift toward the space that mefenamic acid previously occupied. 169 Figure A.9: H-bonds formed between DIDS and protein residues in its binding site during MD simulations. DDS:O1-O6 are the oxygens of the sulfonic acids; DDS:N1 and N2 are nitrogens of the isothiocyanates. Red lines indicate at which time point and between which atom H-bonds were formed. 170 Figure A.10: Clustering of MD trajectories based on DIDS conformations. (A) The clustering dendrogram based on RMSD illustrates the similarity of DIDS conformations in MD trajectories. (B) Centroids from each cluster with the lowest RMSD compared to all other conformations in the cluster were selected and overlayed for comparison. The color of the frame around the panel shows to which cluster the depicted conformation belongs. 171 Figure A.11: Root-mean-square deviations (RMSD) of DIDS from its initial position during MD simulations. (A) ps-IKs, and mutations, (B) K41C, (C) W323A, and (D) Y46C complexes. Individual runs are shown in separate colors, n=5 for each. 172 Figure A.12: Current waveform and G-V changes induced by DIDS for all binding site mutants. (A-E) current traces in control (top) and presence of 100 μM DIDS (bottom). For binding site mutants, cells were held at –80 mV, then pulsed to between –90 mV and +100 mV for 4 s followed by –40 mV for 2 s. G-V plots in control (black) and presence of 100 μM Mef (colors). Error bars are ± SEM. Refer to Table 2.5 for Boltzmann fit values. 173 Appendix B : Supplementary Figures for Chapter 3 Figure B.1: Current waveform changes of WT-EQ induced by mefenamic acid with a 10 s pulse. (A) Current waveform of WT-EQ, control (black) and 100 μM Mef (grey). Cells were depolarized from −80 mV to +60 mV for 10 s and stepped back to −40 mV for 2 s. Interpulse interval was 15 s. The dotted line denotes zero current. (B) Fraction of remaining tail current after application of 100 μM Mef for WT-EQ with a 4 s pulse (n = 6) compared to a 10 s pulse (n = 3). (C) Current amplitude of WT-EQ measured at the end of a 10 s pulse, normalized to the current amplitude measured at the beginning of the pulse in the presence of 100 μM Mef. 174 Figure B.2: Time-dependent effects of mefenamic acid on KCNQ1 and KCNE1 mutants. (A) Current waveform of E43C-E1Q1, control (black), after 270 s (green) and 570 s (violet) of exposure to 100 μM Mef. (B) Current waveform of E1Q1-A300C, control (black), after 45 s (green) and 150 s (maroon) of exposure to 100 μM Mef. (C) Current waveform of Y46A-EQ, control (black), after 15 s (green) and 75 s (beige) of exposure to 100 μM Mef. (D) Current waveform of EQ-W323A, control (black), after 30 s (green) and 270 s (pink) of exposure to 100 μM Mef. Cells were depolarized from −80 mV to +60 mV for 4 s and stepped back to −40 mV for 0.8 s (Y46A-EQ and EQ-W323A) and 2 s (E43C-E1Q1, E1Q1-A300C). Interpulse interval was 15 s. The dotted line denotes zero current. (E) Fraction of remaining tail current after application of 100 μM Mef for Y46A-EQ (n = 5) and EQ-W323A (n = 4). *p < 0.05 vs. control. 175 Figure B.3: G-V and deactivation kinetic changes induced by HMR1556 in E43C-E1Q1. (A) Current waveform of E43C-E1Q1, control (black) after addition of 30 nM HMR1556 (brown). Cells were depolarized from −80 mV to +60 mV for 4 s and stepped back to −40 mV for 2 s. Interpulse interval was 15 s. The dotted line denotes zero current. (B) G-V plots in control (black) and after the application of 30 nM HMR1556 (brown). E43C-E1Q1 HMR1556 (n = 3): V1/2 = 4.2 mV, k = 22.9 mV. Mean V1/2 of activation and slope values for control are reported in Table 3.1. (C) Deactivation time constants versus different membrane potentials for E43C-E1Q1 in the absence (black circles, n = 4) and in the presence of 30 nM HMR1556 (brown circles, n = 3). Figure B.4: Clustering of MD trajectories based on mefenamic acid conformations within ps-IKs W323A model. The clustering dendrogram based on mefenamic acid RMSD illustrates the similarity of Mef conformations in MD trajectories. 176 Figure B.5: Clustering of MD trajectories based on mefenamic acid conformations within the ps-Y46C-IKs model. The clustering dendrogram based on mefenamic acid RMSD illustrates the similarity of Mef conformations in MD trajectories.
Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Fedida, David
Multiple effects of KCNQ1 activators on the delayed cardiac rectifier potassium channel molecular complex
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