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Bauer CK, Bilet A, Harms FL, Bähring R. KCNH3 Loss-of-Function Variant Associated with Epilepsy and Neurodevelopmental Delay Enhances Kv12.2 Channel Inactivation. Int J Mol Sci 2025; 26:4631. [PMID: 40429775 PMCID: PMC12111102 DOI: 10.3390/ijms26104631] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2025] [Revised: 05/06/2025] [Accepted: 05/11/2025] [Indexed: 05/29/2025] Open
Abstract
A de novo missense variant in KCNH3 has been identified in a patient with neurological symptoms including seizures. Here, we confirm the previously reported loss-of-function features for the associated Kv12.2 mutant A371V and investigate the underlying mechanism. Loss of function was not rescued by low temperature during channel biogenesis. Elevated external K+ reduced the rectification of Kv12.2 conductance as predicted by the GHK current equation, allowing the detection of currents mediated by homomeric A371V Kv12.2 channels and a detailed biophysical analysis of the mutant. Compared to wild-type, the voltage dependences of activation and deactivation of A371V Kv12.2 channels were shifted in the positive direction by 15 to 20 mV. Moreover, A371V Kv12.2 channels exhibited accelerated inactivation kinetics combined with a dramatic negative shift in the voltage dependence of inactivation by more than 100 mV. Even in heteromeric wild-type + A371V Kv12.2 channels, inactivation was enhanced, leading to a significant current reduction at physiological potentials. Our Kv12.2 data show similarities to Kv11 channels regarding C-type inactivation and differences regarding the sensitivity to external K+ and pharmacological inhibition of inactivation. The gating modification caused by the A371V amino acid substitution in Kv12.2 renders loss of function voltage-dependent, with a possible impact on neuronal excitability and firing behavior.
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Affiliation(s)
- Christiane K. Bauer
- Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Arne Bilet
- Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Frederike L. Harms
- Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
| | - Robert Bähring
- Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
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2
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Codding SJ, Trudeau MC. Photoinhibition of the hERG potassium channel PAS domain by ultraviolet light speeds channel closing. Biophys J 2024; 123:2392-2405. [PMID: 38796698 PMCID: PMC11365103 DOI: 10.1016/j.bpj.2024.05.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Revised: 04/30/2024] [Accepted: 05/22/2024] [Indexed: 05/28/2024] Open
Abstract
hERG potassium channels are critical for cardiac excitability. hERG channels have a Per-Arnt-Sim (PAS) domain at their N-terminus, and here, we examined the mechanism for PAS domain regulation of channel opening and closing (gating). We used TAG codon suppression to incorporate the noncanonical amino acid 4-benzoyl-L-phenylalanine (BZF), which is capable of forming covalent cross-links after photoactivation by ultraviolet (UV) light, at three locations (G47, F48, and E50) in the PAS domain. We found that hERG-G47BZF channels had faster closing (deactivation) when irradiated in the open state (at 0 mV) but showed no measurable changes when irradiated in the closed state (at -100 mV). hERG-F48BZF channels had slower activation, faster deactivation, and a marked rightward shift in the voltage dependence of activation when irradiated in the open (at 0 mV) or closed (at -100 mV) state. hERG-E50BZF channels had no measurable changes when irradiated in the open state (at 0 mV) but had slower activation, faster deactivation, and a rightward shift in the voltage dependence of activation when irradiated in the closed state (at -100mV), indicating that hERG-E50BZF had a state-dependent difference in UV photoactivation, which we interpret to mean that PAS underwent molecular motions between the open and closed states. Moreover, we propose that UV-dependent biophysical changes in hERG-G47BZF, F48BZF, and E50BZF were the direct result of photochemical cross-linking that reduced dynamic motions in the PAS domain and broadly stabilized the closed state relative to the open state of the channel.
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Affiliation(s)
- Sara J Codding
- Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
| | - Matthew C Trudeau
- Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
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3
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Trudeau MC. A hydrophobic nexus at the heart of hERG K channel gating. Biophys J 2024; 123:1907-1909. [PMID: 38475996 PMCID: PMC11309969 DOI: 10.1016/j.bpj.2024.03.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2024] [Revised: 03/08/2024] [Accepted: 03/08/2024] [Indexed: 03/14/2024] Open
Affiliation(s)
- Matthew C Trudeau
- Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland.
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4
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Stevens-Sostre WA, Flores-Aldama L, Bustos D, Li J, Morais-Cabral JH, Delemotte L, Robertson GA. An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation. Biophys J 2024; 123:2024-2037. [PMID: 38219015 PMCID: PMC11309987 DOI: 10.1016/j.bpj.2024.01.010] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2023] [Revised: 11/17/2023] [Accepted: 01/09/2024] [Indexed: 01/15/2024] Open
Abstract
Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.
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Affiliation(s)
- Whitney A Stevens-Sostre
- Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
| | - Lisandra Flores-Aldama
- Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
| | - Daniel Bustos
- Centro de Investigación de Estudios Avanzados Del Maule (CIEAM), Vicerrectoría de Investigación y Postgrado, Universidad Católica Del Maule, Talca, Chile; Laboratorio de Bioinformática y Química Computacional (LBQC), Departamento de Medicina Traslacional, Facultad de Medicina, Universidad Católica Del Maule, Talca, Chile
| | - Jin Li
- Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
| | - João H Morais-Cabral
- Instituto de Investigação e Inovação Em Saude da Universidade Do Porto (i3S); Instituto de Biologia Molecular e Celular, Universidade Do Porto, Porto, Portugal
| | - Lucie Delemotte
- KTH Royal Institute of Technology, Science for Life Laboratory, Stockholm, Sweden
| | - Gail A Robertson
- Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin.
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5
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Owusu-Mensah A, Treat J, Bernardi J, Pfeiffer R, Goodrow R, Tsevi B, Lam V, Audette M, Cordeiro JM, Deo M. Identification and characterization of two novel KCNH2 mutations contributing to long QT syndrome. PLoS One 2024; 19:e0287206. [PMID: 38181028 PMCID: PMC10769013 DOI: 10.1371/journal.pone.0287206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Accepted: 05/31/2023] [Indexed: 01/07/2024] Open
Abstract
We identified two different inherited mutations in KCNH2 gene, or human ether-a-go-go related gene (hERG), which are linked to Long QT Syndrome. The first mutation was in a 1-day-old infant, whereas the second was in a 14-year-old girl. The two KCNH2 mutations were transiently transfected into either human embryonic kidney (HEK) cells or human induced pluripotent stem-cell derived cardiomyocytes. We performed associated multiscale computer simulations to elucidate the arrhythmogenic potentials of the KCNH2 mutations. Genetic screening of the first and second index patients revealed a heterozygous missense mutation in KCNH2, resulting in an amino acid change (P632L) in the outer loop of the channel and substitution at position 428 from serine to proline (S428P), respectively. Heterologous expression of P632L and S428P into HEK cells produced no hERG current compared to the wild type (WT). Moreover, the co-transfection of WT and P632L yielded no hERG current; however, the co-transfection of WT and S428P yielded partial hERG current. Action potentials were prolonged in a complete or partial blockade of hERG current from computer simulations which was more severe in Purkinje than ventricular myocytes. Three dimensional simulations revealed a higher susceptibility to reentry in the presence of hERG current blockade. Our experimental findings suggest that both P632L and S428P mutations may impair the KCNH2 gene. The Purkinje cells exhibit a more severe phenotype than ventricular myocytes, and the hERG current blockade renders the ventricles an arrhythmogenic substrate from computer modeling.
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Affiliation(s)
- Anthony Owusu-Mensah
- Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, Virginia, United States of America
| | - Jacqueline Treat
- Masonic Medical Research Laboratory, Utica, New York, United States of America
| | - Joyce Bernardi
- Masonic Medical Research Laboratory, Utica, New York, United States of America
| | - Ryan Pfeiffer
- Masonic Medical Research Laboratory, Utica, New York, United States of America
| | - Robert Goodrow
- Masonic Medical Research Laboratory, Utica, New York, United States of America
| | - Bright Tsevi
- Department of Engineering, Norfolk State University, Norfolk, Virginia, United States of America
| | - Victoria Lam
- Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, Virginia, United States of America
| | - Michel Audette
- Department of Computational Modeling and Simulation Engineering, Old Dominion University, Norfolk, Virginia, United States of America
| | - Jonathan M. Cordeiro
- Masonic Medical Research Laboratory, Utica, New York, United States of America
- ICON Laboratory Services Incorporation, Whitesboro, New York, United States of America
| | - Makarand Deo
- Department of Engineering, Norfolk State University, Norfolk, Virginia, United States of America
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Wang YJ, Yeh CJ, Gao ZH, Hwang E, Chen HH, Wu SN. Inhibitory Perturbations of Fluvastatin on Afterhyperpolarization Current, Erg-mediated K + Current, and Hyperpolarization-activated Cation Current in Both Pituitary GH 3 Cells and Primary Embryonic Mouse Cortical Neurons. Neuroscience 2023; 531:12-23. [PMID: 37661016 DOI: 10.1016/j.neuroscience.2023.08.038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Revised: 08/27/2023] [Accepted: 08/29/2023] [Indexed: 09/05/2023]
Abstract
Fluvastatin (FLV), the first synthetically derived 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is a potent inhibitor of cholesterol biosynthesis. While its primary mechanism of action is to reduce cholesterol levels, there is some evidence suggesting that it may also have effects on K+ channels. However, the overall effects of fluvastatin on ionic currents are not yet well understood. The whole-cell clamp recordings were applied to evaluate the ionic currents and action potentials of cells. Here, we have demonstrated that FLV can effectively inhibit the amplitude of erg-mediated K+ current (IK(erg)) in pituitary tumor (GH3) cells, with an IC50 of approximately 3.2 µM. In the presence of FLV, the midpoint in the activation curve of IK(erg) was distinctly shifted to a less negative potential by 10 mV, with minimal modification of the gating charge. However, the magnitude of hyperpolarization-activated cation current (Ih) elicited by long-lasting membrane hyperpolarization was progressively decreased, with an IC50 value of 8.7 µM, upon exposure to FLV. More interestingly, we also found that FLV (5 µM) could regulate the action potential and afterhyperpolarization properties in primary embryonic mouse cortical neurons. Our study presents compelling evidence indicating that FLV has the potential to impact both the amplitude and gating of the ion channels IK(erg) and Ih. We also provide credible evidence suggesting that this drug has the potential to modify the properties of action potentials and the afterhyperpolarization current in electrically excitable cells. However, the assumption that these findings translate to similar in-vivo results remains unclear.
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Affiliation(s)
- Ya-Jean Wang
- Department of Senior Services Industry Management, Minghsin University of Science and Technology, Hsinchu, Taiwan.
| | - Che-Jui Yeh
- Institute of Molecular Medicine and Bioengineering, National Yang Ming Chiao Tung University, Hsinchu, Taiwan
| | - Zi-Han Gao
- Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan
| | - Eric Hwang
- Institute of Molecular Medicine and Bioengineering, National Yang Ming Chiao Tung University, Hsinchu, Taiwan; Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan; Institute of Bioinformatics and Systems Biology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan
| | - Hwei-Hisen Chen
- Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan; Institute of Neuroscience, National Chengchi University, Taipei, Taiwan.
| | - Sheng-Nan Wu
- Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan; Institute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, Taiwan; Department of Medical Research and Education, An Nan Hostpial, China Medical University Hospital, China Medical University, Tainan, Taiwan.
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7
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Zhang HQ, Lin JL, Pan L, Mao L, Pang JL, Yuan Q, Li GY, Yi GS, Lin YB, Feng BL, Li YD, Wang Y, Jie LJ, Zhang YH. Enzastaurin cardiotoxicity: QT interval prolongation, negative inotropic responses and negative chronotropic action. Biochem Pharmacol 2023; 209:115443. [PMID: 36720353 DOI: 10.1016/j.bcp.2023.115443] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2022] [Revised: 01/22/2023] [Accepted: 01/23/2023] [Indexed: 01/31/2023]
Abstract
Several clinical trials observed that enzastaurin prolonged QT interval in cancer patients. However, the mechanism of enzastaurin-induced QT interval prolongation is unclear. Therefore, this study aimed to assess the effect and mechanism of enzastaurin on QT interval and cardiac function. The Langendorff and Ion-Optix MyoCam systems were used to assess the effects of enzastaurin on QT interval, cardiac systolic function and intracellular Ca2+ transient in guinea pig hearts and ventricular myocytes. The effects of enzastaurin on the rapid delayed rectifier (IKr), the slow delayed rectifier K+ current (IKs), transient outward potassium current (Ito), action potentials, Ryanodine Receptor 2 (RyR2) and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) expression and activity in HEK 293 cell system and primary cardiomyocytes were investigated using whole-cell recording technique and western blotting. We found that enzastaurin significantly prolonged QT interval in guinea pig hearts and increased the action potential duration (APD) in guinea pig cardiomyocytes in a dose-dependent manner. Enzastaurin potently inhibited IKr by binding to the human Ether-à-go-go-Related gene (hERG) channel in both open and closed states, and hERG mutant channels, including S636A, S631A, and F656V attenuated the inhibitory effect of enzastaurin. Enzastaurin also moderately decreased IKs. Additionally, enzastaurin also induced negative chronotropic action. Moreover, enzastaurin impaired cardiac systolic function and reduced intracellular Ca2+ transient via inhibition of RyR2 phosphorylation. Taken together, we found that enzastaurin prolongs QT, reduces heart rate and impairs cardiac systolic function. Therefore, we recommend that electrocardiogram (ECG) and cardiac function should be continuously monitored when enzastaurin is administered to cancer patients.
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Affiliation(s)
- He-Qiang Zhang
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Jia-le Lin
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Lei Pan
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Liang Mao
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Key Laboratory of Medical Electrophysiology, Southwest Medical University, Luzhou, Sichuan, China
| | - Jing-Long Pang
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Qian Yuan
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Gui-Yang Li
- Department of Cardiology, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Gang-Si Yi
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Yang-Bin Lin
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Bao-Long Feng
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Yun-da Li
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China
| | - Yan Wang
- Department of Cardiology, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China.
| | - Ling-Jun Jie
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China; Department of Cardiology, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China.
| | - Yan-Hui Zhang
- Institute of Cardiovascular Diseases, Xiamen Cardiovascular Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, China.
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Bassetto CAZ, Costa F, Guardiani C, Bezanilla F, Giacomello A. Noncanonical electromechanical coupling paths in cardiac hERG potassium channel. Nat Commun 2023; 14:1110. [PMID: 36849440 PMCID: PMC9971164 DOI: 10.1038/s41467-023-36730-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Accepted: 02/15/2023] [Indexed: 03/01/2023] Open
Abstract
Voltage-gated potassium channels are involved in many physiological processes such as nerve impulse transmission, the heartbeat, and muscle contraction. However, for many of them the molecular determinants of the gating mechanism remain elusive. Here, using a combination of theoretical and experimental approaches, we address this problem focusing on the cardiac hERG potassium channel. Network analysis of molecular dynamics trajectories reveals the presence of a kinematic chain of residues that couples the voltage sensor domain to the pore domain and involves the S4/S1 and S1/S5 subunit interfaces. Mutagenesis experiments confirm the role of these residues and interfaces in the activation and inactivation mechanisms. Our findings demonstrate the presence of an electromechanical transduction path crucial for the non-domain-swapped hERG channel gating that resembles the noncanonical path identified in domain-swapped K+ channels.
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Affiliation(s)
- Carlos A Z Bassetto
- Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA
| | - Flavio Costa
- Dipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma, Rome, Italy
| | - Carlo Guardiani
- Dipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma, Rome, Italy
| | - Francisco Bezanilla
- Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA.
- Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA.
- Centro Interdisciplinario de Neurociencias, Facultad de Ciencias, Universidad de Valparaiso, Valparaiso, Chile.
| | - Alberto Giacomello
- Dipartimento di Ingegneria Meccanica e Aerospaziale, Sapienza Università di Roma, Rome, Italy.
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9
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Zangerl-Plessl EM, Wu W, Sanguinetti MC, Stary-Weinzinger A. Binding of RPR260243 at the intracellular side of the hERG1 channel pore domain slows closure of the helix bundle crossing gate. Front Mol Biosci 2023; 10:1137368. [PMID: 36911523 PMCID: PMC9996038 DOI: 10.3389/fmolb.2023.1137368] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Accepted: 02/07/2023] [Indexed: 02/25/2023] Open
Abstract
The opening and closing of voltage-dependent potassium channels is dependent on a tight coupling between movement of the voltage sensing S4 segments and the activation gate. A specific interaction between intracellular amino- and carboxyl-termini is required for the characteristically slow rate of channel closure (deactivation) of hERG1 channels. Compounds that increase hERG1 channel currents represent a novel approach for prevention of arrhythmia associated with prolonged ventricular repolarization. RPR260243 (RPR), a quinoline oxo-propyl piperidine derivative, inhibits inactivation and dramatically slows the rate of hERG1 channel deactivation. Here we report that similar to its effect on wild-type channels, RPR greatly slows the deactivation rate of hERG1 channels missing their amino-termini, or of split channels lacking a covalent link between the voltage sensor domain and the pore domain. By contrast, RPR did not slow deactivation of C-terminal truncated hERG1 channels or D540K hERG1 mutant channels activated by hyperpolarization. Together, these findings indicate that ability of RPR to slow deactivation requires an intact C-terminus, does not slow deactivation by stabilizing an interaction involving the amino-terminus or require a covalent link between the voltage sensor and pore domains. All-atom molecular dynamics simulations using the cryo-EM structure of the hERG1 channel revealed that RPR binds to a pocket located at the intracellular ends of helices S5 and S6 of a single subunit. The slowing of channel deactivation by RPR may be mediated by disruption of normal S5-S6 interactions.
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Affiliation(s)
| | - Wei Wu
- Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research & Training Institute, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, UT, United States
| | - Michael C Sanguinetti
- 3 Department of Internal Medicine, Division of Cardiovascular Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt LakeCity, UT, United States
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10
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Ledford HA, Ren L, Thai PN, Park S, Timofeyev V, Sirish P, Xu W, Emigh AM, Priest JR, Perez MV, Ashley EA, Yarov-Yarovoy V, Yamoah EN, Zhang XD, Chiamvimonvat N. Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome. Heart Rhythm 2022; 19:281-292. [PMID: 34634443 PMCID: PMC8810706 DOI: 10.1016/j.hrthm.2021.10.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Revised: 09/20/2021] [Accepted: 10/04/2021] [Indexed: 11/04/2022]
Abstract
BACKGROUND Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)-encoded K+ channel (Kv11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERGT613M) and RNF207 frameshift (RNF207G603fs) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K+ channel subunits. METHODS Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERGT613M proteins for degradation. RNF207G603fs exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERGT613M subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERGA614V, a known dominant-negative mutant subunit. Finally, the presence of RNF207G603fs with hERGT613M results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell-derived cardiomyocytes. CONCLUSION Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K+ channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.
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Affiliation(s)
- Hannah A Ledford
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California
| | - Lu Ren
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California
| | - Phung N Thai
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California
| | - Seojin Park
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California; Department of Physiology and Cell Biology, University of Nevada, Reno, Reno, Nevada
| | - Valeriy Timofeyev
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California
| | - Padmini Sirish
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California; Department of Veterans Affairs, Northern California Health Care System, Mather, California
| | - Wilson Xu
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California
| | - Aiyana M Emigh
- Department of Physiology and Membrane Biology, University of California, Davis, Davis, California
| | - James R Priest
- Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, California
| | - Marco V Perez
- Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, California
| | - Euan A Ashley
- Division of Cardiovascular Medicine, Department of Medicine, Stanford University, Stanford, California
| | - Vladimir Yarov-Yarovoy
- Department of Physiology and Membrane Biology, University of California, Davis, Davis, California
| | - Ebenezer N Yamoah
- Department of Physiology and Cell Biology, University of Nevada, Reno, Reno, Nevada
| | - Xiao-Dong Zhang
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California; Department of Veterans Affairs, Northern California Health Care System, Mather, California.
| | - Nipavan Chiamvimonvat
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, Davis, California; Department of Veterans Affairs, Northern California Health Care System, Mather, California.
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11
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Soohoo SM, Tiwari PB, Suzuki YJ, Brelidze TI. Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance. J Biol Chem 2021; 298:101433. [PMID: 34801551 PMCID: PMC8693265 DOI: 10.1016/j.jbc.2021.101433] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Revised: 11/15/2021] [Accepted: 11/16/2021] [Indexed: 11/29/2022] Open
Abstract
Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.
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Affiliation(s)
- Stephanie M Soohoo
- Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Purushottam B Tiwari
- Department of Oncology, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Yuichiro J Suzuki
- Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Tinatin I Brelidze
- Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, District of Columbia, USA.
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12
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Jie LJ, Li YD, Zhang HQ, Mao L, Xie HB, Zhou FG, Zhou TL, Xie D, Lin JL, Li GY, Cai BN, Zhang YH, Wang Y. Mechanisms of gefitinib-induced QT prolongation. Eur J Pharmacol 2021; 910:174441. [PMID: 34474028 DOI: 10.1016/j.ejphar.2021.174441] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2020] [Revised: 08/20/2021] [Accepted: 08/24/2021] [Indexed: 11/26/2022]
Abstract
Gefitinib, a tyrosine kinase inhibitor, was the first targeted therapy for non-small cell lung cancer (NSCLC). Gefitinib could block human Ether-à-go-go-Related Gene (hERG) channel, an important target in drug-induced long QT syndrome. However, it is unclear whether gefitinib could induce QT interval prolongation. Here, whole-cell patch-clamp technique was used for evaluating the effect of gefitinib on rapidly-activating delayed rectifier K+ current (IKr), slowly-activating delayed rectifier K+ current (IKs), transient outward potassium current (Ito), inward rectifier K+ current (IK1) and on action potentials in guinea pig ventricular myocytes. The Langendorff heart perfusion technique was used to determine drug effect on the ECG. Gefitinib depressed IKr by binding to open and closed hERG channels in a concentration-dependent way (IC50: 1.91 μM). The inhibitory effect of gefitinib on wildtype hERG channels was reduced at the hERG mutants Y652A, S636A, F656V and S631A (IC50: 8.51, 13.97, 18.86, 32.99 μM), indicating that gefitinib is a pore inhibitor of hERG channels. In addition, gefitinib accelerated hERG channel inactivation and decreased channel steady-state inactivation. Gefitinib also decreased IKs with IC50 of 23.8 μM. Moreover, gefitinib increased action potential duration (APD) in guinea pig ventricular myocytes and the corrected QT interval (QTc) in isolated perfused guinea pig hearts in a concentration-dependent way (1-30 μM). These findings indicate that gefitinib could prolong QTc interval by potently blocking hERG channel, modulating kinetic properties of hERG channel. Partial block of KCNQ1/KCNE1 could also contribute to delayed repolarization and prolonged QT interval. Thus, caution should be taken when gefitinib is used for NSCLC treatment.
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Affiliation(s)
- Ling-Jun Jie
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Yun-Da Li
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - He-Qiang Zhang
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Liang Mao
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China; Southwest Medical University, Luzhou, Sichuan, 646000, China
| | - Hua-Bin Xie
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Fa-Guang Zhou
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Tian-Li Zhou
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Dong Xie
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Jia-Le Lin
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Gui-Yang Li
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Bin-Ni Cai
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China
| | - Yan-Hui Zhang
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China.
| | - Yan Wang
- Xiamen Cardiovascular Hospital, School of Medicine, Xiamen University, Xiamen, Fujian, 361004, China.
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13
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Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating. Proc Natl Acad Sci U S A 2021; 118:2108796118. [PMID: 34716268 DOI: 10.1073/pnas.2108796118] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Accepted: 09/22/2021] [Indexed: 11/18/2022] Open
Abstract
The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.
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14
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Codding SJ, Johnson AA, Trudeau MC. Gating and regulation of KCNH (ERG, EAG, and ELK) channels by intracellular domains. Channels (Austin) 2021; 14:294-309. [PMID: 32924766 PMCID: PMC7515569 DOI: 10.1080/19336950.2020.1816107] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The KCNH family comprises the ERG, EAG, and ELK voltage-activated, potassium-selective channels. Distinct from other K channels, KCNH channels contain unique structural domains, including a PAS (Per-Arnt-Sim) domain in the N-terminal region and a CNBHD (cyclic nucleotide-binding homology domain) in the C-terminal region. The intracellular PAS domains and CNBHDs interact directly and regulate some of the characteristic gating properties of each type of KCNH channel. The PAS-CNBHD interaction regulates slow closing (deactivation) of hERG channels, the kinetics of activation and pre-pulse dependent population of closed states (the Cole-Moore shift) in EAG channels and voltage-dependent potentiation in ELK channels. KCNH channels are all regulated by an intrinsic ligand motif in the C-terminal region which binds to the CNBHD. Here, we focus on some recent advances regarding the PAS-CNBHD interaction and the intrinsic ligand.
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Affiliation(s)
- Sara J Codding
- Department of Physiology, University of Maryland School of Medicine , Baltimore, MD, USA
| | - Ashley A Johnson
- Department of Physiology, University of Maryland School of Medicine , Baltimore, MD, USA
| | - Matthew C Trudeau
- Department of Physiology, University of Maryland School of Medicine , Baltimore, MD, USA
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15
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Zequn Z, Jiangfang L. Molecular Insights Into the Gating Kinetics of the Cardiac hERG Channel, Illuminated by Structure and Molecular Dynamics. Front Pharmacol 2021; 12:687007. [PMID: 34168566 PMCID: PMC8217747 DOI: 10.3389/fphar.2021.687007] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2021] [Accepted: 05/20/2021] [Indexed: 11/13/2022] Open
Abstract
The rapidly activating delayed rectifier K+ current generated by the cardiac hERG potassium channel encoded by KCNH2 is the most important reserve current for cardiac repolarization. The unique inward rectification characteristics of the hERG channel depend on the gating regulation, which involves crucial structural domains and key single amino acid residues in the full-length hERG channel. Identifying critical molecules involved in the regulation of gating kinetics for the hERG channel requires high-resolution structures and molecular dynamics simulation models. Based on the latest progress in hERG structure and molecular dynamics simulation research, summarizing the molecules involved in the changes in the channel state helps to elucidate the unique gating characteristics of the channel and the reason for its high affinity to cardiotoxic drugs. In this review, we aim to summarize the significant advances in understanding the voltage gating regulation of the hERG channel based on its structure obtained from cryo-electron microscopy and computer simulations, which reveal the critical roles of several specific structural domains and amino acid residues.
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Affiliation(s)
- Zheng Zequn
- Department of Cardiovascular, Medical College, Ningbo University, Ningbo, China
| | - Lian Jiangfang
- Department of Cardiovascular, Lihuili Hospital Affiliated to Ningbo University, Ningbo, China
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16
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Kurata HT. Unconventional voltage sensing in an inwardly rectifying potassium channel. J Gen Physiol 2021; 153:212077. [PMID: 33956072 PMCID: PMC8105720 DOI: 10.1085/jgp.202112929] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Affiliation(s)
- Harley T Kurata
- Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
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17
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Szendrey M, Guo J, Li W, Yang T, Zhang S. COVID-19 Drugs Chloroquine and Hydroxychloroquine, but Not Azithromycin and Remdesivir, Block hERG Potassium Channels. J Pharmacol Exp Ther 2021; 377:265-272. [PMID: 33674391 DOI: 10.1124/jpet.120.000484] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Accepted: 03/02/2021] [Indexed: 12/27/2022] Open
Abstract
Drug-induced long QT syndrome (LQTS) is an established cardiac side effect of a wide range of medications and represents a significant concern for drug safety. The rapidly and slowly activating delayed rectifier K+ currents, mediated by channels encoded by the human ether-a-go-go-related gene (hERG) and KCNQ1 + KCNE1, respectively, are two main currents responsible for ventricular repolarization. The common cause for drugs to induce LQTS is through impairing the hERG channel. For the recent emergence of COVID-19, caused by severe acute respiratory syndrome coronavirus 2, several drugs have been investigated as potential therapies; however, there are concerns about their QT prolongation risk. Here, we studied the effects of chloroquine, hydroxychloroquine, azithromycin, and remdesivir on hERG channels. Our results showed that although chloroquine acutely blocked hERG current (IhERG), with an IC50 of 3.0 µM, hydroxychloroquine acutely blocked IhERG 8-fold less potently, with an IC50 of 23.4 µM. Azithromycin and remdesivir did not acutely affect IhERG When these drugs were added at 10 µM to the cell culture medium for 24 hours, remdesivir increased IhERG by 2-fold, which was associated with an increased mature hERG channel expression. In addition, these four drugs did not acutely or chronically affect KCNQ1 + KCNE1 channels. Our data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns. SIGNIFICANCE STATEMENT: This work demonstrates that, among off-label potential COVID-19 treatment drugs chloroquine, hydroxychloroquine, azithromycin, and remdesivir, the former two drugs block hERG potassium channels, whereas the latter two drugs do not. All four drugs do not affect KCNQ1 + KCNE1. As hERG and KCNQ1 + KCNE1 are two main K+ channels responsible for ventricular repolarization, and most drugs that induce long QT syndrome (LQTS) do so by impairing hERG channels, these data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns.
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Affiliation(s)
- Mark Szendrey
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Jun Guo
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Wentao Li
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Tonghua Yang
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Shetuan Zhang
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
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18
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Mironenko A, Zachariae U, de Groot BL, Kopec W. The Persistent Question of Potassium Channel Permeation Mechanisms. J Mol Biol 2021; 433:167002. [PMID: 33891905 DOI: 10.1016/j.jmb.2021.167002] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 04/14/2021] [Accepted: 04/15/2021] [Indexed: 02/09/2023]
Abstract
Potassium channels play critical roles in many physiological processes, providing a selective permeation route for K+ ions in and out of a cell, by employing a carefully designed selectivity filter, evolutionarily conserved from viruses to mammals. The structure of the selectivity filter was determined at atomic resolution by x-ray crystallography, showing a tight coordination of desolvated K+ ions by the channel. However, the molecular mechanism of K+ ions permeation through potassium channels remains unclear, with structural, functional and computational studies often providing conflicting data and interpretations. In this review, we will present the proposed mechanisms, discuss their origins, and will critically assess them against all available data. General properties shared by all potassium channels are introduced first, followed by the introduction of two main mechanisms of ion permeation: soft and direct knock-on. Then, we will discuss critical computational and experimental studies that shaped the field. We will especially focus on molecular dynamics (MD) simulations, that provided mechanistic and energetic aspects of K+ permeation, but at the same time created long-standing controversies. Further challenges and possible solutions are presented as well.
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Affiliation(s)
- Andrei Mironenko
- Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
| | - Ulrich Zachariae
- Computational Biology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
| | - Bert L de Groot
- Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
| | - Wojciech Kopec
- Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
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19
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Abstract
Long QT syndrome (LQTS) is a cardiovascular disorder characterized by an abnormality in cardiac repolarization leading to a prolonged QT interval and T-wave irregularities on the surface electrocardiogram. It is commonly associated with syncope, seizures, susceptibility to torsades de pointes, and risk for sudden death. LQTS is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. The availability of therapy for this lethal disease emphasizes the importance of early and accurate diagnosis. Additionally, understanding of the molecular mechanisms underlying LQTS could help to optimize genotype-specific treatments to prevent deaths in LQTS patients. In this review, we briefly summarize current knowledge regarding molecular underpinning of LQTS, in particular focusing on LQT1, LQT2, and LQT3, and discuss novel strategies to study ion channel dysfunction and drug-specific therapies in LQT1, LQT2, and LQT3 syndromes.
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Affiliation(s)
| | - Isabelle Deschênes
- Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio
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20
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Ozturk N, Uslu S, Ozdemir S. Diabetes-induced changes in cardiac voltage-gated ion channels. World J Diabetes 2021; 12:1-18. [PMID: 33520105 PMCID: PMC7807254 DOI: 10.4239/wjd.v12.i1.1] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2020] [Revised: 11/05/2020] [Accepted: 11/13/2020] [Indexed: 02/06/2023] Open
Abstract
Diabetes mellitus affects the heart through various mechanisms such as microvascular defects, metabolic abnormalities, autonomic dysfunction and incompatible immune response. Furthermore, it can also cause functional and structural changes in the myocardium by a disease known as diabetic cardiomyopathy (DCM) in the absence of coronary artery disease. As DCM progresses it causes electrical remodeling of the heart, left ventricular dysfunction and heart failure. Electrophysiological changes in the diabetic heart contribute significantly to the incidence of arrhythmias and sudden cardiac death in diabetes mellitus patients. In recent studies, significant changes in repolarizing K+ currents, Na+ currents and L-type Ca2+ currents along with impaired Ca2+ homeostasis and defective contractile function have been identified in the diabetic heart. In addition, insulin levels and other trophic factors change significantly to maintain the ionic channel expression in diabetic patients. There are many diagnostic tools and management options for DCM, but it is difficult to detect its development and to effectively prevent its progress. In this review, diabetes-associated alterations in voltage-sensitive cardiac ion channels are comprehensively assessed to understand their potential role in the pathophysiology and pathogenesis of DCM.
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Affiliation(s)
- Nihal Ozturk
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Serkan Uslu
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Semir Ozdemir
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
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21
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Influence of Kv11.1 (hERG1) K + channel expression on DNA damage induced by the genotoxic agent methyl methanesulfonate. Pflugers Arch 2021; 473:197-217. [PMID: 33452554 DOI: 10.1007/s00424-021-02517-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 12/22/2020] [Accepted: 01/05/2021] [Indexed: 10/22/2022]
Abstract
Besides their crucial role in cell electrogenesis and maintenance of basal membrane potential, the voltage-dependent K+ channel Kv11.1/hERG1 shows an essential impact in cell proliferation and other processes linked to the maintenance of tumour phenotype. To check the possible influence of channel expression on DNA damage responses, HEK293 cells, treated with the genotoxic agent methyl methanesulfonate (MMS), were compared with those of a HEK-derived cell line (H36), permanently transfected with the Kv11.1-encoding gene, and with a third cell line (T2) obtained under identical conditions as H36, by permanent transfection of another unrelated plasma membrane protein encoding gene. In addition, to gain some insights about the canonical/conduction-dependent channel mechanisms that might be involved, the specific erg channel inhibitor E4031 was used as a tool. Our results indicate that the expression of Kv11.1 does not influence MMS-induced changes in cell cycle progression, because no differences were found between H36 and T2 cells. However, the canonical ion conduction function of the channel appeared to be associated with decreased cell viability at low/medium MMS concentrations. Moreover, direct DNA damage measurements, using the comet assay, demonstrated for the first time that Kv11.1 conduction activity was able to modify MMS-induced DNA damage, decreasing it particularly at high MMS concentration, in a way related to PARP1 gene expression. Finally, our data suggest that the canonical Kv11.1 effects may be relevant for tumour cell responses to anti-tumour therapies.
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22
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Wang M, Gao M, Fang S, Zheng R, Peng D, Luo Q, Yu B. L51P, a novel mutation in the PAS domain of hERG channel, confers long QT syndrome by impairing channel activation. Am J Transl Res 2020; 12:8040-8049. [PMID: 33437379 PMCID: PMC7791479] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Accepted: 10/28/2020] [Indexed: 06/12/2023]
Abstract
The human ether-à-go-go-related gene (hERG) potassium channel mediates the repolarization of ventricular action potentials. Mutations in the KCNH2 cause long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. Here, we functionally analyzed a mutation of hERG potassium channel (p.L51P), gaining novel insights into clinical genotype-phenotype relationships. Potassium currents were recorded by whole-cell patch clamping in HEK293 cells transiently transfected with wild-type and/or mutant hERG potassium channel. Immunofluorescence assay and confocal imaging were undertaken to study the effects of L51P mutation on channel trafficking. The models of the protein structure of hERG and its mutations are predicted by Amber16 software. Molecular dynamics (MD) of individual protein were performed with Particle Mesh Ewald (PME). The production of MD simulations of hERG-WT and hERG-Mut at constant pressure and temperature were carried out with SHAKE. L51 was a conservative amino acid, located in the Per-Arnt-Sim (PAS) domain of the amino terminus. L51P caused loss of function via impairing channel activation. L51P was predicted to destroy hydrophobic structure in the PAS domain, thus causing the failure of channel opening. In summary, the present study identifies L51P as a novel mutation of hERG potassium channel. L51P mutation mechanistically impairs channel activation, reducing channel functionality.
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Affiliation(s)
- Mi Wang
- Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South UniversityChangsha, Hunan, China
- Research Institute of Blood Lipid and Atherosclerosis, Central South UniversityChangsha, Hunan, China
| | - Min Gao
- Department of Pharmacy, The Sixth Affiliated Hospital, Sun Yat-sen UniversityGuangzhou, Guangdong, China
| | - Senbiao Fang
- School of Computer Science and Engineering, Central South UniversityChangsha, Hunan, China
| | - Ruoqian Zheng
- School of Computer Science and Engineering, Central South UniversityChangsha, Hunan, China
| | - Daoquan Peng
- Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South UniversityChangsha, Hunan, China
- Research Institute of Blood Lipid and Atherosclerosis, Central South UniversityChangsha, Hunan, China
| | - Qin Luo
- Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South UniversityChangsha, Hunan, China
- Research Institute of Blood Lipid and Atherosclerosis, Central South UniversityChangsha, Hunan, China
| | - Bilian Yu
- Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South UniversityChangsha, Hunan, China
- Research Institute of Blood Lipid and Atherosclerosis, Central South UniversityChangsha, Hunan, China
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23
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Varró A, Tomek J, Nagy N, Virág L, Passini E, Rodriguez B, Baczkó I. Cardiac transmembrane ion channels and action potentials: cellular physiology and arrhythmogenic behavior. Physiol Rev 2020; 101:1083-1176. [PMID: 33118864 DOI: 10.1152/physrev.00024.2019] [Citation(s) in RCA: 113] [Impact Index Per Article: 22.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Cardiac arrhythmias are among the leading causes of mortality. They often arise from alterations in the electrophysiological properties of cardiac cells and their underlying ionic mechanisms. It is therefore critical to further unravel the pathophysiology of the ionic basis of human cardiac electrophysiology in health and disease. In the first part of this review, current knowledge on the differences in ion channel expression and properties of the ionic processes that determine the morphology and properties of cardiac action potentials and calcium dynamics from cardiomyocytes in different regions of the heart are described. Then the cellular mechanisms promoting arrhythmias in congenital or acquired conditions of ion channel function (electrical remodeling) are discussed. The focus is on human-relevant findings obtained with clinical, experimental, and computational studies, given that interspecies differences make the extrapolation from animal experiments to human clinical settings difficult. Deepening the understanding of the diverse pathophysiology of human cellular electrophysiology will help in developing novel and effective antiarrhythmic strategies for specific subpopulations and disease conditions.
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Affiliation(s)
- András Varró
- Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary.,MTA-SZTE Cardiovascular Pharmacology Research Group, Hungarian Academy of Sciences, Szeged, Hungary
| | - Jakub Tomek
- Department of Computer Science, British Heart Foundation Centre of Research Excellence, University of Oxford, Oxford, United Kingdom
| | - Norbert Nagy
- Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary.,MTA-SZTE Cardiovascular Pharmacology Research Group, Hungarian Academy of Sciences, Szeged, Hungary
| | - László Virág
- Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary
| | - Elisa Passini
- Department of Computer Science, British Heart Foundation Centre of Research Excellence, University of Oxford, Oxford, United Kingdom
| | - Blanca Rodriguez
- Department of Computer Science, British Heart Foundation Centre of Research Excellence, University of Oxford, Oxford, United Kingdom
| | - István Baczkó
- Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary
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24
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Barros F, de la Peña P, Domínguez P, Sierra LM, Pardo LA. The EAG Voltage-Dependent K + Channel Subfamily: Similarities and Differences in Structural Organization and Gating. Front Pharmacol 2020; 11:411. [PMID: 32351384 PMCID: PMC7174612 DOI: 10.3389/fphar.2020.00411] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Accepted: 03/18/2020] [Indexed: 12/17/2022] Open
Abstract
EAG (ether-à-go-go or KCNH) are a subfamily of the voltage-gated potassium (Kv) channels. Like for all potassium channels, opening of EAG channels drives the membrane potential toward its equilibrium value for potassium, thus setting the resting potential and repolarizing action potentials. As voltage-dependent channels, they switch between open and closed conformations (gating) when changes in membrane potential are sensed by a voltage sensing domain (VSD) which is functionally coupled to a pore domain (PD) containing the permeation pathway, the potassium selectivity filter, and the channel gate. All Kv channels are tetrameric, with four VSDs formed by the S1-S4 transmembrane segments of each subunit, surrounding a central PD with the four S5-S6 sections arranged in a square-shaped structure. Structural information, mutagenesis, and functional experiments, indicated that in "classical/Shaker-type" Kv channels voltage-triggered VSD reorganizations are transmitted to PD gating via the α-helical S4-S5 sequence that links both modules. Importantly, these Shaker-type channels share a domain-swapped VSD/PD organization, with each VSD contacting the PD of the adjacent subunit. In this case, the S4-S5 linker, acting as a rigid mechanical lever (electromechanical lever coupling), would lead to channel gate opening at the cytoplasmic S6 helices bundle. However, new functional data with EAG channels split between the VSD and PD modules indicate that, in some Kv channels, alternative VSD/PD coupling mechanisms do exist. Noticeably, recent elucidation of the architecture of some EAG channels, and other relatives, showed that their VSDs are non-domain swapped. Despite similarities in primary sequence and predicted structural organization for all EAG channels, they show marked kinetic differences whose molecular basis is not completely understood. Thus, while a common general architecture may establish the gating system used by the EAG channels and the physicochemical coupling of voltage sensing to gating, subtle changes in that common structure, and/or allosteric influences of protein domains relatively distant from the central gating machinery, can crucially influence the gating process. We consider here the latest advances on these issues provided by the elucidation of eag1 and erg1 three-dimensional structures, and by both classical and more recent functional studies with different members of the EAG subfamily.
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Affiliation(s)
- Francisco Barros
- Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Edificio Santiago Gascón, Oviedo, Spain
| | - Pilar de la Peña
- Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Edificio Santiago Gascón, Oviedo, Spain
| | - Pedro Domínguez
- Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, Edificio Santiago Gascón, Oviedo, Spain
| | - Luisa Maria Sierra
- Departamento de Biología Funcional (Area de Genética), Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Universidad de Oviedo, Oviedo, Spain
| | - Luis A. Pardo
- Oncophysiology Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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25
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He S, Moutaoufik MT, Islam S, Persad A, Wu A, Aly KA, Fonge H, Babu M, Cayabyab FS. HERG channel and cancer: A mechanistic review of carcinogenic processes and therapeutic potential. Biochim Biophys Acta Rev Cancer 2020; 1873:188355. [PMID: 32135169 DOI: 10.1016/j.bbcan.2020.188355] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2019] [Revised: 02/28/2020] [Accepted: 02/28/2020] [Indexed: 12/21/2022]
Abstract
The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.
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Affiliation(s)
- Siyi He
- Department of Surgery, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | | | - Saadul Islam
- Department of Surgery, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Amit Persad
- Department of Surgery, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Adam Wu
- Department of Surgery, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Khaled A Aly
- Department of Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada
| | - Humphrey Fonge
- Department of Medical Imaging, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W8, Canada; Department of Medical Imaging, Royal University Hospital, Saskatoon, Saskatchewan S7N 0W8, Canada
| | - Mohan Babu
- Department of Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada
| | - Francisco S Cayabyab
- Department of Surgery, Neuroscience Research Group, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
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26
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Shi YP, Thouta S, Claydon TW. Modulation of hERG K + Channel Deactivation by Voltage Sensor Relaxation. Front Pharmacol 2020; 11:139. [PMID: 32184724 PMCID: PMC7059196 DOI: 10.3389/fphar.2020.00139] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2019] [Accepted: 01/31/2020] [Indexed: 12/17/2022] Open
Abstract
The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, Ikr, in the heart, which is essential for normal cardiac electrical activity and rhythm. Slow deactivation is one of the hallmark features of the unusual gating characteristics of hERG channels, and plays a crucial role in providing a robust current that aids repolarization of the cardiac action potential. As such, there is significant interest in elucidating the underlying mechanistic determinants of slow hERG channel deactivation. Recent work has shown that the hERG channel S4 voltage sensor is stabilized following activation in a process termed relaxation. Voltage sensor relaxation results in energetic separation of the activation and deactivation pathways, producing a hysteresis, which modulates the kinetics of deactivation gating. Despite widespread observation of relaxation behaviour in other voltage-gated K+ channels, such as Shaker, Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase Ci-VSP, the relationship between stabilization of the activated voltage sensor by the open pore and voltage sensor relaxation in the control of deactivation has only recently begun to be explored. In this review, we discuss present knowledge and questions raised related to the voltage sensor relaxation mechanism in hERG channels and compare structure-function aspects of relaxation with those observed in related ion channels. We focus discussion, in particular, on the mechanism of coupling between voltage sensor relaxation and deactivation gating to highlight the insight that these studies provide into the control of hERG channel deactivation gating during their physiological functioning.
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Affiliation(s)
- Yu Patrick Shi
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada
| | - Samrat Thouta
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada
| | - Thomas W Claydon
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada
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27
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Selectivity filter modalities and rapid inactivation of the hERG1 channel. Proc Natl Acad Sci U S A 2020; 117:2795-2804. [PMID: 31980532 DOI: 10.1073/pnas.1909196117] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.
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28
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Robertson GA, Morais-Cabral JH. hERG Function in Light of Structure. Biophys J 2019; 118:790-797. [PMID: 31669064 DOI: 10.1016/j.bpj.2019.10.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Revised: 09/27/2019] [Accepted: 10/02/2019] [Indexed: 12/25/2022] Open
Abstract
The human ether-a-go-go-related gene1 (hERG) ion channel has been the subject of fascination since it was identified as a target of long QT syndrome more than 20 years ago. In this Biophysical Perspective, we look at what makes hERG intriguing and vexingly unique. By probing recent high-resolution structures in the context of functional and biochemical data, we attempt to summarize new insights into hERG-specific function and articulate important unanswered questions. X-ray crystallography and cryo-electron microscopy have revealed features not previously on the radar-the "nonswapped" transmembrane architecture, an "intrinsic ligand," and hydrophobic pockets off a pore cavity that is surprisingly small. Advances in our understanding of drug block and inactivation mechanisms are noted, but a full picture will require more investigation.
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Affiliation(s)
- Gail A Robertson
- Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin.
| | - João H Morais-Cabral
- i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC- Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
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29
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Kodirov SA, Zhuravlev VL, Brachmann J. Prevailing Effects of Ibutilide on Fast Delayed Rectifier K + Channel. J Membr Biol 2019; 252:609-616. [PMID: 31584122 DOI: 10.1007/s00232-019-00098-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2019] [Accepted: 07/29/2019] [Indexed: 11/27/2022]
Abstract
Effects of ibutilide, a class III antiarrhythmic drug, on delayed rectifier potassium currents (IK) in freshly isolated guinea pig ventricular myocytes were studied. Experiments were performed using the whole-cell configuration of patch-clamp technique under blockade of L-type calcium currents (Cav1). Ibutilide at concentrations ranging between 10 nM and 100 µM inhibited IKr in dose-dependent manner with a half maximal effective concentration of 2.03 ± 0.74 µM (n = 5-10). The amplitude of tail currents activated by prepulse to + 20 mV was decreased from 253 ± 52 to 130 ± 25 pA (n = 8, p < 0.01) in the presence of 1 µM ibutilide. The envelope test revealed time-dependent changes in ratio of IK-tail/ΔIK during 0.2-2 s pulse durations in the absence of drug. With ibutilide, regardless of pulse duration, a relatively constant ratio was estimated, indicative of predominant involvement of IKr component. The slow IKs persisted to greater extent even at 100 μM ibutilide revealing a distinguishable selectivity toward the IKr component.
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Affiliation(s)
- Sodikdjon A Kodirov
- Department of Cardiology, University Hospital Heidelberg, 69120, Heidelberg, Germany. .,Department of Physiology, Saint Petersburg University, Saint Petersburg, Russia, 199034. .,Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteura St., 02-093, Warsaw, Poland. .,Department of Cardiology, Klinikum Coburg, Teaching Hospital of the University of Würzburg, Coburg, Germany.
| | - Vladimir L Zhuravlev
- Department of Cardiology, University Hospital Heidelberg, 69120, Heidelberg, Germany.,Department of Physiology, Saint Petersburg University, Saint Petersburg, Russia, 199034
| | - Johannes Brachmann
- Department of Cardiology, University Hospital Heidelberg, 69120, Heidelberg, Germany.,Department of Cardiology, Klinikum Coburg, Teaching Hospital of the University of Würzburg, Coburg, Germany
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30
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Perissinotti L, Guo J, Kudaibergenova M, Lees-Miller J, Ol'khovich M, Sharapova A, Perlovich GL, Muruve DA, Gerull B, Noskov SY, Duff HJ. The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain. Mol Pharmacol 2019; 96:259-271. [PMID: 31182542 PMCID: PMC6666383 DOI: 10.1124/mol.118.115642] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 05/28/2019] [Indexed: 12/14/2022] Open
Abstract
Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.
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Affiliation(s)
- Laura Perissinotti
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Jiqing Guo
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Meruyert Kudaibergenova
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - James Lees-Miller
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Marina Ol'khovich
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Angelica Sharapova
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - German L Perlovich
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Daniel A Muruve
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Brenda Gerull
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Sergei Yu Noskov
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
| | - Henry J Duff
- Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada (L.P., M.K., S.Y.N.); Libin Cardiovascular Institute of Alberta (J.G., J.-L.M., H.J.D.) and Snyder Institute for Chronic Diseases (D.A.M.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Institute of Solution Chemistry, Russian Academy of Sciences, Ivanovo, Russian Federation (M.O., A.S., G.L.P.); Department of Cardiac Sciences and Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada (B.G.); and Comprehensive Heart Failure Center and Department of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany (B.G.)
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31
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De Zio R, Gerbino A, Forleo C, Pepe M, Milano S, Favale S, Procino G, Svelto M, Carmosino M. Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome. J Cell Mol Med 2019; 23:6331-6342. [PMID: 31361068 PMCID: PMC6714209 DOI: 10.1111/jcmm.14521] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2019] [Revised: 06/02/2019] [Accepted: 06/08/2019] [Indexed: 12/29/2022] Open
Abstract
The K+ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K+ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy.
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Affiliation(s)
- Roberta De Zio
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy
| | - Andrea Gerbino
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy
| | - Cinzia Forleo
- Cardiology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Martino Pepe
- Cardiology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Serena Milano
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy
| | - Stefano Favale
- Cardiology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
| | - Giuseppe Procino
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy
| | - Maria Svelto
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy
| | - Monica Carmosino
- Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.,Department of Sciences, University of Basilicata, Potenza, Italy
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32
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Tschirhart JN, Li W, Guo J, Zhang S. Blockade of the Human Ether A-Go-Go-Related Gene (hERG) Potassium Channel by Fentanyl. Mol Pharmacol 2019; 95:386-397. [PMID: 30665971 DOI: 10.1124/mol.118.114751] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Accepted: 01/12/2019] [Indexed: 11/22/2022] Open
Abstract
The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). Drug-mediated or medical condition-mediated disruption of hERG function is the primary cause of acquired long-QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. Fentanyl abuse poses a serious health concern, with abuse and death rates rising over recent years. As fentanyl has a propensity to cause sudden death, we investigated its effects on the hERG channel. The effects of norfentanyl, the main metabolite, and naloxone, an antidote used in fentanyl overdose, were also examined. Currents of hERG channels stably expressed in HEK293 cells were recorded using the whole-cell voltage-clamp method. When hERG tail currents were analyzed upon -50 mV repolarization after a 50 mV depolarization, fentanyl and naloxone blocked hERG current (IhERG) with IC50 values of 0.9 and 74.3 μM, respectively, whereas norfentanyl did not block. However, fentanyl-mediated block of IhERG was voltage dependent. When a voltage protocol that mimics a human ventricular action potential (AP) was used, fentanyl blocked IhERG with an IC50 of 0.3 μM. Furthermore, fentanyl (0.5 μM) prolonged AP duration and blocked IKr in ventricular myocytes isolated from neonatal rats. The concentrations of fentanyl used in this study were higher than seen with clinical use but overlap with postmortem overdose concentrations. Although mechanisms of fentanyl-related sudden death need further investigation, blockade of hERG channels may contribute to the death of individuals with high-concentration overdose or compromised cardiac repolarization.
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Affiliation(s)
- Jared N Tschirhart
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Wentao Li
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Jun Guo
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
| | - Shetuan Zhang
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada
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Shi YP, Thouta S, Cheng YM, Claydon TW. Extracellular protons accelerate hERG channel deactivation by destabilizing voltage sensor relaxation. J Gen Physiol 2018; 151:231-246. [PMID: 30530765 PMCID: PMC6363419 DOI: 10.1085/jgp.201812137] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 08/23/2018] [Accepted: 11/07/2018] [Indexed: 11/22/2022] Open
Abstract
The human ether-à-go-go–related gene (hERG) encodes a delayed rectifier K+ channel with slow deactivation gating. Shi et al. find that acidic residues on S3 contribute to slow deactivation kinetics by stabilizing the relaxed state of the voltage sensor, which can be mitigated by extracellular protons. hERG channels underlie the delayed-rectifier K+ channel current (IKr), which is crucial for membrane repolarization and therefore termination of the cardiac action potential. hERG channels display unusually slow deactivation gating, which contributes to a resurgent current upon repolarization and may protect against post-depolarization–induced arrhythmias. hERG channels also exhibit robust mode shift behavior, which reflects the energetic separation of activation and deactivation pathways due to voltage sensor relaxation into a stable activated state. The mechanism of relaxation is unknown and likely contributes to slow hERG channel deactivation. Here, we use extracellular acidification to probe the structural determinants of voltage sensor relaxation and its influence on the deactivation gating pathway. Using gating current recordings and voltage clamp fluorimetry measurements of voltage sensor domain dynamics, we show that voltage sensor relaxation is destabilized at pH 6.5, causing an ∼20-mV shift in the voltage dependence of deactivation. We show that the pH dependence of the resultant loss of mode shift behavior is similar to that of the deactivation kinetics acceleration, suggesting that voltage sensor relaxation correlates with slower pore gate closure. Neutralization of D509 in S3 also destabilizes the relaxed state of the voltage sensor, mimicking the effect of protons, suggesting that acidic residues on S3, which act as countercharges to S4 basic residues, are involved in stabilizing the relaxed state and slowing deactivation kinetics. Our findings identify the mechanistic determinants of voltage sensor relaxation and define the long-sought mechanism by which protons accelerate hERG deactivation.
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Affiliation(s)
- Yu Patrick Shi
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Samrat Thouta
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Yen May Cheng
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
| | - Tom W Claydon
- Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
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Steffensen AB, Andersen MN, Mutsaers N, Mujezinovic A, Schmitt N. SUMO co-expression modifies K V 11.1 channel activity. Acta Physiol (Oxf) 2018; 222. [PMID: 28888063 DOI: 10.1111/apha.12974] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2017] [Revised: 08/31/2017] [Accepted: 09/06/2017] [Indexed: 11/27/2022]
Abstract
AIM The voltage-gated potassium channel KV 11.1 is the molecular basis for the IKr current, which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesized that the post-translational modification sumoylation fine-tunes channel activity. METHODS The functional effects of sumoylation on KV 11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analysis of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. RESULTS Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV 11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93 and K116) in the PAS domain as the putative SUMO-targets. CONCLUSION This study indicates KV 11.1 as a sumoylation target and offers three main targets: K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain.
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Affiliation(s)
- A. B. Steffensen
- Department of Biomedical Sciences; Faculty of Health and Medical Sciences; Danish National Research Foundation Centre for Cardiac Arrhythmia; University of Copenhagen; Copenhagen Denmark
| | - M. N. Andersen
- Department of Biomedical Sciences; Faculty of Health and Medical Sciences; Danish National Research Foundation Centre for Cardiac Arrhythmia; University of Copenhagen; Copenhagen Denmark
| | - N. Mutsaers
- Department of Biomedical Sciences; Faculty of Health and Medical Sciences; Danish National Research Foundation Centre for Cardiac Arrhythmia; University of Copenhagen; Copenhagen Denmark
| | - A. Mujezinovic
- Department of Biomedical Sciences; Faculty of Health and Medical Sciences; Danish National Research Foundation Centre for Cardiac Arrhythmia; University of Copenhagen; Copenhagen Denmark
| | - N. Schmitt
- Department of Biomedical Sciences; Faculty of Health and Medical Sciences; Danish National Research Foundation Centre for Cardiac Arrhythmia; University of Copenhagen; Copenhagen Denmark
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Lainez S, Doray A, Hancox JC, Cannell MB. Regulation of Kv4.3 and hERG potassium channels by KChIP2 isoforms and DPP6 and response to the dual K + channel activator NS3623. Biochem Pharmacol 2018; 150:120-130. [PMID: 29378180 PMCID: PMC5906734 DOI: 10.1016/j.bcp.2018.01.036] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2017] [Accepted: 01/22/2018] [Indexed: 12/14/2022]
Abstract
Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ± 1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound’s effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.
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Affiliation(s)
- Sergio Lainez
- School of Physiology, Pharmacology and Neuroscience, Faculty of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
| | - Adélaïde Doray
- School of Physiology, Pharmacology and Neuroscience, Faculty of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
| | - Jules C Hancox
- School of Physiology, Pharmacology and Neuroscience, Faculty of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
| | - Mark B Cannell
- School of Physiology, Pharmacology and Neuroscience, Faculty of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
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The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues. Biophys J 2017; 113:1979-1991. [PMID: 29117522 DOI: 10.1016/j.bpj.2017.09.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2017] [Revised: 08/30/2017] [Accepted: 09/06/2017] [Indexed: 11/21/2022] Open
Abstract
Kv11.1 (hERG) is a voltage-gated potassium channel that shows very slow ionic current activation kinetics, and an unusual underlying biphasic gating charge movement with fast and slow components that differ greatly in time course. The structural basis and role of the fast component of gating charge (Qfast) is unclear, and its relationship to the slow activation of hERG channels is not understood. In this study we have used the cut-open oocyte voltage-clamp technique to investigate the relationship of fast gating charge movement-to-residue interactions between D411 at the bottom of the S1, and lower S4 domain charged and uncharged residues. Neutralization of D411 or K538 and V535A prevented Qfast and greatly accelerated overall charge movement. Voltage-clamp fluorometry showed a loss of a fast component of S4 fluorescence in D411N, V535A, and K538Q upon depolarization, whereas [2-(trimethyl ammonium) ethyl] methanethiosulfonate chloride modification of I521C in the outer S4 was enhanced at more negative potentials and at earlier times in these same mutants. A functional interaction between these regions during activation was suggested by ΔΔGo values >4.2 kJ/mol obtained from double mutant cycle analysis. The data indicate that interactions of S1 residue D411 with lower S4 residues stabilizes early closed states of the channel, and that disruption of these interactions results in both faster rates of activation gating and an elimination of the fast component of gating charge movement and of fluorescence. We propose that the Qfast charge movement during activation accompanies transitions through early closed states of the hERG activation pathway, and that the weak voltage dependence of these transitions limits the overall activation rate of hERG channels. Disruption of the D411-S4 interactions destabilizes these early closed states, leaving hERG channels able to activate at a rate similar to conventional potassium channels.
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37
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Characterization of loperamide-mediated block of hERG channels at physiological temperature and its proarrhythmia propensity. J Pharmacol Toxicol Methods 2017; 88:109-122. [DOI: 10.1016/j.vascn.2017.08.006] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Revised: 08/14/2017] [Accepted: 08/18/2017] [Indexed: 02/05/2023]
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Jeevaratnam K, Chadda KR, Huang CLH, Camm AJ. Cardiac Potassium Channels: Physiological Insights for Targeted Therapy. J Cardiovasc Pharmacol Ther 2017; 23:119-129. [PMID: 28946759 PMCID: PMC5808825 DOI: 10.1177/1074248417729880] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The development of novel drugs specifically directed at the ion channels underlying particular features of cardiac action potential (AP) initiation, recovery, and refractoriness would contribute to an optimized approach to antiarrhythmic therapy that minimizes potential cardiac and extracardiac toxicity. Of these, K+ channels contribute numerous and diverse currents with specific actions on different phases in the time course of AP repolarization. These features and their site-specific distribution make particular K+ channel types attractive therapeutic targets for the development of pharmacological agents attempting antiarrhythmic therapy in conditions such as atrial fibrillation. However, progress in the development of such temporally and spatially selective antiarrhythmic drugs against particular ion channels has been relatively limited, particularly in view of our incomplete understanding of the complex physiological roles and interactions of the various ionic currents. This review summarizes the physiological properties of the main cardiac potassium channels and the way in which they modulate cardiac electrical activity and then critiques a number of available potential antiarrhythmic drugs directed at them.
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Affiliation(s)
- Kamalan Jeevaratnam
- 1 Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.,2 School of Medicine, Perdana University-Royal College of Surgeons Ireland, Serdang, Selangor Darul Ehsan, Malaysia
| | - Karan R Chadda
- 1 Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.,3 Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom
| | - Christopher L-H Huang
- 3 Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom.,4 Division of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - A John Camm
- 5 Cardiac Clinical Academic Group, St George's Hospital Medical School, University of London, Cranmer Terrace, London, United Kingdom
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Barrese V, Cidad P, Yeung SY, López-López JR, McNeish AJ, Ohya S, Pérez-García MT, Greenwood IA. Proliferative Role of Kv11 Channels in Murine Arteries. Front Physiol 2017; 8:500. [PMID: 28747891 PMCID: PMC5506201 DOI: 10.3389/fphys.2017.00500] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Accepted: 06/29/2017] [Indexed: 12/22/2022] Open
Abstract
K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries. Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 μM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly. Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.
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Affiliation(s)
- Vincenzo Barrese
- Vascular Research Centre, Institute of Molecular & Clinical Sciences, St George's University of LondonLondon, United Kingdom
| | - Pilar Cidad
- Departmento de Fisiología, Universidad de ValladolidValladolid, Spain
| | - Shuk Y Yeung
- Vascular Research Centre, Institute of Molecular & Clinical Sciences, St George's University of LondonLondon, United Kingdom
| | | | - Alister J McNeish
- Reading School of Pharmacy, University of ReadingReading, United Kingdom
| | - Susumu Ohya
- Department of Pharmacology, Kyoto Pharmaceutical UniversityKyoto, Japan
| | | | - Iain A Greenwood
- Vascular Research Centre, Institute of Molecular & Clinical Sciences, St George's University of LondonLondon, United Kingdom
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40
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Mauerhöfer M, Bauer CK. Effects of Temperature on Heteromeric Kv11.1a/1b and Kv11.3 Channels. Biophys J 2017; 111:504-523. [PMID: 27508435 DOI: 10.1016/j.bpj.2016.07.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2016] [Revised: 06/30/2016] [Accepted: 07/01/2016] [Indexed: 01/17/2023] Open
Abstract
Kv11.1 channels are crucial in cardiac physiology, and there is increasing evidence of physiological roles of different Kv11 channels outside the heart. The HERG (human Kv11.1a) channel has previously been shown to carry substantially more current at elevated temperatures, and we have now comparably investigated the temperature dependence of neuronal Kv11.3 channels and the more ubiquitous heteromeric Kv11.1a/1b channels. Transiently expressed rat Kv11 channels were studied at 21°C, 30°C, and 35°C. At near-physiological temperature, the maximal sustained outward current density was almost three times the mean value obtained at room temperature for Kv11.1a/1b, and increased by ∼150% for Kv11.3. For both channels, reduced inactivation contributed to the current increase at higher temperature. Elevated temperature moved Kv11.1a/1b isochronal activation curves to more negative potentials, but shifted the potential of half-maximal Kv11.3 channel activation to more depolarized values and reduced its voltage sensitivity. Thus, increased temperature stabilized the open state over the closed state of Kv11.1a/1b channels and exerted the opposite effect on Kv11.3 channel activation. Both Kv11 channels exhibited an overall high temperature sensitivity of most gating parameters, with remarkably high Q10 factors of ∼5 for the rate of Kv11.1a/1b activation. The Q10 factors for Kv11.3 gating were more uniform, but still higher for activation than for inactivation kinetics. The results demonstrate that characteristic differences between Kv11.1a/1b and Kv11.3 determined at room temperature do not necessarily apply to physiological conditions. The data provided here can aid in the design of models that will enhance our understanding of the role of Kv11 currents in excitable cells.
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Affiliation(s)
- Maike Mauerhöfer
- Department of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Christiane K Bauer
- Department of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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41
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Phan K, Ng CA, David E, Shishmarev D, Kuchel PW, Vandenberg JI, Perry MD. The S1 helix critically regulates the finely tuned gating of Kv11.1 channels. J Biol Chem 2017; 292:7688-7705. [PMID: 28280240 DOI: 10.1074/jbc.m117.779298] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 02/26/2017] [Indexed: 11/06/2022] Open
Abstract
Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.
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Affiliation(s)
- Kevin Phan
- From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010.,the St. Vincent's Clinical School, University of New South Wales, New South Wales 2052, and
| | - Chai Ann Ng
- From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010.,the St. Vincent's Clinical School, University of New South Wales, New South Wales 2052, and
| | - Erikka David
- From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010
| | - Dmitry Shishmarev
- the School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia
| | - Philip W Kuchel
- the School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales 2006, Australia
| | - Jamie I Vandenberg
- From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010.,the St. Vincent's Clinical School, University of New South Wales, New South Wales 2052, and
| | - Matthew D Perry
- From the Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, New South Wales 2010, .,the St. Vincent's Clinical School, University of New South Wales, New South Wales 2052, and
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hERG S4-S5 linker acts as a voltage-dependent ligand that binds to the activation gate and locks it in a closed state. Sci Rep 2017; 7:113. [PMID: 28273916 PMCID: PMC5427910 DOI: 10.1038/s41598-017-00155-2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2016] [Accepted: 02/08/2017] [Indexed: 12/27/2022] Open
Abstract
Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5–S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels.
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Jie LJ, Wu WY, Li G, Xiao GS, Zhang S, Li GR, Wang Y. Clemizole hydrochloride blocks cardiac potassium currents stably expressed in HEK 293 cells. Br J Pharmacol 2017; 174:254-266. [PMID: 27886373 DOI: 10.1111/bph.13679] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2016] [Revised: 11/21/2016] [Accepted: 11/22/2016] [Indexed: 12/16/2022] Open
Abstract
BACKGROUND AND PURPOSE Clemizole, a histamine H1 receptor antagonist has a potential therapeutic effect on hepatitis C infection and also potently inhibits TRPC5 ion channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and thus affects cardiac repolarization. EXPERIMENTAL APPROACH Whole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IKs and Kv 1.5 channel current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. Isolated hearts from guinea pigs were used to determine the effect on the ECG. KEY RESULTS Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50 : 0.07 μM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50 : 0.82, 0.89, 1.49 and 2.98 μM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and human Kv 1.5 channel current. Moreover, clemizole increased the duration of the ventricular action potential in guinea pig hearts and the QTc interval in isolated perfused hearts from guinea pigs, in a concentration-dependent manner (0.1-1.0 μM). CONCLUSION AND IMPLICATIONS Our results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs , delays cardiac repolarization and thereby prolongs QT interval. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.
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Affiliation(s)
- Ling-Jun Jie
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
| | - Wei-Yin Wu
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
| | - Gang Li
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
| | - Guo-Sheng Xiao
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
| | - Shetuan Zhang
- Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada
| | - Gui-Rong Li
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
| | - Yan Wang
- Xiamen Cardiovascular Hospital, Xiamen University, Xiamen, China
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Chiamvimonvat N, Chen-Izu Y, Clancy CE, Deschenes I, Dobrev D, Heijman J, Izu L, Qu Z, Ripplinger CM, Vandenberg JI, Weiss JN, Koren G, Banyasz T, Grandi E, Sanguinetti MC, Bers DM, Nerbonne JM. Potassium currents in the heart: functional roles in repolarization, arrhythmia and therapeutics. J Physiol 2017; 595:2229-2252. [PMID: 27808412 DOI: 10.1113/jp272883] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2016] [Accepted: 10/11/2016] [Indexed: 12/19/2022] Open
Abstract
This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.
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Affiliation(s)
- Nipavan Chiamvimonvat
- Department of Internal Medicine, University of California, Davis, Genome and Biomedical Science Facility, Rm 6315, Davis, CA, 95616, USA.,Department of Veterans Affairs, Northern California Health Care System, Mather, CA, 95655, USA
| | - Ye Chen-Izu
- Department of Internal Medicine, University of California, Davis, Genome and Biomedical Science Facility, Rm 6315, Davis, CA, 95616, USA.,Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA.,Department of Biomedical Engineering, University of California, Davis, Genome and Biomedical Science Facility, Rm 2303, Davis, CA, 95616, USA
| | - Colleen E Clancy
- Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA
| | - Isabelle Deschenes
- Department of Physiology and Biophysics, and Biomedical Engineering, Case Western Reserve University, Cleveland, OH, 44109, USA.,Heart and Vascular Research Center, MetroHealth Medical Center, Cleveland, OH, 44109, USA
| | - Dobromir Dobrev
- Institute of Pharmacology, West German Heart and Vascular Center, University Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany
| | - Jordi Heijman
- Department of Cardiology, Cardiovascular Research Institute Maastricht, Faculty of Health, Medicine, and Life Sciences, Maastricht University, Maastricht, The Netherlands
| | - Leighton Izu
- Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA
| | - Zhilin Qu
- Division of Cardiology, Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, 3645 MRL, Los Angeles, CA, 90095, USA
| | - Crystal M Ripplinger
- Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA
| | - Jamie I Vandenberg
- Victor Chang Cardiac Research Institute, 405 Liverpool Street, Darlinghurst, NSW, 2010, Australia
| | - James N Weiss
- Division of Cardiology, Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, 3645 MRL, Los Angeles, CA, 90095, USA
| | - Gideon Koren
- Cardiovascular Research Center, Rhode Island Hospital and the Cardiovascular Institute, The Warren Alpert Medical School of Brown University, Providence, RI, 02903, USA
| | - Tamas Banyasz
- Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Eleonora Grandi
- Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA
| | - Michael C Sanguinetti
- Department of Internal Medicine, University of Utah, Nora Eccles Harrison Cardiovascular Research & Training Institute, Salt Lake City, UT, 84112, USA
| | - Donald M Bers
- Department of Pharmacology, University of California, Davis, Genome and Biomedical Science Facility, Rm 3503, Davis, CA, 95616, USA
| | - Jeanne M Nerbonne
- Departments of Developmental Biology and Internal Medicine, Cardiovascular Division, Washington University Medical School, St Louis, MO, 63110, USA
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Abstract
Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (IhERG) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased IhERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2 Therefore, TBX20 can be considered a KCNH2-modifying gene.
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Bohnen MS, Peng G, Robey SH, Terrenoire C, Iyer V, Sampson KJ, Kass RS. Molecular Pathophysiology of Congenital Long QT Syndrome. Physiol Rev 2017; 97:89-134. [PMID: 27807201 PMCID: PMC5539372 DOI: 10.1152/physrev.00008.2016] [Citation(s) in RCA: 122] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Ion channels represent the molecular entities that give rise to the cardiac action potential, the fundamental cellular electrical event in the heart. The concerted function of these channels leads to normal cyclical excitation and resultant contraction of cardiac muscle. Research into cardiac ion channel regulation and mutations that underlie disease pathogenesis has greatly enhanced our knowledge of the causes and clinical management of cardiac arrhythmia. Here we review the molecular determinants, pathogenesis, and pharmacology of congenital Long QT Syndrome. We examine mechanisms of dysfunction associated with three critical cardiac currents that comprise the majority of congenital Long QT Syndrome cases: 1) IKs, the slow delayed rectifier current; 2) IKr, the rapid delayed rectifier current; and 3) INa, the voltage-dependent sodium current. Less common subtypes of congenital Long QT Syndrome affect other cardiac ionic currents that contribute to the dynamic nature of cardiac electrophysiology. Through the study of mutations that cause congenital Long QT Syndrome, the scientific community has advanced understanding of ion channel structure-function relationships, physiology, and pharmacological response to clinically employed and experimental pharmacological agents. Our understanding of congenital Long QT Syndrome continues to evolve rapidly and with great benefits: genotype-driven clinical management of the disease has improved patient care as precision medicine becomes even more a reality.
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Affiliation(s)
- M S Bohnen
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - G Peng
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - S H Robey
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - C Terrenoire
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - V Iyer
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - K J Sampson
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
| | - R S Kass
- Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York Stem Cell Foundation Research Institute, New York, New York
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Gintant GA, Su Z, Martin RL, Cox BF. Utility of hERG Assays as Surrogate Markers of Delayed Cardiac Repolarization and QT Safety. Toxicol Pathol 2016; 34:81-90. [PMID: 16507548 DOI: 10.1080/01926230500431376] [Citation(s) in RCA: 65] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
HERG (human-ether-a-go-go-related gene) encodes for a cardiac potassium channel that plays a critical role in defining ventricular repolarization. Noncardiovascular drugs associated with a rare but potentially lethal ventricular arrhythmia (Torsades de Pointes) have been linked to delayed cardiac repolarization and block of hERG current. This brief overview will discuss the role of hERG current in cardiac electrophysiology, its involvement in drug-induced delayed repolarization, and approaches used to define drug effects on hERG current. In addition, examples of hERG blocking drugs acting differently (i.e., overt and covert hERG blockade due to multichannel block) together with the utility and limitations of hERG assays as tools to predict the risk of delayed repolarization and proarrhythmia are discussed.
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Affiliation(s)
- Gary A Gintant
- Deptartment of Integrative Pharmacology, Abbott Laboratories, Abbott Park, Illinois 60064-6119, USA.
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48
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Wu W, Gardner A, Sachse FB, Sanguinetti MC. Ginsenoside Rg3, a Gating Modifier of EAG Family K+ Channels. Mol Pharmacol 2016; 90:469-82. [PMID: 27502018 PMCID: PMC5034692 DOI: 10.1124/mol.116.104091] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2016] [Accepted: 08/05/2016] [Indexed: 01/11/2023] Open
Abstract
Ginsenoside 20(S)-Rg3 (Rg3) is a steroid glycoside that induces human ether-à-go-go-related gene type 1 (hERG1, Kv11.1) channels to activate at more negative potentials and to deactivate more slowly than normal. However, it is unknown whether this action is unique to hERG1 channels. Here we compare and contrast the mechanisms of actions of Rg3 on hERG1 with three other members of the ether-à-go-go (EAG) K(+) channel gene family, including EAG1 (Kv10.1), ERG3 (Kv11.3), and ELK1 (Kv12.1). All four channel types were heterologously expressed in Xenopus laevis oocytes, and K(+) currents were measured using the two-microelectrode voltage-clamp technique. At a maximally effective concentration, Rg3 shifted the half-point of voltage-dependent activation of currents by -14 mV for ERG1 (EC50 = 414 nM), -20 mV for ERG3 (EC50 = 374 nM), -28 mV for EAG1 (EC50 = 1.18 μM), and more than -100 mV for ELK1 (EC50 = 197 nM) channels. Rg3 also induced slowing of ERG1, ERG3, and ELK1 channel deactivation and accelerated the rate of EAG1 channel activation. A Markov model was developed to simulate gating and the effects of Rg3 on the voltage dependence of activation of hELK1 channels. Understanding the mechanism underlying the action of Rg3 may facilitate the development of more potent and selective EAG family channel activators as therapies for cardiovascular and neural disorders.
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Affiliation(s)
- Wei Wu
- Nora Eccles Harrison Cardiovascular Research and Training Institute (W.W., A.G., F.B.S., M.C.S.), Department of Bioengineering (F.B.S.), Department of Internal Medicine, Division of Cardiovascular Medicine (M.C.S.), University of Utah, Salt Lake City, Utah
| | - Alison Gardner
- Nora Eccles Harrison Cardiovascular Research and Training Institute (W.W., A.G., F.B.S., M.C.S.), Department of Bioengineering (F.B.S.), Department of Internal Medicine, Division of Cardiovascular Medicine (M.C.S.), University of Utah, Salt Lake City, Utah
| | - Frank B Sachse
- Nora Eccles Harrison Cardiovascular Research and Training Institute (W.W., A.G., F.B.S., M.C.S.), Department of Bioengineering (F.B.S.), Department of Internal Medicine, Division of Cardiovascular Medicine (M.C.S.), University of Utah, Salt Lake City, Utah
| | - Michael C Sanguinetti
- Nora Eccles Harrison Cardiovascular Research and Training Institute (W.W., A.G., F.B.S., M.C.S.), Department of Bioengineering (F.B.S.), Department of Internal Medicine, Division of Cardiovascular Medicine (M.C.S.), University of Utah, Salt Lake City, Utah
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49
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Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain. Proc Natl Acad Sci U S A 2016; 113:9916-21. [PMID: 27516548 DOI: 10.1073/pnas.1601116113] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.
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50
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Lörinczi E, Helliwell M, Finch A, Stansfeld PJ, Davies NW, Mahaut-Smith M, Muskett FW, Mitcheson JS. Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain. J Biol Chem 2016; 291:17907-18. [PMID: 27325704 PMCID: PMC5016179 DOI: 10.1074/jbc.m116.733576] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Indexed: 11/24/2022] Open
Abstract
The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.
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Affiliation(s)
- Eva Lörinczi
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN
| | - Matthew Helliwell
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN, the School of Physiology and Pharmacology, University of Bristol, Bristol BS5 1TD, and
| | - Alina Finch
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN
| | - Phillip J Stansfeld
- the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
| | - Noel W Davies
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN
| | - Martyn Mahaut-Smith
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN
| | - Frederick W Muskett
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN
| | - John S Mitcheson
- From the Department of Molecular and Cell Biology, University of Leicester, Leicester LE1 9HN,
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