title: Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking.
abstract: The antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K(+) external solution ([K(+)]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone's inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A.
authors.affiliation: School of Physiology and Pharmacology and Cardiovascular Research Laboratories, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Charlotte K
authors.initials: CK
authors.lastname: Colenso
authors.affiliation: School of Physiology and Pharmacology and Cardiovascular Research Laboratories, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Aziza
authors.initials: A
authors.lastname: El Harchi
authors.affiliation: School of Physiology and Pharmacology and Cardiovascular Research Laboratories, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Brighton and Sussex Medical School, University of Sussex, Falmer BN1 9PX, UK.
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Witchel
authors.affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address: C.Dempsey@bristol.ac.uk.
authors.forename: Chris E
authors.initials: CE
authors.lastname: Dempsey
authors.affiliation: School of Physiology and Pharmacology and Cardiovascular Research Laboratories, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address: Jules.hancox@bristol.ac.uk.
authors.forename: Jules C
authors.initials: JC
authors.lastname: Hancox
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000235:genetics; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000638:Amiodarone / Q000494:pharmacology*; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D000072237:ERG1 Potassium Channel / Q000037:antagonists & inhibitors* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D009154:Mutation; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D014162:Transfection
chemical_list: D000889:Anti-Arrhythmia Agents; D000072237:ERG1 Potassium Channel; C000606913:KCNH2 protein, human; D000638:Amiodarone; D000409:Alanine
keywords: Amiodarone; Amiodarone hydrochloride (PubChem CID: 441325); Antiarrhythmic; I(Kr); Long QT; QT interval; hERG