Molecular Determinants of Humanether-à-go-go-Related Gene 1 (hERG1) K⁺Channel Activation by NS1643

Abstract

Human ether-à -go-go-related gene 1 (hERG1) channels conduct the rapid delayed rectifier K ϩ current, I Kr , an important determinant of action potential repolarization in mammals, including humans. Reduced I Kr function caused by mutations in KCNH2 or drug block of hERG1 channels prolongs the QT interval of the electrocardiogram and increases the risk of ventricular fibrillation and sudden cardiac death. Several activators of hERG1 channels have been discovered in recent years. These compounds shorten the duration of cardiac action potentials and have been proposed as a new therapeutic approach for the treatment of acquired or congenital long QT syndrome. We defined previously the mechanism of action of 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643), a compound that increases hERG1 currents by shifting the voltage-dependence of inactivation to more positive potentials. Here, we use scanning mutagenesis of hERG1 and functional characterization of 56 mutant channels heterologously expressed in Xenopus laevis oocytes to define the molecular determinants of the binding site for NS1643. Most point mutations did not alter response to the drug; however, 10 mutant channels had reduced sensitivity, and F619A and I567A exhibited enhanced activation by the drug. Some of these residues form a cluster and, together with molecular modeling, suggest that NS1643 binds to a pocket near the extracellular ends of the S5/S6 segments of two adjacent hERG1 channel subunits. This putative binding site differs from the sites described previously for two other hERG1 activators, (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) and 2-(4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino)-benzoic acid (PD-118057).

Structure-Guided Topographic Mapping and Mutagenesis to Elucidate Binding Sites for the Human Ether-a-Go-Go-Related Gene 1 Potassium Channel (KCNH2) Activator NS1643

“…These residues were not exposed to the NS1643 molecule during previous molecular modeling studies. It is noteworthy that the binding site for NS1643 proposed by Grunnet et al (2011) is made up of several amino acid residues predicted to be part of the IC-1 and IC-2 sites reported in the current study. The docking results reported in this study also predicted Ile567 as a site in hERG1 that interacts with NS1643.…”

“…Using a developed model of hERG1, we are able to provide insights into the topology of putative interacting sites for the NS1643. We also discuss structural correlations with published experimental studies (Grunnet et al, 2011) and offer novel insights that may explain the pharmacological action of NS1643. Docking site predictions for NS1643 were prompted by site-directed mutagenesis and patch-clamp experiments to assess the effects of the drug on the mutated channel currents.…”

“…An approach combining site-directed mutagenesis, functional voltage-clamp assays, and subsequent molecular modeling was used by Grunnet et al (2011) to identify interacting sites for NS1643 on hERG1. They proposed a binding site for NS1643 in the vicinity of the EC end of the S5S6 segments of two adjacent hERG1 subunits.…”

“…Cells with a loss of inward rectification had a diminished response to NS1643. Grunnet et al (2011) examined the impact of a set of amino acid mutations in hERG1 with NS1643 treatment. Their study proposed a binding site in the vicinity of the extracellular (EC) end of the S5S6 segments of two adjacent hERG1 subunits.…”

Strong Activation of ether-à-go-go-Related Gene 1 K+ Channel Isoforms by NS1643 in Human Embryonic Kidney 293 and Chinese Hamster Ovary Cells

“…Phe656 of herg1a, a residue that interacts with most classic herg blockers, was suggested as a possible low-affinity binding site. Thus, different binding sites for NS1643 seem to exist in erg1a channels, suggesting that NS1643 possibly acts from both the external (Xu et al, 2008;Grunnet et al, 2011) and internal sides of the membrane. The presence of different binding sites would also well explain the differences in the time course and use dependence of the increase in current amplitude and the shift in the voltage dependence of activation found for erg1b channels in the present study.…”