Stoichiometry of a hERG1 Agonist on Channel Gating

Abstract

Human ether-a-go-go-related gene 1 (hERG1) K+ channels mediate repolarization of action potentials in cardiomyocytes. A refined mechanistic understanding of the action of hERG1 agonists may assist development of novel compounds for treatment of arrhythmia associated with acquired or congenital long QT syndrome. RPR-260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5 trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR) profoundly slows deactivation and modestly attenuates fast C-type inactivation of hERG1 channels without affecting its kinetics. A Markov model of channel gating incorporating two open states reproduced these experimental findings. A binding site for RPR has been localized to a hydrophobic pocket between two adjacent hERG1 subunits. Accordingly, one homotetrameric hERG1 channel contains four identical RPR binding sites. In this study, the stoichiometric basis of RPR-altered hERG1 channel currents was investigated. Concatenated hERG1 tetramers incorporating a variable number of wild-type subunits and mutant subunits (L553A to prevent RPR binding) were constructed and heterologously expressed in Xenopus oocytes. RPR slowed the rate of hERG1 channel deactivation as a function of the number of wild-type subunits (i.e., accessible RPR binding sites) contained within a tetramer. Occupancy of all four available binding sites was required to attain maximum effect. In contrast, occupancy of only three binding sites was sufficient to achieve the maximum shift (+24 mV) in the voltage required for half-maximal inactivation. The distinct subunit stoichiometries associated with RPR-induced changes in hERG1 deactivation and inactivation reflects the different structural basis of the activation (S6 bundle crossing) and inactivation (selectivity filter) gates.