Abstract

Human ether-à-go-go-related gene 1 (hERG1) channels mediate repolarization of cardiac action potentials. Inherited long QT syndrome (LQTS) caused by loss-of-function mutations, or unintended blockade of hERG1 channels by many drugs, can lead to severe arrhythmia and sudden death. Drugs that activate hERG1 are a novel pharmacological approach to treat LQTS. 3-Nitro-n-(4-phenoxyphenyl) benzamide [ICA-105574 (ICA)] has been discovered to activate hERG1 by strong attenuation of pore-type inactivation. Here, we used scanning mutagenesis of hERG1 to identify the molecular determinants of ICA action. Three mutations abolished the activator effects of 30 μM ICA, including L622C in the pore helix, F557L in the S5 segment, and Y652A in the S6 segment. One mutation in S6 (A653M) switched the activity of ICA from an activator to an inhibitor, revealing its partial agonist activity. This was confirmed by showing that the noninactivating mutant hERG1 channel (G628C/S631C) was inhibited by ICA and that the addition of the F557L mutation rendered the channel drug-insensitive. Simulated molecular docking of ICA to homology models of hERG1 corroborated the scanning mutagenesis findings. Together, our findings indicate that ICA is a mixed agonist of hERG1 channels. Activation or inhibition of currents is mediated by the same or overlapping binding site located in the pore module between two adjacent subunits of the homotetrameric channel.