Abstract

Loss-of -function mutations in human ether-a-go-go-related gene 1 (hERG1) is associated with life-threatening arrhythmias. hERG1 activators are being developed as treatments for acquired or genetic forms of long QT syndrome. The locations of the putative binding pockets for activators are still being elucidated. In silico docking of the activator 1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea (NS1643) to an S1-S6 transmembrane homology model of hERG1 predicted putative binding sites. The predictions of the in silico docking guided subsequent in vitro mutagenesis and electrophysiological measurements. The novel interacting site for NS1643 is predicted around Asn629 at the outer mouth of the channel. The applied N629H mutation is the sole amino acid replacement in the literature that abrogates the NS1643-induced left shift of the V_1/2 of activation. In contrast, both N629T and N629D showed pharmacologic responses similar to wild type. Another important interacting pocket is predicted at the intracellular surface in the S4–S5 linker. Mutagenesis of the residues critical to interactions in this pocket had major effects on the pharmacologic response to NS1643. The inward conductance elicited by hyperpolarization of D540K hERG1 was abrogated by NS1643 treatment, suggesting that it alters the inward movement of the S4 segment. The neighboring E544L mutation markedly exaggerated tail-current responses to NS1643. However, an L564A substitution inhibited drug response. Structure-guided mutagenesis identified widespread clusters of amino acids modulating drug-induced shifts in inactivation; such modulation may reflect allosteric changes in tertiary structure. Model-guided mutagenesis led to the discovery of a range of novel interacting residues that modify NS1643-induced pharmacologic responses.