Received for publication November 7, 2007.
Revised December 24, 2007.
Accepted for publication December 27, 2007.

A single amino acid difference between ether-a-go-go-related gene channel subtypes determines differential sensitivity to a small molecule activator

Abstract

Activators of human ether-a-go-go-related gene 1 (hERG1) channels such as RPR260243 ((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) reverse the effect of hERG1 blockers and shorten the duration of cardiac action potentials. RPR260243 (RPR) slows the rate of deactivation and shifts the voltage dependence of channel inactivation to more positive potentials. We recently mapped the binding site for RPR to several residues located near the cytoplasmic ends of the S5 and S6 helices of the hERG1 subunit. These residues are conserved in the highly homologous ether-a-go-go-related gene 3 (ERG3) subunit; however, RPR blocks ERG3 channels. Here, we compare hERG1 and rat ERG3 (rERG3) channels to explore the molecular basis for differential channel sensitivity to RPR. Channels were heterologously expressed in Xenopus oocytes and currents were recorded using the two-electrode voltage clamp technique. Site-directed mutagenesis was used to swap the two residues within the putative binding domain that differed between hERG1 and rERG3. The differential sensitivity of hERG1 and rERG3 channels to the agonist effect of RPR could be accounted for by a single S5 residue (Thr556 in hERG1, Ile558 in rERG3). A Thr in this position favors agonist activity, whereas an Ile reveals a secondary blocking effect of RPR.

Key words: Ion channel regulation, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches, Receptor binding studies