Molecular Determinants of KCNQ1 Channel Block by a Benzodiazepine GUISCARD SEEBOHM, JUN CHEN, NATHALIE STRUTZ, CHRIS CULBERSON, CHRISTIAN LERCHE, and MICHAEL C. SANGUINETTI Departments of Physiology (G.S., J.C., M.C.S.) and Biology (N.S.), University of Utah, Salt Lake City, Utah; Molecular Systems, Merck Research Laboratories, West Point, Pennsylvania (C.C.); and Cardiovascular Diseases, Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany (C.L.) Received December 2, 2002; accepted April 7, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT KCNQ1 channels underlie the slow delayed rectifier K + current, mediate repolarization of cardiac action potentials, and are a potential therapeutic target for treatment of arrhythmia. (E)-(+)-N- [(3R)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiaz- epin-3-yl]-3-(2,4-dichlorophenyl)-2-propenamide [L-735821 (L-7)] is a potent blocker of KCNQ1 channels. Here we describe the structural determinants of KCNQ1 that are critical for high-affinity block by L-7 using site-directed mutagenesis to alter specific residues and voltage clamp to record channel currents in Xenopus laevis oocytes. Chimeric channels were constructed by combina- tion of regions from L-7–sensitive KCNQ1 and L-7–insensitive KCNQ2 channel subunits. This approach localized the drug inter- action site to the pore and S6 domains of KCNQ1. Substitution of single amino acids identified Thr-312 of the pore domain and Ile-337, Phe-339, Phe-340, and Ala-344 of the S6 domain as the most important molecular determinants of channel block. Some mutations also altered the inactivation properties of KCNQ1, but there was no correlation between extent of inactivation and sen- sitivity to block by L-7. Modeling was used to simulate the docking of L-7 to the KCNQ1 channel pore. The docking was consistent with our experimental data and predicts that L-7 blocks K + con- ductance by physically precluding the occupancy of a K + ion to a pore helix-coordinated site within the central hydrated cavity, a crucial step in ion permeation. KCNQ1 (KVLQT1) was discovered by positional cloning (Wang et al., 1996) and is the founding member of the KCNQ voltage-gated K + channel family. KCNQ1 channels are char- acterized by fast activation and a delayed, voltage-dependent inactivation. Coassembly with the -subunit KCNE1 gener- ates a very slowly activating delayed-rectifier K + current, I Ks , with no apparent inactivation (Barhanin et al., 1996; Sanguinetti et al., 1996). KCNQ2 subunits coassemble with KCNQ3 subunits to form channels that conduct the neuronal M-current (Wang et al., 1998). Physiological functions for KCNQ1/KCNE channels include repolarization of cardiac ac- tion potentials, modulation of H + secretion into the stomach, Cl - secretion into the colon, and secretion of K + into the stria media of the inner ear (Neyroud et al., 1997; Mall et al., 1998; Grahammer et al., 2001). A potent class of I Ks blockers was discovered serendipi- tously. Substituted 1,4-benzodiazepines were developed as potent and selective antagonists of cholecystokinin-B re- ceptors (Evans et al., 1987). A side effect of some of these compounds was prolongation of QT interval caused by block of I Ks channels in the heart. Optimization of I Ks block by chemical modification of the cholecystokinin-B antago- nists led to the discovery of L-7 (Fig. 1) and related com- pounds (Selnick et al., 1997). Selective blockers of I Ks were initially developed as class III antiarrhythmic drugs (Busch et al., 1996; Salata et al., 1996) because of their ability to prolong ventricular refractoriness. Other poten- tial therapeutic applications for KCNQ1 blockers include peptic ulcer disease or diarrhea (Grahammer et al., 2001). However, the subsequent finding that mutations in either KCNQ1 (Wang et al., 1996) or KCNE1 (Splawski et al., 1997a,b) cause inherited LQTS understandably dimin- ished interest for development of I Ks blockers as antiar- rhythmic agents. An understanding of the molecular fea- tures of the L-7 binding site of KCNQ1 might facilitate the design of newer drugs that block the channel in a fashion that has less inherent proarrhythmic risk. The goal of this study was to determine the structural basis for functional interaction of L-7 with the KCNQ1 channel. We constructed KCNQ1/KCNQ2 chimera channels to iden- tify important domains of KCNQ1 that mediate block. This was followed by site-directed mutagenesis of the identified domains to determine whether specific residues might mod- This work was supported by National Heart, Lung, and Blood Institute grant HL55236 (to M.C.S.) and Deutcshe Forschungsgemeinschaft fellowship SE 1077/1-1 (to G.S.). ABBREVIATIONS: L-7, L-735821 ((E)-(+)-N-[(3R)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3-(2,4-dichlorophe- nyl)-2-propenamide); MES, 2-[N-morpholino]ethanesulfonic acid; I Ks , slow delayed rectifier K + current; wt, wild type 0026-895X/03/6401-70 –77$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 1 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2310/1074630 Mol Pharmacol 64:70–77, 2003 Printed in U.S.A. 70 at ASPET Journals on November 7, 2016 molpharm.aspetjournals.org Downloaded from