Voltage-Dependent Profile of Human Ether-a-go-go-Related Gene Channel Block Is Influenced by a Single Residue in the S6 Transmembrane Domain JOSE A. S ˘ ANCHEZ-CHAPULA, TANIA FERRER, RICARDO A. NAVARRO-POLANCO, and MICHAEL C. SANGUINETTI Unidad de Investigacio ´ n “Carlos Me ´ ndez” del Centro Universitario de Investigaciones Biome ´ dicas de la Universidad de Colima, Colima, Me ´ xico (J.A.S.-C., T.F., R.A.N.-P.); and Department of Physiology and Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah (M.C.S.) Received November 13, 2002; accepted January 23, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Many common medications block delayed rectifier K + channels and prolong the duration of cardiac action potentials. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) delayed rectifier K + channels expressed in Xenopus laevis oocytes by quinidine, an antiarrhythmic drug, and vesnarinone, a car- diotonic drug. The IC 50 values determined with voltage-clamp pulses to 0 mV were 4.6 M and 57 M for quinidine and quinine, respectively. Block of HERG by quinidine (and its iso- mer quinine) was enhanced by progressive membrane depo- larization and accompanied by a negative shift in the voltage dependence of channel activation. As reported previously for other HERG blockers (e.g., MK-499, cisapride, terfenadine, chloroquine), the potency of quinidine was reduced 100-fold by the mutation of key aromatic residues (Y652, F656) located in the S6 domain. Mutations of Y652 eliminated (Y652F) or reversed (Y652A) the voltage dependence of HERG channel block by quinidine and quinine. These quinolines contain a charged N atom that might bond with Y652 by a cation- interaction. However, similar changes in the voltage-dependent profile for block of Y652F or Y652A HERG channels were observed with vesnarinone, a cardiotonic drug that is un- charged at physiological pH. Together, these results suggest that voltage-dependent block of HERG results from gating- dependent changes in the orientation of Y652, a critical com- ponent of the drug binding site, and not from a transmembrane field effect on a charged drug molecule. HERG (Warmke and Ganetzky, 1994) encodes the pore- forming  subunits of channels that conduct the rapid de- layed rectifier K + current I Kr (Sanguinetti et al., 1995; Trudeau et al., 1995). Blockers of I Kr were developed to treat arrhythmia, but unintended block of HERG K + channels can also be proarrhythmic and is a serious side effect for many otherwise clinically useful drugs. Previously, we used site- directed mutagenesis and voltage clamp of mutant channels expressed in Xenopus laevis oocytes to elucidate the molecu- lar mechanisms of HERG channel block by structurally di- verse drugs, including MK-499, cisapride, and terfenadine (Mitcheson et al., 2000). These studies identified two aro- matic residues (Y652 and F656) in the S6 domain of the HERG channel subunit that are critical for high-affinity binding of drugs. In contrast to high-affinity ligands, low- affinity block of HERG by chloroquine is voltage-dependent (Sanchez-Chapula et al., 2001), with an enhanced block in response to increasing membrane depolarization. The volt- age-dependent profile for block by chloroquine can be re- versed by mutation of Y652 to Ala, whereas mutation to Phe eliminates the voltage dependence of the block (Sanchez- Chapula et al., 2002). These findings suggest that interaction of chloroquine with the phenol of Y652 mediates voltage- dependent block of WT HERG channels. Like chloroquine, micromolar concentrations of quinidine are required for the block of HERG channels expressed in oocytes. Both drugs are substituted quinolines; however, chloroquine has two positively charged alkylamines with pK a values of 8.4 and 10.8, whereas quinidine has a single ter- tiary N in a quinuclidine group with a pK a of 8.6. These N atoms are predominantly protonated at physiological pH and conceivably could mediate cation- interaction with Y652. Here, we also examined the effects of the uncharged drug vesnarinone on Y652A and Y652F HERG channels to deter- mine whether a drug must possess an ionizable N atom to block HERG channels in a voltage-dependent manner. This work was supported by a grant from Abbott Laboratories, grant TW001211 from Fogarty International Research Collaboration, grant HL55236 from National Institutes of Health/NHLBI, and grant 34954-M from CONACyT (Mexico). ABBREVIATIONS: HERG, human ether-a-go-go-related gene; WT, wild type; Mes, 2-(N-morpholino)ethanesulfonic acid; MK-499, [(+)-N-[1'-(6- cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro (2H-1-benzpyran-2,4'-piperidin)-6-yl]methane sulfonamide]-HCl. 0026-895X/03/6305-1051–1058$7.00 MOLECULAR PHARMACOLOGY Vol. 63, No. 5 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2283/1057194 Mol Pharmacol 63:1051–1058, 2003 Printed in U.S.A. 1051 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from