ORIGINAL ARTICLE Block of hERG Channels by Berberine Mechanisms of Voltage- and State-Dependence Probed With Site-Directed Mutant Channels Aldo Rodriguez-Menchaca, MSc,* Tania Ferrer-Villada, PhD,* Jesus Lara, MD, PhD,* David Fernandez, PhD,† Ricardo A. Navarro-Polanco, PhD,* and Jose A. Sanchez-Chapula, MD, PhD* Abstract: Berberine prolongs the duration of cardiac action po- tentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, I Kr and I Ks . Here we have studied the effects of berberine on hERG (I Kr ) and KCNQ1/KCNE1 (I Ks ) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC 50 for berberine was 3.1 6 0.5 mM on hERG compared with 11 6 4% decreases on KCNQ1/KCNE1 channels by 100 mM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC 50 = 80 6 5 mM) compared with KCNQ1/KCNE1 channels (;20% block at 300 mM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by ber- berine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists. Key Words: berberine, HEK-293, hERG, KCNQ1/KCNE1, I Kr ,I Ks , oocytes (J Cardiovasc Pharmacol TM 2006;47:21–29) B erberine is a benzodioxoquinolozine plant alkaloid iso- lated from several species of Berberis and Coptis. Various pharmacological actions have been described for berberine, including antimicrobial and cardiovascular effects. Both clin- ical and animal studies suggested that berberine prevented ischemia-induced ventricular tachyarrhythmia, enhanced the force of cardiac contraction, and decreased peripheral resis- tance and blood pressure. 1 Berberine prolongs action potential duration (APD) of cardiac muscle in several species, without affecting resting membrane potential or action potential amplitude. 2 In cat ventricular myocytes, the increase in APD was due to blockade of the rapid delayed rectifying potassium current, I Kr . 3 However, it was reported that in guinea-pig ventric- ular myocytes, berberine blocked the slow delayed rectifying outward potassium current, I Ks without affecting I Kr . 4 Human ether-a-go-go related gene (hERG) subunits coassemble to form channels that conduct I Kr . 5 KCNQ1 and KCNE1 (KvLQT1 + minK) subunits combine to form chan- nels that conduct I Ks . 6,7 We heterologously expressed these channels in HEK-293 cells and Xenopus oocytes to resolve the apparent controversy regarding the type of cardiac delayed rectifier K + channels affected by berberine. We also deter- mined if berberine preferentially blocked open or inactivated hERG channels because inactivation may induce conforma- tional changes within the inner cavity that maximize interaction with some drugs. 8 Finally, we determined if block was affected by mutation of specific residues (ie, Y652, F656) that are believed to be important for binding of many other drugs to hERG. 9 METHODS Xenopus Oocytes Wild type hERG, KCNQ1, and KCNE1 cRNAs were prepared in the pSP64 plasmid vector (Promega, Madison, WI). Point mutations were introduced into hERG (V625A, Y652A, F656T) in the pSP64 plasmid expression vector (Promega, Madison, WI) as described previously. 9 G628C/S631C hERG was a gift from Dr. Gea-Ny Tseng. Complementary RNAs for injection into oocytes were prepared with SP6 Cap- Scribe (Roche, Mexico, Mexico) following linearization of the expression construct with EcoRI. Isolation and maintenance of Xenopus oocytes, and cRNA injection were performed as described. 10 A GeneClamp 500 amplifier (Molecular Devices Corp) and standard 2 microelectrode voltage-clamp techniques 11 were used to record currents in oocytes. Currents were recorded at room temperature (22°C –24°C), 2 to 4 days after cRNA injection. Glass microelectrodes were filled with 3 M KCl, and their tips broken to obtain resistances of 0.5 to 1 MV. The external low Cl - solution contained: 96 mM Mes-Na (2-[morpholino]ethanesulfonic acid salt sodium), 2 mM Mes- K, 2 mM Mes 2 -Ca, 5 mM, HEPES, 1 mM MgCl 2 , adjusted to pH 7.6 with methanesulfonic acid. Voltage commands were Received for publication August 8, 2005; accepted October 4, 2005. From the *Unidad ‘‘Carlos Mendez’’ del Centro Universitario de Inves- tigaciones Biome ´dicas de la Universidad de Colima, Colima, Mexico; and †Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT. This work was supported by SEP-SESIC (Mexico) Grant No. 2003-06-001- 007 and Fondo Ramon Alvarez-Buylla (Universidad de Colima, Mexico). Reprints: Jose A. Sanchez-Chapula, CUIB, Universidad de Colima, Apdo. Postal 199, CP 28000, Colima, Col. Me ´xico (e-mail: sancheza@cgic.ucol.mx). Copyright Ó 2006 by Lippincott Williams & Wilkins J Cardiovasc Pharmacol ä  Volume 47, Number 1, January 2006 21 Copyright ' Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.