Molecular Pharmacology of the Amiloride Analog 3-Amino-6- chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)- amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a Pan Inhibitor of the Na + -Ca 2+ Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells Agnese Secondo, Anna Pannaccione, Pasquale Molinaro, Paolo Ambrosino, Pellegrino Lippiello, Alba Esposito, Maria Cantile, Priti R. Khatri, Daniela Melisi, Gianfranco Di Renzo, and Lucio Annunziato Division of Pharmacology, Department of Neuroscience, School of Medicine, “Federico II” University of Naples, Naples, Italy (A.S., A.P., P.M., P.A., P.L., A.E., M.C., P.R.K., G.D., L.A.); Fondazione IRCCS SDN, Naples, Italy (L.A.); and Department of Pharmaceutical and Toxicological Chemistry, Faculty of Pharmacy, “Federico II” University of Naples, Naples, Italy (D.M.) Received February 9, 2009; accepted July 13, 2009 ABSTRACT With the help of single-cell microflorimetry, 45 Ca 2+ radiotracer fluxes, and patch-clamp in whole-cell configuration, we exam- ined the effect of the amiloride derivative 3-amino-6-chloro-5- [(4-chloro-benzyl)amino]- N-[[(2,4-dimethylbenzyl)amino]- iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na + /Ca 2+ exchanger (NCX) and on several other membrane currents including voltage- and pH- sensitive ones. This amiloride analog suppressed the bidirec- tional activity of all NCX isoforms in a concentration-dependent manner. The IC 50 values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidi- rectional NCX1, NCX2, and NCX3 activity. Deletion mutagene- sis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the  2 repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma mem- brane currents encoded by voltage-dependent Ca 2+ channels, tetrodotoxin-sensitive Na + channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB- DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels. More than 40 years ago, amiloride and amiloride analogs were described by Cragoe and colleagues (1967), via a biolog- ical screen process, as potent inhibitors of sodium channels in urinary epithelium (ENaC), thus acting as a potassium-spar- ing diuretic. Amiloride and its analogs were subsequently shown to be inhibitors of other membrane transporters. In the same year that amiloride was synthesized, Baker and Blaustein (1968) functionally discovered the existence of the ubiquitous plasma membrane sodium/calcium exchanger (NCX). Amiloride, at very high concentrations, was found to be an effective inhibitor of NCX. Since then, amiloride has been used by numerous investigators as a probe to block NCX function (Sharikabad et al., 1997). Unfortunately, two major This work was supported by COFIN 2006; the “Ministero Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale Fondi Italia-Cina Legge 401/1990 2007, 2008”; Ministero della Salute, Ricerca San- itaria RF-FSL352059 Ricerca finalizzata 2006; Ministero della Salute, Ricerca Oncologica 2006; Ministero della Salute, Progetto Strategico, 2007; Ministero della Salute, Progetto Ordinario, 2007 (all to L.A.); Legge Regionale 5 28/03/ 2002 (to A.S.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.152132. ABBREVIATIONS: CB-DMB, 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino] iminomethyl]-pyrazinecarboxamide; DMSO, dimethyl sulfoxide; MES, 4-morpholineethanesulfonic acid; MTT, 3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide; NCX, Na + -Ca 2+ exchanger; [Ca 2+ ] i , intracellular calcium concentration; BHK, baby kidney hamster; ENaC, epithelial Na + channel; NHE, Na + /H + exchanger; TEA, tetraethylammonium; TTX, tetrodotoxin; VDCC, voltage-dependent calcium channel; SN6, 2-[4-(4-nitrobenzyloxy)benzyl]thiazo- lidine-4-carboxylic acid ethyl ester; KB-R7943, 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate; SEA0400, 2-[4-[(2,5- difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; NMDG, N-methyl-D-glucamine; YM-244769, N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]- phenoxy}nicotinamide. 0022-3565/09/3311-212–221$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 152132/3514700 JPET 331:212–221, 2009 Printed in U.S.A. 212 at ASPET Journals on June 14, 2016 jpet.aspetjournals.org Downloaded from