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 Exchanger Isoforms NCX1, NCX2, and NCX3 in Stably Transfected Cells

With the help of single-cell microflorimetry, Ca radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined 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 exchanger (NCX) and on several other membrane currents including voltageand pHsensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC50 values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis 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 membrane currents encoded by voltage-dependent Ca channels, tetrodotoxin-sensitive Na channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CBDMB, 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 biological screen process, as potent inhibitors of sodium channels in urinary epithelium (ENaC), thus acting as a potassium-sparing 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 401/199