0026-895X/97/050872-10$3.00/0
Copyright © by The American Society for Pharmacology and Experimental Therapeutics
All rights of reproduction in any form reserved.
MOLECULAR PHARMACOLOGY 51:872-881 (1997).

Inhibition of Recombinant Ca²⁺ Channels by Benzothiazepines and Phenylalkylamines: Class-Specific Pharmacology and Underlying Molecular Determinants

Dongming Cai, Jennifer G. Mulle, and David T. Yue

Program in Molecular and Cellular System Physiology, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

To understand the molecular basis of state-dependent pharmacological blockade of voltage-gated Ca²⁺ channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca²⁺ channels (_1C, _1A, and _1E) expressed from cDNA clones transfected into HEK 293 cells. State-dependent blockade figures importantly in the therapeutically desirable property of use-dependent drug action. Verapamil (a phenylalkylamine) and diltiazem (a benzothiazepine) were imperfectly selective, so differences in the state dependence of inhibition could be compared among the various channels. We found only quantitative differences in pharmacological profile of verapamil: half-maximal inhibitory concentrations spanned a 2-fold range (70 µM for _1A, 100 µM for _1E, and 110 µM for _1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of _1C channels; _1A and _1E channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 µM for _1C, 220 µM for _1E, and 270 µM for _1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from _1C and _1A and localized the structural determinants for state dependence to repeats III and IV of _1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant _1C construct by changing three amino acids in IVS6 (Y1490I, A1494S, I1497M) that have been implicated as key coordinating sites for avid benzothiazepine binding. Although these mutations increased the half-maximal inhibitory concentration of diltiazem inhibition by ~10-fold, the state-dependent nature of inhibition was spared. This result points to the existence of physically distinct elements controlling drug binding and access to the binding site, thereby favoring a "guarded-receptor" rather than a "modulated-receptor" mechanism of drug inhibition.