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

To understand the molecular basis of state-dependent pharmacological blockade of voltage-gated Ca channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca channels (a1C, a1A, and a1E) 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 mM for a1A, 100 mM for a1E, and 110 mM for a1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only statedependent block of a1C channels; a1A and a1E channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 mM for a1C, 220 mM for a1E, and 270 mM for a1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from a1C and a1A and localized the structural determinants for state dependence to repeats III and IV of a1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant a1C 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 “guardedreceptor” rather than a “modulated-receptor” mechanism of drug inhibition.