Inhibition of recombinant Ca2+ 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 Ca2+ channels, we systematically characterized phenylalkylamine and benzothiazepine inhibition of three molecular classes of Ca2+ channels (alpha(1C), alpha(1A), and alpha(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 mu M for alpha(1A), 100 mu M for alpha(1E), and 110 mu M for alpha(1C)), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of alpha(1C) channels; alpha(1A) and alpha(1E) channels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 mu M for alpha(1C), 220 mu M for alpha(1E), and 270 mu M for alpha(1E)). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from alpha(1C) and alpha(1A) and localized the structural determinants for state dependence to repeats III and IV of alpha(1C), which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant alpha(1C) construct by changing three amino acids in IVS6 (Y14901, 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 similar to 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.