INHIBITION OF RECOMBINANT CA2- CLASS-SPECIFIC PHARMACOLOGY AND UNDERLYING MOLECULAR DETERMINANTS( CHANNELS BY BENZOTHIAZEPINES AND PHENYLALKYLAMINES )

To understand the molecular basis of state-dependent pharmacological b lockade of voltage-gated Ca2+ channels, we systematically characterize d phenylalkylamine and benzothiazepine inhibition of three molecular c lasses of Ca2+ channels (alpha(1C), alpha(1A), and alpha(1E) expressed from cDNA clones transfected into HEK 293 cells. State-dependent bloc kade figures importantly in the therapeutically desirable property of use-dependent drug action. Verapamil (a phenylalkylamine) and diltiaze m (a benzothiazepine) were imperfectly selective, so differences in th e state dependence of inhibition could be compared among the various c hannels. We found only quantitative differences in pharmacological pro file 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-indepe ndent 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 fr om alpha(1C) and alpha(1A) and localized the structural determinants f or state dependence to repeats III and IV of alpha(1C), which have bee n found to contain the structures required for benzothiazepine binding . We then constructed a mutant alpha(1C) construct by changing three a mino acids in IVS6 (Y14901, A1494S, I1497M) that have been implicated as key coordinating sites for avid benzothiazepine binding. Although t hese 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 phys ically distinct elements controlling drug binding and access to the bi nding site, thereby favoring a ''guarded-receptor'' rather than a ''mo dulated-receptor'' mechanism of drug inhibition.