PT  - JOURNAL ARTICLE
AU  - Cai, Dongming
AU  - Mulle, Jennifer G.
AU  - Yue, David T.
TI  - Inhibition of Recombinant Ca<sup>2+</sup> Channels by Benzothiazepines and Phenylalkylamines: Class-Specific Pharmacology and Underlying Molecular Determinants
AID  - 10.1124/mol.51.5.872
DP  - 1997 May 01
TA  - Molecular Pharmacology
PG  - 872--881
VI  - 51
IP  - 5
4099  - http://molpharm.aspetjournals.org/content/51/5/872.short
4100  - http://molpharm.aspetjournals.org/content/51/5/872.full
SO  - Mol Pharmacol1997 May 01; 51
AB  - 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 (α1C, α1A, and α1E) 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 α1A, 100 μm for α1E, and 110 μm for α1C), and inhibition was state dependent in all channels. In contrast, diltiazem produced only state-dependent block of α1C channels; α1A and α1Echannels demonstrated state-independent block despite similar half-maximal inhibitory concentrations (60 μm for α1C, 220 μm for α1E, and 270 μm for α1A). To explore the molecular basis for the sharp distinction in state-dependent inhibition by diltiazem, we constructed chimeric channels from α1C and α1A and localized the structural determinants for state dependence to repeats III and IV of α1C, which have been found to contain the structures required for benzothiazepine binding. We then constructed a mutant α1C 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.