Roles of L-type Ca^(2+) Channel Subunits of Skeletal Muscle in Dihydropyridine Binding

Abstract

Purified skeletal muscle 1,4-dihydropyridine (DHP) receptors are composed of five polypeptides termed a1,b,¡,a2,and d. Among these, the a, subunit is known to be sufficient to function as a voltage-dependent Ca" channel and a DHP receptor. The a, alone exhibits very similar allosteric regulation of DHP binding to that found in skeletal muscle T-tubules. However, we previously showed that in the absence of Ca2+, DHP binding to a, alone at subsaturating concentrations was reduced, which could be restored by the (-) stereoisomer of a phenylalkylamine, D600. We hypothesized that this difference is due to lack of other regulatory subunits, specifically the (3, y and a26 components that copurify with a1. In order to test our hypothesis, we coexpressed the a1 subunit with non-a1 components in mouse fiboblasts, L cells, and monkey kidney cells, COS.M6 in various combinations .and compared their DHP binding activity for the effect of (-)D600 in the absence of Cat . Coexpression of b with a, did not normalize the abnormal enhancing effect of (-)D600 on DHP binding. Coexpression of either g or a2d partially reduced the enhancing effect of (-)D600. Importantly, coexpression of all the components, that is, when the receptor was composed of a1bga2d completely abrogated the abnormal effect of (-)D600. Thus, our data clearly demonstrate that all the component copurifying with a, are essential to constitute the functional DHP receptor.