MOLECULAR DETERMINANTS OF CARDIAC CA2- SUBUNIT REQUIREMENT FOR THE HIGH-AFFINITY AND ALLOSTERIC REGULATION OF DIHYDROPYRIDINE BINDING( CHANNEL PHARMACOLOGY )

Cardiac L-type Ca2+ channels are multisubunit complexes composed of al pha(1C), alpha(2) delta, and beta(2) subunits, We tested the roles of these subunits in forming a functional complex by characterizing the e ffects of subunit composition on dihydropyridine binding, its alloster ic regulation, and the ability of dihydropyridines to inhibit channel activity, Transfection of COS.M6 cells with cardiac alpha(1C-a) (alpha (1)) led to the appearance of dihydropyridine ([H-3]PN200-110) binding which was increased by coexpression of cardiac beta(2a) (beta), alpha (2) delta(a) (alpha(2)), and the skeletal muscle gamma. Maximum bindin g was achieved when cells expressed alpha(1), beta, and alpha(2). Cell s transfected with alpha(1) and beta had a binding affinity that was 5 -10-fold lower than that observed in cardiac membranes. Coexpression o f alpha(2) normalized this affinity, (-)-D600 and diltiazem both parti ally inhibited PN200-110 binding to cardiac microsomes, but stimulated binding in cells transfected with alpha(1) and beta. Again, coexpress ion of alpha(2) normalized this allosteric regulation. Therefore coexp ression of alpha(1) beta and alpha(2) completely reconstituted high af finity dihydropyridine binding and its allosteric regulation as observ ed in cardiac membranes, Skeletal muscle gamma was not required for th is reconstitution, Expression in Xenopus oocytes demonstrated that coe xpression of alpha(2) with alpha(1) beta increased the potency and max imum extent of block of Ca2+ channel currents by nisoldipine, a dihydr opyridine Ca2+ channel antagonist. Our results demonstrate that alpha( 2) subunits are essential components of the cardiac L-type Ca2+ channe l and predict a minimum subunit composition of alpha(1C)beta(2) alpha( 2) delta for this channel.