COMPARISON OF FURA-2 IMAGING AND ELECTROPHYSIOLOGICAL ANALYSIS OF MURINE CALCIUM-CHANNEL ALPHA-1 SUBUNITS COEXPRESSED WITH NOVEL BETA-2 SUBUNIT ISOFORMS

A polymerase chain reaction product was used to isolate mouse brain cD NA clones coding for isoforms of the beta subunit of voltage-dependent Ca2+ channels. The two mouse brain beta 2 subunit cDNA clones describ ed, beta 2a and beta 2b, differed by alternative splicing within the c oding region but possessed a unique amino terminus not yet reported in other beta 2 subunit cDNAs. Northern blot and RNase protection analys es demonstrated that both mRNA isoforms could be detected in highest a bundance in heart and brain and at lower levels in lung, kidney, and t estis. In a novel assay for beta 2 subunit function, COS-1 cells were transfected with alpha 1 and beta 2 subunit expression vectors and ass ayed for increases in intracellular Ca2+ concentration by using fura-2 imaging. Co-transfection of COS-1 cells with the mouse brain class C- l alpha 1 subunit expression vector and either of the beta 2 subunit e xpression vectors resulted in increases in intracellular Ca2+ concentr ation after stimulation with elevated K+ and the dihydropyridine agoni st Bay K 8644. Transfection of either alpha 1 or beta 2 subunit expres sion vectors alone did not result in an elevation of intracellular Ca2 + concentration. Electrophysiological recording of human embryonic kid ney 293 cells transfected with the expression vector for the alpha 1 s ubunit alone or with either beta 2 subunit demonstrated expression of voltage-dependent Ca2+ channels that were dihydropyridine sensitive. C urrents formed by expression of only the alpha 1 subunit were small an d slowly inactivated. In contrast, the currents formed by coexpression of alpha 1 subunits with either beta 2 subunit were larger and inacti vated more rapidly. Dihydropyridine binding studies demonstrated that coexpression of alpha 1 subunits with beta 2 subunits increased the de nsity of functional receptors, compared with expression of alpha 1 sub units alone. These experiments suggested that coexpression of the alph a 1 and beta 2 subunits produced functional dihydropyridine-sensitive Ca2+ channels and that both beta subunit isoforms had modulatory effec ts on these channels.