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 cDNA clones coding for isoforms of the beta subunit of voltage-dependent Ca2+ channels. The two mouse brain beta 2 subunit cDNA clones described, beta 2a and beta 2b, differed by alternative splicing within the coding region but possessed a unique amino terminus not yet reported in other beta 2 subunit cDNAs. Northern blot and RNase protection analyses demonstrated that both mRNA isoforms could be detected in highest abundance in heart and brain and at lower levels in lung, kidney, and testis. In a novel assay for beta 2 subunit function, COS-1 cells were transfected with alpha 1 and beta 2 subunit expression vectors and assayed for increases in intracellular Ca2+ concentration by using fura-2 imaging. Co-transfection of COS-1 cells with the mouse brain class C-1 alpha 1 subunit expression vector and either of the beta 2 subunit expression vectors resulted in increases in intracellular Ca2+ concentration after stimulation with elevated K+ and the dihydropyridine agonist Bay K 8644. Transfection of either alpha 1 or beta 2 subunit expression vectors alone did not result in an elevation of intracellular Ca2+ concentration. Electrophysiological recording of human embryonic kidney 293 cells transfected with the expression vector for the alpha 1 subunit alone or with either beta 2 subunit demonstrated expression of voltage-dependent Ca2+ channels that were dihydropyridine sensitive. Currents formed by expression of only the alpha 1 subunit were small and slowly inactivated. In contrast, the currents formed by coexpression of alpha 1 subunits with either beta 2 subunit were larger and inactivated more rapidly. Dihydropyridine binding studies demonstrated that coexpression of alpha 1 subunits with beta 2 subunits increased the density of functional receptors, compared with expression of alpha 1 subunits alone. These experiments suggested that coexpression of the alpha 1 and beta 2 subunits produced functional dihydropyridine-sensitive Ca2+ channels and that both beta subunit isoforms had modulatory effects on these channels.
Massa
E
E
Graduate Program in Neuroscience, University of Michigan, Ann Arbor 48109-0720, USA.
Kelly
K M
KM
Yule
D I
DI
MacDonald
R L
RL
Uhler
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000032:analysis* / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000302:isolation & purification; D016257:Fura-2; D017275:Isradipine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D012260:Ribonucleases / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D018076:DNA, Complementary; D012260:Ribonucleases; D002118:Calcium; D016257:Fura-2; D017275:Isradipine