Cloning and expression of the Ca2+ channel alpha1C and beta2a subunits from guinea pig heart.
Complimentary DNA clones encoding the alpha1C and beta2a subunits of guinea-pig cardiac L-type Ca2+ channels were isolated using the PCR method. The open reading frame encoded 2,169 amino acids for the alpha1C and 597 amino acids for the beta2a subunit. The proteins showed 94.2 and 94.8%, respectively, identity to the respective subunit of the rabbit protein. The message size of the guinea pig alpha1C and beta2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the alpha1C subunit is expressed exclusively in the heart, while the beta2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The alpha1C and beta2a subunits are transiently expressed in BHK (baby hamster kidney) cells, and the channel currents were studied using the whole-cell patch clamp technique in medium containing 30 mM Ba2+. In cells expressing alpha1C alone, the Ba2+ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of beta2a with alpha1C did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig alpha1C and rabbit beta1+alpha2/delta, a Ba2+ current comparable to those in native myocytes was observed. The Ba2+ current can be blocked completely by nifedipine and is enhanced 3-fold by Bay K 8644. On the other hand, neither forskolin nor okadaic acid affects the Ba2+ current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.
Ding
S
S
Department of Physiology Kurume University, Kurume, 839-0861, Japan.
Kuroki
S
S
Kameyama
A
A
Yoshimura
A
A
Kameyama
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D015220:Calcium Channels; D017931:DNA Primers; D018076:DNA, Complementary