Cloning and Expression of the Ca super(2+) Channel alpha sub(1C) and beta sub(2a) Subunits from Guinea Pig Heart

Complimentary DNA clones encoding the alpha sub(1C) and beta sub(2a) subunits of guinea-pig cardiac L-type Ca super(2+) channels were isolated using the PCR method. The open reading frame encoded 2,169 amino acids for the alpha sub(1C) and 597 amino acids for the beta sub(2a) 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 alpha sub(1C) and beta sub(2a) subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the alpha sub(1C) subunit is expressed exclusively in the heart, while the beta sub(2a) subunit is expressed in the heart, cerebellum, whole brain, and stomach. The alpha sub(1C) and beta sub(2a) 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 Ba super(2+). In cells expressing alpha sub(1C) alone, the Ba super(2+) current was activated at-30 mV and more positive potentials and peaked at about 10 mV. The co-expression of beta sub(2a) with alpha sub(1C) did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig alpha sub(1C) and rabbit beta sub(1)+ alpha sub(2)/ delta , a Ba super(2+) current comparable to those in native myocytes was observed. The Ba super(2+) 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 Ba super(2+) current, suggesting that cAMP-mediated modulation is not easily reproduced in transfected cells, unlike that seen in native cardiac myocytes.