Cloning and Expression of the Ca2+ Channel α1c and β2a Subunits from Guinea Pig Heart

• Published in: Journal of biochemistry (Tokyo) 1999-04, Vol.125 (4), p.750-759
• Main Authors: Ding, Shan, Kuroki, Sachiko, Kameyama, Asako, Yosbimura, Akihiko, Kameyama, Masaki
• Format: Article
• Language: English
• Subjects: Ca2+ channel; cloning; guinea pig heart; α1c subunit; β2 subunit
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a022346

Complimentary DNA clones encoding the α1c and β2a 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 α1c and 597 amino acids for the β2a, 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 α1c and β2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the α1c subunit is expressed exclusively in the heart, while the β2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The α1c and β2a 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 αc alone, the Ba2+ current was activated at −30 mV and more positive potentials and peaked at about 10 mV. The co-expression of β2a with αlc did not affect the voltage-dependence of the current, but increased the peak current and accelerated current decay. In cells transfected with guinea pig αlc and rabbit β1 + α2/δ, 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.