Cloning and Expression of the Ca²⁺ Channel α_1c and β_2a Subunits from Guinea Pig Heart

Abstract

Complimentary DNA clones encoding the α_1c and β_2a 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 a, c and 597 amino acids for the α_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 α_1c and β_2a subunits was 8.0 and 3.5/4.0 kb, respectively. RT-PCR analysis revealed that the α_1c subunit is expressed exclusively in the heart, while the β_2a subunit is expressed in the heart, cerebellum, whole brain, and stomach. The α_1c and β_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 30mM Ba²⁺. In cells expressing α_1c alone, the Ba²⁺ current was activated at -30 mV and more positive potentials and peaked at about 10 mV. The co-expression of β_2a with α_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 α_1c 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.