Cloning and Characterization of Alternatively Spliced Voltage-Gated Calcium Channel Subunits.

Abstract

Neuronal voltage gated Ca2+ channels are multimeric transmembrane protein structures that consist of at least four subunits, alpha 1, alpha2/delta, and beta. Homology screening of a human spinal cord cDNA library revealed that the alpha 1A and beta 4 subunit subtypes undergo alternative splicing. The pore-forming alpha 1A subunit splice variants differ in C-terminal exon composition. The alpha 1A-C2 variant possesses exon 37a and exon 44 while the alpha 1A-C16 variant has exon 37b but lacks exon 44. Expression studies in Xenopus oocytes in conjunction with beta subunit subtypes beta 1a, beta 1b, beta 3, and beta 4 demonstrated that the inclusion of exon 44 increased the rates of activation, inactivation, and recovery from inactivation of alpha 1A subunits while not affecting the voltage dependence of activation and inactivation.

Two alternatively spliced N-terminal variants of the beta 4 subunit (beta 4a and beta 4b) were isolated from human spinal cord. The novel beta 4a subunit has a 15 amino acid N-terminus while the beta 4b subunit, previously identified in brain, has a 49 amino acid N-terminus. Expression of alpha 1A/beta 4a or alpha 1A/beta 4b complexes in Xenopus oocytes revealed that compared to beta 4a, beta 4b left-shifts the voltage dependence of activation and inactivation, decreases the rate of fast inactivation, increases the rate of slow inactivation, and increases the sensitivity of alpha 1A subunits to the cone snail toxin omega-CTx-MVIIC. Site directed mutagenesis revealed that four amino acids (G10, D13, P15, and P18) of the N-terminus are responsible for conveying the effects of beta 4b on the alpha 1A subunit.