Abstract
Two new short splice variants of the Ca2+ channel beta2 subunit were cloned from human heart poly(A)(+) mRNA. The 410-amino acid beta2f subunit is encoded by exons 1A, 2A, 3, 4, 12, 13, and 14 of the human Cavbeta2 gene and lacks the protein kinase A phosphorylation site, the beta-interaction domain (De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495-503), 40% of the beta-SH3 domain, and 73% of the guanylate kinase domain of the putative membrane-associated guanylate kinases module (McGee, A. W., Nunziato, D. A., Maltez, J. M., Prehoda, K. E., Pitt, G. S., and Bredt, D. S. (2004) Neuron 42, 89-99), and helix alpha3 of the alpha1-subunit binding pocket (Van Petegem F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Nature 429, 671-675). The beta2g transcript has two potential initiation codons. With the second ATG codon, it generates the 164-amino acid beta2Deltag subunit encoded essentially by the distal part of exon 14, and thus beta2Deltag completely lacks any of the above motifs. Immunoprecipitation analysis confirmed stable association of beta2f and beta2Deltag with the alpha1C subunit. The plasma membrane localization of beta2f and beta2Deltag was substantially increased by co-expression of the alpha1C,77 and alpha2delta subunits. In COS1 cells, beta2f and beta2Deltag increased plasma membrane targeting of the pore-forming alpha1C subunit and differentially facilitated (beta2f > beta2Deltag) the voltage gating of otherwise silent Cav1.2 channels. We conclude that it is unlikely that the beta-interaction domain, membrane-associated guanylate kinases module, and the alpha1-subunit binding pocket helix alpha3 are essential for the interaction of the alpha1C and beta2 subunits and suggest that in addition to the alpha1-subunit binding pocket helices alpha5 and alpha8, a yet unresolved C-terminal beta2 region plays a crucial role.
Highlights
Two new short splice variants of the Ca2؉ channel 2 subunit were cloned from human heart poly(A)(؉) mRNA
The most interesting result of this study is that vast deletions in the central region of the 2 subunit that include most of or the entire Src homology 3 (SH3)-guanylate kinase (GK) region do not compromise the -subunit modulation of the Ca2ϩ channel
It is unlikely that the small parts of the SH3 and GK domains remaining in the 2f subunit are sufficient to support their cross-interaction, because the distal loop and strand E of the conserved domain I implemented in this interaction [12] are both absent from 2f
Summary
Two new short splice variants of the Ca2؉ channel 2 subunit were cloned from human heart poly(A)(؉) mRNA. Structural principles underlying the -subunit modulation of Ca2ϩ channels were approached by a search of structural homology with known regulatory proteins It has been found [9] that the vast central conservative region of 2 subunits shares distant homology with the Src homology 3 (SH3)-guanylate kinase (GK) module of membrane-associated guanylate kinases (MAGUKs). This hypothesis was further elaborated by studying mutations of the rat (N terminus-palmitoylated) 2a subunit that interfere with interactions between SH3 and GK and affect inactivation of Ba2ϩ currents [10, 11]. The 2f and 2g transcripts lack exons encoding a single protein kinase A (PKA) phospho-
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