Deletion of the C-terminal phosphorylation sites in the cardiac ß-subunit does not affect the basic ß-adrenergic response of the heart and the Ca(v)1.2 channel.

Phosphorylation of the cardiac ß subunit (Ca(v)ß(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)ß(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ßStop mouse). This mutation prevented translation of the Ca(v)ß(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ßStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the ß-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ßStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAßStop) or S1512A and S1570A (SFßStop) in the C terminus of the α(1C) subunit. The ß-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished ß-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)ß(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.

Truncation of murine CaV1.2 at Asp-1904 results in heart failure after birth.

The carboxyl-terminal intracellular tail of the L-type Ca(2+) channel CaV1.2 modulates various aspects of channel activity.For example, deletion of the carboxyl-terminal sequence at Ser-1905 increased CaV1.2 currents in an expression model. To verify this finding in an animal model, we inserted three stop codons at the corresponding Asp-1904 in the murine CaV1.2 gene. Mice homozygous for the Stop mutation (Stop/Stop mice)were born at a Mendelian ratio but died after birth. Stop/Stop hearts showed reduced beating frequencies and contractions.Surprisingly, Stop/Stop cardiomyocytes displayed reduced IBa and a minor expression of the CaV1.2Stop protein. In contrast,expression of the CaV1.2Stop protein was normal in pooled smooth muscle samples from Stop/Stop embryos. As the CaV1.2 channel exists in a cardiac and smooth muscle splice variant, HK1 and LK1, respectively, we analyzed the consequences of the deletion of the carboxyl terminus in the respective splice variant using the rabbit CaV1.2 clone expressed in HEK293 cells.HEK293 cells transfected with the HK1Stop channel showed a reduced IBa and CaV1.2 expression. Treatment with proteasome inhibitors increased the expression of HK1Stop protein and IBa in HEK293 cells and in Stop/Stop cardiomyocytes indicating that truncation of CaV1.2 containing the cardiac exon 1a amino terminus results in proteasomal degradation of the translated protein. In contrast, HEK293 cells transfected with the LK1Stop channel had normal IBa and CaV1.2 expression. These findings indicate that absence of the carboxyl-terminal tail differentially determines the fate of the cardiac and smooth muscle splice variant of the CaV1.2 channel in the mouse.