Deletion of the C-terminal Phosphorylation Sites in the Cardiac β-Subunit Does Not Affect the Basic β-Adrenergic Response of the Heart and the Cav1.2 Channel

Phosphorylation of the cardiac β subunit (Cavβ2) of the Cav1.2 L-type Ca2+ channel complex has been proposed as a mechanism for regulation of L-type Ca2+ 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 Cavβ2 gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Cavβ2 C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type ICa. The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Cav1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α1C subunit. The β-adrenergic regulation of the cardiac ICa was unaltered in these mouse lines. In contrast, truncation of the Cav1.2 at Asp1904 abolished β-adrenergic up-regulation of ICa in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Cavβ2, Ser1928, Ser1512, and Ser1570 of the Cav1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Cav1.2 channel.Background: β-Adrenergic receptors stimulate cardiac ICa via PKA-dependent phosphorylation.Results: Deletion of the C-terminal phosphorylation sites in the β2 gene did not affect isoproterenol-stimulated ICa.Conclusion: Phosphorylation of the C terminus of the β2 subunit in vivo does not contribute to β-adrenergic regulation of ICa.Significance: The PKA-dependent regulation of ICa does not require the C terminus of the β2 subunit. Phosphorylation of the cardiac β subunit (Cavβ2) of the Cav1.2 L-type Ca2+ channel complex has been proposed as a mechanism for regulation of L-type Ca2+ 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 Cavβ2 gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of the Cavβ2 C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac βStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type ICa. The regulation of the L-type current by stimulation of the β-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). βStop mice were cross-bred with mice expressing the Cav1.2 gene containing the mutation S1928A (SAβStop) or S1512A and S1570A (SFβStop) in the C terminus of the α1C subunit. The β-adrenergic regulation of the cardiac ICa was unaltered in these mouse lines. In contrast, truncation of the Cav1.2 at Asp1904 abolished β-adrenergic up-regulation of ICa in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Cavβ2, Ser1928, Ser1512, and Ser1570 of the Cav1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Cav1.2 channel. Background: β-Adrenergic receptors stimulate cardiac ICa via PKA-dependent phosphorylation. Results: Deletion of the C-terminal phosphorylation sites in the β2 gene did not affect isoproterenol-stimulated ICa. Conclusion: Phosphorylation of the C terminus of the β2 subunit in vivo does not contribute to β-adrenergic regulation of ICa. Significance: The PKA-dependent regulation of ICa does not require the C terminus of the β2 subunit.