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

ABSTRACT: Phosphorylation of the cardiac ? subunit (Ca(v)?(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)?(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ?Stop mouse). This mutation prevented translation of the Ca(v)?(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ?Stop 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 ?-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ?Stop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SA?Stop) or S1512A and S1570A (SF?Stop) in the C terminus of the ?(1C) subunit. The ?-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 ?-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)?(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.

SUBMITTER: Brandmayr J

PROVIDER: S-EPMC3391128 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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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 Ca(v)1.2 channel.

Brandmayr Julia J Poomvanicha Montatip M Domes Katrin K Ding Jie J Blaich Anne A Wegener Jörg W JW Moosmang Sven S Hofmann Franz F

The Journal of biological chemistry 20120515 27

Phosphorylation of the cardiac β subunit (Ca(v)β(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)β(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; βStop mouse). This mutation prevented translation of th ...[more]

PMID: 22589548

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Project description:RationaleChanging activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic.ObjectiveThe primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure.Methods and resultsWe generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVβ to α1C I-II loop and eliminated β-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β2B and Rad or transgenically expressed in cardiomyocytes.ConclusionsCa2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVβ binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α1C.

Dataset Information

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

Publications

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 Ca(v)1.2 channel.

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