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Proteolytic cleavage and PKA phosphorylation of ?1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

ABSTRACT: Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the ?-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of ?-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in ?1C and C-terminal proteolytic cleavage of the ?1C subunit. We generated transgenic mice expressing an ?1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for ?-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute ?-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse ?1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant ?1C Prevention of C-terminal cleavage did not alter ?-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in ?1C in ?-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on ?1C are not redundant and do not each fractionally contribute to the net stimulatory effect of ?-adrenergic stimulation. Further, proteolytic cleavage of ?1C is not required for ?-adrenergic stimulation of CaV1.2.

SUBMITTER: Katchman A

PROVIDER: S-EPMC5576811 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

Katchman Alexander A Yang Lin L Zakharov Sergey I SI Kushner Jared J Abrams Jeffrey J Chen Bi-Xing BX Liu Guoxia G Pitt Geoffrey S GS Colecraft Henry M HM Marx Steven O SO

Proceedings of the National Academy of Sciences of the United States of America 20170807 34

Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We gene ...[more]

PMID: 28784807

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Project description:Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaV?1, CaV?, and CaV?2?1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaV?2?1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaV?2?1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaV?2?1 ?1059-1063) impaired current up-regulation. CaV?2?1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaV?2?1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaV?2?1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaV?2?1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaV?2?1 G1060I and CaV?2?1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaV?2?1 ?1059-1063, but not CaV?2?1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaV?2?1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaV?2?1 averts its cell-surface expression, its interaction with CaV?1, and modulation of CaV1.2 currents.

Dataset Information

Proteolytic cleavage and PKA phosphorylation of ?1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

Publications

Proteolytic cleavage and PKA phosphorylation of α<sub>1C</sub> subunit are not required for adrenergic regulation of Ca<sub>V</sub>1.2 in the heart.

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