Dataset Information

β-adrenergic regulation of the L-type Ca2+ channel does not require phosphorylation of α1C Ser1700.

ABSTRACT:

Rationale

Sympathetic nervous system triggered activation of protein kinase A, which phosphorylates several targets within cardiomyocytes, augments inotropy, chronotropy, and lusitropy. An important target of β-adrenergic stimulation is the sarcolemmal L-type Ca(2+) channel, CaV1.2, which plays a key role in cardiac excitation-contraction coupling. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes, however, are incompletely known. Recently, it has been postulated that proteolytic cleavage at Ala(1800) and protein kinase A phosphorylation of Ser(1700) are required for β-adrenergic modulation of CaV1.2.

Objective

To assess the role of Ala(1800) in the cleavage of α1C and the role of Ser(1700) and Thr(1704) in mediating the adrenergic regulation of CaV1.2 in the heart.

Methods and results

Using a transgenic approach that enables selective and inducible expression in mice of FLAG-epitope-tagged, dihydropyridine-resistant CaV1.2 channels harboring mutations at key regulatory sites, we show that adrenergic regulation of CaV1.2 current and fractional shortening of cardiomyocytes do not require phosphorylation of either Ser(1700) or Thr(1704) of the α1C subunit. The presence of Ala(1800) and the (1798)NNAN(1801) motif in α1C is not required for proteolytic cleavage of the α1C C-terminus, and deletion of these residues did not perturb adrenergic modulation of CaV1.2 current.

Conclusions

These results show that protein kinase A phosphorylation of α1C Ser(1700) does not have a major role in the sympathetic stimulation of Ca(2+) current and contraction in the adult murine heart. Moreover, this new transgenic approach enables functional and reproducible screening of α1C mutants in freshly isolated adult cardiomyocytes in a reliable, timely, cost-effective manner.

SUBMITTER: Yang L

PROVIDER: S-EPMC3864014 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Publications

β-adrenergic regulation of the L-type Ca2+ channel does not require phosphorylation of α1C Ser1700.

Yang Lin L Katchman Alexander A Samad Tahmina T Morrow John J Weinberg Richard R Marx Steven O SO

Circulation research 20130703 7

<h4>Rationale</h4>Sympathetic nervous system triggered activation of protein kinase A, which phosphorylates several targets within cardiomyocytes, augments inotropy, chronotropy, and lusitropy. An important target of β-adrenergic stimulation is the sarcolemmal L-type Ca(2+) channel, CaV1.2, which plays a key role in cardiac excitation-contraction coupling. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes, however, are incompletely known. Recently, it has been postu ...[more]

PMID: 23825359

Similar Datasets

Project description:BackgroundType 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ.Methods and resultsPhosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation.ConclusionsIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

Dataset Information

β-adrenergic regulation of the L-type Ca2+ channel does not require phosphorylation of α1C Ser1700.

Rationale

Objective

Methods and results

Conclusions

Publications

β-adrenergic regulation of the L-type Ca2+ channel does not require phosphorylation of α1C Ser1700.

Similar Datasets

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