β-Adrenergic Regulation of the L-type Ca2+ Channel Does Not Require Phosphorylation of α1C Ser1700
Rationale: Sympathetic nervous system triggered activation of protein kinase A (PKA), which phosphorylates several targets within cardiomyocytes, augments inotropy, chronotropy and lusitopy. An important target of β-adrenergic stimulation is the sarcolemmal L-type Ca2+ 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 Ala1800 and PKA phosphorylation of Ser1700 are required for β-adrenergic modulation of CaV1.2.
Objective: To assess the role of Ala1800 in the cleavage of α1C and the role of Ser1700 and Thr1704 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 Ser1700 or Thr1704 of the α1C subunit. The presence of Ala1800 and the 1798NNAN1801 motif in α1C are 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 PKA phosphorylation of α1C Ser1700 does not have a major role in the sympathetic stimulation of Ca2+ 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 and cost-effective manner.
- molecular electrophysiology
- excitation-contraction coupling
- transgenic mice
- ion channel
- calcium channel
- sympathetic nervous system
- Received May 30, 2013.
- Revision received June 30, 2013.
- Accepted July 3, 2013.