Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response.
L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by β-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The β-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by β-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.
Department of Pharmacology, University of Washington, Seattle, WA 98195-7280.
Ying
Y
Fu
Ruth E
RE
Westenbroek
Todd
T
Scheuer
William A
WA
Catterall
D000704:Analysis of Variance; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D047390:Casein Kinase II / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology*; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008817:Mice, Mutant Strains; D008958:Models, Molecular; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation
D020746:Calcium Channels, L-Type; D047390:Casein Kinase II; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D002118:Calcium