title: Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700.
abstract: L-type calcium (Ca(2+)) currents conducted by voltage-gated Ca(2+) channel CaV1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca(2+) entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700-Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3-4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca(2+) current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280.
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Westenbroek
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280.
authors.forename: Todd
authors.initials: T
authors.lastname: Scheuer
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280 wcatt@uw.edu.
authors.forename: William A
authors.initials: WA
authors.lastname: Catterall
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D019943:Amino Acid Substitution; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D047390:Casein Kinase II / Q000378:metabolism; D004095:Dihydropyridines / Q000494:pharmacology; D017079:Exercise Tolerance / Q000235:genetics / Q000502:physiology; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D017136:Ion Transport / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008958:Models, Molecular; D020125:Mutation, Missense; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology; D032383:Myocytes, Cardiac / Q000502:physiology*; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011943:Receptors, Adrenergic, beta / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D000318:Adrenergic beta-Agonists; C514350:CACNA1C protein, mouse; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D011943:Receptors, Adrenergic, beta; D010768:Phosphoserine; D001464:Barium; C038806:1,4-dihydropyridine; D047390:Casein Kinase II; D007545:Isoproterenol; D002118:Calcium
keywords: cAMP; calcium channel; cardiac hypertrophy; protein kinase; β-adrenergic