title: CaMKII Phosphorylation of Na(V)1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure.
abstract: The cardiac voltage-gated sodium channel, Na(V)1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca(2+))/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of Na(V)1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human Na(V)1.5 has not been systematically analyzed and Na(V)1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human Na(V)1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC-MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of Na(V)1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human Na(V)1.5.
authors: †Department of Pharmacology, University of California Davis, Genome Building 3513, Davis, California 95616, United States.
authors: Anthony W
authors: AW
authors: Herren
authors: §UC Davis Genome Center, University of California Davis, 451 Health Science Drive, Davis, California 95616, United States.
authors: Darren M
authors: DM
authors: Weber
authors: †Department of Pharmacology, University of California Davis, Genome Building 3513, Davis, California 95616, United States.
authors: Robert R
authors: RR
authors: Rigor
authors: ∥Perelman School of Medicine, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104, United States.
authors: Kenneth B
authors: KB
authors: Margulies
authors: §UC Davis Genome Center, University of California Davis, 451 Health Science Drive, Davis, California 95616, United States.
authors: Brett S
authors: BS
authors: Phinney
authors: †Department of Pharmacology, University of California Davis, Genome Building 3513, Davis, California 95616, United States.
authors: Donald M
authors: DM
authors: Bers
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006352:Heart Ventricles / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; D011994:Recombinant Proteins; C568320:SCN5A protein, human; D012694:Serine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: CaMKII; Na channel; NaV1.5; SCN5A; arrhythmia; cardiac; heart failure; mass spectrometry; phosphorylation; sodium