CaMKII Phosphorylation of NaV1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure

• Published in: Journal of proteome research 2015-04, Vol.14 (5 p.2298-2311), p.2298-2311
• Main Authors: Herren, Anthony W, Weber, Darren M, Rigor, Robert R, Margulies, Kenneth B, Phinney, Brett S, Bers, Donald M
• Format: Article
• Language: English
• Subjects: action potentials; antibodies; arrhythmia; calcium; calcium-calmodulin-dependent protein kinase; heart failure; humans; mass spectrometry; phosphorylation; proteome; sodium channels
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.5b00107

The cardiac voltage-gated sodium channel, NaV1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca2+)/calmodulin­(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of NaV1.5 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 NaV1.5 has not been systematically analyzed and NaV1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human NaV1.5 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 NaV1.5 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 NaV1.5.