0279: Phosphoproteomic identification of CAMKII- and SGK1-dependent phosphorylation sites on the native cardiac NAV1.5 channel protein in heart failure

Voltage-gated Na+ (Nav) channels are key determinants of myocardial excitability and defects in Nav channel functioning or regulation, associated with inherited and acquired cardiac disease, increase the risk of life-threatening arrhythmias. In heart failure, the inactivation gating properties of Nav1.5 channels are altered, resulting in decreased channel availability and increased late Na+ current. Although previous studies have suggested roles for CaMKII- and SGK1-dependent Nav1.5 phosphorylation sites, the global native phosphorylation pattern of Nav1.5 channels associated with these pathophysiological alterations is unknown. Mass spectrometric (MS)-based phosphoproteomic analyses were undertaken to identify in situ the native phosphorylation sites on the Nav1.5 protein purified from ventricles isolated from WT and CaMKIIdc- Tg mice overexpressing CaMKIIdc in the heart. Quantitative analyses of Nav1.5 phosphopeptides allowed comparing the relative abundances of Nav1.5 phosphorylation sites in CaMKIIdc-Tg, versus WT, ventricles. A total of seventeen Nav1.5 phosphorylation sites were identified, seven of which are novel as compared with those reported in our previous MS analyses. Thirteen of these sites are located in the first loop, one in the second loop, one in the N-terminus and two in the Cterminus of Nav1.5. Interestingly, out of these seventeen phosphorylation sites, the C-terminal phosphoserine pS1938 is present in the CaMKIIdc-Tg IPs (n=3/4) and absent in the WT IPs (n=0/4), and pS1989 is 9-fold more represented (p