Abstract 13658: Identification of Two Native Nav1.5 Phosphorylation Sites as Potential Determinants of the Increased Late Na+ Current 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 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 CaMKII-dependent Nav1.5 phosphorylation, the global native phosphorylation pattern of Nav1.5 channels associated with these alterations is unknown. Phosphoproteomic analyses were undertaken to identify and quantify in situ the native phosphorylation sites on the Nav1.5 proteins purified from wild-type and CaMKIIdc-overexpressing (CaMKIIdc-Tg) mouse ventricles. A total of 18 phosphosites were identified, 8 of which are novel compared with our previous MS analyses. Of these 18 phosphosites, the C-terminal phosphoserines pS1937/pS1938 are present in the CaMKIIdc-Tg IPs (n=3/4) and absent in the WT IPs (n=0/4). In addition, pS1989 is 9-fold more represented (p<0.05, n=4 in each condition) in the CaMKIIdc-Tg, than in the WT, IPs. To explore the possibility that phosphorylation at these C-terminal sites regulates the gating properties of Nav1.5 channels, the orthologous human (serine to glutamate) double phosphomutant Nav1.5-S1933E-S1984E was generated and investigated by whole cell voltage-clamp analyses in HEK293 cells. These analyses revealed that the relative percentage of the TTX-sensitive late Na+ current, compared with the peak Na+ current, is significantly (p<0.01) higher for the double phosphomutant (0.07 ± 0.007%, n=24), compared with the WT (0.04 ± 0.004%, n=20), currents. In addition, although the fast and slow time constants of inactivation are similar, the relative contribution of the fast inactivating component of the peak Na+ current is significantly (p<0.05) lower with the phosphomutant, compared with the WT channels. Together, these analyses provide 8 novel native cardiac Nav1.5 phosphorylation sites, 2 of which are in the C-terminus of Nav1.5 and selectively modulate the late component of Na+ current, suggesting a role for modulation at these sites in heart failure.
Author Disclosures: F. Coyan: None. S. Burel: None. M.R. Meyer: None. C.F. Lichti: None. J.H. Brown: None. F. Charpentier: None. J.M. Nerbonne: None. R.R. Townsend: None. L.S. Maier: None. C. Marionneau: None.
- © 2014 by American Heart Association, Inc.