Proteomic and functional mapping of cardiac Na_V1.5 channel phosphorylation sites

Phosphorylation of the voltage-gated Na⁺ (Na_V) channel Na_V1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed Na_V1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on Na_V1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic Na_V1.5 mutants revealed the roles of three phosphosites in regulating Na_V1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface Na_V1.5 expression and peak Na⁺ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular Na_V1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of Na_V1.5 channels is highly complex, site specific, and dynamic.