Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating

Phosphorylation of NaV1.5 channels regulates cardiac excitability, yet the phosphorylation sites regulating channel function and the underlying mechanisms remain largely unknown. Using a systematic quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from non-failing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines-664 and -667 regulate the voltage-dependence of channel activation in a cumulative manner, whereas phosphorylation of the nearby serine-671, which is increased in failing hearts, decreases cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, the results demonstrate that ventricular NaV1.5 is highly phosphorylated, and that the phosphorylation-dependent regulation of NaV1.5-encoded channels is highly complex, site-specific and dynamic.