Voltage-gated Na⁺ (Na_V) channels are key regulators of myocardial excitability, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in Na_V1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of Na_V1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the Na_V1.5 proteins purified from adult WT and failing CaMKIIδ_c-overexpressing (CaMKIIδ_c-Tg) mouse ventricles. Of 19 native Na_V1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδ_c-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of Na_V1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases Na_V1.5 channel availability and decreases late Na⁺ current, two effects that were abrogated with Na_V1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to Na_V1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with Na_V1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of Na_V1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδ_c-dependent arrhythmogenic disorders in failing hearts.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.