S-Palmitoylation of the sodium channel Nav1.6 regulates its activity and neuronal excitability

S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys¹¹⁶⁹, Cys¹¹⁷⁰, and Cys¹⁹⁷⁸) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys¹⁹⁷⁸ is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys¹⁹⁷⁸S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.