Sodium channel β1 subunits are post-translationally modified by tyrosine phosphorylation, S-palmitoylation, and regulated intramembrane proteolysis

Voltage-gated sodium channel (VGSC) ?1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC ? subunits and participate in cell?cell and cell?matrix adhesion, all with important implications for intracellular signal transduction, cell migration, and differentiation. Human loss-of-function variants inSCN1B, the gene encoding the VGSC ?1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that ?1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that ?1 subunits undergo regulated intramembrane proteolysis via the activity of ?-secretase 1 and ?-secretase, resulting in the generation of a soluble intracellular domain, ?1-ICD, which modulates transcription. Here, we report that ?1 subunits are phosphorylated by FYN kinase. Moreover, we show that ?1 subunits areS-palmitoylated. Substitution of a single residue in ?1, Cys-162, to alanine prevented palmitoylation, reduced the level of ?1 polypeptides at the plasma membrane, and reduced the extent of ?1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of ?1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of ?1-p.C162A to WT levels. Despite these observations, palmitoylation-null ?1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration ofS-palmitoylation of a VGSC ? subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.