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Voltage-gated sodium channel (VGSC) β1 subunits are multifunctional proteins that modulate the biophysical properties and cell-surface localization of VGSC α 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 in SCN1B, the gene encoding the VGSC β1 subunits, are linked to severe diseases with high risk for sudden death, including epileptic encephalopathy and cardiac arrhythmia. We showed previously that β1 subunits are post-translationally modified by tyrosine phosphorylation. We also showed that β1 subunits undergo regulated intramembrane proteolysis via the activity of β-secretase 1 and γ-secretase, resulting in the generation of a soluble intracellular domain, β1-ICD, which modulates transcription. Here, we report that β1 subunits are phosphorylated by FYN kinase. Moreover, we show that β1 subunits are S-palmitoylated. Substitution of a single residue in β1, Cys-162, to alanine prevented palmitoylation, reduced the level of β1 polypeptides at the plasma membrane, and reduced the extent of β1-regulated intramembrane proteolysis, suggesting that the plasma membrane is the site of β1 proteolytic processing. Treatment with the clathrin-mediated endocytosis inhibitor, Dyngo-4a, re-stored the plasma membrane association of β1-p.C162A to WT levels. Despite these observations, palmitoylation-null β1-p.C162A modulated sodium current and sorted to detergent-resistant membrane fractions normally. This is the first demonstration of S-palmitoylation of a VGSC β subunit, establishing precedence for this post-translational modification as a regulatory mechanism in this protein family.

Loss-of-function (LOF) variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including developmental and epileptic encephalopathy and cardiac arrhythmia. β1 Subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming α subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Here, we investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase and show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA sequencing, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b-null cardiac tissue, which lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. We propose that human disease variants resulting in SCN1B LOF cause transcriptional dysregulation that contributes to altered excitability. Moreover, these results provide important insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multifunctionality of sodium channel β1 subunits.