Voltage-Gated Sodium Channel B1 Subunit Processing by BACE1 and y-Secretase: Regulatory Mechanisms and Downstream Signaling

Abstract

Voltage-gated sodium channels (VGSCs) are heterotrimeric proteins comprised of one pore-forming, alpha subunit and two non-pore forming, beta subunits. Variants in SCN1B, the gene which encodes VGSC beta1 subunits, are linked to human diseases with high incidence of sudden death including Early Infantile-Developmental and Epileptic Encephalopathy (EI-DEE) and cardiac arrhythmia. Scn1b-null mice are a model of EI-DEE, including a cardiac phenotype, and 100% of mice die by approximately postnatal day 21. Beta1 subunits are classically known for their role in modulating the gating, kinetics, and localization of the ion channel pore. In addition, beta1 is a member of the immunoglobulin super family of cell adhesion molecules (CAMs). Beta1 functions in both homophilic and heterophilic cell adhesion with downstream roles in neurite outgrowth and neuronal pathfinding and fasciculation. Although these functions explain a portion of SCN1B-linked disease, the underlying mechanisms for differential gene expression observed in the Scn1b-null model of EI-DEE remained poorly understood. Studies presented in this thesis sought to understand the contribution of beta1 to transcriptional changes in Scn1b-null animals. We and others have shown, in addition to the aforementioned functions of beta1, beta1 is a substrate for sequential cleavage by Beta-site APP cleaving enzyme 1 (BACE1) and gamma-secretase. We created a cell culture based assay to study the molecular mechanisms that regulate cleavage and its subsequent downstream signaling. Using biochemical approaches, we demonstrated beta1 is post-translationally palmitoylated, the addition of a 16-carbon fatty acid chain to a cysteine residue by a thioester bond, at p.C162. Beta1 palmitoylation promotes beta1 localization to the plasma membrane and consequently promotes beta1 processing by BACE1 at the plasma membrane. Sequential cleavage generates a small, soluble peptide, the beta1-intracellular domain (beta1-ICD). Beta1-ICD is translocated to the nucleus where it modulates transcription. Expression of the Beta1-ICD generally downregulates gene expression of genes implicated in proliferation, the immune response, calcium ion binding, and voltage-gated potassium channels. Interestingly, these same Gene Ontology (GO) groups are upregulated in cardiac ventricle isolated from Scn1b-null mice, where this pathway is deleted, compared to wild-type. Subsequent increases in potassium current are observed in acutely isolated ventricular myocytes from Scn1b-null mice. In heterologous cells, expression of the beta1-ICD increases Scn4a and Scn5a transcripts and decreases Scn3a transcripts. Expression of the beta1-ICD does not lead to increases in sodium current density, nor does it act directly on the Nav1.5 alpha subunit when the peptide is profused during patch clamp. This work identified a novel function of a species of the beta1 subunit, beta1-ICD, as both a transcriptional modulator. Identification of this pathway provides a direct mechanism by which loss of beta1 in the Scn1b-null model of EI-DEE, and potentially in patients with loss-of-function SCN1B variants, results in changes in gene expression and altered excitability.