Abstracts

Rationale: Dravet Syndrome (DS) is a pediatric developmental and epileptic encephalopathy that typically onsets in the first year of life. DS presents with intractable seizures, behavioral and developmental delay, ataxia, and an increased risk of sudden unexpected death in epilepsy. Homozygous mutations in SCN1B, which encodes voltage-gated sodium channel (VGSC) β1 and β1B subunits, are linked to DS. Scn1b null mice are a model of DS. β1 modulates the gating and kinetics of the ion channel pore, functions in cell adhesion, cell signaling, and neuronal pathfinding and fasciculation. β1 is a substrate for sequential cleavage by BACE1 and γ-secretase which generates a soluble intracellular domain (ICD). We investigated the biochemical mechanisms and downstream signaling of β1 cleavage in vitro and in vivo to understand how this signaling pathway, or loss thereof, may contribute to cellular function and thus human disease. Methods: We used heterologous expression of V5-tagged β1 cDNA constructs to study biochemical mechanisms regulating β1 cleavage and its downstream signaling. β1 cleavage was assayed by immunoblot with an antibody against the V5 tag. Imaging was performed using confocal immunofluorescence microscopy. RNA-Sequencing (RNA-Seq) analysis was completed from Chinese Hamster Lung (CHL) cells stably overexpressing either soluble enhanced Green Fluorescent Protein (eGFP) or bicistronic β1-ICD-V5-2AeGFP and from Scn1b wild-type (WT) and Scn1b-null mouse cardiac ventricle. Results: CHL cells, which were previously shown not to express VGSC α or β1 subunit mRNA, endogenously express BACE1 and γ-secretase at levels sufficient to drive β1 cleavage in vitro. Using CHL cells, we showed β1 sequential cleavage can be blocked with BACE1 and γ-secretase inhibitors. We hypothesized β1 was palmitoylated at p.C162. Using Acyl-RAC and Acyl-PEG, we determined that β1 is singly palmitoylated at p.C162. Expression of a β1-palmitoylation deficient mutant, β1-p.C162A, resulted in a significant reduction in the amount of β1 cleavage product generated. β1-p.C162A localizes to the plasma membrane and modulates Nav1.5-generated sodium current in heterologous cells similarly to WT. The β1-ICD protein localized to the nucleus, suggesting the ICD generated is translocated to this organelle. We hypothesized the β1-ICD may function as a transcriptional modulator. To nominate genes regulated by the β1-ICD, we completed RNA-seq from CHL cells stably transfected with eGFP or β1-ICD-V5-2AeGFP. Gene Ontology analysis revealed β1-ICD expression was sufficient to downregulate expression of subsets of calcium ion binding, the immune response, and cellular proliferation genes. Although the β1 signaling pathway may be relevant in many tissue types, we narrowed our examination of changes in gene expression to mouse cardiac ventricle because of potential cardiac contributions to SUDEP. Scn1b deletion, and thus deletion of the signaling pathway initiated by the β1-ICD, results in increased expression of the same subsets of genes, suggesting that the β1-ICD may normally act as a molecular break on expression of these gene groups in vivo. Conclusions: We showed that β1 subunits are substrates for sequential cleavage by BACE1 and γ-secretase, resulting in the generation of a soluble ICD. We found that β1 S-palmitoylation promotes cleavage. RNA-seq experiments identified gene pathways that are downregulated by β1-ICD overexpression but upregulated in Scn1b null tissue, suggesting that the β1-ICD may normally act as molecular break on gene expression. These results give important new insights into the mechanisms of SCN1B-linked channelopathies. Funding: NIH-T32 Systems and Integrative Physiology Training Grant GM008322-26 and NIH F31HL144047 to AAB, NIH R37NS076752 to LLI, NIH-T32 Pharmacological Sciences Training Grant GM007767 to JMP and NE.