Abstracts

Rationale: Voltage-gated Na+ channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. VGSC β1 subunits (encoded by SCN1B) modulate VGSC cell surface expression, voltage dependence, and kinetics, while also functioning as immunoglobulin superfamily cell adhesion molecules (CAMs) that regulate cellular aggregation, migration, neurite outgrowth and fasciculation. Mutations in SCN1B result in the Genetic Epilepsy with Febrile Seizures plus (GEFS+) spectrum of epilepsies, including Dravet Syndrome, in human patients. Scn1b null mice exhibit a phenotype that includes spontaneous severe seizures and ataxia. These mice have defects in hippocampal and cerebellar microorganization prior to the onset of hyperexcitability, suggesting that VGSC β1 subunits contribute to the regulation of cell-cell adhesion in vivo, further, that defects in β1-mediated neuronal patterning may lead to hyperexcitability rather than result from it. We demonstrated, using cerebellar granule neurons (CGNs), that β1-β1 trans homophilic cell adhesion promotes neurite outgrowth via a mechanism that requires fyn kinase. We postulated that tyrosine phosphorylation of β1 may act as a molecular switch for downstream signal transduction in response to β1-β1 trans homophilic adhesion. Previous studies provided support for this hypothesis with the observations that both β1-mediated anykrin recruitment in response to cell-cell adhesion and β1-mediated regulation of Na+ current are modulated by tyrosine phosphorylation. Methods: Here, we used CGNs as a model system to investigate the role of β1 tyrosine phosphorylation in β1-mediated neurite outgrowth. In this study, a BacMam baculovirus system was used to express β1WT or β1Y181E (a phosphomimetic substitution) in Scn1b null CGNs to ask if the tyrosine phosphomimetic mutation affected neurite outgrowth. In addition, a cell aggregation model of β1-β1 trans homophilic adhesion was used to examine the effect of aggregation on the extent of tyrosine phosphorylation of β1. Results: Expression of β1Y181E in Scn1b null cerebellar granule neurons resulted in significantly increased neurite length relative to the expression of β1WT. Using a cell aggregation model of β1-β1 trans homophilic adhesion, we demonstrated that transfection of β1WT induced cellular aggregation at 37C while GFP-transfected cells did not aggregate. Subsequent immunoprecipitation of aggregated cells with anti-pY20 antibody demonstrated that β1WT is tyrosine phosphorylated following 30 minutes of aggregation. Conclusions: These results suggest a model in which β1 subunits become tyrosine phosphorylated as a result of of β1-β1 trans homophilic adhesion. Further, the tyrosine phosphorylated form of β1 drives neurite outgrowth.