The voltage-gated sodium channel β3 subunit modulates C6 glioma cell motility independently of channel activity

Abstract

Voltage-gated sodium channels (VGSCs) initiate action potentials in nerve and muscle cells and are regulated by auxiliary β subunits. VGSC β subunits are also expressed in some cancer types, suggesting potential functions distinct from their role in electrophysiological excitability. This study investigated the occurrence and functional implications of the VGSC β3 subunit (the product of SCN3B gene) in glioma, focusing on the role of its extracellular immunoglobulin domain (β3 Ig). Data mining explored associations between β3 expression and glioma severity at patient, tissue, and single-cell levels. Using C6 glioma cells expressing β3 or β3 without its Ig domain, we examined the effects on cell viability, mobility, and actin-based cell protrusions. A single-chain variable fragment (scFv) antibody targeting the β3 Ig was selected by phage display to interfere with its functions. The interacting proteins with β3 Ig were identified by immunoprecipitation-mass spectrometry. Data mining revealed negative correlations between β3 expression and glioma severity and aggressiveness. Expression of β3 in C6 cells reduced cell migration and invasion without affecting cell viability. Filopodia were significantly increased while lamellipodia/ruffles were decreased, producing striking cell morphological changes. These effects were abrogated by expression of the β3 subunit lacking the β3 Ig domain or exogenous application of an scFv targeting β3 Ig. Most of the plasma membrane-associated proteins immunoprecipitated with the β3 subunit are known regulators of actin polymerization. Our data reveals a novel and unexpected role for the VGSC β3 subunit in orchestrating actin organization and negatively regulating cell migration in glioma cells which may potentially explain clinical correlations with glioma severity. SCN3B expression increased occurences of filopodia (functioning as sensors for cell migration) and reduced lamellipodia/ruffles (providing the mechanical force for cell migration). These together decreased cell migration. This effect is abrogated when the extracellular domain of SCN3B is absent. A single-chain variable fragment (scFv) antibody targetting the extracellular domain of VGSC β3, selected by phage display, also disrupted this effect of SCN3B, implicating its extracellular domain in this process. We emphasize the novelty and value of our paper for several reasons: • This is the first paper reporting the regulatory effect of VGSC β3 subunit on cell migration. • We generated customized scFv to study the function of a specific domain of the protein. • We have conducted high-resolution confocal microscopy to systematically analyzed correlation of protein level and cell protrusion. • We capture the visible difference in imaging of β3-expressing and β3-non-expressing cells under microscopy. • Our conclusion in clinical glioma is supported by tissue IHC (supplementary materials for review only).

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