Role of Voltage-gated Sodium Channel b Subunit Signaling in Neuronal Development and Cancer.

Abstract

Voltage-gated sodium channels (VGSCs) in brain are composed of a single pore-forming alpha subunit and two beta subunits. VGSC beta subunits are multifunctional, with conducting roles as ion channel regulatory subunits and non-conducting roles as immunoglobulin superfamily cell adhesion molecules. This thesis focused on three important issues in VGSC beta subunit signaling. First, while VGSC alpha subunits have been shown to play a role in breast cancer metastasis, a role for beta subunits in this process was not understood. We demonstrated that beta1 expression is more abundant in weakly metastatic MCF-7 than in strongly metastatic MDA-MB-231 human breast cancer cells. Knockdown of beta1 expression in MCF-7 cells reduced adhesion and increased migration. Expression of beta1 in MDA-MB-231 cells reduced lateral motility and proliferation. Thus, VGSC beta1 may be a novel cell adhesion molecule in human breast cancer. Second, we hypothesized that tyrosine phosphorylation of beta1 is a critical step in the neurite outgrowth signaling cascade initiated by beta1-beta1 trans homophilic adhesion. Here, we showed that expression of a construct mimicking the tyrosine-phosphorylated form of beta1 drives neurite outgrowth independent of trans homophilic cell-cell adhesion. Cellular aggregation resulted in elevated levels of beta1 tyrosine phosphorylation. In a cell-free kinase assay, we identified beta1 as a fyn kinase substrate. These data suggest that tyrosine phosphorylation of beta1 is a critical component of the beta1-mediated neurite outgrowth signaling cascade. Third, it was shown previously that the final step in VGSC biosynthesis in central neurons is concomitant alpha-beta2 disulfide linkage and insertion into the plasma membrane. Here, mutagenesis was used to demonstrate that a single cysteine-to-alanine substitution at extracellular residue cysteine-26, located within the beta2 immunoglobulin domain, abolishes the covalent linkage between alpha and beta2 subunits. Loss of alpha-beta2 covalent complex formation disrupts the targeting of beta2 to nodes of Ranvier and to the axon initial segment, suggesting that linkage with alpha is required for normal beta2 subcellular localization in vivo. My thesis work has improved our understanding of how VGSC β subunits regulate important cell processes such as cell motility and how β subunits, in turn, are regulated by other proteins.