AbstractThe voltage-gated Na+channel β1 subunit, encoded bySCN1B, regulates cell surface expression and gating of α subunits, and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. AbnormalSCN1Bexpression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na+current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD was necessary and sufficient to increase Na+current measured at the plasma membrane. Importantly, although the endogenous Na+current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. In addition, β1-GFP increased mRNA levels of the TTX-sensitive α subunitsSCN1A/Nav1.1 andSCN9A/Nav1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer.