Voltage-gated Na+ channels (VGSCs), composed of a pore-forming, Na+-conducting α-subunit and auxiliary β-subunits (β1-β4), are responsible for the inward Na+ current (INa) underlying neuronal action potentials. β-subunits regulate α-subunit function by promoting plasma membrane localisation, thereby increasing INa, and through alteration of α-subunit activation and inactivation kinetics. Perturbations in β-subunit function contribute to various channelopathies. For example, mutations in β1 have been reported in generalised epilepsy with febrile seizures (GEFS+) and Brugada syndrome. Also, VGSC subunits, including β1, promote breast cancer invasion and metastasis. However, the interaction between β-subunit domains and α-subunit function is incompletely understood. Several α-subunit interaction sites are within the β1 extracellular domain (ECD) and intracellular domain (ICD). β1 is sequentially processed by β- and γ-secretase, releasing the ECD and ICD respectively, yet the impact of this proteolysis on β1-mediated regulation of α-subunit function has yet to be delineated. Here, we investigated how secretases influence β1-mediated regulation of INa in MDAMB231 (231) cells using whole-cell patch clamp recording. Results are expressed as mean ± SEM, statistical significance determined using ANOVA with Tukey’s post hoc tests. Treatment of 231 cells expressing β1-GFP with a γ-secretase inhibitor significantly decreased INa from -15.2 ± 1.3 pA/pF to -8.0 ± 1.4 pA/pF (n=12-16; P<0.01) without affecting 231 cells expressing GFP alone (-6.9 ± 1.1 pA/pF vs. -5.9 ± 1.1 pA/pF; n=14-16; P=0.996). This result suggests that γ-secretase is involved in regulating β1-mediated modulation of α-subunit activity. To determine whether β1ICD is directly involved in regulating α-subunit function, β1ICD-GFP was expressed in 231 cells and INa compared to 231-GFP and 231-β1GFP cells. 231-β1ICDGFP cells displayed a significantly larger INa than 231-GFP cells (-9.3 ± 1.0 pA/pF vs. -4.4 ± 0.6 pA/pF; P<0.01; n=14-16). Furthermore, the INa in 231-β1ICD-GFP cells was not significantly different from that of 231-β1GFP cells (-11.7 ± 0.9 pA/pF; n=15; P=0.2). These data support the notion that cleaved β1ICD is involved in regulating INa. To further investigate the importance of secretase cleavage, a cleavage-resistant β1 (SRβ1), lacking the β-secretase site (amino acids 142-147), was expressed in 231 cells. We anticipated that if β1ICD is necessary for increasing INa, then SRβ1 should not increase INa compared to 231-GFP control cells. Surprisingly, SRβ1 expression significantly increased INa compared to 231-GFP cells (-12.9 ± 1.2 pA/pF vs. -5.9 ± 0.8 pA/pF; n=15; P<0.05). The INa of 231-SRβ1-GFP cells was also significantly smaller than the INa in 231-β1GFP cells (-21.2 ± 2.8 pA/pF; n=15; P<0.01). ln conclusion, β1 regulates INa by both secretase-dependent and secretase-independent mechanisms. Further work is required to elucidate the molecular pathways involved.