Voltage-gated Na+ channels (VGSCs) are composed of a pore-forming α subunit and one or more auxiliary β subunits. The β subunits regulate channel gating and are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules. Although classically expressed in excitable cells, VGSC α and β subunits are also present in cancer cells, including breast cancer (BCa). In the metastatic MDA-MB-231 BCa cell line, Na+ influx through Nav1.5 increases invasion and migration. β1 expression is up-regulated in BCa specimens relative to control tissue. β1 over-expression also increases tumour growth, invasion and metastasis in an orthotopic tumour model. In addition, β1 promotes neurite-like process outgrowth from BCa cells, and this is dependent on the presence of the Ig loop (1). Therefore, targeting and inhibiting the adhesive function of β1 may provide a novel therapeutic method for inhibiting metastasis. The purpose of this study was to test the hypothesis that the Ig loop is required for β1 to regulate adhesion and Na+ current in BCa cells. We studied cell-cell adhesion using an aggregation assay. Briefly, tumour cells were maintained in suspension for up to 2 h with gentle shaking, aliquots were taken at 30 min intervals, placed on glass slides and aggregation was monitored under a microscope. The number of particles per field of view was counted. A particle was defined as a single cell or cluster of cells of any size. Increased adhesion was therefore reflected in a decrease in particle number. After 30 minutes, the number particles per field of view was 52.5±2.4 (mean±SEM) in β1 over-expressing cells, compared to 79.4±1.4 in control MDA-MB-231 cells, in agreement with previous observations (2) (t-test, n=4, P<0.001). In contrast, over-expression of a truncated β1 mutant lacking the Ig loop (β1Δ40-124) did not increase adhesion compared to control cells (t-test, n=3, P=0.26). Therefore, the Ig loop is necessary for the increase in adhesion of MDA-MB-231 cells. We next studied Na+ currents by whole-cell patch clamp recording in voltage clamp mode. The peak Na+ current density increased from -6.7±0.3 pA/pF in control MDA-MB-231 cells to -15.3±2.4 pA/pF in β1 over-expressing cells, in agreement with previous observations (P<0.05) (2). However, the peak Na+ current density was not significantly altered in MDA-MB-231 cells overexpressing the β1Δ40-124 truncation mutant, compared to control cells (control, n=14; MDA-MB-231-β1, n=15; MDA-MB-231-β1Δ40-124, n=13; Kruskal-Wallis with post-hoc Dunn’s tests). These data suggest that the Ig loop of β1 is required to modulate both adhesion and Na+ current in BCa cells. Thus, the Ig loop of β1 may enhance invasion and metastasis through dual roles, and may therefore represent a useful novel therapeutic target.