Voltage-gated Na+ channel (VGSC), predominantly neonatal Nav1.5 (nNav1.5), activity can potentiate a variety of cellular behaviours involved in the metastatic cascade (Fraser et al.,2005). We have shown previously that the strongly metastatic human breast cancer (BCa) MDA-MB-231 cells express VGSC α-subunit (VGSCα) protein both in plasma membrane (PM) and intracellularly (Chioni and Djamgoz, 2005). In the present study, we determined if VGSC expression involved self-regulation. Western blots and confocal microscopy identified VGSC α protein and its subcellular location (quantified by confocal densitometry) in MDA-MB-231 cells. A novel polyclonal antibody specific for nNav1.5 was used (Chioni et al, 2005). Proliferation, transwell migration and matrigel invasion were measured as before (Fraser et al.,2005). Cells were treated with TTX (1-10 μ)M in normal medium. Data are presented as mean ± SEM; paired t-test was used in statistical analyses. Under normal conditions, migrated cells were seen to express noticeably more VGSC protein, compared with non-migrated cells. Treatment with TTX (up to 10 μM) for 48 hours had no effect on total VGSC? protein expression. However, 10 μM TTX decreased nNav1.5 protein in PM by 46 ±3 % (p<0.01; n=3 experiments/100 cells). Treatment with TTX (10 μM; 48 h) during assaying decreased invasiveness of MDA-MB-231 cells by 49 ± 10 % (n=9; p<0.001). TTX also reduced migration in a dose-dependent way. Thus, 5 μM TTX reduced MDA-MB-231 cell migration by 31 ± 10 % (n= 7; p0.05). The effect of pre-treating the cells with TTX (10 μM; 48 h) was studied in migration assays. TTX pre-treatment reduced migration by 55 ± 8 %, compared with non-pre-treated cells (n=6; p<0.05). TTX (10 μM) reduced migration of non-pre-treated cells by 42 ± 5 % (n=7; p0.05; n=6). TTX had no effect on proliferation of pre-treated or non-pre-treated cells. These results are consistent with auto-regulation of functional VGSC expression in MDA-MB-231 cells. Thus, blocking VGSC activity with TTX would induce trafficking of nNav1.5 protein from PM into the cell, leading to suppression of metastatic cell behaviour.