The β3 subunit of the cardiac voltage gated sodium channel (Nav1.5) has been demonstrated to influence both trafficking and gating of Nav1.5 [1]. Indeed, β3 mutations or its knock-out have been associated with cardiac arrhythmias such as Brugada Syndrome (BrS) [2]. We have previously demonstrated an ability of β3 to form dimers and trimers at the plasma membrane via its extracellular immunoglobulin (Ig) domain [3,4]. However, the stability and importance of this oligomerisation for potential complexes with the Nav1.5-α subunit and the functional implication is unclear. HEK293F cells ± stable Nav1.5 expression (HEK and HEK-Nav1.5) were used throughout this study and transiently transfected with tagged β3 or Nav1.5α-subunits, as appropriate. Interactions between β3-subunits was determined by chemical (BS3) cross-linking and between α- and β3-subunits by co-immunoprecipitation (Co-IP) and western blotting. To investigate Nav1.5 channel oligomerisation in vivo we used super-resolution STORM imaging with cluster analysis; reporting cluster radii and number of molecules in each cluster [5]. Comparisons of radii and nearest neighbour distributions for clusters of Nav1.5±β3 in reconstructed STORM images were tested for statistical significance using the Kolmogorov-Smirnov (KS) test. Whole cell patch clamp was used to measure Na+ currents with steady-state activation and inactivation, and recovery from inactivation protocols. Western-blots of cross-linked β3-myc or β3-GFP showed bands at molecular weights indicative of monomeric, dimeric and trimeric forms of β3. Super-resolution STORM images demonstrated expression of Nav1.5α at the plasma membrane. However, these were not randomly dispersed; rather, ~40% of Nav1.5α-subunits assembled into large molecular clusters. Although the proportion of Nav1.5α in these clusters was unaffected by the β3-subunit, the proportion of larger-radii clusters was significantly increased (KS test, P = 4 x 10-9) as well as nearest neighbour distances. Co-expression of β3-GFP did not affect Nav1.5 steady-state activation parameters or peak Na+ current. However, it did result in a depolarising shift of V1/2 of steady-state inactivation (-96.14±0.72 mV vs -90.64±1.02 mV with β3, n = 20 and 27, P<0.001) and an accelerated recovery from inactivation (5.29±0.92 vs 2.97±0.82 with β3, n = 7 and 10, P<0.001). Our results demonstrate that even when expressed alone in HEK cells Nav1.5 α-subunits co-assemble into larger oligomeric clusters. While the β3-subunit is not required to facilitate this clustering, it does significantly alter the geometry of these complexes, functionally shifting channel inactivation to significantly more depolarised potentials and accelerating recovery from inactivation. This would be expected to increase channel availability, therefore loss of β3 could contribute to BrS.