The cardiac sodium channel, Nav1.5, drives the rising phase of the cardiac action potential. It comprises an ion-conducting α-subunit together with one or more regulatory β-subunits of which there are four main isoforms, β1-β4. The β3-subunit is thought to influence Nav1.5 trafficking and gating; its mutations or knock-outs are associated with a variety of cardiac arrhythmic conditions including Brugada Syndrome (BrS; 1, 2). Available cryo-EM structures suggest multiple points of interaction in α-β subunit combinations. Previous evidence highlights a particularly important role for the extracellular immunoglobulin (Ig) domain of β3 in functional modulation of Nav1.5 (3). We have utilized a combination of electrophysiological,  biochemical and cell-biological techniques to evaluate the functional behaviour of the Nav1.5-β3-subunit complex in vivo (4, 5). We show that the β3-subunit can form monomers, dimers and trimers within the plasma membrane independently of its binding of the Nav1.5 α-subunit. Using super-resolution stochastic optical reconstruction microscopy (STORM), we show that the Nav1.5 α-subunit assembles into localised, higher-order, two-dimensional clusters within the plasma membrane both in the presence and absence of the β3-subunit. Although the β3-subunit did not affect the average number of α-subunits within these clusters, it significantly increased the cluster radii (KS test, P = 4 x 10-9), suggesting that orientation and cluster packing of the Nav1.5 α-subunits is sensitive to the presence of the β3-subunit. Whole-cell patch-clamp experiments identified a depolarising shift of steady-state inactivation in the presence of β3 as well as an accelerated recovery from inactivation which would act to enhance channel availability. Following on from this we have used site-directed mutagenesis to investigate two unusual mutations in structurally distinct regions of the β3-subunit. One mutation is a novel in-frame threonine deletion within the extracellular Ig domain recently identified in a BrS patient in the absence of any other causative mutations. Whole cell patch-clamp experiments highlighted a reduction in peak current compared to wild-type β3 and a loss of the depolarising shift of steady-state inactivation. Yet this mutation did not abolish the Nav1.5-β3 interaction. The second mutation is targeted at an unusually positioned and highly conserved glutamic acid residue within the transmembrane domain of the β3-subunit. This mutation selectively abrogated the acceleration of recovery from inactivation that is normally exerted by wild-type β3. Our work highlights multiple structural and functional interactions between Nav1.5 and β3-subunits in which the latter plays crucial roles, both in regulating normal Nav1.5 function and in the higher-order organisation of Nav1.5 within the plasma membrane. Future experiments will investigate the stoichiometry of these interactions and how mutations affect the oligomeric structures.