Brugada Syndrome (BrS) is characterised by right precordial ST-segment and T-wave changes and an increased risk of potentially fatal ventricular arrhythmias. Several mutations in the SCN3b gene encoding the cardiac voltage-gated sodium channel (Nav1.5) β3 subunit have been identified in patients with BrS [1,2]. Here, we investigate the role of a novel SCN3b mutation in producing BrS phenotypes. A 54-year old man presented with palpitations and dizziness. The diagnostic type 1 Brugada pattern was present and subsequently seen intermittently on a 24-hour ECG. Next-generation sequencing analysis of a 12 gene panel revealed a heterozygous 3-base deletion (ACG) from SCN3b position 412 to 414. The variant yields an in-frame deletion of a single threonine at position 138 (T138), localised within the Greek key motif-β sheet of the extracellular immunoglobulin-like domain of the β3-subunit. HEK293 cells stably expressing Nav1.5 were transiently transfected with eGFP labelled full-length β3 (β3-WT), T138 deleted-β3 (β3-∆T138) or empty (eGFP) vectors as appropriate. Whole cell patch clamp protocols imposed initial, 100 ms duration, activating steps to test voltages varied in 5 mV increments between -140 and + 45 mV from a -120 mV holding potential to measure Na+ current (INa) activation. A further 50 ms duration step to a fixed -40 mV voltage assessed the resulting INa inactivation. Data (means ± SEM) were compared with One-Way ANOVA followed by Tukey post-hoc tests. β3-∆T138 expressing cells showed significantly decreased peak Na current densities INa compared to β3-WT (-213.0±62.0 (n=8) vs -499.7±111.0 pA/pF (n=7); P=0.043), but similar voltages of half maximal steady state activation V1/2. WT-β3 showed depolarized shifts in V1/2 of steady-state inactivation relative to that of the eGFP cells (-72.67±1.38 (n=9) vs -80.77±1.85 mV (n=11); P=0.042), but this was not observed with the ∆T138-β3 mutant (eGFP vs β3-∆T138: -80.77±1.85 mV (n=11) vs -79.95±1.96 mV, (n=13), P=0.990). To analyse the functional impact of the mutant in the heterozygous state, INa recordings were made in cells co-expressing β3-WT and β3-∆T138 (β3-WT/∆T138) at a 1:1 ratio. Peak INa was rescued by β3-WT/∆T138 (β3-WT vs β3WT/∆T138: -499.7±111.0 pA/pF (n=7) vs -462.6±41.7 pA/pF, (n=6), P=0.938). The V1/2 of steady-state activation was unaffected with the β3-WT/∆T138 heterozygous mixture. The V1/2 of steady-state inactivation of β3-WT/∆T138 showed increased variation but was not significantly different from β3-∆T138 (-80.77±1.85 mV (n=11) vs -78.40±2.33 mV, (n=11), respectively, P=0.986). Co-immunoprecipitation and western blotting confirmed that the ∆T138 mutation did not disrupt the Nav1.5-β3 interaction. In addition, biotinylation experiments revealed that the ∆T138 mutation did not prevent trafficking to and expression of the β3 or Nav1.5 subunits on the cell surface membrane. Our results demonstrate that the T138 deletion in the β3 subunit reduced peak INa density and shifted channel inactivation to less depolarised potentials, hence reducing channel availability, contributing to a BrS phenotype. Nevertheless, the heterozygous expression of this mutant protein seems to ameliorate the effect on peak INa density, while maintaining more hyperpolarised channel inactivation possibly explaining the relatively mild phenotype exhibited by the patient.