Short QT syndrome (SQTS) is a rare but potentially fatal cardiac channelopathy (Hancox et al, 2018; Harrell et al, 2015).  The T618I mutation in the hERG channel pore has been termed a “hotspot” mutation as it is the mutation observed most frequently in successfully genotyped cases (Hu et al, 2017; Sun et al, 2011).  A distinct U wave has been observed in the electrocardiogram of ~70% of T618I carriers and these patients are vulnerable to ventricular fibrillation; however, atrial fibrillation has not been reported (Hu et al, 2017).  This study was undertaken to compare the effect of the T618I mutation on hERG current (IhERG) elicited by action potential (AP) waveforms from different cardiac regions.  HEK293 cells were transiently transfected with wild-type (WT) or T618I hERG constructs and whole-cell patch-clamp measurements of IhERG were made at 37 oC, using ventricular, Purkinje fibre (PF) and atrial AP waveforms as previously described (McPate et al, 2009).  Data are presented as mean ± SEM. Under ventricular AP clamp, WT IhERG attained a maximal amplitude of 125.4 ± 34.5 pA/pF during AP repolarization, peaking at -23.5 ± 1.8 mV (n=5).  During the same waveform, T618I IhERG attained a maximal amplitude of 565.9 ± 155.8 pA/pF (n=6; p< 0.05 vs WT, unpaired t test), whilst peaking earlier during the AP (at +19.0 ± 2.6 mV; p0.05 vs ventricular; atrial p<0.01 vs ventricular, repeated measures 1-way ANOVA, with Tukey post-test).  Normalizing peak repolarizing current to that elicited by the ventricular AP command showed maximal repolarizing WT IhERG current during the PF waveform to be 84 ± 6% of maximal ventricular repolarizing current and for the atrial AP command the comparable value was 41 ± 6 % (n=5).   For T618I, maximal IhERG during the PF and atrial APs was 282.0 ± 94.6 pA/pF and 103.9 ± 33.3 pA/pF respectively (n=6; p<0.05 and p<0.01 vs ventricular AP respectively).  When normalised similarly to WT data, peak repolarizing current during the PF and atrial APs were respectively 49 ± 4 % (p<0.001 vs WT; unpaired t test) and 18.8 ± 2 % (p<0.01 vs WT) of peak ventricular AP repolarizing current.  Collectively, these results indicate that the T618I mutation increases repolarizing ventricular IhERG, whilst it also augments differences in IhERG between PF and ventricular APs.  This in turn may contribute to heterogeneity of ventricular-PF repolarization and consequently to the U waves seen in T618I carriers.  The comparatively brief duration and lack of pronounced plateau of the atrial AP is likely to account for the disparity in T618I effect between atrial and ventricular APs.