Long QT syndrome (LQTS) and short QT syndrome (SQTS) are arrhythmogenic disorders, characterised by altered cardiac action potential (AP) repolarization and subsequent risk of arrhythmia and sudden death. The human Ether-à-go-go-Related Gene (hERG/KCNH2) encodes the pore forming subunit of channels carrying the ‘rapid’ delayed rectifier current IKr, a key constituent of AP repolarization. Loss of function (LOF) mutations in hERG act to delay repolarization and cause LQTS type 2 (LQT2) (Schwartz et al, 2009). Conversely, gain of function (GOF) hERG mutations act to accelerate repolarization and underlie SQTS Type 1 (SQT1) (Hancox et al, 2018). LOF hERG mutations can reduce IKr current via different mechanisms: abnormal transcription/translation, defective protein trafficking, defective channel gating and/or altered ion permeation (Delisle et al, 2004). For LQT2 the most common mechanism is defective trafficking (Anderson et al, 2014). SQT1-causing GOF hERG mutations have been shown to modify the kinetic properties of IKr (Hancox et al, 2018), but their effects on trafficking are not well established. To investigate whether GOF hERG mutations can alter hERG channel trafficking we utilised LI-COR® based ‘On-Cell’ and ‘In-Cell’ Western assays to quantitatively measure the effects of two GOF mutations (N588K and T618I) and two LOF mutations (G601S and A561V) on the cell surface ‘On-Cell’ and total ‘In-Cell’ expression level of the hERG1a channel in transiently transfected Human Embryonic Kidney 293 cells. The trafficking status of mutant hERG channels was assessed under both homozygous and heterozygous expression to mimic patient phenotype. For full methodological details of this assay please refer to (Al-Moubarak et al, 2020). Data are from five independent repeats (n=5) and are presented as mean ± S.E.M. Statistical analysis was performed using One Way ANOVA and a Dunnett’s multiple comparison test. Our data show that the GOF SQT1 causing mutants N588K and T618I significantly increased cell surface expression of the hERG channel when compared to the wild-type (WT) level (homozygous: 1.05±0.12 arbitrary fluorescent units (x10-7) and heterozygous: 1.09±0.13) under both homozygous (N588K: 156.5% of WT level, 1.64±0.16, p<0.05; T618I: 193.3% of WT level; 2.03±0.17, p<0.001) and heterozygous (N588K+WT: 139.9% of WT level, 1.53±0.11, p<0.05; T618I+WT: 152.4% of WT level; 1.66±0.10, p<0.05) expression. In contrast, the loss of function mutants G601S and A561V significantly reduced the cell surface expression level of the hERG channel under both expression states (homozygous: G601S: 32.5% of WT level, 0.34±0.04, p<0.05; A561V: 36.3% of WT level, 0.38±0.05, p<0.05) (heterozygous: G601S: 59.1% of WT level, 0.65±0.04, p<0.05; A561V: 51.6% of WT level, 0.56±0.08, p0.05) different from the WT channel, indicating that these mutations do not act to perturb translation. The alterations to the trafficking status of the hERG channel when expressed in the heterozygous state highlights that these mutants exert dominant phenotypes. In addition, our study provides evidence that augmented trafficking may be an important contributor to the disease mechanism for specific SQT1 causing GOF hERG mutations.