hERG (human Ether-à-go-go Related Gene) is responsible for ion channels mediating rapid delayed rectifier potassium current, IKr, which is key to cardiac action potential repolarization. Gain-of-function hERG mutations give rise to the SQT1 variant of the Short QT Syndrome (SQTS). Reggae mutant zebrafish, with a S4 zERG mutation (Leucine499Proline; L499P), display arrhythmic features analogous to those seen in the SQTS. The affected S4 domain ERG residue is highly conserved. This study was executed to determine how the homologous hERG mutation (L532P) influences channel function at 37 °C. Whole-cell measurements of current (IhERG) were made from HEK 293 cells expressing WT or L532P hERG. The half maximal activation voltage (V0.5) of L532P IhERG was positively shifted by ~+36 mV compared to WT IhERG; however at negative voltages a pronounced L532P IhERG was observed. Both activation and deactivation time-courses were accelerated for L532P IhERG. The inactivation V0.5 for L532P IhERG was shifted by ~+32 mV. Under action potential (AP) voltage-clamp, L532P IhERG exhibited a dome-shaped current peaking at ~+16 mV, compared to ~−31 mV for WT-IhERG. The L532P mutation produced an ~ 5-fold increase in the IC50 for dronedarone inhibition of IhERG. Homology modeling indicated that the L532 residue within the S4 helix lies closely apposed to the S5 region of an adjacent hERG subunit. Alterations to the S4 domain structure and, potentially, to interactions between adjacent hERG subunits are likely to account for the functional effects of this mutation.