The hERG potassium channel is critical for normal cardiac repolarisation. hERG channel block underlies acquired long QT syndrome. Recent evidence suggests that S5, in close apposition to S6, may enable novel drug binding modes to hERG, in particular F557 in the S5 domain has been suggested to be a significant determinant of binding of some hERG inhibitors and activators [1-3]. Cisapride is a high affinity hERG channel inhibitor, and binding to the hERG channel of cisapride has been mapped to residues within the channel’s central cavity [4]. The aim of this study was to probe whether mutation of F557 influences hERG channel kinetics and cisapride block using a mammalian expression system at physiological temperature, in concert with computational docking to a realistic hERG channel structure. Whole cell patch-clamp recordings of hERG current (IhERG) were made at 37°C from Human Embryonic Kidney (HEK 293) cells either stably expressing WT hERG or transiently transfected with wild-type (WT) or F557L hERG [2], Docking was performed using the recent cryo-EM open channel structure of hERG (Protein Data Bank code 5VA1) [5]. Using a standard current-voltage (I-V) protocol, mean I-V relations for IhERG tails showed no difference in half-maximal activation voltage (V0.5) and k (slope factor) values between WT and F557L, inactivation V0.5 and k values from availability plots for WT and F557L IhERG also did not differ significantly from one-another. The time-courses for development of both inactivation and deactivation of WT and F557L IhERG did not differ significantly. The mean time constant of recovery from inactivation showed a modest, but significant reduction for F557L hERG (1.22±0.26ms (n=6) for F557L and 1.77±0.04ms (n=7) for WT; mean ± SEM; p<0.05, unpaired t test). The potency of cisapride block of IhERG tails at -40 mV following depolarization to +20 mV was evaluated. IC50 and nH values generated from concentration-response relations were 5.19±1.17nM, 0.68±0.10 for WT and 46.3±10.2nM, 0.51±0.07 for F557L (right-ward shift of IC50 by ~9 fold compared to WT; n= 4-6 cells for each cisapride concentrations; values are mean±SEM). Docking of cisapride to the recent cryo-EM derived hERG structure [5] showed that cisapride can interact directly with F557 on the S5 domain as well as Y652 and F656 on S6. Our results show that F557L IhERG kinetics are largely similar to those of WT IhERG, although recovery of F557L IhERG from inactivation is somewhat faster. As F557L has a limited effect on hERG channel kinetics, its effect in reducing cisapride potency is most likely to be due to direct interaction of the drug with F557 and/or coordination between F557 and inner cavity high affinity binding sites.