Disopyramide (DISO) is a class Ia antiarrhythmic drug that delays ventricular repolarisation; this action associated with inhibition of the native cardiac rapid delayed rectifier K+ current (IKr) and of recombinant hERG channels (the IKr channel α subunit clone;(Virag et al, 1998; Paul et al, 2001; McPate et al, 2006)). In comparison with the known situation for high-affinity hERG blocking drugs, hERG channel inactivation may not be obligatory for DISO to exert its inhibitory effect, suggestive of distinct mechanisms of inhibition and/or binding to the hERG channel (Paul et al, 2001, McPate et al, 2008). To-date the binding site on the hERG channel for DISO has not been mapped. Here the sensitivity to DISO of alanine and threonine mutants of key residues located either in the S6 segment or pore-helix of hERG were investigated at 37oC using conventional voltage clamp protocols. Human embryonic kidney (HEK-293) cells stably expressing the hERG mutant Y652A and Chinese Hamster Ovary cells transiently transfected with the S620T mutant (generated using Quikchange® mutagenesis) were used. Whole-cell recordings were made using a standard external Tyrode’s solution and K+-based pipette solution. Numerical data are expressed as mean ± S.E.M values. Previous studies from our laboratory have found WT hERG current (IhERG) to be inhibited by DISO with half-maximal inhibition (IC50) values of between ~7 to 10 μM (Paul et al, 2001; McPate et al, 2006). Here, DISO was found to inhibit Y652A IhERG tails recorded at -40 mV following test pulses to +20 mV with a high IC50 (402.6 ± 0.04 μM; n=5-7 cells per concentration; 5 concentrations between 10 μM and 3 mM were tested). The S620T inactivation-deficient mutant has been proposed to provide a means of assessing drug affinity for the hERG channel open state (Perrin et al, 2008). S620T IhERG tails were inhibited by DISO with an IC50 of 67.7 ± 0.06 μM (n=5-7 cells per concentration; 3 concentrations between 10 μM and 3 mM were tested). This is concordant with a measurable, though comparatively modest effect of channel inactivation on DISO binding (cf Paul et al, 2001; McPate et al, 2008, Perrin et al, 2008). The results of preliminary docking analysis using a homology model built onto the KvAP crystal structure template suggest that in low-energy binding structures the aromatic groups of DISO may reside at the lower end of the pore cavity. Docking analysis has also implicated residue F656 as a potentially important binding determinant. Collectively, our experimental data and docking simulations suggest that DISO interacts with S6 helix aromatic residues (Y652 and F656) on hERG channel gating. Additional in vitro investigations of hERG-mutants are ongoing to elucidate further the nature of DISO-hERG binding.