The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K+ channels there is little mechanistic information on this action. We have investigated in detail hERG K+ channel current (I-hERG) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell IhERG measurements were made at 37 C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I-hERG tails at -40 mV following a 2 s depolarization to +20 mV with an IC50 value of 80 nM. Comparable levels of I-hERG inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I-hERG was only weakly voltage-dependent, though the time dependence of IhERG inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I-hERG inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K+ channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore. (c) 2006 Elsevier Inc. All rights reserved.