Vesnarinone, a cardiotonic agent, blocks I(Kr) and, unlike other I(Kr) blockers, produces a frequency-dependent prolongation of action potential duration (APD). To elucidate the mechanisms, we studied the effects of vesnarinone on HERG, the cloned human I(Kr) channel, heterologously expressed in Xenopus laevis oocytes. Vesnarinone caused a concentration-dependent inhibition of HERG currents with an IC(50) value of 17.7 +/- 2.5 microM at 0 mV (n = 6). When HERG was coexpressed with the beta-subunit MiRP1, a similar potency for block was measured (IC(50): 15.0 +/- 3.0 microM at 0 mV, n = 5). Tonic block of the HERG channel current was minimal (<5% at 30 microM, n = 5). The rate of onset of block and the steady-state value for block of current were not significantly different for test potentials ranging from -40 to +40 mV [time constant (tau) = 372 +/- 76 ms at +40 mV, n = 4]. Recovery from block at -60, -90, and -120 mV was not significantly different (tau = 8.5 +/- 1.5 s at -90 mV, n = 4). Vesnarinone produced similar effects on inactivation-removed mutant (G628C/S631C) HERG channels. The IC(50) value was 10.7 +/- 3.7 microM at 0 mV (n = 5), and the onset and recovery from block of current findings were similar to those of wild-type HERG. Amino acids important for the binding of vesnarinone were identified using alanine-scanning mutagenesis of residues believed to line the inner cavity of the HERG channel. Six important residues were identified, including G648, F656, and V659 located in the S6 domain and T623, S624, and V625 located at the base of the pore helix. These residues are similar but not identical to those determined previously for MK-499, an antiarrhythmic drug. In conclusion, vesnarinone preferentially blocks open HERG channels, with little effect on channels in the rested or inactivated state. These actions may contribute to the favorable frequency-dependent prolongation in APD.