The human ether-à-go-go related gene product (HERG) channel is essential for electrical activity of heart cells, and block of this channel by many drugs leads to lethal arrhythmias. Tyr(652) and Phe(656) of the sixth transmembrane helix are candidates for the drug binding site. In the tetrameric HERG channel, a drug with asymmetric structure should interact unevenly with multiple residues from different subunits. To elucidate the topology of the drug-binding site, we constructed tandem dimers of HERG channels and the aromatic Tyr(652) and Phe(656) residues were replaced by alanine singly or doubly. Eight types of HERG channels, including homotetrameric mutants, having different numbers and arrangements of aromatic residues at the blocking site, were studied. Effects of cisapride on channels expressed in Xenopus laevis oocytes were examined electrophysiologically. The inhibition constants (K(i)) were increased significantly as the diagonal Tyr(652) were deleted, whereas those for the diagonal Phe(656)-deleted mutant were not changed. These results suggest that Tyr(652) residues from adjacent subunits contributed to the binding. Two types of double mutants of tandem dimers showed significantly distinct affinities, suggesting that the coexistence of Tyr(652) and Phe(656) on a subunit in diagonal position is crucial to having a high affinity. Thermodynamic double-mutant cycle analyses revealed interactions between Tyr(652) and Phe(656) upon binding. The kinetics and voltage-dependence of blocking suggested transitions of the binding site from low to high affinity. These approaches using a set of mutant HERG channels gave a dynamic picture of the spatial arrangements of residues that contribute to the drug-channel interaction.