Molecular determinants of HERG potassium channel blockade by domiphen bromide and benzethonium chloride

Domiphen bromide (DMP) and benzethonium chloride (BZT) are synthetic quaternary ammonium compounds widely used as disinfectants. Both agents are potent inhibitors of the human ether-à-go-go-related gene (HERG) potassium channel, a key contributor to cardiac repolarization. Dysfunction of HERG is associated with long QT syndrome and arrhythmias, yet the molecular mechanisms underlying DMP and BZT inhibition remain incompletely understood. In this study, we employed site-directed mutagenesis and whole-cell patch-clamp recording to identify key residues mediating DMP and BZT binding. Wild-type and nine mutant HERG channels were expressed in HEK-293 T cells, targeting residues in the pore helix (T623A, S624A, V625A), S6 helix (G648A, Y652A, F656A), S5-pore linker (S631A), and S5-S6 connector (N588K), including a double mutant (N588K/S631A). DMP exhibited strong dependence on S624, V625, Y652, N588, and S631, whereas BZT primarily involved S624, V625, and Y652. Computational docking revealed that DMP forms hydrogen bonds, π-cation, and π-π interactions with S624 and Y652, while BZT interacts through π-cation and π-π stacking with Y652 and hydrophobic contacts with S624. Importantly, our data highlight the quaternary ammonium group as a critical pharmacophore, mediating strong interactions with serine and aromatic residues via π-cation, electrostatic, and hydrogen bonding mechanisms, contributing to high-affinity channel blockade. In conclusion, this study defines the molecular determinants underlying DMP and BZT binding to the HERG channel and provides mechanistic insight that may guide the design of safer therapeutics with minimized HERG liability.