The low-potency, voltage-dependent HERG blocker propafenone--molecular determinants and drug trapping.

The molecular determinants of high-affinity human ether-a-go-go-related gene (HERG) potassium channel blockade by methanesulfonanilides include two aromatic residues (Phe656 and Tyr652) on the inner helices (S6) and residues on the pore helices that face into the inner cavity, but determinants for lower-affinity HERG blockers may be different. In this study, alanine-substituted HERG channel mutants of inner cavity residues were expressed in Xenopus laevis oocytes and were used to characterize the HERG channel binding site of the antiarrhythmic propafenone. Propafenone's blockade of HERG was strongly dependent on residue Phe656 but was insensitive or weakly sensitive to mutation of Tyr652, Thr623, Ser624, Val625, Gly648, or Val659 and did not require functional inactivation. Homology models of HERG based on KcsA and MthK crystal structures, representing the closed and open forms of the channel, respectively, suggest propafenone is trapped in the inner cavity and is unable to interact exclusively with Phe656 in the closed state (whereas exclusive interactions between propafenone and Phe656 are found in the open-channel model). These findings are supported by very slow recovery of wild-type HERG channels from block at -120 mV, but extremely rapid recovery of D540K channels that reopen at this potential. The experiments and modeling suggest that the open-state propafenone binding-site may be formed by the Phe656 residues alone. The binding site for propafenone (which may involve pi-stacking interactions with two or more Phe656 side-chains) is either perturbed or becomes less accessible because of closed-channel gating. This provides further evidence for the existence of gating-induced changes in the spatial location of Phe656 side chains.