MODULATION OF HERG AFFINITY FOR E-4031 BY [K-TYPE INACTIVATION(](O) AND C)

Rectification of HERG is due to a rapid inactivation process that has been labeled C-type inactivation and is believed to be due to closure of the external mouth of the pore. We examined the effects of mutation of extracellular residues that remove C-type inactivation on binding of the intracellularly acting methanesulfonanilide drug E-4031. Remova l of inactivation through mutation reduced drug affinity by more than an order of magnitude, Elevation of [K+](o) in the wild-type channel r educes channel affinity for E-4031. Elevation of [K+](o) also interfer es with the extracellular pore mouth closure associated with C-type in activation through a 'foot in the door' mechanism. We examined the pos sibility that [K+](o) elevation reduces drug binding through inhibitio n of C-type inactivation by comparing drug block in the wild-type and inactivation-removed mutant channels. Elevation of [K+](o) decreased a ffinity in both channel constructs by a roughly equal amount. These re sults suggest that [K+](o) alters drug binding affinity independently of its effects on C-type inactivation. They further suggest that inhib ition of pore mouth closure by elevated [K+](o) does not have same eff ect on drug affinity as mutations removing C-type inactivation. (C) 19 97 Federation of European Biochemical Societies.