Modulation of WERG affinity for E-4031 by [K⁺](o) and C-type inactivation

Rectification of MERG is due to a rapid inactivation process that has been labeled C-type inactivation and is believed to he 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 methanosulfonanilide drug E4031. Removal of inactivation through mutation reduced drug affinity by more than an order of magnitude. Elevation of [K⁺](o) in the wild-type channel reduces channel affinity for E-4031. Elevation of [K⁺](o) also interferes with extracellular pore mouth closure associated with C-type inactivation through a 'foot in the door' mechanism. We exnmined the possibility that [K⁺](o) elevation reduces drug binding through inhibition of C-type inactivation by comparing drug block in the wild-type and inactivation-removed mutant channels. Elevation of [K⁺[(o)], decreased affinity in both channel constructs by a roughly equal amount. These results suggest that [K⁺](o) alters drug binding affinity independently of its effects on C-type inactivation. They further suggest that inhibition of pore mouth closure by elevated [K⁺](o) does not have same effect on drug affinity as mutations removing C-type inactivation.