Modulation of HERG affinity for E‐4031 by [K+]o and C‐type inactivation

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Abstract
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. 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 the extracellular pore mouth closure associated with C‐type inactivation through a `foot in the door' mechanism. We examined 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.