Interaction of charybdotoxin with permeant ions inside the pore of a K + channel

Abstract

Charybdotoxin (CTX) blocks high conductance Ca 2+-activated K + channels by binding to a receptor site in the externally facing “mouth.” Toxin bound to the channel can be destabilized from its site by K + entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca 2+-activated K + channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K + with CTX. If position 27 carries a positively charged residue, internal K + accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gin is substituted at this position, the dissociation rate is completely insensitive to either internal K + or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K + destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K +-specific binding site on the external end of the conduction pathway and that a K + ion occupying this site destabilizes CTX via direct electrostatic repulsion with the ε-amino group of Lys-27.