Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin.

Electrostatic interactions between charybdotoxin (CTX), a specific peptide pore blocker of K+ channels, and a Ca(2+)-activated K+ channel were investigated with a genetically manipulable recombinant CTX. Point mutations at certain charged residues showed only small effects on the binding affinity of the toxin molecule: Lys11, Glu12, Arg19, His21, Lys31, and Lys32. Replacement by Gln at Arg25, Lys27, or Lys34 strongly decreased the affinity of the toxin. These affinity changes were mainly due to large increases of toxin dissociation rates without much effect on association rates, as if close-range interactions between the toxin and its receptor site of the channel were disrupted. We also found that the neutralization of Lys27 to Gln removed the toxin's characteristic voltage dependence in dissociation rate. Mutation and functional mapping of charged residues revealed a molecular surface of CTX which makes direct contact with the extracellular mouth of the K+ channel.