Proton probing of the charybdotoxin binding site of Shaker K+ channels.

We have investigated the interaction of charybdotoxin (CTX) with Shaker K channels. We substituted a histidine residue for the wild-type phenylalanine (at position 425) in an inactivation-removed channel. The nature of the imidazole ring of the histidine provides the ability to change the charge on this amino acid side chain with solution hydrogen ion concentration. Wild-type, recombinant CTX blocked wild-type Shaker channels in a bimolecular fashion with a half-blocking concentration (Kd) of 650 nM (at a membrane potential of 0 mV). The F425H mutant channels were much more sensitive to CTX block with an apparent Kd (at pH 7.0) of 75 nM. Block of F425H but not wild-type channels was strongly pH sensitive. A pH change from 7 to 5.5 rendered the F425H channels >200-fold less sensitive to CTX. The pH dependence of CTX block was steeper than expected for inhibition produced by H+ ions binding to identical, independent sites. The data were consistent with H+ ions interacting with subunits of the channel homotetrameric structure. The in situ pK for the imidazole group on the histidine at channel position 425 was determined to be near 6.4 and the dissociation constant for binding of toxin to the unprotonated channel was near 50 nM. We estimate that the binding of a H+ ion to each subunit adds 0.8 kcal/mol or more of interaction energy with CTX. We used mutant toxins to test electrostatic and steric interactions between specific CTX residues and channel position 425. Our results are consistent with a model in which protons on F425H channel subunits interact with three positive charges on CTX at an effective distance 6-7 A from this channel position.