PROTON PROBING OF THE CHARYBDOTOXIN BINDING-SITE OF SHAKER K+ CHANNELS

We have investigated the interaction of charybdotoxin (CTX) with Shake r K channels. We substituted a histidine residue for the wild-type phe nylalanine (at position 425) in an inactivation-removed channel. The n ature of the imidazole ring of the histidine provides the ability to c hange 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 (K-d) Of 650 nM (at a membrane potential of 0 mV). The F425H mutant ch annels were much more sensitive to CTX block with an apparent K-d (at pH 7.0) of 75 nM. Block of F425H but not wild-type channels was strong ly 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 s teeper than expected for inhibition produced by H+ ions binding to ide ntical, independent sites. The data were consistent with H+ ions inter acting with subunits of the channel homotetrameric structure. The in s itu pK for the imidazole group on the histidine at channel position 42 5 was determined to be near 6.4 and the dissociation constant for bind ing of toxin to the unprotonated channel was near 50 nM. We estimate t hat 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 electros tatic and steric interactions between specific CTX residues and channe l position 425. Our results are consistent with a model in which proto ns on F425H channel subunits interact with three positive charges on C TX at an effective distance 6-7 Angstrom from this channel position.