The external vestibules of many K+ channels carry a high-affinity receptor for charybdotoxin, a peptide of known structure. Point mutations of a recombinant toxin identified the residues directly involved in the interaction with a Ca(2+)-activated K+ channel. The interaction surface is formed from 8 of the 37 residues and covers about 25% of the peptide's molecular surface. The shape of the toxin permits a deduced picture of the complementary receptor site in the external vestibule of the K+ channel.

Basic electrophysiological properties of the KcsA K+ channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na+ to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.

Cysteine mutagenesis for the purpose of chemical labelling was applied to the K+ channel neurotoxin charybdotoxin, a 37-residue peptide with six functionally essential cysteines. An additional 'spinster cysteine' was introduced at a position far away in space from the toxin's known interaction surface where it contacts its K+ channel receptor. Despite the presence of the extra unpaired cysteine residue, the toxin still folds efficiently and may be labelled by fluorescent and radioactive reagents to give a functionally competent toxin.

No abstract