Revised Manuscript Received July 7, 1995® abstract: High-conductance calcium-activated potassium (maxi-K) channels are composed of two subunits, a and (3. The pore-forming a subunit is a member of the mSlo family of K+ channels, whereas the [3 subunit is a novel protein that modulates the biophysical and pharmacological properties of the channel complex. In the presence of a bifunctional cross-linking reagent, monoiodotyrosine charybdotoxin ([125I] ChTX) is covalently incorporated specifically into Lys69 of the/3 subunit, which is located in a large extracellular loop of this protein. Using variants of ChTXwhich retain their channel-blocking activity and in which individual Lys residues have been mutated, we have identified the corresponding amino acid in ChTX that is involved in the cross-linking reaction. All of the ChTX mutants investigated bind to the maxi-K channel and display the same pharmacological profile as native ChTX in competition binding experiments. Whereas substitution of amino acids at positions 11 and 31 of ChTXyields wild-type cross-linking patterns, the peptide without a Lys at position 32 fails to incorporate into the (3 subunit of the maxi-K channel. Given the model for the interaction between ChTX and the outer vestibule of the maxi-K channel that has beenproposed (Stampe et al., 1994), our data constrain the maximum distance between the pore of this channel and the region in the extracellular loop of the j3 subunit where the cross-linking reaction takes place to 11 A. This topological limit helps define structural features of the maxi-K channel that may aide in probing the functional interaction between a and (3 subunits of the channel complex.

Large-conductance Ca2+-activated K+(maxi-K) 1 channels are a family of channel proteins that display both high selectivity for and high conductance of potassium ion (Latorre etal., 1989). These channels are gated by increasing intracellular Ca2+ levels and by membranedepolarization. Maxi-K channels are potently blocked by a number of peptidyl toxins [eg, charybdotoxin (ChTX), iberiotoxin (IbTX), and limbatustoxin] that have been isolated from scorpion venoms[for a review, see Garcia et al.(1993)]. A large body of evidence suggests that these toxins bind in the outer vestibule of the channel to physically occlude the