CROSS-LINKING OF CHARYBDOTOXIN TO HIGH-CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM CHANNELS - IDENTIFICATION OF THE COVALENTLY MODIFIED TOXIN RESIDUE

High-conductance calcium-activated potassium (maxi-K) channels are com posed of two subunits, alpha and beta. The pore-forming alpha subunit is a member of the mSlo family of K+ channels, whereas the beta subuni t is a novel protein that modulates the biophysical and pharmacologica l properties of the channel complex. In the presence of a bifunctional cross-linking reagent, monoiodotyrosine charybdotoxin ([I-125]ChTX) i s covalently incorporated specifically into LyS(69) Of the beta subuni t, which is located in a large extracellular loop of this protein. Usi ng variants of ChTX which retain their channel-blocking activity and i n which individual Lys residues have been mutated, we have identified the corresponding amino acid in ChTX that is involved in the cross-lin king reaction. All of the ChTX mutants investigated bind to the maxi-K channel and display the same pharmacological profile as native ChTX i n competition binding experiments. Whereas substitution of amino acids at positions 11 and 31 of ChTX yields wild-type cross-linking pattern s, the peptide without a Lys at position 32 fails to incorporate into the beta subunit of the maxi-K channel. Given the model for the intera ction between ChTX and the outer vestibule of the maxi-K channel that has been proposed (Stampe et al., 1994), our data constrain the maximu m distance between the pore of this channel and the region in the extr acellular loop of the beta subunit where the cross-linking reaction ta kes place to 11 Angstrom. This topological limit helps define structur al features of the maxi-K channel that may aide in probing the functio nal interaction between alpha and beta subunits of the channel complex .