Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2853-2858). To identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on 125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of beta was demonstrated after comparison of reactivities of native beta and single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta, when mutated individually or in combinations, had no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the alpha subunit that allows high affinity ChTX binding.