The β Subunit of the High Conductance Calcium-activated Potassium Channel

Abstract

Coexpression of and subunits of the high conduct- ance 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 subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munu- jos, 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 subunit that are responsible for this change in binding affinity, Ala scan- ning mutagenesis was carried out along the extracellu- lar loop of , and the resulting effects on 125I-ChTX bind- ing were determined after coexpression with the 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 was demonstrated after comparison of reactivities of native and single- Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of , when mutated indi- vidually or in combinations, had no effect on toxin bind- ing with the exception of Glu94, whose alteration modi- fies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual resi- dues between Cys76 and Cys103 indicate that four posi- tions, Leu90, Tyr91, Thr93, and Glu94 are critical in con- ferring high affinity 125I-ChTX binding to the subunit complex. Mutations at these positions cause large ef- fects on the kinetics of ligand association and dissocia- tion, but they do not alter the physical interaction of with the subunit. All these data, taken together, sug- gest that the large extracellular loop of the maxi-K chan- nel subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate con- formation within the subunit that allows high affinity ChTX binding.