Genetically engineered metal ion binding sites on the outside of a channel's transmembrane beta-barrel

We are exploring the ability of genetically engineered versions of the Stap hylococcus aureus alpha-hemolysin (alpha HL) ion channel to serve as ration ally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel curre nt of wild-type alpha HL was affected by [Zn(ll)] less than or equal to 1 m M. Binding sites for the divalent cations were formed by altering the numbe r and location of coordinating side chains, e.g., histidines and aspartic a cids, between positions 126 and 134, inclusive. Several mutant alpha HLs ex hibited Zn(ll)-induced current noise that varied with Zn(ll) concentration. At a fixed divalent cation concentration, the current fluctuation kinetics depended on the analyte type, e.g., Zn(il), Cu(ll), Ni(ll), and Co(ll). We also show that the ability of Zn(ll) to change the mutant channel current suggests that the pore's topology is beta-sheet and that position 130 is ne ar the turn at the trans mouth. Both conclusions are consistent with the cr ystal structure of WT-alpha HL oligomerized in detergent. Our results, in t he context of the channel's crystal structure, suggest that conductance blo ckades were caused by Zn(ll) binding to the outside surface of the pore. Th us, analyte-induced current blockades alone might not establish whether an analyte binding site is inside a pore.