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

We are exploring the ability of genetically engineered versions of the Staphylococcus aureus alpha-hemolysin (alpha HL) ion channel to serve as rationally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel current of wild-type alpha HL was affected by [Zn(ll)] less than or equal to 1 mM. Binding sites for the divalent cations were formed by altering the number and location of coordinating side chains, e.g., histidines and aspartic acids, between positions 126 and 134, inclusive. Several mutant alpha HLs exhibited 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 near the turn at the trans mouth. Both conclusions are consistent with the crystal structure of WT-alpha HL oligomerized in detergent. Our results, in the context of the channel's crystal structure, suggest that conductance blockades were caused by Zn(ll) binding to the outside surface of the pore. Thus, analyte-induced current blockades alone might not establish whether an analyte binding site is inside a pore.