Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore

Cyclodextrins act as noncovalent molecular adapters when lodged in the lume n of the alpha-hemolysin (alpha HL) pore. The adapters act as binding sites for channel blockers, thereby offering a basis for the detection of a vari ety of organic molecules with alpha HL as a biosensor element. To further s uch studies, it is important to find conditions under which the dwell rime of cyclodextrins in the lumen of the pore is extended. Here, we use single- channel recording to explore the pH- and voltage-dependence of the interact ion of beta-cyclodextrin (beta CD) with alpha HL.beta CD can access its bin ding site only from the trans entrance of pores inserted from the cis side of a bilayer. Analysis of the binding kinetics shows that there is a single binding site for beta CD, with an apparent equilibrium dissociation consta nt that varies by >100-fold under the conditions explored. The dissociation rate constant for the neutral beta CD molecule varies with pH and voltage, a result that is incompatible with two states of the alpha HL pore, one of high and the other of low affinity. Rather, the data suggest that the actu al equilibrium dissociation constant for the alpha HL.beta CD complex varie s continuously with the transmembrane potential.