Channel inhibition by alkanols occurs at a binding site on the nicotinic acetylcholine receptor

The mechanism by which normal alkanols longer than ethanol inhibit cation flux through the transient open state of the nicotinic acetylcholine receptor (nAcChoR) is unknown. They might act nonspecifically either by perturbing the lipid bilayer or by binding to many low affinity sites. Alternatively, they might act in a mutually exclusive manner at a well defined site on the protein. To address this problem, a rapid assay of agonist-induced 86Rb+ efflux from nAcChoR-rich Torpedo membrane vesicles was used that enabled the anesthetic-induced inhibition to be measured on a millisecond time scale, under conditions where the concentration of all ligands was raised in < 1 msec, thereby avoiding complications due to desensitization. By measuring the inhibition constant of one agent as a function of the fixed concentration of a second agent, it is possible to distinguish between nonspecific action and mutually exclusive action. Our data are inconsistent with the hypothesis that 1-octanol and 1-heptanol act in a nonspecific manner, but they are consistent with the hypothesis that these two alkanols act in a mutually exclusive manner at a well defined site. The data suggest that the alkanols sterically compete for the site, but experimental limitations prevented a less plausible model, in which there is a strong negative allosteric interaction between separate octanol and heptanol sites, from being ruled out. Should the latter interaction occur, the data indicate that occupation of one alkanol site would decrease the affinity of the other by about 50-fold. The local anesthetic procaine is known to act in a mutually exclusive manner with the agonist self-inhibition site. We found that octanol and procaine acted as separate sites, which exhibited a negative heterotrophic interaction such that octanol reduced the affinity of procaine 6-fold. We conclude that octanol and heptanol inhibit cation flux through the channel of the nAcChoR by binding to a site (or a set of sites of equal affinity) whose location is distinct from, but allosterically coupled to, the agonist self-inhibition site.