CHANNEL INHIBITION BY ALKANOLS OCCURS AT A BINDING-SITE ON THE NICOTINIC ACETYLCHOLINE-RECEPTOR

The mechanism by which normal alkanols longer than ethanol inhibit cat ion flux through the transient open state of the nicotinic acetylcholi ne receptor (nAcChoR) is unknown. They might act nonspecifically eithe r by perturbing the lipid bilayer or by binding to many low affinity s ites. Alternatively, they might act in a mutually exclusive manner at a well defined site on the protein. To address this problem, a rapid a ssay of agonist-induced Rb-86(+) efflux from nAcChoR-rich Torpedo memb rane 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 tl msec, thereby avoiding complications due to desensitization. By measuring the inhibition cons tant of one agent as a function of the fixed concentration of a second agent, it is possible to distinguish between nonspecific action and m utually exclusive action. Our data are inconsistent with the hypothesi s 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 mu tually exclusive manner at a well defined site. The data suggest that the alkanols sterically compete for the site, but experimental limitat ions prevented a less plausible model, in which there is a strong nega tive allosteric interaction between separate octanol and heptanol site s, from being ruled out. Should the latter interaction occur, the data indicate that occupation of one alkanol site would decrease the affin ity of the other by about 50-fold. The local anesthetic procaine is kn own to act in a mutually exclusive manner with the agonist self-inhibi tion site. We found that octanol and procaine acted at separate sites, which exhibited a negative heterotrophic interaction such that octano l reduced the affinity of procaine 6-fold. We conclude that octanol an d heptanol inhibit cation flux through the channel of the nAcChoR by b inding to a site (or a set of sites of equal affinity) whose location is distinct from, but allosterically coupled to, the agonist self-inhi bition site.