Quinacrine noncompetitive inhibitor binding site localized on the Torpedo acetylcholine receptor in the open state

Open-channel blockers of the nicotinic acetylcholine receptor (nAcChR) are widely thought to act sterically by entering and "plugging" the open channel of the nAcChR. However, quinacrine, a fluorescent open-channel blocker, has been recently shown to bind to the nAcChR at a site near the lipid bilayer while the receptor is in a closed, desensitized state, suggesting that at least one open-channel blocker might act allosterically outside the channel [Valenzuela et al. (1992) J. Biol. Chem. 267, 8238]. To determine whether or not quinacrine also binds near the lipid bilayer when the receptor is in an open state, a short-range lipophilic quencher (5-doxylstearate, 5-SA) was used to assess the proximity of the nAcChR-bound quinacrine to the lipid bilayer while the receptor was transiently open by an agonist. Initial experiments using a stopped-flow instrument established the conditions required to monitor a portion of the changes in quinacrine fluorescence associated with its binding to the receptor in the open state. 5-SA (80 microM) reduced the amplitude of the rapid agonist-induced change in quinacrine emission to 44% +/- 12% of the control value, indicating that the quinacrine was binding to a site proximal to the membrane-partitioned 5-SA. Control experiments established that 5-SA had no effect on the ability of the receptor to undergo agonist-induced conformational changes, suggesting that little, if any, 5-SA distributed into the channel lumen and perturbed the functional activity of the receptor. Together, the results indicate that quinacrine binds to a site on the open receptor that is in contact with the lipid bilayer and not in the channel lumen.