Multiple Affinity States for Noncompetitive Blockers Revealed by [³H]Phencyclidine Binding to Acetylcholine Receptor Rich Membrane Fragments from Torpedo marmorata

Phencyclidine, a potent noncompetitive blocker of the permeability response to acetylcholine, binds selectively (below 20 µM concentration) to the high-affinity allosteric site of the membrane-bound acetylcholine receptor from Torpedo marmorata electric organ [Heidmann, T., Oswald, R. E., & Changeux, J.-P. (1983) Biochemistry (preceding paper in this issue)]. The kinetics of [³H]phencyclidine interaction with this particular site (present as a single copy per receptor light form) were investigated by a manual filtration technique and fluorescence stopped-flow measurements in the presence, and in the absence, of cholinergic ligands specific for the acetylcholine receptor site. When the membrane fragments were equilibrated with the agonist carbamoylcholine prior to the addition of [³H] phencyclidine, the association and dissociation rate constants of [³H] phencyclidine increased respectively from (1.22 ± 0.05) ϗ 10³ M⁻¹ s⁻¹ to (7.2 ± 0.5) × 10³ M⁻¹ s⁻¹ and from (7.5 ± 0.1) × 10⁻³ s⁻¹ to (9.8 ± 0.7) × 10⁻³ s⁻¹. On the other hand, preincubation with erabutoxin decreased both rates by a factor of about 2 from their value measured in the absence of effector. When carbamoylcholine was mixed with acetylcholine receptor rich membranes simultaneously with [³H] phencyclidine, the association rate of [³H] phencyclidine increased 10³–10⁴-fold and the dissociation rate 4–10-fold. These effects were not observed with the competitive antagonist d-tubocurarine. The data are interpreted in terms of a four-state model of the allosteric transitions of the acetylcholine receptor where D is a high-affinity slowly desensitized state, I an intermediate rapidly desensitized state, and A an active state, the only one where the ion channel is open. Rate constants for the interaction of [³H]phencyclidine with these different conformations of the acetylcholine receptor are estimated. The results suggest that, in addition to its stabilization of the D state reported in the previous paper, phencyclidine binds with the fastest rate to the A state where the channel is open and accelerates a transition toward the I state.