MOLECULAR ENVIRONMENT OF THE PHENCYCLIDINE BINDING-SITE IN THE NICOTINIC ACETYLCHOLINE-RECEPTOR MEMBRANE

Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclidine, 4-phenyl-4-(1-piperidinyl)-2,2,6,6-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits Rb-86 flux (IC50 = 6.6-mu-M), and [H-3]phencyclidine binding to both resting and desensitized acetylcholine receptor (IC50 = 17-mu-M and 0.22-mu-M, respectively). From an indirect Hill plot of the inhibition of [H-3]phencyclidine binding by PPT, a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in alpha-bungarotoxin-treated preparations. Taken together, these results indicate that PPT mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2-3 units.