Identification of Polypeptides of the Phencyclidine Receptor of Rat Hippocampus by Photoaffinity Labeling with [³H]Azidophencyclidine

Polypeptide components of the phencyclidine (PCP) receptor present in rat hippocampus were identified with the photolabile derivative of phencyclidine [³H]azidophencyclidine ([³H]AZ-PCP). The labeled affinity probe was shown to reversibly bind to specific sites in the dark. The number of receptor sites bound is equal to those labeled by [³H]PCP, and their pharmacology and stereospecificity are identical with those of the PCP/σ-opiate receptors. The dissociation constant of [³H] AZ-PCP from these receptors is 0.25 ± 0.08 μM. Photolysis of hippocampus membranes preequilibrated with [³H] AZ-PCP, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the existence of five major labeled bands of which a M_r90000 band and a M_r 33 000 band were heavily labeled. Inhibition experiments, in which membranes were incubated with [³H] AZ-PCP in the presence of various PCP analogues and opiates, indicate that labeling of both the M_r90000 band and the M_r33 000 band is sensitive to relatively low concentrations (10 μM) of potent PCP/σ receptor ligands, while similar concentrations of levoxadrol, naloxone, morphine, D-Ala-D-Leu-enkephalin, atropine, propranolol, and serotonin were all ineffective. Stereoselective inhibition of labeling of the M_r 90 000 band and of the M_r33 000 band was also observed by the use of dexoxadrol and levoxadrol. The M_r33 000 band was not as sensitive as the M_r90000 band to inhibition by the selective PCP receptor ligands N- [l-(2-thienyl)cyclohexyl]piperidine and PCP. Strong inhibition of labeling of the M_r33 000 band by less selective PCP receptor ligands such as N-[1-(3-hydroxyphenyl)cyclohexyl]piperidine and (±)-N-allylnormetazocine was also observed. The labeling of the other three polypeptides (M_r62 000, 49 000, and 40 000) was only mildly affected by dexoxadrol and (±)-N-allylnormetazocine, suggesting that this interaction does not have a classical PCP/σ receptor pharmacology. Thus, these labeled bands could be constituents of a second PCP receptor.