Localization of phencyclidine binding sites on alpha and beta subunits of the nicotinic acetylcholine receptor from Torpedo ocellata electric organ using azido phencyclidine

Abstract

A photolabile derivative of phencycliding (PCP), azido phencyclidine (AZ-PCP), was synthesized and used to localize PCP binding sites on the acetylcholine receptor from Torpedo ocellata electric organ. In the dark, the binding of micromolar concentrations of [3H]AZ-PCP to a receptor-enriched membrane preparation fits a single dissociation constant (& = 2.65 PM) and is very similar to the binding of ["HIPCP. The agonist carbamylcholine increases the association rate (and the affinity) of these ligands to the receptor, but it does not alter the total number of available binding sites.Following UV irradiation and gel electrophoresis, [3H]AZ-PCP was found to label specifically the (Y and fl subunits of the receptor. The labeling of the cx subunit band was heavier, and it was inhibited by tetracaine and PCP but not by a-bungarotoxin (a-Bgt). The addition of carbamylcholine enhanced the labeling of the /3 subunit; this effect was diminished by cr-Bgt. The labeling of the p subunit was also inhibited by tetracaine and PCP. The effect of carbamylcholine, which binds to the LY subunit, could be the result of an induced conformational change, which is propagated to the p subunit and increases its labeling by [3H]AZ-PCP.A simple model which accommodates the binding and photoaffinity labeling data is described. According to the model, the high affinity PCP binding site is located between the (Y and /I receptor subunits, and the drug thus becomes attached simultaneously to both. Hypothetical overlapping recognition sites for PCP on these receptor subunits would allow binding (and labeling) with increased affinity in the presence of carbamylcholine with no increase in the number of available sites.

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

“…In attempts to locate the binding sites for noncompetitive inhibitors, several groups have photoaffinity-labeled Torpedo receptors efficiently with azido or mustard derivatives of noncompetitive inhibitors (Oswald & Changeux, 1981a; Kaldany & Karlin, 1983;Haring et al, 1984) or weakly by UV irradiation of the unmodified inhibitors (Oswald & Changeux, 1981b;Heidmann et al, 1983). Different inhibitors label different subunits, although the ability of chlorpromazine to label all four subunits with roughly equal efficiency and the displacement of chlorpromazine by other inhibitors suggest that the binding site for the inhibitors may exist in an area that is accessible to all receptor subunits, possibly in or near the central hydrophilic crevice, where the distances to all five receptor subunits are minimal (Changeux et al, 1984).…”

Identification of polypeptides of the phencyclidine receptor of rat hippocampus by photoaffinity labeling with [3H]azidophencyclidine

“…Although the sensitivity of labeling to various pharmacological active drugs was not reported, these authors indicated that several polypeptides were labeled by [3H]AZ-PCP, in agreement with the results reported here. In this connection, it should be noted that photoaffinity labeling of other receptors (e.g., nicotinic cholinergic receptors or opioid receptors) have also yielded labeling of multiple bands (Oswald & Changeux, 1981;Haring et al, 1983aHaring et al, , 1984Bidlack et al, 1981), which in the case of the nicotinic receptors are constituents of the receptor molecule. Interestingly, polypeptides with molecular weights similar to those labeled by [3H]AZ-PCP were also identified by affinity labeling or affinity chromatography of opioid receptors.…”

“…In this paper we describe the binding characteristics and affinity labeling of rat hippocampal PCP receptors, determined by employing the photoactivatable analogue of PCP N-[ 1 -(3-azidophenyl)cyclohexyl]piperidine (azidophencyclidine, AZ-PCP). This drug was used in our previous studies to localize the site of noncompetitive blockers of cholinergic receptors from Torpedo electric organ (Haring et al, 1983a(Haring et al, , 1984.…”

Acetylcholine Receptor: An Allosteric Protein

“…Both 3H-labeled perhydrohistrionicotoxin and phencyclidine, under ultraviolet irradiation (107), gave similar results. However, with T. californica and quinacrine mustard (36,109) [or triphenylmethylphosphonium (110)] or with T. ocellata and azidophencyclidine (111), labeling occurred primarily at level of the a or the ,3 chains, respectively. With T. marmorata, 3H-labeled chlorpromazine labeled all four chains, suggesting that they all contribute to the single highaffinity site present per oligomer (104,107).…”