Association of a spin-labeled local anesthetic with the allosterically coupled noncompetitive inhibitor site on the acetylcholine receptor

A Palma, JM Herz, HH Wang and P Taylor

Radioligand binding and ESR were used to study the association of a spin-labeled local anesthetic, 2-[N,N-dimethyl-N-(2,2,6,6- tetramethylpiperidinooxyl)] ethyl-4-hexyloxybenzoate iodide (C6SLMel), with acetylcholine receptor-enriched membranes from Torpedo californica. In the presence of carbamylcholine, we found that C6SLMel competitively inhibits [3H]phencyclidine binding with high affinity (KD = 8.7 X 10(-7) M for C6SLMel), whereas in the presence of alpha-toxin or in the absence of agonist, C6SLMel binds with lower affinity (KD = 2 X 10(-5) M). At concentrations lower than 1 X 10(-5) M, C6SLMel does not bind to the agonist site but enhances [3H]acetylcholine binding. These findings show that C6SLMel binds to the allosterically coupled noncompetitive inhibitor site which is regulated by agonist binding. In addition, C6SLMel preferentially associates with the desensitized receptor state known to exhibit high affinities for agonists and local anesthetics. ESR measurements of C6SLMel bound to receptor membranes in the absence of agonist show moderately immobilized spectra. Addition of carbamylcholine results in the appearance of a strongly immobilized component. Prior exposure to alpha-toxin blocks the carbamylcholine- induced, strongly immobilized component in the ESR spectrum. Furthermore, in the presence of carbamylcholine, back-titration of bound C6SLMel with phencyclidine decreases the highly immobilized component at concentrations consistent with the KD for phencyclidine. These findings indicate that C6SLMel detects conformational changes between the resting and desensitized acetylcholine receptor states that occur at the noncompetitive inhibitor binding site. The strongly mobilized component is not affected by ferricyanide addition, suggesting that the binding site is in a region not readily accessible to anion collision from the aqueous phase.

Volume 30, Issue 3, pp. 243-251, 09/01/1986
Copyright © 1986 by American Society for Pharmacology and Experimental Therapeutics