Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic

Abstract

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [3H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [3H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC50= 70 μM) than in the closed channel state. In addition, both drugs between 0.1 and 1 mM produced a concentration-dependent enhancement of [3H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [3H]azietomidate photoincorporation into the nAChR a and δ subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of aGlu-262 and δGln-276 at the extracellular end and δSer-258 and δSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [3H]azietomidate photolabeled αTyr-93, aTyr-190, and aTyr-198 in the site at the α-γ interface and δAsp-59 (but not the homologous position, γGlu-57). Increasing [3H]azietomidate concentration from 1.8 to 150 μM increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [3H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.