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[摘要]：Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 mu M. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (> 10 mu M) increased the submaximal response to acetylcholine (10 mu M) with a 2.5-fold increase at 60 mu M. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [H-3] tetracaine and [3H] phencyclidine to Torpedo nAChR-rich membranes (IC50 values of 0.8 mM). nAChR-rich membranes were photolabeled with [3H] TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (delta Leu-265) and M2-13 (alpha Val-255 and delta Val-269), whereas labeling of alpha M2-10 (alpha Ser-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gamma M3 (gamma Met-299), a residue that contributes to the same pocket in the nAChR structure as alpha M2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alpha M2-10 and gamma Met-299) likely to be a site for positive allosteric modulation. |
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