Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

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作者：

Selwyn，S.，Jayakar，William，P.，Dailey，Roderic，G.，Eckenhoff，Jonathan

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摘要：

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPmstabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [3H]AziPm, and labeled amino acids were identified by Edman degradation. [3H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18' and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (-agonist) amino acids in the M2 helices (αM2-6', βM2-6' and -13', and δM2-13') that line the channel lumen (with photolabeling reduced by>90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10'. Propofol enhanced [3H]AziPm photolabeling at αM2-10'. Propofol inhibited [3H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50=40 μM) than it inhibited ion channel photolabeling (IC50=125μM). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.

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关键词：

GATED ION-CHANNEL NONCOMPETITIVE ANTAGONIST GENERAL-ANESTHETICS GABA(A) RECEPTOR ETOMIDATE POTENTIATION DESENSITIZATION SUBUNIT STATE