title: Identification of propofol binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.
abstract: 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, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [(3)H]AziPm, and labeled amino acids were identified by Edman degradation. [(3)H]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 [(3)H]AziPm photolabeling at αM2-10'. Propofol inhibited [(3)H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC50 = 40 μm) than it inhibited ion channel photolabeling (IC50 = 125 μm). 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.
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Selwyn S
authors.initials: SS
authors.lastname: Jayakar
authors.forename: William P
authors.initials: WP
authors.lastname: Dailey
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000818:Animals; D001665:Binding Sites; D029941:Fish Proteins / Q000737:chemistry*; D010777:Photochemistry / Q000379:methods; D015742:Propofol / Q000031:analogs & derivatives* / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014101:Torpedo
chemical_list: D029941:Fish Proteins; D011978:Receptors, Nicotinic; D015742:Propofol