title: Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.
abstract: Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABAAR modulation. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ∼ 50%. When the amino acids photolabeled by [(3)H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [(3)H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.
authors.affiliation: From the Departments of Neurobiology and.
authors.forename: Selwyn S
authors.initials: SS
authors.lastname: Jayakar
authors.affiliation: the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: From the Departments of Neurobiology and.
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.affiliation: the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Dostalova
authors.affiliation: the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, and.
authors.forename: Pavel Y
authors.initials: PY
authors.lastname: Savechenkov
authors.affiliation: the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, and.
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.affiliation: the Department of Chemistry, University of Pennsylvania and.
authors.forename: William P
authors.initials: WP
authors.lastname: Dailey
authors.affiliation: the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Miller
authors.affiliation: Department of Anesthesiology and Critical Care, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104.
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
authors.affiliation: From the Departments of Neurobiology and jonathan_cohen@hms.harvard.edu.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D019920:Photoaffinity Labels; D015742:Propofol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C546240:GABRA1 protein, human; C546273:GABRB3 protein, human; D019920:Photoaffinity Labels; D011963:Receptors, GABA-A; D015742:Propofol
keywords: Anesthesia; GABA Receptor; General Anesthetics; Nicotinic Acetylcholine Receptors (nAChR); Photoaffinity Labeling; Propofol; Protein Drug Interaction
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