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• W1972584910 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. [3H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[3H]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 ([3H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ∼50%. When the amino acids photolabeled by [3H]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 [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[3H]mTFD-MPAB. The propofol-inhibitable [3H]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. 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. [3H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[3H]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 ([3H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ∼50%. When the amino acids photolabeled by [3H]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 [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[3H]mTFD-MPAB. The propofol-inhibitable [3H]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. Propofol, a widely used intravenous general anesthetic, acts as a positive allosteric modulator of inhibitory GABA type A receptors (GABAAR), 2The abbreviations used are: GABAARγ-aminobutyric acid type A receptorAziPm2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenolo-PDo-propofol diazirineazietomidate2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylateetomidate2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylateTDBzl-etomidate4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylatemTFD-MPAB5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acidnAChRnicotinic acetylcholine receptorTMDtransmembrane domainEndoGlu-CS. aureus endopeptidase Glu-CEndoLys-CL. enzymogenes endoproteinase Lys-CrpHPLCreversed-phase high pressure liquid chromatographyOPAo-phthalaldehydeBNPS-skatole3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indolePPFpropofolPTHphenylthiohydantoin. an interaction that determines its anesthetic potency in vivo (1.Belelli D. Lambert J.J. Peters J.A. Wafford K. Whiting P.J. The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11031-11036Crossref PubMed Scopus (348) Google Scholar, 2.Krasowski M.D. Jenkins A. Flood P. Kung A.Y. Hopfinger A.J. Harrison N.L. General anesthetic potencies of a series of propofol analogs correlate with potency for potentiation of γ-aminobutyric acid (GABA) current at the GABA(A) receptor but not with lipid solubility.J. Pharmacol. Exp. Ther. 2001; 297: 338-351PubMed Google Scholar, 3.Jurd R. Arras M. Lambert S. Drexler B. Siegwart R. Crestani F. Zaugg M. Vogt K.E. Ledermann B. Antkowiak B. Rudolph U. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor β3 subunit.FASEB J. 2003; 17: 250-252Crossref PubMed Scopus (505) Google Scholar, 4.Franks N.P. General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal.Nat. Rev. Neurosci. 2008; 9: 370-386Crossref PubMed Scopus (910) Google Scholar). However, the number and location of GABAAR-binding sites for propofol remain uncertain. GABAARs are members of the superfamily of pentameric ligand-gated ion channels formed by five identical or homologous subunits that associate around a central axis that forms the ion channel (5.Miller P.S. Smart T.G. Binding, activation and modulation of Cys-loop receptors.Trends Pharmacol. Sci. 2010; 31: 161-174Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 6.Sigel E. Steinmann M.E. Structure, function, and modulation of GABAA receptors.J. Biol. Chem. 2012; 287: 40224-40231Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Each subunit is made up of an N-terminal extracellular domain, a transmembrane domain (TMD) formed by a loose bundle of four transmembrane helices (M1–M4), with amino acids on one face of each M2 helix contributing to the lumen of the ion channel, and an intracellular domain formed by the amino acids between the M3 and M4 helices. γ-aminobutyric acid type A receptor 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol o-propofol diazirine 2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid nicotinic acetylcholine receptor transmembrane domain S. aureus endopeptidase Glu-C L. enzymogenes endoproteinase Lys-C reversed-phase high pressure liquid chromatography o-phthalaldehyde 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole propofol phenylthiohydantoin. GABAAR residues that may contribute to propofol-binding sites, identified by analyses of the functional properties of mutant receptors, include positions 15 of the β subunit M2 helix (βM2–15′) and four of the M3 helix (βM3–4′, β3Met-286), numbered relative to the conserved Arg and Asp near the N terminus of each subunit's M2 and M3 helices, respectively (7.Krasowski M.D. Nishikawa K. Nikolaeva N. Lin A. Harrison N.L. Methionine 286 in transmembrane domain 3 of the GABA(A) receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics.Neuropharmacology. 2001; 41: 952-964Crossref PubMed Scopus (112) Google Scholar, 8.Bali M. Akabas M.H. Defining the propofol-binding site location on the GABAA receptor.Mol. Pharmacol. 2004; 65: 68-76Crossref PubMed Scopus (147) Google Scholar). In addition, positions in βM4 (9.Richardson J.E. Garcia P.S. O'Toole K.K. Derry J.M. Bell S.V. Jenkins A. A conserved tyrosine in the β2 subunit M4 segment is a determinant of γ-aminobutyric acid type A receptor sensitivity to propofol.Anesthesiology. 2007; 107: 412-418Crossref PubMed Scopus (44) Google Scholar) and in the α subunit cytoplasmic domain (10.Moraga-Cid G. Yevenes G.E. Schmalzing G. Peoples R.W. Aguayo L.G. A single phenylalanine residue in the main intracellular loop of α1 γ-aminobutyric acid type A and glycine receptors influences their sensitivity to propofol.Anesthesiology. 2011; 115: 464-473Crossref PubMed Scopus (31) Google Scholar) have been identified as propofol sensitivity determinants. These propofol sensitivity determinants can be located in models of heteromeric GABAARs constructed by homology from the recently solved structure of a homomeric β3 GABAAR (11.Miller P.S. Aricescu A.R. Crystal structure of a human GABAA receptor.Nature. 2014; (10.1038/nature13293)Crossref Scopus (527) Google Scholar) or the structures of other pentameric ligand-gated ion channels, including the Torpedo nicotinic acetylcholine receptor (nAChR) (12.Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1413) Google Scholar), the prokaryotic proton-gated channel GLIC (13.Hilf R.J. Dutzler R. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.Nature. 2009; 457: 115-118Crossref PubMed Scopus (469) Google Scholar), the amine-gated channel ELIC (14.Zimmermann I. Dutzler R. Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC.PLoS Biol. 2011; 9: e1001101Crossref PubMed Scopus (92) Google Scholar), and the invertebrate glutamate-gated channel GluCl (15.Hibbs R.E. Gouaux E. Principles of activation and permeation in an anion-selective Cys-loop receptor.Nature. 2011; 474: 54-60Crossref PubMed Scopus (787) Google Scholar). In these models, βM2–15′ and βM3–4′, positions that are also sensitivity determinants for the intravenous anesthetic etomidate, are present in a pocket at the interface between the β and α subunits that contains the transmitter-binding sites in the extracellular domain (referred to as the β+-α− interface) (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar, 17.Bertaccini E.J. Yoluk O. Lindahl E.R. Trudell J.R. Assessment of homology templates and an anesthetic-binding site within the γ-aminobutyric acid receptor.Anesthesiology. 2013; 119: 1087-1095Crossref PubMed Scopus (27) Google Scholar), although the other sensitivity determinants are not within that intersubunit pocket. That etomidate binds to this intersubunit site was established by the etomidate-inhibitable photoincorporation of reactive etomidate analogs into βM3–4′ and α1Met-236 in αM1 in a heterogeneous population of GABAARs purified from bovine brain (18.Li G.-D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. Identification of a GABAA receptor anesthetic-binding site at subunit interfaces by photolabeling with an etomidate analog.J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (250) Google Scholar) and in purified human α1β3 GABAAR (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar). Recently, photoaffinity labeling studies with R-[3H]mTFD-MPAB, a photoreactive barbiturate, identified a second class of general anesthetic-binding sites in human α1β3γ2 GABAARs at the β−-α+ and β−-γ+ subunit interfaces (19.Chiara D.C. Jayakar S.S. Zhou X. Zhang X. Savechenkov P.Y. Bruzik K.S. Miller K.W. Cohen J.B. Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.J. Biol. Chem. 2013; 288: 19343-19357Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Although etomidate bound selectively to the β+ interface sites and certain barbiturates bound selectively at the β− interface sites, propofol inhibited photolabeling at both classes of sites, but only at concentrations (IC50 ∼40 µm) that were ∼10-fold higher than the concentrations necessary to potentiate GABA responses. This discrepancy suggests that propofol may bind with higher affinity to other, as yet unidentified, sites in the GABAAR. A reactive propofol analog (o-propofol diazirine (o-PD)) was recently shown to photoincorporate in expressed α1β3 GABAAR into β3His-267 (βM2–17′), an amino acid in the β subunit M2 helix in proximity to the R-mTFD-MPAB site, but projecting into the lumen of the ion channel near the interface between the extracellular and transmembrane domains (20.Yip G.M. Chen Z.W. Edge C.J. Smith E.H. Dickinson R. Hohenester E. Townsend R.R. Fuchs K. Sieghart W. Evers A.S. Franks N.P. A propofol-binding site on mammalian GABAA receptors identified by photolabeling.Nat. Chem. Biol. 2013; 9: 715-720Crossref PubMed Scopus (174) Google Scholar). In this report, we identify propofol-binding sites in a purified human α1β3 GABAAR using 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm), a photoreactive propofol analog that potentiates GABA responses and acts as a general anesthetic (Fig. 1) (21.Hall M.A. Xi J. Lor C. Dai S. Pearce R. Dailey W.P. Eckenhoff R.G. m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol.J. Med. Chem. 2010; 53: 5667-5675Crossref PubMed Scopus (61) Google Scholar). Propofol and AziPm are nAChR inhibitors, and photoaffinity labeling of the Torpedo nAChR established propofol-inhibitable photoincorporation of [3H]AziPm into two sites in the TMD as follows: an intrasubunit site in the δ subunit helix bundle, and a site in the ion channel (22.Jayakar S.S. Dailey W.P. Eckenhoff R.G. Cohen J.B. Identification of propofol-binding sites in a nicotinic acetylcholine receptor with a photoreactive propofol analog.J. Biol. Chem. 2013; 288: 6178-6189Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Propofol and AziPm are also inhibitors of GLIC, and in GLIC crystals propofol binds in the TMD in the intrasubunit pocket formed by the four transmembrane helices (23.Nury H. Van Renterghem C. Weng Y. Tran A. Baaden M. Dufresne V. Changeux J.P. Sonner J.M. Delarue M. Corringer P.J. X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel.Nature. 2011; 469: 428-431Crossref PubMed Scopus (356) Google Scholar). In purified GLIC in detergent solution, propofol inhibited [3H]AziPm photolabeling of amino acids in that binding pocket (24.Chiara D.C. Gill J.F. Chen Q. Tillman T. Dailey W.P. Eckenhoff R.G. Xu Y. Tang P. Cohen J. Photoaffinity labeling the propofol-binding site in GLIC.Biochemistry. 2014; 53: 135-142Crossref PubMed Scopus (34) Google Scholar). Based upon the identification of the GABAAR amino acids photolabeled by [3H]AziPm and the effects of propofol, AziPm, and o-PD on GABAAR photolabeling by [3H]azietomidate and R-[3H]mTFD-MPAB, we found in this study that propofol, AziPm, and o-PD bind in the α1β3 GABAAR to the same intersubunit sites as etomidate and R-mTFD-MPAB, i.e. the homologous sites at the β+-α−, α+-β−, and β+-β− subunit interfaces. We found no evidence of [3H]AziPm photolabeling of GABAAR amino acids that would be located in intrasubunit binding pockets or in the ion channel. Nonradioactive AziPm was synthesized as described (21.Hall M.A. Xi J. Lor C. Dai S. Pearce R. Dailey W.P. Eckenhoff R.G. m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol.J. Med. Chem. 2010; 53: 5667-5675Crossref PubMed Scopus (61) Google Scholar), and [3H]AziPm (10 Ci/mmol) was prepared by AmBios (Newington, CT) by ring iodination followed by catalytic reduction with tritium gas. Nonradioactive R-mTFD-MPAB and R-[3H]mTFD -MPAB (38 Ci/mmol) were prepared previously (25.Savechenkov P.Y. Zhang X. Chiara D.C. Stewart D.S. Ge R. Zhou X. Raines D.E. Cohen J.B. Forman S.A. Miller K.W. Bruzik K.S. Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.J. Med. Chem. 2012; 55: 6554-6565Crossref PubMed Scopus (41) Google Scholar), as was [3H]azietomidate (12 Ci/mmol) (26.Husain S.S. Ziebell M.R. Ruesch D. Hong F. Arevalo E. Kosterlitz J.A. Olsen R.W. Forman S.A. Cohen J.B. Miller K.W. 2-(3-methyl-3H-diaziren-3-yl) ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: a derivative of the stereoselective general anesthetic etomidate for photolabeling ligand-gated ion channels.J. Med. Chem. 2003; 46: 1257-1265Crossref PubMed Scopus (81) Google Scholar), which was also resynthesized at 19 Ci/mmol by catalytic reduction of m-bromoazietomidate with tritium gas. o-PD was synthesized from 2-isopropyl-6-trifluoroacetylphenol as described (20.Yip G.M. Chen Z.W. Edge C.J. Smith E.H. Dickinson R. Hohenester E. Townsend R.R. Fuchs K. Sieghart W. Evers A.S. Franks N.P. A propofol-binding site on mammalian GABAA receptors identified by photolabeling.Nat. Chem. Biol. 2013; 9: 715-720Crossref PubMed Scopus (174) Google Scholar) with the purity >96% as judged by 1H and 19F NMR. As reported (20.Yip G.M. Chen Z.W. Edge C.J. Smith E.H. Dickinson R. Hohenester E. Townsend R.R. Fuchs K. Sieghart W. Evers A.S. Franks N.P. A propofol-binding site on mammalian GABAA receptors identified by photolabeling.Nat. Chem. Biol. 2013; 9: 715-720Crossref PubMed Scopus (174) Google Scholar), the UV spectrum was characterized by an absorption maximum at 280 nm (extinction coefficient, ϵ280 = 2417 ± 24 m−1 cm−1) and an unresolved long wavelength shoulder (ϵ350 = 66 ± 1 m−1 cm−1), and when stored in ethanol at −20 °C, o-PD was stable for more than 1 month. n-Dodecyl-β-d-maltopyranoside, sodium cholate, and CHAPS were from Anatrace-Affymetrix (Anagrade quality). (R)-Etomidate was from Organon Laboratories. Soybean asolectin, FLAG peptide (DYKDDDDK), γ-aminobutyric acid (GABA), propofol, 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BNPS-skatole), and cyanogen bromide (CNBr) were from Sigma. o-Phthalaldehyde (OPA) was from Alfa Aesar. Lysobacter enzymogenes lysine-C endopeptidase (EndoLys-C) was from Roche Applied Science, and Staphylococcus aureus glutamic-C endopeptidase (EndoGlu-C) was from Princeton Separations. α1β3 GABAARs containing the FLAG epitope at the N terminus of the α1 subunit were purified from a tetracycline-inducible, stably transfected HEK293S cell line (27.Dostalova Z. Liu A. Zhou X. Farmer S.L. Krenzel E.S. Arevalo E. Desai R. Feinberg-Zadek P.L. Davies P.A. Yamodo I.H. Forman S.A. Miller K.W. High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABA(A) and serotonin receptors.Protein Sci. 2010; 19: 1728-1738Crossref PubMed Scopus (39) Google Scholar). Briefly, membrane fractions containing 6–10 nmol of [3H]muscimol-binding sites, collected from cells growing on 40–60 tissue culture dishes (15 cm), were resuspended at 1 mg of protein/ml and solubilized overnight in 300 ml of a purification buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 2 mm CaCl2, 5 mm KCl, 5 mm MgCl2, 4 mm EDTA, 20% glycerol, pepstatin, chymostatin, and leupeptin (10 µg/ml each), 2 µg/ml aprotinin, and 1 mm phenylmethanesulfonyl fluoride) supplemented with 2.5 mm n-dodecyl-β-d-maltopyranoside. Solubilized FLAG-α1β3 GABAARs were purified by elution from an anti-FLAG M2 affinity resin in elution buffer (purification buffer supplemented with 11.5 mm cholate, 0.86 mm asolectin, and 1.5 mm FLAG peptide). For competition photolabeling studies, GABAAR was also purified by solubilization for 2.5 h in purification buffer supplemented with 30 mm n-dodecyl-β-d-maltopyranoside followed by elution from the anti-FLAG affinity resin after washing in purification buffer supplemented with 5 mm CHAPS and 0.2 mm asolectin, as described for the α1β3γ2 GABAAR (19.Chiara D.C. Jayakar S.S. Zhou X. Zhang X. Savechenkov P.Y. Bruzik K.S. Miller K.W. Cohen J.B. Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.J. Biol. Chem. 2013; 288: 19343-19357Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). For both protocols, typical purification yields were ∼1.5 nmol of purified receptor (50–60 nm binding sites) in 15–25 ml of elution buffer. For the GABAAR purified in 0.86 mm asolectin, 11.5 mm cholate, IC50 values (total concentration) for inhibition of [3H]azietomidate, R-[3H]mTFD-MPAB, or [3H]AziPm photolabeling were 2–4-fold higher than for GABAAR purified in 0.2 mm asolectin, 5 mm CHAPS. Aliquots of FLAG-α1β3 GABAARs in elution buffer were used for analytical and preparative scale photolabeling (40–80 µl and 1 ml of α1β3 GABAAR, per condition, respectively). Appropriate volumes of radiolabeled, photoreactive anesthetic solutions in methanol were transferred to glass tubes, and solvent was evaporated under an argon stream. Freshly thawed GABAAR in elution buffer was added to the tube, and radioligand was resuspended with gentle vortexing during 30 min on ice to a final [3H]AziPm concentration of ∼5 µm (2.5 µCi per condition) for analytical or ∼10 µm (90 µCi per condition) for preparative scale experiments. GABAAR was equilibrated with [3H]azietomidate or R-[3H]mTFD-MPAB at final concentrations of 0.9 or 1.5 µm, respectively. Receptors were then equilibrated for 10 min with 1 mm GABA before addition of appropriate concentrations of nonradioactive anesthetic. After further incubation on ice for 30 min, the aliquots were transferred to 96-well plastic plates (Corning catalog number 2797) or 3.5-cm diameter plastic Petri dishes (Corning catalog number 3001) for analytical or preparative photolabelings, respectively, and irradiated on ice for 30 min at a distance of 0.5 to 1 cm with a 365 nm lamp (Spectroline Model EN-16, Spectronics Corp, Westbury, NJ). Stock solutions of nonradioactive AziPm (200 mm), propofol (1 m), R-mTFD-MPAB (60 mm), and etomidate (60 mm) were prepared in methanol, and all samples were photolabeled at a methanol concentration of 0.5% (v/v). Following irradiation, samples were mixed with an equal volume of electrophoresis sample buffer (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar), incubated for 30–60 min at room temperature, and then fractionated by SDS-PAGE on a 6% Tris-glycine gel. For analytical scale labeling, samples were loaded onto wells 2 cm deep, 0.8 cm wide, and 0.15 cm thick (sample volume, 150 µl). Preparative scale labeling samples were loaded onto wells 2 cm deep, 11.3 cm wide, and 0.15-cm thick (sample volume, 1.5 ml). Subunits resolved by SDS-PAGE were visualized by Coomassie Brilliant Blue stain and excised to measure incorporated 3H (for analytical scale experiments) or eluted and digested to generate peptide fragments for sequence analysis. For analytical scale experiments, the excised subunits were incubated overnight with 200 µl of deionized water and 500 µl of TS-2 tissue solubilizer (Research Products), and then 3H incorporation was determined by liquid scintillation counting after adding 5 ml of Ecoscint A (National Diagnostics). After photolabeling on a preparative scale, GABAAR subunits were recovered from the excised gel bands as described (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar) and resuspended in 200 µl of digestion buffer (15 mm Tris, 500 µm EDTA, and 0.1% SDS (pH 8.5)). Aliquots (∼90 µl) from gel bands enriched in α1 or β3 subunits were digested at room temperature with 0.5 units of EndoLys-C for 14 days or 2.5 µg of EndoGlu-C for 2–4 days, following which the digests were fractionated by HPLC or directly subjected to protein microsequence analysis. For chemical cleavage at the C terminus of methionines, samples immobilized on PVDF sequencing filters were treated with cyanogen bromide as described (28.Scott M.G. Crimmins D.L. McCourt D.W. Tarrand J.J. Eyerman M.C. Nahm M.H. A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinyldifluoride membranes.Biochem. Biophys. Res. Commun. 1988; 155: 1353-1359Crossref PubMed Scopus (63) Google Scholar, 29.Hamouda A.K. Kimm T. Cohen J.B. Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit Interfaces of a nicotinic acetylcholine receptor.J. Neurosci. 2013; 33: 485-494Crossref PubMed Scopus (45) Google Scholar). For chemical cleavage at the C terminus of tryptophans, samples on PVDF filters were treated with BNPS-skatole as described (30.Crimmins D.L. McCourt D.W. Thoma R.S. Scott M.G. Macke K. Schwartz B.D. In situ chemical cleavage of proteins immobilized to glass-fiber and polyvinylidene difluoride membranes: cleavage at tryptophan residues with 2-(2′-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine to obtain internal amino acid sequence.Anal. Biochem. 1990; 187: 27-38Crossref PubMed Scopus (58) Google Scholar), except that after precipitation of excess BNPS-skatole, the digestion solution was loaded onto a second PVDF filter, and material on the two filters was sequenced simultaneously (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar). α1β3 GABAAR amino acids photolabeled by R-[3H]mTFD-MPAB were identified as described for the α1β3γ2 GABAAR (19.Chiara D.C. Jayakar S.S. Zhou X. Zhang X. Savechenkov P.Y. Bruzik K.S. Miller K.W. Cohen J.B. Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.J. Biol. Chem. 2013; 288: 19343-19357Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The concentration dependence of inhibition of 3H incorporation into GABAAR subunits was fit by nonlinear least squares using SigmaPlot to a single site model, Equation 1,f(x)=(f0−fns)/(1+x/IC50)+fns(Eq. 1) where f(x) is the 3H counts/min (cpm) incorporated into a subunit at the inhibitor total concentration x; f0 is the subunit 3H in the absence of inhibitor; IC50 is the total inhibitor concentration reducing photolabeling by 50%, and fns is the nonspecific subunit photolabeling. IC50 was the adjustable parameter. For [3H]azietomidate and R-[3H]mTFD-MPAB, fns was the residual subunit photolabeling in the presence of 300 µm etomidate or 2.5 mm pentobarbital, respectively. For [3H]AziPm the fns was determined in the presence of either 300 µm etomidate or R-mTFD-MPAB. Although 3H incorporation was determined separately for the three gel bands, IC50 values were determined for inhibition of [3H]azietomidate photoincorporation in the ∼56-kDa α subunit band that reflects photolabeling of α1Met-236 and for R-[3H]mTFD-MPAB (16.Chiara D.C. Dostalova Z. Jayakar S.S. Zhou X. Miller K.W. Cohen J.B. Mapping general anesthetic-binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-etomidate, a photoreactive etomidate analogue.Biochemistry. 2012; 51: 836-847Crossref PubMed Scopus (92) Google Scholar) in the 59- and 61-kDa β subunit bands that reflect photolabeling of β3Met-227 (see under “Results”). For [3H]AziPm, inhibition of photolabeling was quantified only for the β subunit bands, as pharmacologically specific photolabeling was too small a component of α subunit photolabeling. For GABAAR purified in 0.2 mm asolectin, 5 mm CHAPS, photolabeling in the presence of nonradioactive AziPm at concentrations >30 µm resulted in GABAAR aggregation, as evidenced by decreased stain intensity of GABAAR subunit gel bands after SDS-PAGE. IC50 values for AziPm were determined from data at concentrations ≤30 µm. Subunit fragments generated by enzymatic digestion were fractionated by reversed phase HPLC (rpHPLC) on an Agilent 1100 binary HPLC system, using a Brownlee C4-Aquapore column (100 × 2.1 mm, 7 µ particle size) at 40 °C. Solvent A was 0.08% trifluoroacetic acid in water, and solvent B was 0.05% trifluoroacetic acid in 60% acetonitrile and 40% 2-propanol. A nonlinear eluti" @default.
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• W1972584910 title "Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog" @default.
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