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Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*

      GABA type A receptors (GABAAR), the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. How such structurally diverse agents can act similarly as positive allosteric modulators of GABAARs remains unclear. Previously, photoreactive etomidate analogs identified two equivalent anesthetic-binding sites in the transmembrane domain at the β+ subunit interfaces, which also contain the GABA-binding sites in the extracellular domain. Here, we used R-[3H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB), a potent stereospecific barbiturate anesthetic, to photolabel expressed human α1β3γ2 GABAARs. Protein microsequencing revealed that R-[3H]mTFD-MPAB did not photolabel the etomidate sites at the β+ subunit interfaces. Instead, it photolabeled sites at the α+ and γ+ subunit interfaces in the transmembrane domain. On the (+)-side, α1M3 was labeled at Ala-291 and Tyr-294 and γ2M3 at Ser-301, and on the (−)-side, β3M1 was labeled at Met-227. These residues, like those in the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. The selectivity of R-etomidate for the β+ interface relative to the α++ interfaces was >100-fold, whereas that of R-mTFD-MPAB for its sites was >50-fold. Each ligand could enhance photoincorporation of the other, demonstrating allosteric interactions between the sites. The structural heterogeneity of barbiturate, etomidate, and propofol derivatives is accommodated by varying selectivities for these two classes of sites. We hypothesize that binding at any of these homologous intersubunit sites is sufficient for anesthetic action and that this explains to some degree the puzzling structural heterogeneity of anesthetics.
      Background: General anesthetics of diverse chemical structure potentiate GABAA receptors by binding to unknown sites.
      Results: A photoreactive barbiturate identifies intersubunit-binding sites distinct from, but homologous to, sites identified by photoreactive etomidate analogs.
      Conclusion: Propofol, barbiturates, and etomidate analogs bind with variable selectivities to two classes of sites.
      Significance: This study helps define the diversity of GABAA receptor general anesthetic-binding sites.

      Introduction

      General anesthetics of diverse structures, including volatile anesthetics, propofol, etomidate, barbiturates, steroids, and alcohols, potentiate inhibitory GABA type A receptors (GABAAR)
      The abbreviations used are: GABAAR, GABA type A receptor; nAChR, nicotinic acetylcholine receptor; mTFD-MPAB, 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid; EndoGlu-C, S. aureus endoproteinase Glu-C; EndoLys-C, L. enzymogenes endoproteinase Lys-C; PVDF, polyvinylidene fluoride; BNPS-skatole, 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole; rpHPLC, reversed-phase high-performance liquid chromatography; OPA, o-phthalaldehyde; MPPB, 1-methyl-5-phenyl-5-propyl barbituric acid; PTH, phenylthiohydantoin.
      in vitro with a pharmacology and concentration dependence that suggest this receptor is a major contributor to the anesthetic state (
      • Macdonald R.L.
      • Olsen R.W.
      GABAA receptor channels.
      ,
      • Hemmings Jr., H.C.
      • Akabas M.H.
      • Goldstein P.A.
      • Trudell J.R.
      • Orser B.A.
      • Harrison N.L.
      Emerging molecular mechanisms of general anesthetic action.
      ,
      • Franks N.P.
      General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal.
      ). The importance of GABAARs for anesthesia in vivo was demonstrated by the decreased sensitivity of “knock-in” mice bearing a single substitution at position 15 in the GABAAR β3 subunit transmembrane helix 2 (β3M2–15′), a substitution that reduced GABAAR sensitivity to propofol and etomidate in vitro (
      • 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.
      ). These mice had greatly reduced sensitivity to the immobilizing and hypnotic anesthetic effects of etomidate, propofol, and pentobarbital, with little change in sensitivity to volatile or steroid anesthetics (
      • 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 GABAA receptor β3 subunit.
      ,
      • Zeller A.
      • Arras M.
      • Jurd R.
      • Rudolph U.
      Identification of a molecular target mediating the general anesthetic actions of pentobarbital.
      ,
      • Drexler B.
      • Antkowiak B.
      • Engin E.
      • Rudolph U.
      Identification and characterization of anesthetic targets by mouse molecular genetics approaches.
      ).
      The locations of anesthetic sensitivity determinants in GABAARs have been predicted by use of homology models derived from the structures of other members of the Cys-loop superfamily of pentameric ligand-gated ion channels, the nicotinic acetylcholine receptor (nAChR) (
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.
      ), the prokaryotic homologs ELIC (
      • Hilf R.J.
      • Dutzler R.
      X-ray structure of a prokaryotic pentameric ligand-gated ion channel.
      ) and GLIC (
      • Hilf R.J.
      • Dutzler R.
      Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.
      ), and an invertebrate glutamate-gated chloride channel (
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      ). Each subunit contains an N-terminal extracellular domain, a transmembrane domain made up of a loose bundle of four transmembrane helices (M1–M4), and an intracellular domain formed by the primary structure between the M3 and M4 helices. In an (α)2(β)2γ GABAAR, the transmitter-binding sites are in the extracellular domain at the β+ subunit interfaces, with amino acids from the β and α subunits forming the principal (+) and complementary (−) surfaces of the binding pocket, respectively (Fig. 1). The benzodiazepine-binding site is at an equivalent position at the α+ subunit interface (
      • Sigel E.
      The benzodiazepine recognition site on GABAA receptors.
      ,
      • Sieghart W.
      • Ramerstorfer J.
      • Sarto-Jackson I.
      • Varagic Z.
      • Ernst M.
      A novel GABAA receptor pharmacology: drugs interacting with the α+β interface.
      ). In the transmembrane domain, M2 helices from each subunit associate around a central axis to form the ion channel, and amino acids from the M1 and M3 helices of adjacent subunits contribute to the subunit interfaces. The etomidate-binding sites, identified by photoaffinity labeling of amino acids in βM3 and αM1, are in the two β+ subunit interfaces about 50 Å below the agonist sites (
      • 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.
      ,
      • 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.
      ).
      Figure thumbnail gr1
      FIGURE 1Locations in an (α)2(β)2γ GABAAR of binding sites for GABA, benzodiazepines (BZD), and etomidate.
      We reported recently that the R-enantiomer of 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB) is an extremely potent, photoreactive barbiturate that rivals etomidate in potency and stereoselectivity (
      • 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.
      ). Here, we report that R-[3H]mTFD-MPAB photolabels new anesthetic-binding sites in human α1β3γ2 GABAARs at the α+ and γ+ subunit interfaces. These sites are distinct from but homologous to the R-[3H]azietomidate sites at the two β+ interfaces, as all are located at the same depth in the transmembrane domain. R-[3H]mTFD-MPAB and R-[3H]azietomidate are highly selective for their own sites. We used the ability of derivatives of etomidate, propofol, and barbituric acid to inhibit photolabeling to determine their relative affinities for these two classes of sites. Our results begin to explain how such diverse structures can exert the same action on GABAARs.

      EXPERIMENTAL PROCEDURES

       Materials

      [3H]Muscimol (26 or 36 Ci/mmol) was from PerkinElmer Life Sciences. The detergents n-dodecyl β-d-maltopyranoside and CHAPS were from Anatrace-Affymetrix (Anagrade quality). Soybean asolectin was from Sigma. R- and S-MPAB (5-allyl-1-methyl-5-phenyl-barbituric acid), R-(−)- and S-(+)-mTFD-MPAB (5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid), and [3H] R-mTFD-MPAB (38 Ci/mmol) were synthesized as described (
      • 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.
      ). Racemic MPAB and MPPB (5-propyl-1-methyl-5-phenylbarbituric acid) were synthesized by phenylation of diethyl allylmalonate and diethyl propylmalonate, respectively, with diphenyliodonium trifluoroacetate. Additionally, R- and S-MPPB were prepared from the corresponding isomers of MPAB by catalytic reduction with hydrogen gas on Pd/C. Racemic mBr-MPAB (5-allyl-1-methyl-5-(m-bromophenyl-barbituric acid) was synthesized analogously to the recently described methods (
      • 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.
      ) by reaction of 5-allyl-1-methyl barbiturate with (3-bromophenyl)(4-methoxyphenyl)iodonium. Other barbiturates were from commercial sources. R-(+)- and S-(−)-etomidate were from Organon Labs. Azietomidate and R-[3H]azietomidate (12 Ci/mmol) (
      • 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.
      ), R-TDBzl-etomidate (
      • Husain S.S.
      • Nirthanan S.
      • Ruesch D.
      • Solt K.
      • Cheng Q.
      • Li G.D.
      • Arevalo E.
      • Olsen R.W.
      • Raines D.E.
      • Forman S.A.
      • Cohen J.B.
      • Miller K.W.
      Synthesis of trifluoromethylaryl diazirine and benzophenone derivatives of etomidate that are potent general anesthetics and effective photolabels for probing sites on ligand-gated ion channels.
      ), and R- and S-pTFD-etomidate (
      • Husain S.S.
      • Stewart D.
      • Desai R.
      • Hamouda A.K.
      • Li S.G.
      • Kelly E.
      • Dostalova Z.
      • Zhou X.
      • Cotten J.F.
      • Raines D.E.
      • Olsen R.W.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      p-Trifluoromethyldiazirinyl-etomidate: A potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels.
      ) were prepared previously. Propofol was from Sigma, 2,6-di-tert-butylphenol from Acros, and 2,6-di-sec-butylphenol from Chiron. Saccharomyces aureus endoproteinase Glu-C (EndoGlu-C) and Lysobacter enzymogenes endoproteinase Lys-C (EndoLys-C) were from Worthington and Roche Applied Science, respectively.

       Purification of Human α1β3γ2 GABAAR

      α1β3γ2 GABAARs with a FLAG epitope at the N terminus of the α1 subunit were expressed in a tetracycline-inducible, stably transfected HEK293S cell line and purified on an anti-FLAG affinity resin with modifications of procedures used to purify a previously characterized tetracycline-inducible FLAG-α1β3 GABAAR (
      • 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.
      ,
      • 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: GABAA and serotonin receptors.
      ). Membranes harvested from 60 15-cm plates (4–8 nmol of [3H]muscimol-binding sites) were solubilized in 30 mm n-dodecyl β-d-maltopyranoside (instead of 2.5 mm) for 2.5 h at 4 °C, and the wash and elution buffers contained 5 mm CHAPS, 0.2 mm asolectin, or 10 mm CHAPS, 0.86 mm asolectin (instead of 13 mm cholate, 0.86 mm asolectin). Aliquots from pooled elution fractions were characterized for [3H]muscimol-binding sites and modulation by etomidate. Individual preparations, starting from membranes containing 2–4 nmol of [3H]muscimol-binding sites (15–20 pmol/mg of protein), typically resulted in 0.5–1.5 nmol of purified receptor (30–60 nm binding sites) in 15–25 ml of elution buffer. Aliquots of purified GABAAR were frozen and stored at −80 °C.

       Radioligand Binding Assays

      [3H]Muscimol binding to purified GABAAR was measured by filtration after precipitation with polyethylene glycol (
      • 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.
      ). The total concentration of sites was determined at 500 nm [3H]muscimol and with 1 mm GABA to determine nonspecific binding. Anesthetic modulation of 2–3 nm [3H]muscimol binding was measured as described (
      • 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.
      ,
      • 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: GABAA and serotonin receptors.
      ), except that samples were incubated for 60 min at room temperature before addition of polyethylene glycol and γ-globulins and then filtered after a 30-min incubation at room temperature. The modulation results are presented as the percentage of the specifically bound [3H]muscimol over that without modulators.

       GABAAR Photolabeling

      Purified GABAAR in elution buffer was photolabeled on an analytical scale (40–80-μl aliquots containing ∼3 pmol of [3H]muscimol sites) to characterize photolabeling at the subunit level and to quantify the effects of nonradioactive anesthetics (or agonist) on photolabeling. To identify photolabeled amino acids, GABAAR was photolabeled on a preparative scale (1.5–2.5-ml aliquots containing ∼90 pmol of [3H]muscimol sites). Appropriate amounts of R-[3H]mTFD-MPAB or R-[3H]azietomidate were transferred to glass tubes, and solvent (methanol) was evaporated under an argon stream. Freshly thawed GABAAR in elution buffer was added to the tube, and the radioligand was resuspended at 4 °C with gentle vortexing for 30 min to a final concentration of 0.5–1 μm (∼1 μCi per analytical sample and 25–45 μCi per preparative sample). Drugs of interest were added to the aliquots, and samples were incubated for 30 min. Samples were then placed in the wells of a 96-well plastic microtiter plate (analytical photolabeling) or in a plastic 3.5-cm Petri dish (preparative photolabeling) and irradiated on ice for 30 min with a 365-nm lamp (Spectroline 280L) at a distance of ∼1 cm. Samples were then solubilized at room temperature in an equal volume of sample buffer (9 parts of 40% sucrose, 10% SDS, 2% glycerol, 0.0125% bromphenol blue, 0.3 m Tris, pH 6.8, and 1 part β-mercaptoethanol) and fractionated by Laemmli SDS-PAGE (6% acrylamide, 0.24% bisacrylamide resolving gel). The large sample volumes in preparative photolabelings (3–5 ml) necessitated the use of 1.5-mm thick slab gels that were 12 cm long and 14 cm wide, with a 5-cm stacker layer (4% acrylamide) and wells 6 cm deep and 12 cm wide. SDS-polyacrylamide gels were stained with Coomassie Blue after electrophoresis. Prior to UV irradiation, all samples were incubated in glass vials, and anesthetic additions were made using glass microcaps. Anesthetic stock solutions were prepared in methanol, and final methanol concentrations were ≤0.5% (v/v).
      In analytical scale photolabeling, the 3H incorporation into GABAAR subunits was visualized by fluorography using En3hance (PerkinElmer Life Sciences) and quantified by liquid scintillation counting of excised gel bands that had been incubated in 0.1 ml of water, 0.5 ml of tissue solubilizer TS-II (RPI) overnight before addition of scintillation fluid (EcoScint A, National Diagnostics). In preparative scale photolabeling experiments, the GABAAR subunit bands excised from the stained gels were eluted individually into 12 ml of buffer (100 mm NH4HCO3, 0.1% SDS, and 2.5 mm dithiothreitol, pH 8.4) for 3 days at 20 °C with gentle agitation. The eluates were filtered, concentrated, acetone-precipitated, and resuspended in 100–200 μl of digestion buffer (15 mm Tris and 0.1% SDS, pH 8.5).
      Photolabeled amino acids were identified in three preparative photolabeling experiments using purified α1β3γ2 GABAAR (∼60 nm [3H]muscimol sites, eluted in a buffer containing 10 mm CHAPS and 0.86 mm asolectin). GABAARs were photolabeled with the following: (i) 1 μm R-[3H]azietomidate ± 100 μm etomidate (+1 mm GABA); (ii) 0.6 μm R-[3H]mTFD-MPAB ± 1 mm pentobarbital (+1 mm GABA); and (iii) 0.9 μm R-[3H]mTFD-MPAB in the absence of other drugs or in the presence of 1 mm GABA or 100 μm etomidate.

       Quantification of Anesthetic and GABA Modulation of Photolabeling

      Modulation of R-[3H]mTFD-MPAB and R-[3H]azietomidate photolabeling by general anesthetics and GABA was quantified in analytical photolabeling experiments. Although 3H incorporation in all three subunit bands was determined, parameters were determined for the concentration dependence of drug modulation of R-[3H]mTFD-MPAB photoincorporation in the 59- and 61-kDa bands that reflect photolabeling primarily of β3Met-227 and of R-[3H]azietomidate photolabeling in the 56-kDa band that reflects photolabeling of α1Met-236 (see under “Results”). The level of nonspecific 3H incorporation in subunits was determined in the presence of 1 mm pentobarbital for R-[3H]mTFD-MPAB and 1 mm etomidate for R-[3H]azietomidate. Subunit photolabeling was quantified as a function of the total concentration of nonradioactive anesthetics. Because GABAARs were photolabeled in solutions containing 5 mm CHAPS, 0.4 mm asolectin, the anesthetic free concentrations will be substantially lower than the total concentrations and dependent upon the anesthetic lipophilicity (oil/buffer partition coefficient).
      For conditions when an anesthetic only inhibited GABAAR photolabeling, the data were fit to a single site model for competitive inhibition, as shown in Equation 1,
      f1(x)=(f0fns)/(1+x/IC50)+fns
      (Eq. 1)


      where f1(x) is the 3H counts/min incorporated in a subunit at anesthetic concentration x; f0 is the subunit counts/min in the absence of inhibitor; fns is the nonspecific subunit photolabeling, and IC50 is the total drug concentration reducing photolabeling by 50%. When a drug only enhanced photolabeling, data were fit to Equation 2,
      f2(x)=(fmaxf0)/(1+EC50/x)+f0
      (Eq. 2)


      where f2(x) is the counts/min incorporated at drug concentration x; fMAX is the maximal level of photolabeling in counts/min; f0 is the subunit photolabeling in counts/min in the absence of drug, and EC50 is the total drug concentration producing 50% of maximal enhancement. If an anesthetic at low concentrations produced an enhancement of photolabeling and then inhibition at high concentrations, data were fit to a model assuming anesthetic binding to independent potentiating and inhibitory sites (Equation 3),
      f3(x)=(f2(x)fns)/(1+x/IC50)+fns
      (Eq. 3)


       Chemical and Enzymatic Fragmentation

      Aliquots isolated from gel bands enriched in either α1 or β3 subunits were digested at 20 °C with either 100 μg of endoproteinase Glu-C (EndoGlu-C, Worthington) for 2 days or 0.5 units of endoproteinase Lys-C (EndoLys-C, Roche Applied Science) for 2 weeks. For chemical cleavage at the C terminus of methionines, samples immobilized on PVDF sequencing filters were treated with cyanogen bromide as described (
      • 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.
      ,
      • 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.
      ). For chemical cleavage at the C terminus of tryptophans, samples on PVDF filters were treated with BNPS-skatole as described (
      • 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 polyvinylidenedifluoride membranes: Cleavage at tryptophan residues with 2-(2′-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine to obtain internal amino acid sequence.
      ), except that after precipitation of the excess BNPS-skatole, the digestion solution was loaded onto a second PVDF filter, and material on the two filters was sequenced simultaneously (
      • 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.
      ).

       HPLC Purification and Protein Microsequencing

      Reversed-phase HPLC was performed as described (
      • Ziebell M.R.
      • Nirthanan S.
      • Husain S.S.
      • Miller K.W.
      • Cohen J.B.
      Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic.
      ) on an Agilent 1100 binary pump system using a Brownlee Aquapore BU-300 column. Samples were eluted at 0.2 ml/min with increasing concentrations of 60% isopropyl alcohol, 40% acetonitrile, 0.05% TFA. Elution of peptides was monitored by the absorbance at 215 nm and by liquid scintillation counting of a 10% aliquot of each 0.5-ml fraction.
      Samples were sequenced on an Applied Biosystems Procise 492 protein sequencer modified to collect two-thirds of each cycle for PTH-derivative detection/quantification and one-third for 3H determination by liquid scintillation counting. For direct sequencing of intact subunits or subunit digests containing SDS, samples were loaded onto Prosorb PVDF filters (Applied Biosystems) following the manufacturer's instructions. HPLC fractions for sequence analysis were drop-loaded at 45 °C onto TFA-treated glass fiber filters that were then treated with BiobreneTM. For selected samples, the sequencer was paused after the designated cycles, and the sample filter was treated with o-phthalaldehyde (OPA) before resuming sequencing as follows: (i) to block all free N termini before treatment of the filter with cyanogen bromide or (ii) to chemically isolate for further sequencing only those fragments containing a proline in the designated cycle. OPA reacts with primary amines, but not with secondary amines, and treatment with OPA prevents further sequencing of fragments not containing a proline at that cycle, thereby confirming that any subsequent peak of 3H release originated from the proline-containing peptide (
      • Brauer A.W.
      • Oman C.L.
      • Margolies M.N.
      Use of ophthalaldehyde to reduce background during automated Edman degradation.
      ,
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine-binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ). PTH-derivatives were quantitated by peak heights over background, and the actual picomole quantities and counts/min detected are plotted in the figures. The amount of a peptide sequenced was determined by fitting the individual residues detected to Equation 4,
      Ix=I0×Rx
      (Eq. 4)


      where Ix is the picomoles detected in cycle x; I0 is the initial amount of peptide, and R is the repetitive yield. Cys, Ser, His, and Trp were not used for the fit due to known problems with their quantifications. The efficiency of amino acid photolabeling in counts per min/pmol (cpm/pmol) was calculated by Equation 5,
      E(x)=2×(cpmxcpm(x1))/I0×Rx
      (Eq. 5)


      where cpmx is the counts/min in cycle x.

       Molecular Modeling

      Comparison of structural models for α1β3 GABAAR constructed by homology with GLIC and GluCl (
      • 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.
      ) established that the positions of amino acids of βM3/αM1 and αM3/βM1 contributing to the β+ and α+ interfaces, respectively, were the same. Compared with the GLIC-derived structure, there was an increased distance in the GluCl structure between the M3 and M1 helices where the allosteric potentiator ivermectin is bound in GluCl. Because etomidate could be docked within the more constrained intersubunit pocket of the GLIC-derived model, we constructed a β3α1β3α1γ2 GABAAR homology model based on a GLIC structure (Protein Data Bank code 3P50) as described (
      • 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.
      ), with the exception that the human γ2 subunit sequence replaced the third β3 subunit sequence in that β3α1β3α1β3 model. In the GLIC structure, Tyr-263, the M3 residue homologous to the GABAAR-photolabeled residues α1Tyr-294, β3Phe-289, and γ2Phe-304, is within 4 Å of the residue in the M1 helix across the interface, Pro-204. Therefore, to accommodate R-mTFD-MPAB or R-azietomidate in their interface binding pockets in the β3α1β3α1γ2 GABAAR homology model, the aromatic side chains (α1Tyr-294, β3Phe-289, and γ2Phe-304) were rotated out of the interface. A membrane force field was calculated for the structure using the Discovery Studio molecular modeling package (Accelrys Inc.), and the model was energy-minimized with anesthetics occupying each of the five transmembrane interface pockets. R-mTFD-MPAB was placed horizontally in the α+, γ+, and α+ interfaces, and R-azietomidate was placed horizontally in the two β+ interfaces, with their diazirines protruding between the M3+ and M1 α-helices in close proximity to the photolabeled residues. CDocker, a CHARMm-based molecular dynamics simulated annealing program, was used to dock R-mTFD-MPAB at each interface pocket using an 11-Å radius binding-site sphere centered on each minimized anesthetic. The best 200–300 solutions were collected for each interface starting from 50 random orientations of 50 molecular dynamics-altered anesthetic structures. Solutions were obtained at all interfaces.
      For R-mTFD-MPAB (volume, 275 Å3) at the α+ interface site, all 300 solutions were oriented similarly, with the major difference being the location of the diazirine as determined by a 180° rotation of the phenyl group around the C5-phenyl bond. The Connolly surface, determined by a probe of radius 1.4 Å, for the 300 solutions defined a volume of 535 Å3. The lowest energy solution was positioned with the diazirine carbon within 4.5 Å from the photolabeled residues β3Met-227 and α1Tyr-294, the phenyl ring stacked with α1Tyr-294, the N-methyl of barbituric acid within 4 Å of α1Ser-270 (αM2–15′) and α1Tyr-294, and the C5 allyl within 4 Å of β3Ile-264 (β3M2–14′). CDocker interaction energies overlapped for the two orientations, with the lowest 15 solutions differing by 2 kcal/mol and all 300 solutions differing by 10 kcal/mol.
      At the γ+ interface, R-mTFD-MPAB was docked in two orientations with overlapping CDocker interaction energies that differed by <5 kcal/mol. For 77 of 200 solutions (volume of 329 Å3), R-mTFD-MPAB was docked as at the α+ interface, with diazirine carbon within 5 Å of β3Met-227 and the NH of barbituric acid ∼3 Å from γ2Ser-280 (γM2–15′). In the second solution, R-mTFD-MPAB was oriented vertically within the interface, with the diazirine pointing up, ∼5 Å from γ2Ser-280 and γ2Ser-301, but 8 Å from β3Met-227. The NH of barbituric acid was pointing down, 4 Å from γ2Phe-304.

      RESULTS

       Photolabeling α1β3 and α1β3γ2 GABAARs with R-[3H]Azietomidate and R-[3H]mTFD-MPAB

      The FLAG-α1β3γ2 GABAAR purified in asolectin/CHAPS contained γ2 subunits, as evidenced by the ratio of [3H]muscimol to [3H]flunitrazepam-binding sites (1.2 ± 0.6). Energetic coupling was preserved between anesthetic sites in the transmembrane domain and the agonist site in the extracellular domain. R-Etomidate and barbiturates (pentobarbital or R- or S-mTFD-MPAB) potentiated [3H]muscimol binding to the same extent (Table 1).
      TABLE 1The enhancement by general anesthetics of [3H]muscimol binding to purified α1β3γ2 GABAAR
      CompoundConcentrationRS
      μm
      mTFD-MPAB100179 ± 19%166 ± 8%
      (+/−)-Pentobarbital1,000170 ± 24%
      Etomidate10174 ± 29%
      To begin characterizing anesthetic-binding sites in the α1β3γ2 GABAAR, we photolabeled samples with R-[3H]azietomidate or R-[3H]mTFD-MPAB (Fig. 2) at anesthetic concentrations and compared the patterns of subunit photolabeling to those seen for the α1β3 GABAAR (
      • 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.
      ,
      • 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.
      ). When GABAAR subunits were resolved by SDS-PAGE after photolabeling, the two preparations appeared essentially the same based upon Coomassie Blue stain, with three bands migrating at ∼56, ∼59, and ∼61 kDa (Fig. 2A). For the α1β3 GABAAR, the N-terminal sequence analyses had established that the ∼56-kDa band contained the FLAG-tagged α1 subunit, whereas the 59- and 61-kDa bands contained β3 subunits differing in their glycosylation patterns, with β3 subunit in the α1 band and α1 subunit in the β3 bands at ∼15% levels (
      • 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.
      ). N-terminal sequencing of material eluted from each band of the α1β3γ2 GABAAR (Fig. 2C) established that the 56-kDa band also contained primarily the α1 subunit, and the 59- and 61-kDa bands contained primarily the β3 subunit, although the γ2 subunit (beginning at Lys-2) was distributed in all three bands. When the subunit incorporation of R-[3H]azietomidate or R-[3H]mTFD-MPAB was monitored by fluorography (Fig. 2B), for α1β3 and α1β3γ2 GABAARs, R-[3H]azietomidate and R-[3H]mTFD-MPAB were incorporated primarily, but not exclusively, into the 56- and 59-kDa bands, respectively. For R-[3H]azietomidate-photolabeled α1β3γ2 GABAAR, sequence analysis of subunit fragments isolated from digests enriched in α1 or β3 subunits identified etomidate-inhibitable photolabeling of the same amino acids (α1Met-236 in αM1 and β3Met-286 in βM3) as in the GABAAR purified from bovine brain (
      • 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.
      ) or in the α1β3 GABAAR (data not shown) (
      • 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.
      ).
      Figure thumbnail gr2
      FIGURE 2R- [3H]mTFD-MPAB and R- [3H]azietomidate photolabeling of α1β3 and α1β3γ2 GABAARs. GABAARs were photolabeled with 0.8 μm R-[3H]azietomidate or 0.3 μm R-[3H]mTFD-MPAB in the absence and presence of 1 mm pentobarbital (PB), and aliquots (∼5 pmol of [3H]muscimol sites/lane) were fractionated by SDS-PAGE. A, Coomassie Blue (Coo Blue) stain of representative gel lanes, with the mobilities of molecular weight markers and the calculated masses of the stained gel bands indicated. B, 3H incorporation into GABAAR subunits, as determined by fluorography (1 month exposure). C, quantitation of the α1, β3, and γ2 subunit distributions, as determined by Edman sequence analysis of materials extracted from the stained gel bands. α1 was concentrated in the 56-kDa band and β3 in the 59- and 61-kDa bands. The γ2 subunit is distributed diffusely in all three bands.

       R-[3H]mTFD-MPAB Photolabels an Anesthetic-binding Site Distinct from, but Coupled Energetically to, the Etomidate- and Agonist-binding Sites in the α1β3γ2 GABAAR

      R-mTFD-MPAB acts as a potent tadpole general anesthetic, characterized by an EC50 of 3.7 μm and as a GABAAR potentiator at anesthetic concentrations, whereas S-mTFD-MPAB was 10-fold less potent as an anesthetic and potentiated weakly GABAAR responses (
      • 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.
      ). In the absence of GABA, R- and S-mTFD-MPAB each produced a concentration-dependent inhibition of R-[3H]mTFD-MPAB photolabeling, characterized by IC50 values of 1.4 ± 0.2 and 34 ± 6 μm, respectively, with high concentrations of either enantiomer inhibiting subunit incorporation by >95% (Fig. 3A). In contrast, R-mTFD-MPAB at concentrations up to 10 μm increased R-[3H]azietomidate photolabeling of the GABAAR by ∼25% (EC50 = 1.4 μm), with inhibition seen only at higher concentrations (IC50 = 63 ± 8 μm) (Fig. 3B). S-mTFD-MPAB only inhibited R-[3H]azietomidate photolabeling (IC50 = 50 ± 12 μm).
      Figure thumbnail gr3
      FIGURE 3R-[3H]mTFD-MPAB binds to site(s) in the α1β3γ2 GABAAR that are distinct from, but coupled energetically to, the etomidate and GABA-binding sites. GABAARs in the absence (●, ○, ■) or presence (□) of GABA were photolabeled on an analytical scale with R-[3H]mTFD-MPAB (A, C, and E) or R-[3H]azietomidate (B, D, and F) in the presence of increasing concentrations of nonradioactive R-mTFD-MPAB (●) or S-mTFD-MPAB (○) (−GABA; A and B), R-etomidate (C and D), or pentobarbital (E and F), and 3H incorporation into GABAAR subunits was determined by SDS-PAGE and liquid scintillation counting. The concentration dependences of inhibition (IC50) and potentiation (EC50) were fit as described under “Experimental Procedures,” and the values of IC50/EC50 of the plotted lines are included under the “Results.” The amounts of R-[3H]mTFD-MPAB incorporation in the presence of 1 mm pentobarbital or R-[3H]azietomidate incorporation in the presence of 1 mm R-etomidate are indicated by long dashed lines.
      In the presence of GABA, R-etomidate at 1 mm inhibited R-[3H]mTFD-MPAB photolabeling by <20%, although it completely inhibited R-[3H]azietomidate photolabeling with an IC50 of 7 ± 1 μm (Fig. 3, C and D). In the absence of GABA, R-etomidate inhibited R-[3H]azietomidate photolabeling with an IC50 of 21 ± 1 μm, although it increased R-[3H]mTFD-MPAB photolabeling by ∼100%, to the level seen in the presence of GABA. The concentration dependence of enhancement (EC50 = 9 ± 4 μm) was close to the IC50 of 20 μm for R-etomidate inhibition of R-[3H]azietomidate photolabeling. In the presence of GABA, R-etomidate at 1 mm inhibited R-[3H]mTFD-MPAB photolabeling by <15%, which indicated that the affinity of R-etomidate for those binding sites is >100-fold weaker than for the R-[3H]azietomidate sites. The GABA enhancement of R-[3H]mTFD-MPAB photolabeling (EC50 = 50 μm, not shown) established that there was positive allosteric coupling between the GABA-binding sites in the extracellular domain and the R-[3H]mTFD-MPAB-binding sites, as seen previously for R-[3H]azietomidate sites in brain GABAARs (
      • 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.
      ). The etomidate potentiation of R-[3H]mTFD-MPAB photolabeling and the reciprocal R-mTFD-MPAB potentiation of R-[3H]azietomidate photolabeling (in the absence of GABA) established that there was also positive allosteric coupling between those binding sites.
      Pentobarbital was ∼8-fold more potent as an inhibitor of R-[3H]mTFD-MPAB photolabeling than of R-[3H]azietomidate photolabeling (Fig. 3, E and F). Pentobarbital, which anesthetizes tadpoles with EC50 = 150 μm (
      • Lee-Son S.
      • Waud B.E.
      • Waud D.R.
      A comparison of the potencies of a series of barbiturates at the neuromuscular junction and on the central nervous system.
      ), inhibited R-[3H]mTFD-MPAB photolabeling with IC50 values of 75 ± 6 and 106 ± 18 μm in the presence and absence of GABA, respectively. In contrast, it inhibited R-[3H]azietomidate photolabeling with IC50 values of 600 ± 120 and 1,700 ± 230 μm.

       R-[3H]mTFD-MPAB Photolabels β3Met-227 in β3M1

      Because nonradioactive R-mTFD-MPAB was 60-fold more potent as an inhibitor of R-[3H]mTFD-MPAB photolabeling than of R-[3H]azietomidate photolabeling, the high affinity R-mTFD-MPAB-binding site must be distinct from the R-[3H]azietomidate/etomidate-binding site. To identify the labeled residues, α1β3γ2 GABAAR was photolabeled with R-[3H]mTFD-MPAB (0.6 μm) on a preparative scale in the absence or presence of 1 mm pentobarbital, and we used previously developed strategies (
      • 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.
      ,
      • 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.
      ) to identify photolabeling within each of the four transmembrane helices of the β3 subunit, the subunit with the highest 3H incorporation. When an EndoLys-C digest of material enriched in β3 subunits (eluted from the 59- and 61-kDa gel bands) was fractionated by reversed-phase HPLC (Fig. 4A), all 3H was recovered in hydrophobic fractions, consistent with photolabeling restricted to the GABAAR transmembrane domain. N-terminal sequencing of the pool of the fractions containing the peak of 3H identified a fragment beginning at β3Arg-216 near the beginning of the M1 helix as the primary sequence, with a peak of 3H release in cycle 12 consistent with photolabeling of β3Met-227 (Fig. 4B). Based upon the amounts of 3H and β3Met-227 released at that cycle, β3Met-227 was photolabeled at a calculated efficiency of 980 cpm/pmol (∼3% of β3 labeled), and photolabeling was inhibited by >80% in the presence of pentobarbital.
      Figure thumbnail gr4
      FIGURE 4R-[3H]mTFD-MPAB photolabels β3Met-227 in the β3M1 transmembrane helix. GABAARs were photolabeled on a preparative scale in the presence of 1 mm GABA with R-[3H]mTFD-MPAB (0.6 μm) in the absence (●, □) or presence (○) of 1 mm pentobarbital, and GABAAR subunits were isolated by SDS-PAGE. A, rpHPLC fractionation of EndoLys-C digests of β3 subunits (59–61-kDa gel bands). Fractions 28–29 containing the peak of 3H were pooled for sequencing (B). B and C, 3H (●, ○) and picomoles of PTH-derivatives (□) released during Edman sequencing of β3 subunit fragments beginning at β3Arg-216 (B) and β3His-191 (C). B, primary sequence began at β3Arg-216 (35 pmol, both conditions), and the peak of 3H release in cycle 12 indicated photolabeling of β3Met-227 in βM1 at 980 cpm/pmol (−pentobarbital) and 160 cpm/pmol (+pentobarbital). C, aliquots of β3 subunit from the same preparative labeling were digested with EndoGlu-C and sequenced without fractionation. The sequencing filters were treated with OPA prior to cycle 16, and thereafter, the only sequence detected originally began at β3His-191 (4–5 pmol). The peak of 3H release in cycle 37 confirmed R-[3H]mTFD-MPAB photolabeling of β3Met-227 at 920 cpm/pmol in the absence and 170 cpm/pmol in the presence of pentobarbital.
      Because the sequenced samples also contained a fragment beginning at β3Ala-280, before βM3, at ∼15% the level of the primary sequence, we used an alternative sequencing strategy to confirm the pentobarbital-inhibitable photolabeling of β3Met-227. With β3Met-227 positioned in the subunit primary structure 37 amino acids after β3Glu-190 and a proline (β3Pro-206) in between, we took advantage of the fact that OPA, which reacts with primary amines but not proline, a secondary amine, can be used to prevent further Edman degradation of any peptide not containing a proline at the cycle of treatment (
      • Brauer A.W.
      • Oman C.L.
      • Margolies M.N.
      Use of ophthalaldehyde to reduce background during automated Edman degradation.
      ,
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine-binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ). When an EndoGlu-C digest of material enriched in photolabeled β3 subunit was sequenced, after treatment with OPA at cycle 16, the only sequence remaining began originally at β3His-191. The observed peak of 3H release in cycle 37 confirmed that β3Met-227 was photolabeled at 920 cpm/pmol and that 1 mm pentobarbital reduced its labeling by ∼80% to 170 cpm/pmol (Fig. 4C).

       R-[3H]mTFD-MPAB Photolabels α1Ala-291 and α1Tyr-294 in α1M3

      Although the amino acids photolabeled by R-[3H]azietomidate (β3Met-286 in βM3 and α1Met-236 in αM1) are located in the GABAAR structure at the β+ subunit interfaces (
      • 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.
      ), the amino acid photolabeled by R-[3H]mTFD-MPAB, β3Met-227, is located within the β3M1 helix at the α+ and γ+ subunit interfaces in proximity to amino acids from α1M2/M3 or γ2M2/M3. To determine whether there was also photolabeling of amino acids in α1M3, we devised a strategy to sequence a fragment beginning at α1Asp-287 at the M3 N terminus that entailed the rpHPLC fractionation of an EndoGlu-C digest of material enriched in α1 subunits, the use of OPA, and digestion with cyanogen bromide to cleave after methionines (Fig. 5, A–C). When the fragment beginning at α1Asp-287 was sequenced (Fig. 5A), the peaks of 3H release in cycles 5 and 8 indicated R-[3H]mTFD-MPAB photolabeling of α1Ala-291 and α1Tyr-294 at photolabeling efficiencies of ∼50 cpm/pmol. This identification was confirmed by sequencing for 50 cycles a fragment beginning at αSer-251 before α1M2, produced by digestion with EndoGlu-C, with OPA treatments prior to cycles 3 and 28 corresponding to α1Pro-253 and α1Pro-278. Peaks of 3H release in cycles 41 and 44 confirmed photolabeling of α1Ala-291 (∼90 cpm/pmol) and α1Tyr-294 (∼60 cpm/pmol), and 1 mm pentobarbital inhibited incorporation into both residues by >80% (data not shown).
      Figure thumbnail gr5
      FIGURE 5R-[3H]mTFD-MPAB photolabels α1Ala-291, α1Tyr-294, and γ2Ser-301 in the α1 and γ2 M3 transmembrane helices. A, 3H (●) and picomoles of PTH-derivatives (□) released during Edman sequencing of a GABAAR subunit fragment beginning at α1Asp-287 (7 pmol). The peaks of 3H release in cycles 5 and 8 indicated photolabeling of α1Ala-291 and α1Tyr-294. For this sequencing experiment, material isolated by rpHPLC from an EndoGlu-C digest of α1 subunit (B, fractions 25–27) was sequenced for four cycles, establishing that the primary sequence began at α1Ser-251 before α1M2, a fragment predicted to extend to α1Glu-313 near the C terminus of α1M3 (C). After cycle 4, the sample was treated with OPA to block all free N termini, which was confirmed by five more cycles of Edman degradation, and then treated with cyanogen bromide to cleave at methionines before sequencing for 15 additional cycles. D, 3H (●) and picomoles of PTH-derivatives (□) released during Edman sequencing of a GABAAR subunit fragment beginning at γ2Asp-297 (0.6 pmol). The peak of 3H release in cycle 5 indicated labeling of γ2Ser-301. Material isolated by rpHPLC from an EndoGlu-C digest of 59–61-kDa gel bands (E, fractions 28–29)) was sequenced for 10 cycles, establishing the presence of the fragment beginning at γ2Val-212 (F) as a secondary sequence along with the primary sequence beginning at β3His-191. The sample was treated with OPA after cycle 10 to block all free N termini, sequenced an additional 5 cycles to confirm block, then treated with cyanogen bromide, and sequenced for an additional 15 cycles. The efficiencies of photolabeling of the residues are tabulated in .

       R-[3H]mTFD-MPAB Photolabels γ2Ser-301 in γ2M3

      To characterize photolabeling in γ2M3, we sequenced the fragment beginning at γ2Asp-297 by use of a protocol similar to that used to sequence the homologous α1Asp-287 fragment (Fig. 5, D–F). Material recovered from an rpHPLC fractionation of an EndoGlu-C digest of labeled subunits was sequenced, N-terminally blocked, treated with cyanogen bromide, and resequenced. To maximize the amount of γ2 and minimize the amount of α1 subunit, material was used from the β subunit gel bands that contain more γ2 than α1 subunit. Rather than use the rpHPLC fractions where the α1Ser-251 fragment had eluted (Fig. 5B), we used fractions eluting at higher organic solvent that contained the peak of 3H (from photolabeled β3Met-227 in the β3His-191 fragment) and the γ2Val-212 fragment that begins before γ2M1 and extends through M3 (Fig. 5, E and F). When that material was sequenced after cyanogen bromide digestion (Fig. 5D), the fragment beginning at γ2Asp-296 was present as a secondary sequence, with the primary sequence beginning at β3Pro-228 and no detectable α1 subunit sequences. There was a peak of 3H release in cycle 5, the cycle that contained β3Ile-232 from the primary sequence and γ2Ser-301 from the secondary sequence. Because there was no evidence of photolabeling of β3Ile-232 (Fig. 4B, cycle 17), the peak of 3H release in cycle 5 indicated R-[3H]mTFD-MPAB photolabeling of γ2Ser-301, the amino acid in γM3 homologous to α1Ala-291. We confirmed this identification by using a protocol that took advantage of the unique distributions of Trp and Pro in the three subunits in the M2-M3 region to chemically isolate γ2M3 during sequencing. When labeled subunits from gel bands enriched in either α1 or β3 were treated with BNPS-skatole to cleave at tryptophans and sequenced for 50 cycles with OPA treatment at cycle 7, a peak of 3H release was seen in cycle 45 that confirmed R-[3H]mTFD-MPAB photolabeling of γ2Ser-301 at ∼100 cpm/pmol (data not shown).

       R-[3H]mTFD-MPAB Photolabeling in Other Transmembrane Helices

      By sequencing appropriate rpHPLC fractions from EndoLys-C digests of β3 subunits (
      • 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.
      ), we found that within β3M3 R-[3H]mTFD-MPAB photolabeled β3Met-286, the amino acid photolabeled by R-[3H]azietomidate, and β3Phe-289. However, those residues were photolabeled at ∼20 cpm/pmol, i.e. ∼2% the efficiency of photolabeling of β3Met-227 from the same photolabeling experiment. Any photolabeling within α1M1, if it occurred, was at <3% the efficiency of β3Met-227.
      The sequencing protocols used to characterize photolabeling in α1M3 and γ2M3 (Fig. 5) involved sequencing through α1M2, γ2M2, and β3M2, and any photolabeling within the M2 helices, if it occurred, was at <3% the efficiency of β3Met-227. Sequence analyses of fragments beginning at β3Ile-414 before βM4 and αThr-377 before αM4 that were isolated by rpHPLC from proteolytic digests of β3-enriched material established that photolabeling, if it occurred, within β3M4 was at <0.3% and within α1M4 at <1% the efficiency of labeling of β3Met-227.

       R-[3H]mTFD-MPAB Binds to Sites at the α+ and γ+ Subunit Interfaces Equivalent to the Etomidate-binding Site at the β+ Subunit Interfaces

      The high degree of amino acid sequence conservation between the GABAAR M1–M4 helices and those of GLIC or GluCl allows simple and consistent alignment of those GABAAR regions in homology models based upon GLIC or GluCl (
      • 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.
      ). In an α1β3γ2 GABAAR homology model based upon the structure of GLIC (Fig. 6), the residues photolabeled by R-[3H]mTFD-MPAB are located in two different subunit interfaces (α+ and γ+) (Fig. 6C). In the α+ interface, β3Met-227 in the M1 helix is opposite both α1Ala-291 and α1Tyr-294 in the M3 helix and located between them on an axis perpendicular to the membrane, whereas in the γ+ interface it is opposite γ2Ser-301 in M3 and slightly below it. In both cases there is a pocket between the subunits that is large enough to accommodate R-mTFD-MPAB (volume of 275 Å3). Shown in Fig. 6, D–F, are expanded views of these binding sites with R-mTFD-MPAB docked in the lowest energy orientation predicted by computational docking. R-mTFD-MPAB was predicted to bind with its reactive diazirine positioned in close proximity to the photolabeled amino acids in β3M1 and α1M3 or γ2M3, the NCH3 group of barbituric acid oriented toward α1M2–15′ or γ2M2–15′ (α1Ser-270/γ2Ser-280), and the C5 allyl group oriented toward β3M2–10′ and β3M2–14′ (β3Thr-260/β3Ile-264). β3Pro-228 in β3M1 is predicted to be a major determinant of the shape of this binding pocket, as noted previously for the homologous proline in α1M1 (α1Pro-233) in the etomidate-binding site at the β+ interface (
      • 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.
      ).
      Figure thumbnail gr6
      FIGURE 6R-mTFD-MPAB binds in the GABAAR transmembrane domain to sites at the α+ and γ+ interfaces that are homologous to the etomidate-binding sites at the β+ interfaces. A, side view of an α1β3γ2 GABAAR homology model built using a GLIC crystal structure (Protein Data Bank code 3P50), with α-helices displayed as cylinders, β-sheets as ribbons, and subunits color-coded as follows: α1, light yellow; β3, light blue, and γ2, light green. B and C, views down the ion channel of the GABAAR extracellular (B) and transmembrane (C) domains. A–C, locations are indicated of the pockets containing the binding sites for GABA (green), benzodiazepine (blue), etomidate (brown), and R-mTFD-MPAB (red). D and E, views of R-mTFD-MPAB docked in the pocket at the α+ interface, viewed from the lipid (D) and from the base of the extracellular domain (E). F and G, views from the lipid of R-mTFD-MPAB docked at the γ+ interface (F) and R-azietomidate docked at the β+ interface (G). D–G, docked anesthetic is shown in stick format in its lowest energy orientation, color-coded by element (carbon, gray; oxygen, red; nitrogen, blue; and fluorine, light blue) within the Connolly surface representation of the volumes defined by the ensemble of the 100 lowest energy-minimized docking solutions. Residues photolabeled by R-[3H]mTFD-MPAB are shown in stick format and color-coded as follows: β3Met-227, red; α1Ala-291, magenta; α1Tyr-294, purple; γ2Ser-301, orange; β3Met-286, cyan, and β3Phe-289, yellow-green. Residues photolabeled by R-[3H]azietomidate/[3H]TDBzl-etomidate (G only) are color-coded as follows: β3Met-286, cyan; β3Val-290, dark green; and α1Met-236, lime green. Also color-coded in G are β3Asn-265 (brown, M2–15′), the in vivo etomidate/propofol/pentobarbital sensitivity determinant (
      • 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 GABAA receptor β3 subunit.
      ,
      • Zeller A.
      • Arras M.
      • Jurd R.
      • Rudolph U.
      Identification of a molecular target mediating the general anesthetic actions of pentobarbital.
      ) and β3Phe-301 (blue), the residue photolabeled by an anesthetic steroid in a homopentameric β3 GABAAR (
      • Chen Z.W.
      • Manion B.
      • Townsend R.R.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      • Sieghart W.
      • Fuchs K.
      • Evers A.S.
      Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABAA receptor.
      ). The color-coded residues of D–G are also highlighted in the aligned GABAAR subunit sequences spanning the M1–M3 helices (bottom of figure).
      The binding sites for R-mTFD-MPAB at the α+ and γ+ subunit interfaces are homologous to the etomidate-binding site at the β+ subunit interface identified by photolabeling with R-[3H]azietomidate and R-[3H]TDBzl-etomidate (
      • 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.
      ), which is shown in Fig. 6G with R-azietomidate docked in its predicted lowest energy orientation. The alignment of α1, β3, and γ2 M1–M3 transmembrane helices (45% identity, Fig. 6, bottom) illustrates that α1Ala-291 and γ2Ser-301 occupy the same positions in the R-mTFD-MPAB-binding sites as β3Met-286 (labeled by R-azietomidate) in the etomidate-binding sites. Similarly, β3Met-227 occupies the same position in the R-mTFD-MPAB-binding sites as α1Leu-232 does in the etomidate sites, 4 amino acids or one helical turn above α1Met-236 (also labeled by R-azietomidate). Thus, the R-mTFD-MPAB and R-etomidate binding pockets are at different subunit interfaces, but they are located at the same depth in the transmembrane domain.
      In addition to the four intersubunit-binding sites identified by R-[3H]azietomidate and R-[3H]mTFD-MPAB, there is a fifth potential site in the transmembrane domain at the α+ subunit interface, the same interface that in the extracellular domain contains the benzodiazepine site. This site may be photolabeled by R-[3H]mTFD-MPAB, because the residues it photolabeled in αM3 at the α+ interface are present in the second α subunit at the α+ interface. We have not yet been able to characterize photolabeling in γM1, which is necessary to determine whether this fifth intersubunit site is also photolabeled.

       Selectivities of Etomidates and Barbiturates for Intersubunit-binding Sites

      To determine whether the >50-fold selectivity of R-mTFD-MPAB and ∼10-fold selectivity of pentobarbital for the anesthetic-binding sites at α++ interfaces and the >100-fold selectivity of R-etomidate for the sites at the β+ interfaces were general properties of barbiturates and etomidates, we screened other etomidates (Table 2) and barbiturates (Table 3) as inhibitors of R-[3H]azietomidate and R-[3H]mTFD-MPAB photolabeling. With this assay, only qualitative comparisons can be made of the potencies of different anesthetics, because IC50 values were determined from total rather than free concentrations, and the anesthetics vary greatly in lipophilicity, as evidenced by the ∼100-fold range of partition coefficients (TABLE 2, TABLE 3). However, the ratio of IC50 values of stereoisomer pairs for a site or of each anesthetic for the two binding sites will not depend on the differences in anesthetic partition coefficients.
      TABLE 2Affinities of etomidates for GABAAR anesthetic-binding sites at the β+−α (3HR−azietomidate) and α++−β (R-3HmTFD-MPAB) subunit interfaces a
      Figure thumbnail fx1
      TABLE 3Affinities of barbiturates for GABAAR anesthetic-binding sites at the β+−α (R-3Hazietomidate) and α++−β (R 3HmTFD-MPAB) subunit interfacesa
      Figure thumbnail fx2
      S-Etomidate binds preferentially to the same site as R-etomidate, but with 10-fold lower affinity. Not surprisingly, R-azietomidate and R-TDBzl-etomidate bind with >25-fold selectivity to the sites at the β+ interfaces. However, the presence of a bulky substituent on the etomidate phenyl ring is not tolerated, as the affinity of S- or R-pTFD-etomidate for the β+ sites was reduced by 50-fold compared with R-TDBzl-etomidate, and both isomers bound with 2-fold higher affinity to the α++ interface sites than to the β+ sites.
      Similar to pentobarbital, phenobarbital bound with ∼10-fold selectivity to the binding sites at α++ interfaces, but addition of bulk to the ring, as in thiopental, reduced the selectivity to only 1.6-fold, and brallobarbital bound with 3-fold higher affinity to the “etomidate”-binding site. In contrast to the ∼60-fold binding selectivity of R-mTFD-MPAB, S-mTFD-MPAB bound with lower affinity and nonselectively to both classes of sites. We also examined the effects of stereoisomers of MPPB, as R-MPPB acts as an anesthetic and GABAAR potentiator, although S-MPPB acts as a convulsant and GABAAR inhibitor (
      • Ticku M.K.
      • Rastogi S.K.
      • Thyagarajan R.
      Separate site(s) of action of optical isomers of 1-methyl-5-phenyl-5-propylbarbituric acid with opposite pharmacological activities at the GABA receptor complex.
      ,
      • Kamiya Y.
      • Andoh T.
      • Furuya R.
      • Hattori S.
      • Watanabe I.
      • Sasaki T.
      • Ito H.
      • Okumura F.
      Comparison of the effects of convulsant and depressant barbiturate stereoisomers on AMPA-type glutamate receptors.
      ). The anesthetic isomer bound with 9-fold higher affinity to the sites at the α++ interfaces than at the β+ interfaces, and similarly to R- and S-mTFD-MPAB, the difference between and R- and S-MPPB was the decreased affinity of S-MPPB for the sites at the α++ interfaces.
      For the barbiturates studied, R-mTFD-MPAB possessed the highest affinity and selectivity for the α++ sites. Comparison with R-MPAB indicates that the m-TFD substituent of R-mTFD-MPAB is important for both site selectivity and binding affinity. R-mTFD-MPAB had 30-fold higher affinity than R-MPAB at the α++ sites and only 5-fold higher affinity at the β+ sites. Substitution of mBr (R- mBr-MPAB) increased binding affinity at all interfaces by 5-fold compared with R-MPAB.

       Binding of Propofol and Propofol Analogs to Intersubunit-binding Sites

      Propofol at 300 μm inhibited both R-[3H]mTFD-MPAB (Fig. 7A) and R-[3H]azietomidate (Fig. 7B) photolabeling by >90%, consistent with competitive inhibition at both sites. In the absence or presence of GABA, propofol was ∼2–3-fold more potent as an inhibitor of R-[3H]azietomidate photolabeling. In three photolabeling experiments in the presence of GABA using different GABAAR purifications, the IC50 values (±S.E.) for R-[3H]azietomidate and R-[3H]mTFD-MPAB were 32 ± 12 and 49 ± 10 μm, respectively, with the ratio of IC50(Aziet)/IC50(TFD-MPAB) for paired experiments equal to 0.6 ± 0.1. In the absence of GABA, the IC50 values for R-[3H]azietomidate and R-[3H]mTFD-MPAB were 25 ± 13 and 92 ± 46 μm, respectively, with the ratio of IC50 values for paired experiments equal to 0.28 ± 0.03. In the absence of GABA, propofol at low concentrations produced a small enhancement (∼50%) of R-[3H]mTFD-MPAB photolabeling with the concentration dependences of enhancement and inhibition consistent with EC50 = 12 μm, the IC50 for propofol inhibition of R-[3H]azietomidate photolabeling in the absence of GABA, and IC50 = 40 μm, the IC50 for R-[3H]mTFD-MPAB inhibition (+GABA) (Fig. 7A, dashed line).
      Figure thumbnail gr7
      FIGURE 7Modulation of R-[3H]mTFD-MPAB and R-[3H]azietomidate GABAAR photolabeling by propofol, propofol analogs, alphaxalone, and octanol. α1β3γ2 GABAARs were equilibrated with R-[3H]mTFD-MPAB (A, C, E, and G) or R-[3H]azietomidate (B, D, F, and H) in the absence (●) or presence (▿) of GABA and varying concentrations of propofol (A and B), 2,6-di-sec-butylphenol (○) or 2,6-di-tert-butylphenol (♢) (C and D (+GABA)), alphaxalone (E and F), or octanol (G and H). The concentration dependences of potentiation (EC50) and inhibition (IC50) were fit as described under “Experimental Procedures,” and the values of IC50/EC50 of the plotted lines are included under the “Results.” For each experiment, the amounts of R-[3H]mTFD-MPAB or R-[3H]azietomidate incorporation in the presence of 1 mm pentobarbital or 1 mm R-etomidate are indicated by dotted lines.
      We also determined the inhibition of R-[3H]mTFD-MPAB and R-[3H]azietomidate photolabeling in the presence of GABA by 2,6-di-sec-butylphenol, a propofol analog that is similar in potency to propofol as a GABAAR potentiator and anesthetic (EC50 = 2 μm), and 2,6-di-tert-butylphenol, which at 300 μm was inactive as a GABAAR modulator or anesthetic and did not alter responses to propofol (
      • 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 GABAA receptor but not with lipid solubility.
      ). 2,6-di-sec-Butylphenol was equipotent as an inhibitor of photolabeling by both photoprobes (IC50 = 90 μm), although the inactive isomer, 2,6-di-tert-butylphenol, at 300 μm inhibited photolabeling by <10% (Fig. 7, C and D). Because our experimental IC50 values are determined from total, rather than free, drug concentrations and the partition coefficient of 2,6-di-sec-butylphenol (or 2,6-di-tert-butylphenol) is 6-fold greater than that of propofol (Table 4), it is not possible to determine from our data whether 2,6-di-sec-butylphenol is actually more or less potent than propofol. However, differences in hydrophobicity (partition coefficient) cannot account for the capacity of 2,6-di sec-butylphenol to act as an anesthetic and bind to the GABAAR intersubunit-binding sites, although 2,6-di-tert-butyl phenol neither acts as an anesthetic nor binds to the intersubunit anesthetic-binding sites.
      TABLE 4Affinities of propofol analogs for GABAAR anesthetic-binding sites at the β+ − α (R-3Hazietomidate) and α++ − β (R-3HmTFD-MPAB) subunit interfacesa
      Figure thumbnail fx3

       Interactions of Alphaxalone and Octanol with Intersubunit Anesthetic-binding Sites

      In the presence of GABA, alphaxalone, a synthetic anesthetic steroid, at concentrations up to 30 μm had little or no effect on photolabeling by R-[3H]mTFD-MPAB (Fig. 7E) or R-[3H]azietomidate (Fig. 7F). In the absence of GABA, alphaxalone increased photolabeling at both sites with EC50 values of ∼500 nm, similar to the potentiation of R-[3H]azietomidate photolabeling of brain GABAAR by alphaxalone or neurosteroids (
      • Li G.-D.
      • Chiara D.C.
      • Cohen J.B.
      • Olsen R.W.
      Neurosteroids allosterically modulate binding of the anesthetic etomidate to γ-aminobutyric acid type A receptors.
      ). Alphaxalone binds neither to the R-[3H]azietomidate nor R-[3H]mTFD-MPAB-binding site, which is consistent with early studies demonstrating additive effects of alphaxalone and pentobarbital (
      • Turner D.M.
      • Ransom R.W.
      • Yang J.S.
      • Olsen R.W.
      Steroid anesthetics and naturally occurring analogs modulate the γ-aminobutyric acid receptor complex at a site distinct from barbiturates.
      ).
      Octanol acts as an anesthetic and GABAAR potentiator with EC50 values of ∼60 μm (
      • Husain S.S.
      • Forman S.A.
      • Kloczewiak M.A.
      • Addona G.H.
      • Olsen R.W.
      • Pratt M.B.
      • Cohen J.B.
      • Miller K.W.
      Synthesis and properties of 3-(2-hydroxyethyl)-3-n-pentyldiazirine, a photoactivable general anesthetic.
      ). In the presence of GABA, octanol at 1 mm inhibited R-[3H]mTFD-MPAB (Fig. 7G) photolabeling by 70% and R-[3H]azietomidate (Fig. 7H) photolabeling by 50%. If we assume this inhibition is competitive, analysis yields IC50 values of 450 ± 70 and 1,600 ± 400 μm for R-[3H]mTFD-MPAB and R-[3H]azietomidate, respectively. However, in the absence of GABA, octanol at concentrations up to 1 mm had no effect on photolabeling.

       Effects of GABA and Etomidate on R-[3H]mTFD-MPAB Photoincorporation at the Amino Acid Level

      For R-[3H]azietomidate-photolabeled GABAAR purified from bovine brain, the enhancement of photolabeling seen at the subunit level in the presence of GABA or a neurosteroid, as well as the inhibition of photolabeling in the presence of propofol, was also seen at the level of the photolabeled amino acids (
      • 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.
      ,
      • Li G.-D.
      • Chiara D.C.
      • Cohen J.B.
      • Olsen R.W.
      Neurosteroids allosterically modulate binding of the anesthetic etomidate to γ-aminobutyric acid type A receptors.
      ,
      • Li G.D.
      • Chiara D.C.
      • Cohen J.B.
      • Olsen R.W.
      Numerous classes of general anesthetics inhibit etomidate binding to γ-aminobutyric acid type A (GABAA) receptors.
      ). To determine whether this was also true for the R-[3H]mTFD-MPAB site or whether novel amino acids were photolabeled when subunit photolabeling was enhanced, we characterized photolabeling in βM1, α1M3, γ2M3, and β3M3 for α1β3γ2 GABAARs photolabeled in three conditions as follows: control (no additional drug), +1 mm GABA, or +100 μm etomidate (Table 5). The incorporation at β3Met-277 within β3M1, the residue that accounts for >80% of GABAAR photolabeling, closely paralleled the labeling seen at the subunit level. GABA and etomidate increased photolabeling efficiency by ∼50%, and no novel residues were photolabeled in β3M1. The complex sequencing protocols required to identify photolabeling in α1M3 or γ2M3 made quantification more difficult. Qualitatively, GABA increased photolabeling of α1Ala-291, α1Tyr-294, and γ2Ser-301, and no other amino acids were photolabeled. Additional labeling experiments would be necessary to assess the smaller effects of etomidate on those residues.
      TABLE 5Pharmacological specificity of R-[3H]mTFD-MPAB photoincorporation into residues in the α1β3γ2 GABAAR (cpm/pmol of PTH-derivative)
      Amino acidExperiment 1 +GABAExperiment 2
      Control+Pentobarbital (1 mm)Control+GABA (1 mm)+Etomidate (100 μm)
      β3M1 Met-227950 ± 30168 ± 5420640 ± 50660
      α1M3 Ala-29192<5285041
      α1M3 Tyr-29456<5364626
      γ2M3 Ser-301105 ± 5ND83130115
      β3M3 Met-28625ND713<2
      β3M3 Phe-28918ND1210<2
      We also quantified R-[3H]mTFD-MPAB photolabeling of amino acids in the etomidate-binding site (β3Met-286 and β3Phe-289 in the β3M3 helix), which were labeled at ∼2% the efficiency of β3Met-227. Etomidate inhibited photolabeling of β3Met-286 and β3Phe-289 by >90%, as expected for the presence of those amino acids in the etomidate-binding site, although it enhanced photolabeling of β3Met-227 in the R-mTFD-MPAB site.

      DISCUSSION

      In this report we provide the first demonstration that there are two structurally related, but pharmacologically distinct, classes of intersubunit general anesthetic-binding sites in the transmembrane domain of human α1β3γ2 GABAARs. The binding sites for R-[3H]mTFD-MPAB, a photoreactive barbiturate that acts as a potent, stereoselective GABAAR potentiator and general anesthetic, are located at the α+ and γ+ subunit interfaces, centered three helical turns down from the extracellular end of β3M3 (Fig. 6). At anesthetic concentrations, R-mTFD-MPAB does not bind at the previously characterized etomidate-binding sites (
      • 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.
      ,
      • 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.
      ), which are located at the two β+ subunit interfaces and are also centered three turns down from the extracellular end of α1M3. Conversely, R-etomidate does not bind at the R-mTFD-MPAB-binding sites. Thus, R-mTFD-MPAB binds to homologous but distinct sites from etomidate and its photoreactive derivatives.

       Pharmacology of the Two Classes of General Anesthetic-binding Sites

      R-mTFD-MPAB and R-etomidate each bind with >50-fold selectivity to their preferred sites, with IC50 values similar to the EC50 values for GABAAR potentiation in vitro or anesthesia in vivo. Displacing these ligands with nonradioactive anesthetics (see IC50 values in TABLE 2, TABLE 3, TABLE 4) lead to the conclusion that the two classes of sites are not simply etomidate or “barbiturate” sites. For example, pentobarbital and phenobarbital bound to the α++ sites with ∼10-fold selectivity, whereas thiopental and S-mTFD-MPAB bound with similar affinity to both sites. Furthermore, the barbiturate brallobarbital had an ∼3-fold higher preference for the etomidate (β+) site, and pTFD-etomidate had 2-fold preference for the barbiturate (α++) site. Thus, we refer to these sites by their subunit interface designations. There is precedent for a pharmacological class of anesthetics not binding to isosteric sites in the Cys loop ligand-gated ion channel superfamily. Although some barbiturates that inhibited currents in muscle type nAChRs fully displaced [14C]amobarbital binding, others bound to an unidentified site (
      • Dodson B.A.
      • Urh R.R.
      • Miller K.W.
      Relative potencies for barbiturate binding to the Torpedo acetylcholine receptor.
      ).
      Propofol bound with little selectivity at both classes of sites, suggesting it has at least four binding sites. Although the IC50 values for R-azietomidate or R-mTFD-MPAB binding are close to anesthetic concentrations, the IC50 values for propofol binding to either class of sites (∼40 μm) are ∼20-fold higher than GABA modulatory or anesthetic concentrations (
      • Stewart D.S.
      • Savechenkov P.Y.
      • Dostalova Z.
      • Chiara D.C.
      • Ge R.
      • Raines D.E.
      • Cohen J.B.
      • Forman S.A.
      • Bruzik K.S.
      • Miller K.W.
      p-(4-Azipentyl)propofol: A potent photoreactive general anesthetic derivative of propofol.
      ). This discrepancy might result if propofol binds with higher affinity to as yet unidentified sites in the GABAAR. However, 2,6-di-sec-butyl phenol, which is equipotent with propofol as an anesthetic and GABAAR modulator (
      • 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 GABAA receptor but not with lipid solubility.
      ), binds with potency similar to propofol to the two classes of intersubunit anesthetic-binding sites, although 2,6-di-tert-butylphenol, which is inactive as an anesthetic and GABAAR modulator, did not bind to either class of sites (Table 4). These results make it likely that the four intersubunit sites identified by R-[3H]azietomidate and R-[3H]mTFD-MPAB are the binding sites important for propofol's anesthetic effects. Interestingly, the potentiation and direct activation by propofol, which has little or no subunit interface selectivity, is best fit with a model that requires three equivalent binding sites, whereas etomidate only requires two (
      • Rüsch D.
      • Zhong H.
      • Forman S.A.
      Gating allosterism at a single class of etomidate sites on α1β2γ2L GABAA receptors accounts for both direct activation and agonist modulation.
      ,
      • Ruesch D.
      • Neumann E.
      • Wulf H.
      • Forman S.A.
      An allosteric coagonist model for propofol effects on the α1β2γ2L γ-aminobutyric acid type A receptors.
      ).
      The fact that propofol binds nonselectively to four sites was unexpected, as previous mutational analyses identified propofol sensitivity determinant positions (βM2–15′ and β3Met-286) that in our GABAAR homology model are in the anesthetic-binding sites at the β+ interfaces (
      • Siegwart R.
      • Krähenbühl K.
      • Lambert S.
      • Rudolph U.
      Mutational analysis of molecular requirements for the actions of general anaesthetics at the γ-aminobutyric acid A receptor subtype, α1β2γ2.
      ,
      • Bali M.
      • Akabas M.H.
      Defining the propofol-binding site location on the GABAA receptor.
      ), although the homologous α subunit substitutions in the α+-binding site had little if any effect (
      • Krasowski M.D.
      • Koltchine V.V.
      • Rick C.E.
      • Ye Q.
      • Finn S.E.
      • Harrison N.L.
      Propofol and other intravenous anesthetics have sites of action on the γ-aminobutyric acid type A receptor distinct from that for isoflurane.
      ). However, there are two β+ interface-binding sites in an αβγ GABAAR and only one α+ interface site. In future studies it will be important to determine the effects of simultaneous substitutions at the α++ interface sites on the sensitivity to propofol or other anesthetics binding to those sites.
      Alphaxalone, which potentiated R-[3H]azietomidate and R-[3H]mTFD-MPAB photolabeling, was the only anesthetic tested that did not bind to either site. However, neurosteroids may bind near these intersubunit anesthetic-binding sites but more at the lipid interface, because an anesthetic steroid photolabeled β3Phe-301 (Fig. 6G) in βM3 in homomeric β3 GABAARs (
      • Chen Z.W.
      • Manion B.
      • Townsend R.R.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      • Sieghart W.
      • Fuchs K.
      • Evers A.S.
      Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABAA receptor.
      ).

       Anesthetic-binding Sites at α++ Subunit Interfaces

      In the α1β3γ2 homology model (Fig. 6), the amino acids photolabeled by R-[3H]mTFD-MPAB are in a pocket formed by residues from α1M2/M3 (or γ2M2/M3) and β3M2/M1. R-mTFD-MPAB is predicted by computational docking to bind with its reactive diazirine in close proximity to the photolabeled residues, the NCH3 of barbituric acid oriented toward αM2–15′ or γM2–15′, and the C5-allyl oriented toward βM2–10′/βM2–14′.
      Previous mutational analyses provided evidence that pentobarbital sensitivity determinants were contained within βM1/βM2 (
      • Serafini R.
      • Bracamontes J.
      • Steinbach J.H.
      Structural domains of the human GABAA receptor β3 subunit involved in the actions of pentobarbital.
      ), including β3Pro-228 (
      • Greenfield Jr., L.J.
      • Zaman S.H.
      • Sutherland M.L.
      • Lummis S.C.
      • Niemeyer M.I.
      • Barnard E.A.
      • Macdonald R.L.
      Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement.
      ) that is adjacent to the photolabeled β3Met-227 in βM1 and predicted to be a key determinant of the anesthetic binding pocket's shape (Fig. 6F). Comparison of the amino acids contributing to the α++ and β+ binding pockets identifies nonconserved positions likely to contribute to the strong site selectivities of R-mTFD-MPAB and R-etomidate. Most notable is the difference at position M2–15′, with α1Ser-270/γ2Ser-280 in the R-mTFD-MPAB-binding sites and β3Asn-265 in the R-etomidate-binding sites, because the β3N265S substitution reduces etomidate sensitivity by 10-fold (
      • 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.
      ). Additional differences in M3 positions can be found in the sequence alignments of Fig. 6.
      Because βM2–15′ is predicted to be an important determinant of the shape of the etomidate-binding site at the β+ interface (Fig. 6G) (
      • 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.
      ) and pentobarbital binds with ∼8-fold higher selectivity to the α++ sites, it is surprising that the anesthetic responses of pentobarbital are reduced in the β3N265M knock-in mouse (
      • Zeller A.
      • Arras M.
      • Jurd R.
      • Rudolph U.
      Identification of a molecular target mediating the general anesthetic actions of pentobarbital.
      ). This may indicate that the β+ sites make a greater energetic contribution to the stabilization of GABAAR in the open state. Characterization of the anesthetic effects of R-mTFD-MPAB on the β3N265M GABAAR in vitro and in vivo will clarify whether the substitution prevents transduction of changes initiated by binding to the α++ subunit interfaces.

       Intrasubunit Sites?

      Propofol inhibits the nAChR and the prokaryotic homolog GLIC, and in those proteins it binds to intrasubunit-binding sites within the pocket formed by the transmembrane helix bundle (
      • 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.
      ,
      • 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.
      ). Our studies with R-[3H]mTFD-MPAB (this work) and R-[3H]azietomidate and [3H]TDBzl-etomidate (
      • 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.
      ) provided no evidence of GABAAR intrasubunit-binding sites for those anesthetics, even though we sequenced through each of the α and β subunit transmembrane helices. In these peptides, we observed minor labeling of β3Met-286 and Phe-289 in the β+ anesthetic-binding site at ∼2% the efficiency of β1Met-227. Thus, we can state that, if any intrasubunit labeling occurred, it must be at levels below this.

       Anesthetics and GABAAR Conformational Equilibria

      At lower concentrations, most general anesthetics potentiate GABA responses, and at higher concentrations, they directly activate GABAARs in the absence of GABA. Direct activation and potentiation of nAChRs and GABAARs can be well accounted for by allosteric models that assume that receptors exists in multiple, interconvertible conformational states (
      • Monod J.
      • Wyman J.
      • Changeux J.P.
      On the nature of allosteric transitions: A plausible model.
      ,
      • Auerbach A.
      The gating isomerization of neuromuscular acetylcholine receptors.
      ,
      • Forman S.A.
      Monod-Wyman-Changeux allosteric mechanisms of action and the pharmacology of etomidate.
      ,
      • Changeux J.P.
      Allostery and the Monod-Wyman-Changeux model after 50 years.
      ). Activators and potentiators shift the conformational equilibria toward the open channel state because they bind with higher affinity to open states than to resting, closed channel states. In purified GABAAR in detergent/lipid micelles, positive energetic coupling between the extracellular and transmembrane domains is preserved as evidenced by anesthetic enhancement of [3H]muscimol binding and GABA enhancement of R-[3H]azietomidate/R-[3H]mTFD-MPAB photolabeling. Furthermore, in the absence of GABA, R-etomidate enhances R-[3H]mTFD-MPAB photolabeling, and reciprocally, R-mTFD-MPAB enhances R-[3H]azietomidate photolabeling. Our studies provide no information about the state-dependent differences in affinity for anesthetics binding at either class of sites. However, smaller differences in binding affinity between open (Ko) and closed states (Kc) are required for anesthetics binding to four rather than two sites, because the shift in conformational equilibria will be proportional to (Ko/Kc)n, where n is the number of sites.
      Because R-etomidate does not bind to the α++ sites even at 1 mm, our results provide further evidence that R-etomidate directly activates GABAARs (
      • 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.
      ) by binding solely to the β+ interfaces that also contain the agonist-binding sites in the extracellular domain (
      • 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.
      ). The selective binding of R-mTFD-MPAB to the α++ subunit interfaces provides the first evidence that potentiation and direct activation (
      • 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.
      ) can result from anesthetic binding at interfaces not containing the transmitter-binding site.

       Conclusions

      Our novel finding is that it is possible to synthesize general anesthetics that are selective for sites between specific subunits in the transmembrane domain of pentameric GABAARs. A wide range of general anesthetic structures target these four sites but with variable selectivity, which offers an explanation of the puzzling lack of well defined structure activity relationships among general anesthetics (
      • Meyer H.H.
      Zur theorie der alkoholnarkose. Der einfluss wechselnder temperatur auf wirkungsstärke und theilungscoefficient der narcotica.
      ,
      • Overton C.E.
      ,
      • Rudolph U.
      • Antkowiak B.
      Molecular and neuronal substrates for general anaesthetics.
      ). These observations suggest that it may be possible to develop agents with novel intersubunit specificity that can be used to target specific nerve pathways and behaviors in a subunit-dependent manner (
      • Drexler B.
      • Antkowiak B.
      • Engin E.
      • Rudolph U.
      Identification and characterization of anesthetic targets by mouse molecular genetics approaches.
      ). A similar strategy has recently been proposed for the extracellular domain where a potentiator site has been identified at the α+ interface in a pocket equivalent to the transmitter and benzodiazepine sites at the β+ and α+ subunit interfaces (
      • Sieghart W.
      • Ramerstorfer J.
      • Sarto-Jackson I.
      • Varagic Z.
      • Ernst M.
      A novel GABAA receptor pharmacology: drugs interacting with the α+β interface.
      ,
      • Ramerstorfer J.
      • Furtmüller R.
      • Sarto-Jackson I.
      • Varagic Z.
      • Sieghart W.
      • Ernst M.
      The GABAA receptor α+β-interface: A novel target for subtype selective drugs.
      ,
      • Firestone L.L.
      • Miller J.C.
      • Miller K.W.
      ).

      Acknowledgments

      We thank Dr. Ayman Hamouda for useful comments on the manuscript.

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