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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*

Open AccessPublished:May 15, 2013DOI:https://doi.org/10.1074/jbc.M113.479725
      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.

      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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