Stewart DS , Pierce DW , Hotta M , Stern AT , Forman SA .

P01 GM058448 NIGMS NIH HHS , P41 RR-01081 NCRR NIH HHS , R01 GM089745 NIGMS NIH HHS , R01GM089745 NIGMS NIH HHS , P41 RR001081 NCRR NIH HHS

	Figure 2. Electrophysiological characterization of α1β2N265Cγ2L GABAA receptors.Panel A: GABA concentration response in oocytes. Data points are mean ± sd (n ≥4) peak currents normalized to maximal GABA (1 mM) responses. Lines through data represent fits to logistic equations (Eq. 1, Methods). Open symbols: GABA alone; EC50 = 31±4.7 µM; nH = 1.75±0.088. Filled symbols: GABA plus 3.2 µM etomidate; EC50 = 30±3.2; nH = 1.67±0.068; maximum response = 1.06±0.01. Panel B: Etomidate agonism concentration response in oocytes. Data points are mean ± sd (n ≥4) peak currents normalized to maximal GABA (1 mM) responses. The line represents a logistic fit. EC50 = 35±5.7 µM; nH = 2.5±1.1; maximum response = 0.020±0.007. Panel C: Spontaneous channel gating activity in an oocyte, assessed using picrotoxin (PTX). No outward current during PTX application was observed. Current elicited with 1 mM GABA in the same cell is also displayed. Panel D: Estimation of maximal GABA efficacy in oocytes. GABA (3 mM; white bar) alone elicits a current that is enhanced about 5% with co-application of alphaxalone (2 µM; black bar). Average results for GABA efficacy are reported in Table 1.
	Figure 3. pCMBS modification and lack of etomidate protection in oocyte-expressed α1β2N265Cγ2L GABAA receptors.Panel A: The first trace is elicited with EC10 GABA (10 µM; white bar) and subsequent traces were recorded during sequential 10 s exposures to 0.1 mM p-chloromercuribenzenesulfonate (pCMBS; arrows; *), followed by wash. Basal currents increase with incremental exposure to pCMBS. Panel B: Traces are from another oocyte. Arrows (**) indicate 10 s exposures to 0.1 mM pCMBS plus 300 µM etomidate, followed by wash. Panel C: Rate analysis of current data from panels A (open symbols) and B (solid symbols) is shown, plotted against cumulative pCMBS exposure time. The line represents a nonlinear least squares single exponential fit to control data (no etomidate). The fitted rate constant is 1200±50 M−1s−1. The fitted rate for protection data (+ etomidate) is 1300±270 M−1s−1. Panel D: Average ± sd pCMBS modification rates in the absence and presence of 1 mM GABA and/or 300 µM etomidate.
	Figure 4. Modification and protection at α1M236C reflects anesthetic site occupancy.Panel A) Data represent mean ± SD peak current responses to GABA from oocytes (n = 4) expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, normalized to maximal GABA responses. Lines represent logistic fits to responses using GABA alone (solid circles; EC50 = 58 µM) and GABA with 3.2 µM etomidate (open circles; EC50 = 3.2 µM). Panel B) Data represent mean ± SD peak current responses to etomidate from oocytes (n = 3) expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, normalized to maximal GABA responses. The line represents a logistic fit with etomidate EC50 = 47 µM. Panel C) A single voltage-clamp current trace illustrating maximal GABA (3 mM; white bar above trace) efficacy in oocyte-expressed β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, enhanced with addition of 10 µM etomidate (black bar). Panel D) Data from panels A and B were renormalized to maximal GABA efficacy (methods; Eq. 2) and fitted with a global MWC equilibrium co-agonist equation (methods; Eq. 3). Lines through data points represents the fitted MWC model: L0 = 10,000; KG = 42±8.7 µM; c = 0.0075±0.00048; KE = 50±12 µM; d = 0.0089±0.00096. Panel E) Traces are from a single oocyte expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors, demonstrating the effects of repeated pCMBS applications on the relative responses to low versus high GABA stimulation. Panel F) Points represent response ratios to low (EC10) vs. high GABA, normalized to pre-modification control values. Lines through data represent linear fits used to determine relative bimolecular modification rates: GABA+pCMBS (circles; 1200±57 M−1s−1); GABA+pCMBS +5 µM alphaxalone (triangles; 3700±270 M−1s−1); and GABA+pCMBS +32 µM etomidate (squares; 106±8.4 M−1s−1). Panel G) Summary of modification rate results (mean ± se) for all oocytes expressing β3-α1M236C/β3-α1M236C-γ2L GABAA receptors under different conditions. The rate with pCMBS alone is significantly accelerated with addition of GABA and GABA/alphaxalone. Relative to GABA+alphaxalone, modification in the presence of GABA is slowed 65% by 3 µM etomidate, 95% by 32 µM etomidate, and 91% by 30 µM propofol. * p<0.05; ** p<0.01.
	Figure 5. The βN265M mutation reduces anesthetic-dependent site occupancy.Panel A) Voltage-clamp current traces are from a single oocyte expressing β3N265M-α1M236C/β3N265M-α1M236C-γ2L GABAA receptors, showing the effects of repeated pCMBS+GABA applications on responses to low versus high GABA. Panel B) Points represent response ratios to low (EC10) vs. high GABA, normalized to pre-modification control values, for one oocytes exposed to: GABA+pCMBS (solid squares) followed by GABA+pCMBS +300 µM etomidate (open circles). Lines through data represent linear fits; labels are the apparent bimolecular modification rates (slopes). Panel C) Summary of modification rate results for all oocytes expressing β3N265M-α1M236C/β3N265M-α1M236C-γ2L GABAA receptors modified with pCMBS, in the absence vs. presence of etomidate. Etomidate (300 µM) reduced the apparent modification rate by 51%. Panel D) Summary of modification rate results for GABA-activated receptors (modified in the presence of 1 mM GABA). Modification is inhibited 54% by 300 µM etomidate. Propofol (30 µM) does not significantly inhibit modification.
	Figure 6. The βN265M mutation reduces the channel gating effects of a α1M236W mutation that mimics etomidate.Panel A) Data are reproduced from Guitchounts et al [38] showing mean ± SD GABA concentration-response, normalized to maximal currents, from oocytes expressing concatenated receptor dimer and trimer constructs. Lines through data points represent non-linear least squares logistic fits (Eq. 1, methods). Solid squares represent β2-α1/γ2L-β2-α1 receptors (GABA EC50 = 36 µM) and open circles represent β2-α1M236W/γ2L-β2-α1M236W receptors (EC50 = 1.0 µM). Panel B) Data points are mean ± SD current responses to GABA, normalized to maximal currents. Solid diamonds represent β2N265M-α1/γ2L-β2N265M-α1 receptors (GABA EC50 = 76 µM; 95% CI = 63 to 92 µM) and open triangles represent β2N265M-α1M236W/γ2L-β2N265M-α1M236W- receptors (EC50 = 25 µM; 95% CI = 22 to 29 µM).
	Figure 7. Effects of βN265M mutation on etomidate in inactive versus active GABAA receptors.Monod-Wyman-Changeux equilibrium allosteric models are diagrammed for both α1M236Cβ2γ2L (A) and α1M236Cβ2N265Mγ2L (B) receptors, each with two equivalent etomidate sites. Etomidate dissociation constants for both inactive (R; KE) and GABA-activated (O; dKE) receptors are rounded estimates based on both functional analysis and modification/protection results (Figs. 6 and 7). Etomidate-sensitive α1M236Cβ2γ2L receptors bind etomidate 100-fold more avidly in the active vs. inactive state, resulting in a 10,000-fold shift in the open-closed equilibrium constant (d2L0 vs. L0) when both sites are occupied. In contrast, etomidate-insensitive α1M236Cβ2N265Mγ2L receptors display low affinity (KE ≈ dKE ≈ 300 µM) for etomidate in both resting and GABA-activated states. Thus, high etomidate concentrations result in only partial site occupancy and weak modulatory effects.
	Figure 1. Functional and structural models of GABAA receptor interactions with etomidate. Panel A: A side-on ribbon depiction of a structural homology model for α1β3γ2L GABAA receptors based on the glutamate-gated chloride channel (GluCl) from Caenorhabditis elegans [20]. Both the extracellular domains and transmembrane domains are shown in relation to membrane lipids. Subunits are color-coded (α1 = blue; β3 = yellow; γ2L = green). βN265 and other residues involved in etomidate and propofol binding are depicted as red stick structures in one of two β+/α− transmembrane interfacial sites. Panel B: A view of the transmembrane domains from the extracellular space shows the structure of each subunit’s four-helix bundle and the arrangement of subunits around the central chloride channel (grey circle). Residues in one interfacial anesthetic site are depicted as red stick structures. Panel C: A close-up view of one β+/α− transmembrane inter-subunit etomidate binding site in the homology model. Helix backbones are depicted as solid cylinders. The βN265 residue and six anesthetic contact residues identified by photolabeling or cysteine modification/protection are highlighted as labeled space-filling structures. Panel D: A Monod-Wyman-Changeux two-state (inactive = R; active = O) equilibrium co-agonist scheme with two equivalent orthosteric agonist (GABA; G) sites and two equivalent allosteric agonist (etomidate; E) sites is depicted [1]. For simplicity, states with only one occupied agonist site are omitted. The model is defined by five equilibrium parameters (see Eq. 3, methods): L0 is a basal gating equilibrium (C/O); KG and KE are dissociation constants for respectively, GABA and etomidate binding to inactive receptors; c and d quantify the binding affinity ratios for respectively, GABA and etomidate to active vs. inactive receptors. Maximal agonist efficacies for GABA and etomidate are respectively, (1+L0c2)−1 and (1+L0d2)−1.

Atucha, Structure-activity relationship of etomidate derivatives at the GABA(A) receptor: Comparison with binding to 11beta-hydroxylase. 2009, Pubmed

Atucha, Structure-activity relationship of etomidate derivatives at the GABA(A) receptor: Comparison with binding to 11beta-hydroxylase. 2009, Pubmed
Bali, Defining the propofol binding site location on the GABAA receptor. 2004, Pubmed , Xenbase
Bali, GABA-induced intersubunit conformational movement in the GABAA receptor alpha 1M1-beta 2M3 transmembrane subunit interface: experimental basis for homology modeling of an intravenous anesthetic binding site. 2009, Pubmed , Xenbase
Bali, Gating-induced conformational rearrangement of the γ-aminobutyric acid type A receptor β-α subunit interface in the membrane-spanning domain. 2012, Pubmed , Xenbase
Baumann, Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement. 2002, Pubmed , Xenbase
Baumann, Subunit arrangement of gamma-aminobutyric acid type A receptors. 2001, Pubmed , Xenbase
Belelli, The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid. 1997, Pubmed , Xenbase
Bertaccini, Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor. 2013, Pubmed
Borghese, GABA(A) receptor transmembrane amino acids are critical for alcohol action: disulfide cross-linking and alkyl methanethiosulfonate labeling reveal relative location of binding sites. 2014, Pubmed , Xenbase
Borghese, Mutations M287L and Q266I in the glycine receptor α1 subunit change sensitivity to volatile anesthetics in oocytes and neurons, but not the minimal alveolar concentration in knockin mice. 2012, Pubmed , Xenbase
Chiara, Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue. 2012, Pubmed
Chiara, Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor. 2013, Pubmed
Colquhoun, Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. 1998, Pubmed
Corringer, Structure and pharmacology of pentameric receptor channels: from bacteria to brain. 2012, Pubmed
Desai, Gamma-amino butyric acid type A receptor mutations at beta2N265 alter etomidate efficacy while preserving basal and agonist-dependent activity. 2009, Pubmed , Xenbase
Foadi, Lack of positive allosteric modulation of mutated alpha(1)S267I glycine receptors by cannabinoids. 2010, Pubmed
Forman, Anesthetic sites and allosteric mechanisms of action on Cys-loop ligand-gated ion channels. 2011, Pubmed
Forman, Mutations in the GABAA receptor that mimic the allosteric ligand etomidate. 2012, Pubmed , Xenbase
GODEFROI, DL-1-(1-ARYLALKYL)IMIDAZOLE-5-CARBOXYLATE ESTERS. A NOVEL TYPE OF HYPNOTIC AGENTS. 1965, Pubmed
Galzi, The multiple phenotypes of allosteric receptor mutants. 1996, Pubmed
Guitchounts, Two etomidate sites in α1β2γ2 γ-aminobutyric acid type A receptors contribute equally and noncooperatively to modulation of channel gating. 2012, Pubmed , Xenbase
Hibbs, Principles of activation and permeation in an anion-selective Cys-loop receptor. 2011, Pubmed
Hill-Venning, Subunit-dependent interaction of the general anaesthetic etomidate with the gamma-aminobutyric acid type A receptor. 1997, Pubmed , Xenbase
Husain, 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. 2003, Pubmed , Xenbase
Husain, 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. 2006, Pubmed , Xenbase
Husain, p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels. 2010, Pubmed , Xenbase
Jansen, State-dependent cross-linking of the M2 and M3 segments: functional basis for the alignment of GABAA and acetylcholine receptor M3 segments. 2006, Pubmed , Xenbase
Jayakar, Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog. 2014, Pubmed
Jurd, General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. 2003, Pubmed
Kaur, Unanticipated structural and functional properties of delta-subunit-containing GABAA receptors. 2009, Pubmed , Xenbase
Krasowski, Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. 2001, Pubmed
Li, Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. 2006, Pubmed
Li, Numerous classes of general anesthetics inhibit etomidate binding to gamma-aminobutyric acid type A (GABAA) receptors. 2010, Pubmed
Li, The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. 2011, Pubmed
Lynagh, Opposing effects of the anesthetic propofol at pentameric ligand-gated ion channels mediated by a common site. 2014, Pubmed , Xenbase
Lynagh, Ivermectin binding sites in human and invertebrate Cys-loop receptors. 2012, Pubmed
McCracken, A transmembrane amino acid in the GABAA receptor β2 subunit critical for the actions of alcohols and anesthetics. 2010, Pubmed , Xenbase
Mihic, Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. 1997, Pubmed , Xenbase
Miller, Crystal structure of a human GABAA receptor. 2014, Pubmed
Nishikawa, Volatile anesthetic actions on the GABAA receptors: contrasting effects of alpha 1(S270) and beta 2(N265) point mutations. 2002, Pubmed
Nury, X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. 2011, Pubmed
Olsen, International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update. 2008, Pubmed
Pejo, Analogues of etomidate: modifications around etomidate's chiral carbon and the impact on in vitro and in vivo pharmacology. 2014, Pubmed , Xenbase
Ruesch, An allosteric coagonist model for propofol effects on α1β2γ2L γ-aminobutyric acid type A receptors. 2012, Pubmed , Xenbase
Rüsch, Gating allosterism at a single class of etomidate sites on alpha1beta2gamma2L GABA A receptors accounts for both direct activation and agonist modulation. 2004, Pubmed , Xenbase
Sauguet, Structural basis for potentiation by alcohols and anaesthetics in a ligand-gated ion channel. 2013, Pubmed
Savechenkov, Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic. 2012, Pubmed
Siegwart, Molecular determinants for the action of general anesthetics at recombinant alpha(2)beta(3)gamma(2)gamma-aminobutyric acid(A) receptors. 2002, Pubmed
Siegwart, Mutational analysis of molecular requirements for the actions of general anaesthetics at the gamma-aminobutyric acidA receptor subtype, alpha1beta2gamma2. 2003, Pubmed
Spurny, Multisite binding of a general anesthetic to the prokaryotic pentameric Erwinia chrysanthemi ligand-gated ion channel (ELIC). 2013, Pubmed , Xenbase
Stewart, Tryptophan mutations at azi-etomidate photo-incorporation sites on alpha1 or beta2 subunits enhance GABAA receptor gating and reduce etomidate modulation. 2008, Pubmed , Xenbase
Stewart, State-dependent etomidate occupancy of its allosteric agonist sites measured in a cysteine-substituted GABAA receptor. 2013, Pubmed , Xenbase
Stewart, Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors. 2013, Pubmed , Xenbase
Yip, A propofol binding site on mammalian GABAA receptors identified by photolabeling. 2013, Pubmed
Zeller, Mapping the contribution of beta3-containing GABAA receptors to volatile and intravenous general anesthetic actions. 2007, Pubmed