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The Journal of Neuroscience, 2001, 21:RC136:1-4

RAPID COMMUNICATION
Evidence for a Common Binding Cavity for Three General Anesthetics within the GABAA Receptor

Andrew Jenkins1, Eric P. Greenblatt2, Howard J. Faulkner3, Edward Bertaccini4, Adam Light5, Audrey Lin5, Alyson Andreasen1, Anna Viner1, James R. Trudell4, and Neil L. Harrison1

1 C. V. Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology, Weill Medical College of Cornell University, New York, New York 10021, 2 Department of Anesthesia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, 3 Imperial College School of Medicine, London SW7 2AZ, United Kingdom, 4 Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305, and 5 The University of Chicago, Chicago, Illinois 60637


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The GABAA receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABAA receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABAA receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 Å3. The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABAA receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha  subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

Key words: anesthetic; GABA; binding site; allosteric; receptor; molecular volume


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Many general anesthetics with simple chemical structures prolong the duration of synaptic inhibition via allosteric regulation of GABAA receptors (Jones and Harrison, 1993; Nishikawa and MacIver, 2000). Reciprocal synaptic connections between cortical interneurons can generate high-frequency (30-60 Hz) oscillatory activity (Jefferys et al., 1996), and entrainment of these local inhibitory microcircuits results in synchronization of these "gamma oscillations" (Whittington et al., 1995). Such high-frequency activity has been linked to cognitive functions such as selective attention (Bragin et al., 1995), and these oscillations are highly sensitive to disruption by central depressants (Faulkner et al., 1998). GABAergic inhibition plays a fundamental role in the timing of these high-frequency oscillations, so that drugs that prolong the time course of synaptic inhibition also decrease the characteristic frequency of these oscillations (Whittington et al., 1996), eventually imposing slow, high-amplitude waves in both the EEG and in vitro model systems. Decreases in the EEG spectral edge frequency accompany decrements in cognitive function during the induction of anesthesia (Rampil, 1998). GABAA receptors therefore represent one of the major neurobiological substrates for the action of general anesthetics, especially with regard to the ability of these drugs to induce hypnosis and loss of consciousness (Franks and Lieb, 1994).

Inhaled anesthetics alter the function of GABAA receptors in acutely dissociated neurons (Nakahiro et al., 1989; Wakamori et al., 1991; Li and Pearce, 2000) and in heterologous expression systems (Harrison et al., 1993; Mihic et al., 1997; Jenkins et al., 1999). The modulatory action of these inhaled agents, unlike that of diazepam, is independent of the gamma  subunit of the GABAA receptor (Harrison et al., 1993; Krasowski et al., 1998; Koltchine et al., 1999). In this study, we have therefore examined the action of these anesthetics on GABAA receptors consisting only of alpha  and beta  subunits. GABAA receptor modulation by the anesthetic ether isoflurane is abolished by a point mutation at S270 in the TM2 segment of the alpha  subunit (Mihic et al., 1997; Krasowski et al., 1998), whereas the effects of diazepam and other sedative-hypnotic benzodiazepines are abolished by mutating H101 to arginine in the large extracellular N-terminal domain of the alpha  subunit (Rudolph et al., 1999; McKernan et al., 2000). However, although it is known that flunitrazepam binds to H101 but not R101 (Duncalfe et al., 1996), it is not known why S270 permits receptor modulation by isoflurane, whereas an isoleucine residue at this position does not. The present study probes the involvement of S270 and of two other residues in greater detail and extends our analysis of anesthetic action to include the alkanes halothane and chloroform.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Site-directed mutagenesis and electrophysiology. To create the mutant series at GABAA receptor alpha 2 Ser270, mutations were introduced into the cDNA encoding the human GABAA receptor alpha 2 subunit at bases 890 to 892, with simultaneous loss of a DdeI restriction site (Koltchine et al., 1999). Additional mutations were created at Ala291 and Leu232 using either the Unique Site Elimination method (Amersham Pharmacia Biotech, Arlington Heights, IL) or a Pfu polymerase/DpnI selection method (QuikChange; Stratagene, La Jolla, CA). The sequences of all cDNA inserts were confirmed throughout by double-stranded sequencing. Human embryonic kidney (HEK) 293 cells were maintained in culture on glass coverslips and transfected with cDNAs encoding wild-type or mutant alpha 2 subunits and wild-type beta 1 subunits. Recordings were made using the whole-cell patch-clamp technique as described previously (Koltchine et al., 1999). All drugs and solutions were applied rapidly to the cell by local perfusion using a motor-driven solution exchange device; recordings were made at room temperature (20-22°C). Bath concentrations of the anesthetics were measured using gas chromatography and represent 90-95% of the total applied drug concentration. Numerical data are presented throughout as mean ± SE. Concentration-response curves were determined for the wild-type and each mutant GABAA receptor (Koltchine et al., 1999). Potentiation of a submaximal GABA response by each anesthetic was then calculated as the percentage increase above the control (EC20) response to GABA in the presence of anesthetic (Koltchine et al., 1999).

Molecular volume calculation. Molecules were built in Spartan V5.1 (Wavefunction, San Diego, CA) and optimized with the Merck MMFF94 forcefield. The calculated volumes are van der Waals volumes and are given in Å3.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Different mutations are required to remove sensitivity to different anesthetics

The wild-type alpha 2beta 1 GABAA receptor is potentiated by isoflurane and halothane (Fig. 1a). The mutant receptor alpha 2(S270H)beta 1 is insensitive to isoflurane (Krasowski et al., 1998) but shows normal sensitivity to the alkane anesthetic halothane (Fig. 1b). This finding initially suggested to us that S270 in TM2 might participate in binding isoflurane, but not halothane. The TM1 segment of the homologous glycine receptor was recently inferred to confer sensitivity to halothane (Greenblatt and Meng, 1999); we subsequently observed that the mutant GABAA receptor alpha 2(L232F)beta 1 was insensitive to halothane, whereas sensitivity to isoflurane remained (Fig. 1c). This finding suggested initially that L232 in TM1 might participate in binding halothane, but not isoflurane, and pointed to the possibility that these two anesthetics might act at different sites on the GABAA receptor. However, when a larger Trp residue was introduced at S270, the mutant receptor alpha 2(S270W)beta 1 was shown to lack sensitivity to halothane, as well as isoflurane (Fig. 1d). Interestingly, this receptor retained sensitivity to a third anesthetic, chloroform (Fig. 1e). Mutation at A291 in TM3 of the alpha  subunit has previously been shown to abolish receptor regulation by isoflurane (Krasowski et al., 1998), and we therefore added a second Trp residue at this position to create the double mutant alpha 2(S270W;A291W)beta 1 receptor. This receptor was completely insensitive to chloroform (Fig. 1e), isoflurane, and halothane, but retained sensitivity to propofol (data not shown), an anesthetic believed to exert its actions via a site on the beta  subunit (Sanna et al., 1995; Krasowski et al., 1998).



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  		Figure 1.   Effects of three general anesthetics on wild-type and mutant GABAA receptors. All receptors were expressed in HEK 293 cells and studied using whole-cell voltage-clamp recording at -60 mV (Krasowski et al., 1998). Currents were activated by EC20 concentrations of GABA. a, Isoflurane and halothane enhance responses to GABA in wild-type alpha 2beta 1 GABAA receptors. b, In mutant alpha 2(S270H)beta 1 receptors, the enhancing effect of isoflurane is absent, whereas the halothane effect is preserved. c, In mutant alpha 2(L232F)beta 1 receptors, the enhancing effect of halothane is absent, whereas the isoflurane effect is preserved. d, In mutant alpha 2(S270W)beta 1 receptors, neither halothane nor isoflurane enhances GABA responses. e, Effects of chloroform on wild-type and mutant GABAA receptors. Chloroform potentiated the actions of GABA in wild-type alpha 2beta 1 GABAA receptors, and this effect was preserved in mutant alpha 2(S270W)beta 1 receptors but abolished in the double mutant alpha 2(S270W;A291W)beta 1 receptor. The anesthetic concentrations used in the figures were as follows: isoflurane, 0.5 mM; halothane, 0.32 mM; and chloroform, 0.9 mM. Calibration: 50 pA (vertical axis), 10 sec (horizontal axis).

The idea that the three residues (L232, S270, and A291) could be mutated to selectively or completely remove anesthetic modulation was then tested further. The S270W mutation was sufficient to remove isoflurane and halothane sensitivity, whereas the L232F mutation was sufficient to block potentiation by halothane but not isoflurane; in light of these observations, we also mutated L232 to tryptophan. The alpha 2(L232W)beta 1 receptor expressed at normal levels and, like the alpha 2(S270W)beta 1 receptor, was insensitive to potentiation by both halothane (-1.5 ± 4.8%; n = 20 cells) and isoflurane (5.8 ± 4.6%; n = 20 cells).

Anesthetics of dissimilar size have different cutoffs

The isoflurane sensitivity of the GABAA receptor appears to show a cutoff effect when the receptor is mutated to increase the size of the residue at alpha 270 (Koltchine et al., 1999). Specifically, in the series of mutant receptors alpha 2(S270X)beta 1, these receptors become insensitive to isoflurane (molecular volume 144 Å3; Fig. 2a), because the volume of the side-chain is increased beyond the volume of threonine, i.e., after a change in volume delta V > 30 Å3 (Fig. 2a). We repeated this analysis with the physically smaller anesthetics halothane (molecular volume 110 Å3) and chloroform (molecular volume 90 Å3), and in each case, a cutoff phenomenon was again observed. However, the cutoff occurred at larger side chain volumes than for isoflurane. For halothane this occurs between delta V = 100-140 Å3 (Fig. 2b), whereas for chloroform the cutoff occurs between delta V = 240-280 Å3 (Fig. 2c).



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  		Figure 2.   The effect of the addition of molecular volume by mutagenesis at positions 270 or 291 on GABAA receptor regulation by three anesthetics. Above the figure are representations of the molecular volumes of the three anesthetics. a, Addition of molecular volume produces a cutoff for isoflurane at partial V = 30-40 Å3. b, A cutoff is observed for halothane at partial V = 140 Å3. c, A cutoff for chloroform is not observed until partial V = 280 Å3. The data for each anesthetic at each mutant receptor are expressed as the mean ± SEM of the percentage enhancement (potentiation) of the current response to an EC20 concentration of GABA appropriate to that receptor, plotted against partial V, the increase in molecular volume produced by mutagenesis.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Multiple cutoffs suggest a common interaction site for three general anesthetics

We explain these experimental data by proposing that the side-chains at the key positions L232, S270, and A291 (in transmembrane segments TM1, TM2, and TM3, respectively, of the alpha  subunit) define a binding cavity for small anesthetic molecules. Given our knowledge of the molecular volumes of the anesthetics and the cutoff data from our experiments, the volume of this hypothetical cavity can be estimated as between 250 and 370 Å3. The introduction by mutagenesis of bulky side chains at the critical sites presumably decreases the molecular volume of the anesthetics that can be accommodated within such a cavity. It should be noted that these cutoff effects are independent of anesthetic concentration (Koltchine et al., 1999).

The present results highlight the importance of the TM domains for receptor activation and regulation; mutating selected residues in the TM1 (Thompson et al., 1999), TM2 (Koltchine et al., 1999), and TM3 (Krasowski and Harrison, 2000) domains often results in changes in GABA EC50. These mutations also affect the sensitivity of the receptor to a variety of allosteric modulators. For example, in addition to the changes in anesthetic sensitivity reported here, a mutation in TM1 alpha 1(T230I) renders alpha 1*beta 3gamma 2s receptors more sensitive to the alpha 6-specific noncompetitive antagonist furosemide; a similar result can be achieved by mutating (N265) in the beta 3 subunit (Thompson et al., 1999).

Three additional features of the experimental data are noteworthy. First, the data for the L232F and S270H mutants suggest that the anesthetics do not fit into the putative binding cavity in an identical manner; isoflurane (unlike halothane) is active at the L232F mutant receptor, whereas halothane (unlike isoflurane) is active at the S270H mutant. This could be explained if halothane makes a closer contact with L232 than does isoflurane, which may instead lie closer to S270, accounting for the very sharp cutoff in Figure 2a. The loss of sensitivity to both isoflurane and halothane after additional volume is added in the L232W or S270W mutants supports this idea and suggests that these two residues are part of the same cavity. Second, whereas the ability of isoflurane to enhance GABA currents falls off drastically as side-chain volume is increased at the 270 position, from serine to phenylalanine, halothane actually increases in effectiveness as the residue at 270 is expanded, before reaching the cutoff point at tryptophan. It is possible that for halothane (unlike isoflurane), the binding interactions with TM2 may be suboptimal in the wild-type receptor and are actually improved by the addition of a bulky side chain at 270. Finally, there is a marked discontinuity in the data for chloroform, at ~partial V = 40 Å3. One explanation for this observation would be a change from a cavity occupied by two chloroform molecules in the Asn mutant to a singly occupied cavity in the Glu mutant. If a cavity of >250 Å3 does exist within the wild-type alpha  subunit, it should be possible for two molecules of chloroform (90 Å3) to bind simultaneously within such a cavity. There are existing precedents for double occupancy of cavities by general anesthetic molecules; for example, two molecules of halothane arranged in a head-to-head orientation are found within one of the anesthetic binding sites in human serum albumin (HSA) (Bhattacharya et al., 2000).

Small cavities bind general anesthetic molecules

Small cavities have been demonstrated to exist in a variety of globular proteins, both natural (Tilton et al., 1984; Eriksson et al., 1992; Kono et al., 2000) and synthetic (Johansson et al., 2000) in origin, and these can be altered in size and shape by mutagenesis of appropriate residues (Eriksson et al., 1992; Brunori et al., 2000; Kono et al., 2000; Lee et al., 2000). Anesthetics and other small gas molecules have been demonstrated to occupy such cavities, using a variety of techniques (Tilton et al., 1984; Franks et al., 1998; Brunori et al., 2000; Johansson et al., 2000). The binding of the anesthetic bromoform within a cavity in the enzyme firefly luciferase has been demonstrated using x-ray crystallography (Franks et al., 1998). Binding of such small ligands to protein cavities is often accompanied by the displacement of bound water molecules (Rashin et al., 1986; Matthews et al., 1995), and the interactions are further stabilized by low-energy (<= 2-4 kcal/mol) van der Waals interactions with the cavity surface. The resulting free energy changes, although small, are commensurate with the relatively small energy changes associated with the gating of ligand-gated ion channels and are consistent with the low potencies for the anesthetic agents studied here (10-4-10-3 M). More recently, Bhattacharya et al. (2000) have obtained the high-resolution (2.4 Å) structure of halothane bound to HSA. In all of these cases, anesthetic binding produced no change in local or global protein structure. Instead, the halothane molecules simply occupied pre-existing cavities and made contacts with small polar and apolar amino acids forming the cavity walls.

An anesthetic binding cavity of defined volume in the GABAA receptor

The existence of a similar cavity or "crevice" within the GABAA receptor is supported by data from scanning cysteine accessibility mutagenesis experiments on TM3 in the alpha  subunit (Williams and Akabas, 1999). Moreover, recent experiments using cysteine substitutions in the transmembrane domain of the alpha  subunit also indicate that Ser270 is likely to be involved in binding molecules such as ethanol and isoflurane. Mascia et al. (2000) showed that in the alpha 2(S270C) mutant, an alkanethiol anesthetic and the sulfhydryl reagent propyl methanethiosulfonate produced an irreversible enhancement of receptor function. Furthermore, once alkylated, the alpha 2(S270C) mutant was insensitive to isoflurane. These data suggest that alpha 2(S270) is indeed involved in the binding of these anesthetic molecules.

The cutoff data reported here predict that the receptor should be modulated by molecules larger than isoflurane. This is indeed the case; halogenated ether anesthetics (sevoflurane; 154 Å3) and long-chain alcohols (decanol; 234Å3) both potentiate GABAA receptor function (Dildy-Mayfield et al., 1996; Krasowski and Harrison, 2000). Interestingly, Ser270 is also critical for the action of alcohols (Mihic et al., 1997; Mascia et al., 2000) and so it is important to note that the cutoff for the actions of the n-alkanols on the GABAA receptor occur between decanol and dodecanol, i.e., somewhere between 234 and 276 Å3 (Dildy-Mayfield et al., 1996); mutations at the homologous I307 residue in human GABA rho 1 or the S267 residue in human GlyR alpha 1 alter alcohol cutoff in these related receptors (Wick et al., 1998).

In conclusion, we suggest that modulation of GABAA receptor function by small general anesthetic molecules results from occupation of a cavity of volume ~250-370 Å3, the surface of which is partly defined by L232, S270, and A291. It is therefore of considerable interest that cutoff data from in vivo experiments also point to a maximal volume for anesthetic activity of ~340 Å3 (Curry et al., 1991).


    FOOTNOTES

Received Aug. 21, 2000; revised Jan. 10, 2001; accepted Jan. 17, 2001.

This work was supported by National Institutes of Health Grants GM 45129 to N.L.H. and GM 57062 to E.P.G. We thank Peter Goldstein for critical reading of this manuscript and Suzanne Finn and Irene Paraskevakis for mutant cDNAs.

Correspondence should be addressed to Andrew Jenkins, C. V. Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology A-1050, Joan and Sanford I. Weill Medical College of Cornell University, 525 East 68th Street, Box 124, New York, NY 10021. E-mail: anj2005{at}med.cornell.edu.

This article is published in The Journal of Neuroscience, Rapid Communications Section, which publishes brief, peer-reviewed papers online, not in print. Rapid Communications are posted online approximately one month earlier than they would appear if printed. They are listed in the Table of Contents of the next open issue of JNeurosci. Cite this article as: JNeurosci, 2001, 21:RC136 (1-4). The publication date is the date of posting online at www.jneurosci.org.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Does Natural Selection Explain the Universal Response of Metazoans to Volatile Anesthetics?
Anesth. Analg., September 1, 2008; 107(3): 862 - 863.
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 Anesth. Analg.Home page
L. Yang and J. M. Sonner
Anesthetic-Like Modulation of Receptor Function by Surfactants: A Test of the Interfacial Theory of Anesthesia
Anesth. Analg., September 1, 2008; 107(3): 868 - 874.
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A. Jenkins, I. A. Lobo, D. Gong, J. R. Trudell, K. Solt, R. A. Harris, and E. I. Eger II
General Anesthetics Have Additive Actions on Three Ligand Gated Ion Channels
Anesth. Analg., August 1, 2008; 107(2): 486 - 493.
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 Sci SignalHome page
R. A. Harris, J. R. Trudell, and S. J. Mihic
Ethanol's Molecular Targets
Sci. Signal., July 15, 2008; 1(28): re7 - re7.
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 Br J AnaesthHome page
J. R. Sneyd and D. M. Mathews
Memory and awareness during anaesthesia
Br. J. Anaesth., June 1, 2008; 100(6): 742 - 744.
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 Anesth. Analg.Home page
L. Yang and J. M. Sonner
The Anesthetic-Like Effects of Diverse Compounds on Wild-Type and Mutant {gamma}-Aminobutyric Acid Type A and Glycine Receptors
Anesth. Analg., March 1, 2008; 106(3): 838 - 845.
[Abstract] [Full Text] [PDF]

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 Anesth. Analg.Home page
E. W. Kelly, K. Solt, and D. E. Raines
Volatile Aromatic Anesthetics Variably Impact Human {gamma}-Aminobutyric Acid Type A Receptor Function
Anesth. Analg., November 1, 2007; 105(5): 1287 - 1292.
[Abstract] [Full Text] [PDF]

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 J. Pharmacol. Exp. Ther.Home page
W. Li, X. Jin, D. F. Covey, and J. H. Steinbach
Neuroactive Steroids and Human Recombinant {rho}1 GABA Receptors
J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 236 - 247.
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J. R. Atack, P. Scott-Stevens, J. S. Beech, T. D. Fryer, J. L. Hughes, M. C. Cleij, J.-C. Baron, J. C. Clark, R. J. Hargreaves, and F. I. Aigbirhio
Comparison of Lorazepam [7-Chloro-5-(2-chlorophenyl)-1,3-dihydro-3-hydroxy-2H-1,4-benzodiazepin-2-one] Occupancy of Rat Brain {gamma}-Aminobutyric AcidA Receptors Measured Using in Vivo [3H]Flumazenil (8-Fluoro 5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic Acid Ethyl Ester) Binding and [11C]Flumazenil Micro-Positron Emission Tomography
J. Pharmacol. Exp. Ther., March 1, 2007; 320(3): 1030 - 1037.
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 J. Pharmacol. Exp. Ther.Home page
C. M. Borghese, D. F. Werner, N. Topf, N. V. Baron, L. A. Henderson, S. L. Boehm II, Y. A. Blednov, A. Saad, S. Dai, R. A. Pearce, et al.
An Isoflurane- and Alcohol-Insensitive Mutant GABAA Receptor {alpha}1 Subunit with Near-Normal Apparent Affinity for GABA: Characterization in Heterologous Systems and Production of Knockin Mice
J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 208 - 218.
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 J. Pharmacol. Exp. Ther.Home page
J. Ogata, M. Shiraishi, T. Namba, C. T. Smothers, J. J. Woodward, and R. A. Harris
Effects of Anesthetics on Mutant N-Methyl-D-Aspartate Receptors Expressed in Xenopus Oocytes
J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 434 - 443.
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B. L. Jones, P. J. Whiting, and L. P. Henderson
Mechanisms of anabolic androgenic steroid inhibition of mammalian {varepsilon}-subunit-containing GABAA receptors
J. Physiol., June 15, 2006; 573(3): 571 - 593.
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 Contin Educ Anaesth Crit Care PainHome page
C. J Weir
The molecular mechanisms of general anaesthesia: dissecting the GABAA receptor
CEACCP, April 1, 2006; 6(2): 49 - 53.
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 Mol. Pharmacol.Home page
J. H. Streiff, N. O. Juranic, S. I. Macura, D. O. Warner, K. A. Jones, and W. J. Perkins
Saturation Transfer Difference Nuclear Magnetic Resonance Spectroscopy As a Method for Screening Proteins for Anesthetic Binding
Mol. Pharmacol., October 1, 2004; 66(4): 929 - 935.
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 J. Biol. Chem.Home page
I. A. Lobo, M. P. Mascia, J. R. Trudell, and R. A. Harris
Channel Gating of the Glycine Receptor Changes Accessibility to Residues Implicated in Receptor Potentiation by Alcohols and Anesthetics
J. Biol. Chem., August 6, 2004; 279(32): 33919 - 33927.
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 J. Biol. Chem.Home page
A. Miko, E. Werby, H. Sun, J. Healey, and L. Zhang
A TM2 Residue in the {beta}1 Subunit Determines Spontaneous Opening of Homomeric and Heteromeric {gamma}-Aminobutyric Acid-gated Ion Channels
J. Biol. Chem., May 28, 2004; 279(22): 22833 - 22840.
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 J. Biol. Chem.Home page
D. Rusch, H. Zhong, and S. A. Forman
Gating Allosterism at a Single Class of Etomidate Sites on {alpha}1{beta}2{gamma}2L GABAA Receptors Accounts for Both Direct Activation and Agonist Modulation
J. Biol. Chem., May 14, 2004; 279(20): 20982 - 20992.
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 Br J AnaesthHome page
B. Haelewyn, A. Yvon, J. L. Hanouz, E. T. MacKenzie, P. Ducouret, J. L. Gerard, and S. Roussel
Desflurane affords greater protection than halothane against focal cerebral ischaemia in the rat
Br. J. Anaesth., September 1, 2003; 91(3): 390 - 396.
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 NEJMHome page
J. A. Campagna, K. W. Miller, and S. A. Forman
Mechanisms of Actions of Inhaled Anesthetics
N. Engl. J. Med., May 22, 2003; 348(21): 2110 - 2124.
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R. Darbandi-Tonkabon, W. R. Hastings, C.-M. Zeng, G. Akk, B. D. Manion, J. R. Bracamontes, J. H. Steinbach, S. J. Mennerick, D. F. Covey, and A. S. Evers
Photoaffinity Labeling with a Neuroactive Steroid Analogue. 6-AZI-PREGNANOLONE LABELS VOLTAGE-DEPENDENT ANION CHANNEL-1 IN RAT BRAIN
J. Biol. Chem., April 4, 2003; 278(15): 13196 - 13206.
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D. B. Williams and M. H. Akabas
Structural Evidence that Propofol Stabilizes Different GABAA Receptor States at Potentiating and Activating Concentrations
J. Neurosci., September 1, 2002; 22(17): 7417 - 7424.
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