Introduction

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GABA_A and glycine receptors are the major inhibitory neurotransmitter receptors in the vertebrate nervous system. Both of these inhibitory receptors are positively modulated by clinical concentrations of a wide range of chemically diverse general anesthetics and sedative/hypnotics (Franks and Lieb, 1994; Zimmerman et al., 1994; Harris et al., 1995; Whiting et al., 1995). General anesthetics and sedative/hypnotics known to potentiate the actions of GABA at GABA_A receptors include barbiturates (Study and Barker, 1981), propofol (Hales and Lambert, 1991), steroid anesthetics (Harrison and Simmonds, 1984; Harrison et al., 1987; Peters et al., 1988), etomidate (Uchida et al., 1995), n-alkanols (Nakahiro et al., 1991; Aguayo and Pancetti, 1994; Dildy-Mayfield et al., 1996) and halogenated volatile ether anesthetics (Nakahiro et al., 1989; Wakamori et al., 1991; Jones et al., 1992). Glycine receptors are positively modulated by clinical concentrations of volatile anesthetics (Harrison et al., 1993; Downie et al., 1996) and n-alkanols (Mascia et al., 1996a; Mascia et al., 1996b) but are much less sensitive to barbiturates (Koltchine et al., 1996; Mascia et al., 1996a), propofol and etomidate (Mascia et al., 1996a).

GABA_A and glycine receptors are both members of a ligand-gated ion channel superfamily that also includes the serotonin₃, GABA  (GABA_C) and nicotinic acetylcholine receptors (Betz, 1992; Unwin, 1993; Ortells and Lunt, 1995). Similar to GABA_A and glycine receptors, some general anesthetics have potent actions on serotonin₃ receptors (Jenkins et al., 1996) and on neuronal, but not muscle, nicotinic acetylcholine receptors (Flood et al., 1997; Violet et al., 1997).

The GABA_A receptor is a heteromeric complex assembled from different glycoprotein subunits (_1-6, _1-4, _1-4, , ) that combine to form a chloride channel (reviewed by Macdonald and Olsen, 1994; Whiting et al., 1995). GABA_A receptors in vivo probably consist mostly of pentameric complexes of ,  and subunits with a stoichiometry of (Chang et al., 1996), although receptors lacking the  subunit ( receptors) can be expressed and are fully sensitive to general anesthetics and n-alkanols (Levitan et al., 1988; Pritchett et al., 1989; Harrison et al., 1993; Mihic et al., 1994). Native glycine receptors consist of two different subunits ( and ), which assemble in vivo with a proposed stoichiometry of 3:2 (Langosch et al., 1988; Betz, 1991). Glycine  subunits, which contain the glycine agonist and strychnine binding sites (Vandenberg et al., 1992), can form functional homomeric complexes in heterologous expression systems that are sensitive to strychnine, general anesthetics and n-alkanols (Sontheimer et al., 1989; Harrison et al., 1993; Taleb and Betz, 1994; Mascia et al., 1996a).

To date, three GABA  subunits have been cloned (_1-3), each showing 30% sequence homology with the GABA_A receptor subunits (Cutting et al., 1991, 1992; Ogurusu and Shingai, 1996). In contrast to GABA_A receptors, GABA_C receptors assembled from  subunits are insensitive to benzodiazepines, barbiturates (Shimada et al., 1992) and steroid anesthetics (Feigenspan et al., 1993). n-Alkanols strongly inhibit the currents produced by low concentrations of GABA at the ₁ receptor (Mihic and Harris, 1996).

The dissimilar pharmacology of the GABA  receptor to general anesthetics and sedative/hypnotics can be exploited to identify sites of modulator interaction. Recently, Mihic et al. (1997) identified amino acid residues within TM2 and TM3 of the GABA_A and glycine receptors crucial for modulation by n-alkanols and by the halogenated volatile ether anesthetic enflurane (see fig. 3). We hypothesized that modulation of GABA_A and glycine receptors by other compounds also might depend on the same residues. A sedative/hypnotic structurally related to the n-alkanols is TCEt, the principal active metabolite of chloral hydrate (Breimer, 1977; Hobbs et al., 1996). TCEt enhances the actions of GABA in hippocampal neurons (Lovinger et al., 1993; Peoples and Weight, 1994) and in recombinant GABA_A receptors expressed in stably transfected fibroblast cell lines (Krasowski et al., 1997) .

To study the molecular mechanism of the potentiating action of TCEt, we investigated the effects of TCEt on wild-type GABA_A, glycine and GABA  subunits transiently expressed in HEK 293 cells. The experiments with mutated GABA_A and glycine receptors were performed to test whether the specific amino acid residues within TM2 and TM3 known to be critical for positive modulation by volatile anesthetics and n-alkanols are also necessary for positive modulation by TCEt.

	Methods

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Site-directed mutagenesis. The amino acid sequence alignment of TM2 and TM3 from human GABA_{A 2} (Hadingham et al., 1993a), ₁ (Hadingham et al., 1993b), GABA ₁ (Cutting et al., 1991) and glycine ₁ (Grenningloh et al., 1987) receptor subunits is shown (see fig. 3). The S²⁷⁰I, S²⁷⁰H and A²⁹¹W mutations of the GABA_{A 2} subunit, the M²⁸⁶W mutation of the GABA_{A 1} subunit and the S²⁶⁷I and A²⁸⁸W mutations of the glycine ₁ subunit were introduced by the unique site elimination method (Deng and Nickoloff, 1992) using the USE kit (Pharmacia Biotech, Piscataway, NJ). The method uses a two-primer system, in which one oligonucleotide primer encodes the desired mutation and the other alternates a unique SspI restriction site on the pCIS2 plasmid to an MluI site. The mutagenic reaction mixtures were digested with SspI restriction endonuclease to eliminate the parental template. Positive clones then were screened for the appearance of the MluI site, which were further confirmed by double-stranded sequencing (Sequenase 2.0; United States Biochemical, Cleveland, OH). Both MluI and SspI restriction enzymes were from New England Biolabs (Beverly, MA). The sequences and locations of the mutagenic primers (Operon Technologies, Alameda, CA) are GABA_{A 2}(S²⁷⁰I), 5-GACAACTCTAATCATCAGTGCTCGGAATTC-3, corresponding to bases 879-908 of the ₂ cDNA sequence; GABA_{A 2}(S²⁷⁰H), 5-GACAACTCTACACATCAGTGCTCGGAATTC-3, corresponding to bases 879-908 of the ₂ cDNA sequence; GABA_{A 2}(A²⁹¹W), 5-CATGGACTGGTTTATTTGGGTTTGTTATGCATTTG-3, corresponding to bases 936-970 of the ₂ cDNA sequence; GABA_{A 1}(M²⁸⁶W), 5-GATTGATATTTATCTGTGGGGTTGCTTTGTG-3, corresponding to bases 915-945 of the ₁ cDNA sequence; glycine ₁(S²⁶⁷I), 5-CATGACCACCCAGATCTCCGGCTCTCGAG-3, corresponding to bases 870-898 of the glycine ₁ cDNA; and glycine ₁(A²⁸⁸W), 5-CATTTGGATGTGGGTTTGCCTGCTCTTTGTG-3, corresponding to bases 936-966 of the glycine ₁ cDNA.

Cell culture and transfection. Wild-type or mutant receptor cDNAs were expressed via the vector pCIS2, which contains one copy of the strong promoter from cytomegalovirus and a polyadenylation sequence from SV40. HEK 293 cells (American Type Culture Collection, Rockville, MD) were maintained in culture and passaged weekly by trypsin treatment for a maximum of 20 times before being discarded and replaced with early passage cells. HEK 293 cells were maintained in Eagle's minimum essential medium (Sigma Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), L-glutamine (0.292 µg/ml; GIBCO BRL, Grand Island, NY), penicillin G sulfate (100 units/ml; GIBCO BRL) and streptomycin sulfate (100 µg/ml; GIBCO BRL). For electrophysiological experiments, cells were plated onto glass coverslips coated with poly-D-lysine (Sigma). Each coverslip of cells was transfected according to the calcium phosphate precipitation technique as described previously (Okayama and Chen, 1987; Harrison et al., 1993). Each transfection required 1-5 µg of each cDNA; the cDNA was in contact with the cells for 24 hr under an atmosphere containing 3% CO₂ before being removed and replaced with fresh culture medium in an atmosphere of 5% CO₂.

Electrophysiology. Electrophysiological recordings were performed at room temperature using the whole-cell patch-clamp technique as described previously (Harrison et al., 1993; Krasowski et al., 1997). The coverslips were transferred 24-96 hr after removal of the cDNA to a large chamber that was continuously perfused (2-3 ml/min) with extracellular medium containing (in mM): 145 NaCl, 3 KCl, 1.5 CaCl₂, 1 MgCl₂, 5.5 D-glucose and 10 HEPES, pH 7.4, osmolarity 320-330 mOsM. The electrode solution contained (in mM): 145 N-methyl-D-glucamine hydrochloride, 5 K₂ATP, 5 HEPES/KOH, 2 MgCl2, 0.1 CaCl₂ and 1.1 EGTA, pH 7.2, osmolarity 315 mOsM. Pipette-to-bath resistance was 4-6 M. Cells were voltage-clamped at 60 mV. Because the intracellular and extracellular solutions contained symmetrical chloride concentrations, the chloride equilibrium potential was 0 mV.

Data analysis. Drug-induced potentiation of an agonist-induced current was defined as the percentage increase of the control agonist response (defined as the average of the predrug and postdrug agonist-induced currents). Concentration-response data were fitted (KaleidaGraph; Synergy Software, Reading, PA) with the logistic equation: I/I_max = 100 * [drug]ⁿ/{[drug]ⁿ + (EC₅₀ )ⁿ}, where I/I_max is the percentage of the maximum obtainable response, EC₅₀ is the concentration producing a half-maximal response and n is the Hill coefficient (n_H). Pooled data are presented throughout as mean ± S.E.M. Statistical significance was determined by Student's two-tailed, unpaired t test.

Drugs. Stock solutions of GABA, glycine, TCEt (all from Sigma) and picrotoxin (Research Biochemicals, Natick, MA) were diluted into the extracellular solution daily before use.

	Results

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Contrasting modulation of GABA_A, glycine and ₁ receptors by TCEt. The amplitudes of submaximal (EC₂₀) GABA currents at wild-type GABA_{A 21} receptors were significantly potentiated by coapplication with TCEt (0.1-10 mM; fig. 1A). In these and all other experiments with TCEt in this study, TCEt was preapplied to the cells before the coapplication of TCEt and agonist. This allowed the TCEt to be at equilibrium before GABA application and allowed for the monitoring of any direct action of TCEt (see below).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Contrasting effects of TCEt on wild-type GABAA 21, glycine 1 and GABA 1 receptors. A, TCEt potentiates submaximal GABA-induced currents in GABAA 2 1 receptors. B, TCEt inhibits submaximal GABA-induced currents in the GABA 1 receptor. C, TCEt potentiates submaximal glycine-induced currents in the wild-type glycine 1 receptor. Traces shown in A, B and C are individual recordings from HEK 293 cells transfected with cDNAs encoding the indicated receptor subunit combination. D, Pooled concentration-response relationships for TCEt modulation of currents in response to submaximal (EC20) concentrations of agonist at wild-type GABAA 21, glycine 1 and GABA 1 receptors. Significant potentiation of submaximal GABA responses at GABAA 21 receptors occurs at all TCEt concentrations of 0.1 mM TCEt (P < .05 for each concentration 0 .1 mM; 5  n  11). TCEt potentiates submaximal GABA currents in GABAA 21 receptors with an EC50 value of 0.38 ± 0.03 mM and a Hill slope of 1.6 ± 0.2. Significant inhibition of submaximal GABA currents at GABA 1 receptors occurs at 5 and 10 mM TCEt (P < .05 for both concentrations);at concentrations below 5 mM, TCEt had no effect on submaximal GABA currents at 1 receptors (4  n  7). Significant enhancement of submaximal glycine responses at wild-type glycine 1 receptors occurs at all TCEt concentrations 1 mM (P < .05 for each concentration 1 mM; 7  n  12). The EC50 values and Hill slopes for TCEt potentiation of glycine 1 receptors are 1.0 ± 0.1 mM and 3.2 ± 1.4, respectively. The effect of TCEt at GABA 1 receptors was significantly different from that at GABAA 21 receptors at all TCEt concentrations of 0.1 mM (P < .05 for each concentration 0 .1 mM) and different from that at glycine 1 receptors at all TCEt concentrations of 2 mM (P < .05 for each concentration 2 mM). Error bars indicate mean ± S.E.M. of data from multiple experiments.

Effect of coapplication of TCEt on agonist concentration-response relationships. The coapplication of TCEt (5 mM) caused parallel leftward shifts in the agonist concentration-response curves for GABA_{A 21} and glycine ₁ receptors (fig. 2, A and B). In contrast, the coapplication of TCEt resulted in a parallel rightshift in the GABA concentration-response curve for the GABA ₁ receptor (fig. 2C). TCEt did not significantly alter the Hill slope or maximal current response (E_max) to agonist in any of the three wild-type receptors studied.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Concentration-response relationships for wild-type GABAA 21, glycine 1 and GABA 1 receptors in the presence and absence of TCEt. A, Coapplication of TCEt (5 mM) shifts the GABA concentration-response curve of wild-type GABAA 21 receptors to the left (4  n  9 for all data points). EC50 and Hill slope values determined from the curve fits are GABAA 21 wild-type in absence (EC50 = 9.8 ± 0.6 µM, nH = 2.3 ± 0.4) and presence of 5 mM TCEt (EC50 = 1.1 ± 0.05 µM, nH = 2.9 ± 0.3). TCEt did not alter the maximal current response (Emax) to GABA at GABAA 21 receptors. B, Coapplication of TCEt (5 mM) shifts the glycine concentration-response curve of wild-type glycine 1 receptors to the left, although the shift is less than that at GABAA 21 receptors (5  n  10 for all data points). EC50 and Hill slope values determined from the curve fits are glycine 1 wild-type receptors in absence (EC50 = 46.3 ± 2.5 µM, nH = 1.7 ± 0.1) and presence of 5 mM TCEt (EC50 = 11.7 ± 0.5 µM, nH = 1.8 ± 0.1). TCEt did not alter the Emax to glycine at glycine 1 receptors. C, Coapplication of TCEt (5 mM) shifts the GABA concentration-response curve of wild-type GABA 1 receptors to the right (5  n  7 for all data points). EC50 and Hill slope values determined from the curve fits are GABA 1 wild-type receptors in absence (EC50 = 3.9 ± 0.3 µM, nH = 1.5 ± 0.2) and presence of 5 mM TCEt (EC50 = 13.7 ± 2.2 µM, nH = 1.1 ± 0.2). TCEt did not alter the Emax to GABA at 1 receptors.

Concentration-response and receptor characteristics for wild-type and mutant receptors analyzed in this study. Previous work by Mihic et al. (1997) demonstrates that specific mutations at two specific residues, one each within TM2 and TM3 (fig. 3), dramatically reduce or abolish the sensitivity to n-alkanols and enflurane of GABA_A and glycine receptors expressed in either HEK 293 cells or Xenopus laevis oocytes. After transient expression in HEK 293 cells, all of the GABA_A wild-type and mutant receptors studied here produced inward currents in 30% of cells tested in response to the application of GABA. There have been recent reports of endogenous GABA_A subunit activity in HEK 293 cells (Ueno et al., 1996). Our experience with HEK 293 cells to date does not concur with this finding. In fact, similar to results from other laboratories (Davies et al., 1997), we do not observe significant GABA_A receptor-mediated currents in untransfected HEK 293 cells or in cells transfected with either GABA_A  or subunit cDNAs alone (Koltchine et al., 1996).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence alignment of TM2 and TM3 from human glycine 1, GABAA 2, 1 and GABA 1 receptor subunits. Residue positions in bold type within TM2 and TM3 of glycine 1 subunits (S267 and A288), GABAA 2 (residues S270 and A291) and GABAA 1 subunits (S265 and M286) were investigated in the present study of TCEt.

Mutations within TM2 or TM3 of the glycine ₁ receptor abolish potentiation by low concentrations of TCEt and result in inhibition by high concentrations of TCEt. EC₂₀ glycine currents elicited at the glycine ₁(S²⁶⁷I) receptor were unaffected by low concentrations of TCEt (2 mM) but inhibited by higher concentrations of TCEt (5 and 10 mM; fig. 4A) in a fashion similar to that seen at the GABA ₁ receptor. In contrast to the effects of TCEt at ₁ receptor-mediated currents, the coapplication of TCEt did not appear qualitatively to alter the kinetics of submaximal glycine currents at either the wild-type glycine ₁ or the S²⁶⁷I and A²⁸⁸W mutant receptors. Concentration-response curves for TCEt modulation of submaximal glycine currents at glycine ₁(S²⁶⁷I) and ₁(A²⁸⁸W) receptors differ markedly from that of wild-type glycine ₁ receptors (fig. 4B) and instead appear to be very similar to the actions of TCEt at GABA ₁ receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Mutations in either TM2 or TM3 of the glycine 1 receptor abolish positive modulation by TCEt. A, TCEt at high concentrations inhibits submaximal glycine-induced currents in the glycine 1(S267I) mutant receptor. Recordings shown are from an HEK 293 cell transfected with cDNAs encoding the glycine 1(S267I) mutant receptor. B, Concentration-response data for TCEt modulation of submaximal glycine currents at glycine 1 receptor mutants 1(S267I) and 1(A288W) are shown with the TCEt modulation curve for the wild-type glycine 1 receptor included for comparison. Coapplication of TCEt has a significant inhibitory effect on submaximal glycine currents in the 1(S267I) mutant receptor at TCEt concentrations of 5 and 10 mM (P < 0.05 for both concentrations; 4  n  7). TCEt concentrations of 2 mM have inhibitory effects on the glycine 1(A288W) mutant receptor (P < 0.05 for all concentrations 2 mM; 4  n  7). The magnitude of potentiation by TCEt was significantly lower than that at wild-type glycine 1 receptors at all TCEt concentrations of 2 mM for the glycine 1(S267I) mutant receptors (P < 0.01 for all concentrations 2 mM) and at all TCEt concentrations of 1 mM for the glycine 1(A288W) mutant receptor (P < 0.01 for each concentration 1 mM).

A point mutation within TM3 of the ₁ subunit of the GABA_A receptor ablates potentiation by TCEt. Most of the GABA_A receptor mutants analyzed in this study exhibited normal potentiation by 5 mM TCEt (fig. 5A). Submaximal GABA currents at one mutant receptor, ₂₁(M²⁸⁶W), however, showed no enhancement by 5 mM TCEt (fig. 5A). At a TCEt concentration of 10 mM, inhibition of the GABA response was seen (fig. 5B).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   A mutation in TM3 of the GABAA 1 subunit ablates positive modulation by TCEt. A, Summary of the effects of mutations in TM2 and TM3 of GABAA receptor 2 and 1 subunits on potentiation by TCEt. The ordinate depicts percentage change of an EC20 test concentration of GABA by coapplication with TCEt (where 0% = no potentiation). TCEt (5 mM) significantly enhances EC20 GABA applications for all receptors (P < 0.05 for each receptor) except the 21(M286W) mutant receptor. Potentiation at the 21(M286W) mutant receptor by TCEt is significantly less than that at the wild-type 21 receptor (P < 0.01). Potentiation of all other mutant receptors does not differ from that of the wild-type 21 receptor. Numbers in parentheses above bars indicate number of experiments contributing to mean values. B, Concentration-response data are illustrated for TCEt modulation of submaximal GABA currents at the GABAA 21(M286W) mutant receptor with TCEt modulation curves for wild-type GABAA 21 and GABA 1 receptors included for comparison. Coapplication of TCEt has no significant effect on submaximal GABA currents in 21(M286W) mutant receptors at all TCEt concentrations of 5 mM; 10 mM TCEt, however, has an inhibitory effect (P < 0.05; 4  n  6 for all points). The magnitude of potentiation by TCEt at GABAA 21(M286W) mutant receptors was significantly lower than that at wild-type GABAA 21 receptors at all TCEt concentrations of 0.2 mM (P < 0.05 for each concentration 2 mM). TCEt modulation of GABAA 21(M286W) receptors differed from that at the 1 receptor only at 5 mM TCEt (P < 0.05 for 5 mM only).

Direct actions of TCEt. Although TCEt in the absence of applied agonist has been reported to produce small GABA_A receptor-mediated currents in neuronal GABA_A receptors (Peoples and Weight, 1994), no direct gating effects were evident in our experiments on recombinant GABA_{A 21}, glycine ₁ and GABA ₁ receptors at TCEt concentrations up to 10 mM.

	Discussion

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As described for the barbiturates (Levitan et al., 1988; Pritchett et al., 1989), halogenated volatile ether anesthetics (Harrison et al., 1993) and propofol (Jones et al., 1995), modulation of GABA_A receptors by TCEt does not require the presence of the subunit. The efficacy and apparent affinity of TCEt in enhancing the function of GABA_{A 21} receptors in this study are comparable with those observed for TCEt at neuronal GABA_A receptors (Peoples and Weight, 1994) and at GABA_{A 132} receptors stably expressed in mouse fibroblasts (Krasowski et al., 1997).

The concentrations of TCEt that caused potentiation of GABA_{A 21} and glycine ₁ receptor-mediated currents in this study correspond well with anesthetic concentration ranges determined in vivo. TCEt anesthetizes dogs and humans at concentrations in the range of 0.2-5 mM (Garrett and Lambert, 1973; Breimer, 1977; Owen and Taberner, 1980; Hobbs et al., 1996). As in previous studies of TCEt modulation of GABA_A receptors (Peoples and Weight, 1994; Krasowski et al., 1997), the estimated EC₅₀ values for TCEt potentiation of GABA_{A 21} and glycine ₁ receptor-mediated currents are within the anesthetic concentration range. These data are consistent with the notion that enhancement of GABA_A and/or glycine receptor function may contribute to the hypnotic/anesthetic effects of TCEt.

The dissimilar anesthetic pharmacology of GABA_A, glycine and GABA  receptors has been used to identify receptor domains critical for drug modulation. Recently, Mihic et al. (1997) used glycine ₁/GABA ₁ receptor chimeras to identify a 45-residue domain within the glycine ₁ receptor subunit as being necessary for positive modulation by n-alkanols and volatile anesthetics. This led to the identification of amino acid residues in TM2 and TM3 that appear to be necessary to confer positive modulation of GABA_A and glycine receptors by n-alkanols and the volatile anesthetic enflurane.

As reported for the n-alkanols, TCEt enhances submaximal agonist currents at GABA_A and glycine receptors but inhibits submaximal GABA currents at the GABA ₁ receptor (Mihic and Harris, 1996). In addition, the results of this study show that specific mutations within TM2 and/or TM3 of GABA_A and glycine receptor subunits dramatically affect modulation of submaximal agonist responses by TCEt. Except for the GABA_{A 2}(S²⁷⁰H)₁ mutant receptor,² all of the mutations in this study were the result of the replacement of a GABA_A or glycine receptor subunit residue by the analogous residue in the GABA ₁ receptor. Two specific mutations within either TM2 or TM3 of the glycine ₁ subunit ablated positive modulation by TCEt and resulted in inhibition by high concentrations of TCEt. In the GABA_A receptor, a mutation in TM3 of the ₁ subunit abolished positive modulation of submaximal GABA currents by all concentrations of TCEt studied and introduced inhibition at 10 mM TCEt. By contrast, mutation in either TM2 or TM3 of the GABA ₂ subunit eliminates ethanol and enflurane enhancement of GABA-induced currents at GABA_{A 21} receptors (Mihic et al., 1997). Interestingly, the M²⁸⁶W mutation in TM3 of the GABA_A1 subunit also ablates potentiation of submaximal GABA currents by propofol at GABA_{A 21} and _212S receptors. In fact, similar to TCEt, positive modulation by propofol is affected only by the ₁(M²⁸⁶W) mutation and not by mutations in the ₂ subunit or by mutation in TM2 of the ₁ subunit (Krasowski et al., in press).

The lack of effect of mutations within the GABA_{A 2} subunit on positive modulation by TCEt demonstrates that the molecular substrate for positive modulation by TCEt is different from that for modulation by n-alkanols and volatile ether anesthetics. However, these anesthetics have common requirements for specific residues within TM2 and TM3 for the positive modulation of GABA_A and glycine receptors. In future experiments, we will attempt to delineate additional amino acids that play a role in positive modulation of GABA_A and glycine receptors by these anesthetics.