Etomidate is a potent hypnotic agent that acts via γ-aminobutyric acid receptor type A (GABA(A)) receptors. Evidence supports the presence of two etomidate sites per GABA(A) receptor, and current models assume that each site contributes equally and noncooperatively to drug effects. These assumptions remain untested.


We used concatenated dimer (β2-α1) and trimer (γ2-β2-α1) GABA(A) subunit assemblies that form functional α1β2γ2 channels, and inserted α1M236W etomidate site mutations into both dimers (β2-α1M236W) and trimers (γ2-β2-α1M236W). Wild-type or mutant dimers (D(wt) or D(αM236W)) and trimers (T(wt) or T(αM236W)) were coexpressed in Xenopus oocytes to produce four types of channels: D(wt)T(wt), D(αM236W)T(wt), D(wt)T(αM236W), and D(αM236W)T(αM236W). For each channel type, two-electrode voltage clamp was performed to quantitatively assess GABA EC(50), etomidate modulation (left shift), etomidate direct activation, and other functional parameters affected by αM236W mutations.


Concatenated wild-type D(wt)T(wt) channels displayed etomidate modulation and direct activation similar to α1β2γ2 receptors formed with free subunits. D(αM236W)T(αM236W) receptors also displayed altered GABA sensitivity and etomidate modulation similar to mutated channels formed with free subunits. Both single-site mutant receptors (D(αM236W)T(wt) and D(wt)T(αM236W)) displayed indistinguishable functional properties and equal gating energy changes for GABA activation (-4.9 ± 0.48 vs. -4.7 ± 0.48 kJ/mol, respectively) and etomidate modulation (-3.4 ± 0.49 vs. -3.7 ± 0.38 kJ/mol, respectively), which together accounted for the differences between D(wt)T(wt) and D(αM236W)T(αM236W) channels.


These results support the hypothesis that the two etomidate sites on α1β2γ2 GABA(A) receptors contribute equally and noncooperatively to drug interactions and gating effects.

  • Etomidate binds to two sites on the γ-aminobutyric acid receptor type A (GABAA) receptor, but the contribution of each site to etomidate effects remains untested

  • Etomidate interactions at each of its GABAAreceptor sites produce equal and additive gating modulation effects

IN central nervous system neurons, γ-aminobutyric acid type A (GABAA) receptors are inhibitory receptors, and major targets of general anesthetics such as etomidate and propofol. GABAAreceptors contain five homologous subunits arranged around a chloride-conducting pore. Each subunit includes a large extracellular domain and four transmembrane domains (M1–M4).1The isoform containing two α1, two β2, and one γ2 is the most common, and displays physiology and pharmacology similar to synaptic receptors.2 

Etomidate is a potent sedative/hypnotic drug that acts by potentiating GABAAreceptor activity.3,,5Etomidate enhances α1β2γ2 receptor currents elicited by low GABA concentrations, whereas high concentrations of etomidate directly activate these channels.6,7Structural and pharmacological studies show that both of these effects are mediated by a single class of etomidate binding sites,8and quantitative model analysis suggests that two equivalent etomidate sites are present on each GABAAreceptor.7A photo-activatable etomidate analog, [3H]azi-etomidate, labels affinity-purified bovine brain GABAAreceptors at both αM236 (located in the M1 domain) and βM286 (located in the M3 domain).9Etomidate inhibits photolabeling at αM236 and βM286 in parallel, suggesting that both residues abut a common etomidate site. Homology modeling based on the Torpedo  nicotinic acetylcholine receptor structure suggests that both photolabeled residues face intersubunit clefts,9,10and subunit stoichiometry studies indicate that each receptor contains two α/β interfacial pockets.11,12GABAAreceptor mutations to bulky hydrophobic tryptopan (W) at either α1M236 or β2M286 mimic etomidate's effects by reducing GABA EC50, increasing spontaneous activity, and increasing maximal GABA efficacy, while reducing modulation by etomidate.13Together, these results support the hypothesis that etomidate binds within transmembrane interfacial pockets between α-M1 and β-M3 domains, and that there are two etomidate sites on each α1β2γ2 receptor (fig. 1).

Current models of etomidate action assume that the two binding sites have identical structure and contribute equivalently and independently to modulation of receptor gating,7but these features remain untested. Asymmetry could be caused by the different spatial relationship of the γ2 subunit to each α/β interfacial etomidate site, which could induce unequal etomidate effects at each site. Furthermore, either positive or negative cooperative interactions may occur between etomidate sites.

In this study, we used constrained receptor assembly to test whether the individual etomidate binding sites produce equivalent and independent effects on receptor function. Concatenated-subunit wild-type dimer (β2-α1 = Dwt) and trimer (γ2-β2-α1 = Twt) polypeptides or concatemers containing α1M236W mutations (β2-α1M236W = DαM236W; γ2-β2-α1M236W = TαM236W) were coexpressed in Xenopus  oocytes to produce functional GABAAreceptors with five subunits arranged β2-α1/γ2-β2-α1 counterclockwise when viewed from the synaptic cleft (fig. 1).12,14We compared multiple functional characteristics of all four receptors formed with concatenated dimers and trimers (DwtTwt, DαM236WTαM236W, DαM236WTwt, and DwtTαM236W), with particular focus on whether the two single-site mutants, DαM236WTwtand DwtTαM236W, displayed equivalent versus  nonequivalent properties. To determine if etomidate sites interact cooperatively, we also tested whether adding together DαM236WTwtand DwtTαM236Weffects fully accounted for the large differences in GABA and etomidate sensitivities between DwtTwtreceptors and DαM236WTαM236Wreceptors.

Animal Use

Oocytes for electrophysiology were harvested from Xenopus laevis  frogs, as described previously.7,13Animals were housed in a veterinarian-supervised facility and used with approval of the Massachusetts General Hospital Subcommittee on Research and Animal Care, in accordance with federal and institutional guidelines.


Plasmids encoding the concatenated rat dimer (β2-α1) and trimer (γ2-β2-α1) subunit proteins were a gift from Erwin Sigel, Ph.D. (Professor, Institute for Biochemistry & Molecular Medicine, University of Bern, Bern, Switzerland).12,14These plasmids encode polypeptides that insert a 26-residue linker (QQQQQAAAPAQQAAAPAAQQQQQ) between the C-terminus of β2 and the N-terminus of the mature α1 (without its leader sequence). The trimer also contains a 23-residue linker (QQQQQAAAPAQQQAQAAAPAAQQQQQ) between the C-terminus of γ2 and the N-terminus of the mature β2. DNA mutations encoding a tryptophan substitution at residue 236 of the mature α1 sequence (α1M236W) were introduced into plasmids using Quickchange (Stratagene, La Jolla, CA), as described previously,13and confirmed by complete DNA sequencing of the coding regions. The αM236W mutation was chosen because it impacts multiple receptor function parameters (spontaneous activity, GABA EC50, GABA efficacy, etomidate modulation, and etomidate direct activation), and we aimed to determine how single etomidate site mutations affect each of these parameters. R-etomidate was obtained from Bedford Laboratories (Bedford, OH) as a 2 mg/ml clinical formulation in 35% propylene glycol:water (v/v). GABA and other chemicals (more than 99% pure) were purchased from Sigma-Aldrich (St. Louis, MO).

GABAAReceptor Expression in Xenopus  Oocytes

Messenger RNA was synthesized on linearized coding DNA templates using commercial kits for both DNA transcription and polyadenylation (Ambion, Austin, TX). After purification, mixtures of messenger RNAs (dimer:trimer molar ratio = 1:1) were injected into Xenopus  oocytes, which were then incubated at 18°C for 1–3 days. All four receptor types formed by combining wild-type or mutant dimers and trimers, DwtTwt, DαM236WTwt, DwtTαM236W, and DαM236WTαM236W(fig. 1), were studied electrophysiologically.

Oocyte Electrophysiology

Receptor-mediated currents were measured in oocytes at 21–22°C using the two-electrode voltage clamp (Model OC-725, Warner Instruments) technique. Perfusion and data acquisition were computer-controlled using Clampex8.1 software via  a Digidata 1440 interface (both from Molecular Devices, Cupertino, CA). During electrophysiological experiments, oocytes were placed in a 30 μl flow-chamber and perfused at a rate of 3 ml/min with recording buffer ND96 (96 mM NaCl, 2 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.5). GABA or etomidate solutions in ND96 were applied until currents reached a steady maximum or displayed desensitization. Wash-out times ranged from 1 min (low GABA concentrations) to 5 min (high GABA or low etomidate concentrations), or 10 min (high etomidate concentrations). In different sets of oocytes, GABA concentration-responses were measured either in the absence or presence of etomidate (3.2 μM, for comparison with previous studies of receptors formed from free subunits).7,13Picrotoxin (2 mM in ND96), a potent GABAAreceptor inhibitor, was used to assess spontaneous channel activity. Maximal GABA efficacy was estimated in two ways. For etomidate-sensitive channels (DwtTwt, DαM236WTwt, and DwtTαM236W), the response to maximal GABA (1–3 mM) was compared with responses with maximal GABA plus etomidate (3.2 μM). For DαM236WTαM236Wchannels that displayed very weak etomidate modulation, alphaxalone (10 μM) was shown to enhance (at least fivefold) currents elicited with low (EC10–20) GABA. DαM236WTαM236Wreceptor currents activated with maximal GABA (0.1 mM) were compared in the same cells with currents elicited with maximal GABA plus alphaxalone. We presume that maximal GABA plus the positive modulators activate all receptors.

Statistical Analysis

Peak currents were digitally filtered (lowpass Gaussian 5–10 Hz), baseline corrected, and measured offline using Clampfit software (Molecular Devices). All currents were normalized to maximal GABA response in the same cell, which was measured at the beginning and intermittently throughout experiments on each oocyte. At least four measurements in different cells were made for each type of experiment and experimental condition. Normalized current data from all cells in each type of experiment were combined for nonlinear least-squares method analysis. Agonist concentration-response curves were fitted to logistic (Hill) functions with variable slope (eq. 1) using Graphpad Prism 5.1 (La Jolla, CA):

Agonist is either GABA or etomidate, EC50is the concentration eliciting a current halfway between minimum and maximum, and nH is the Hill slope.

For the three mutated channels, apparent Gibbs free energy shifts (ΔG) for GABA-induced gating were calculated from fitted log (GABA EC50) values associated with mutant versus  wild-type (DwtTwt) concatenated channels (eq. 2):

R is the universal gas constant (8.314 J/mol · K) and T is absolute temperature.

For all four channel types, etomidate modulation of GABA currents was assessed as the ratio of GABA EC50in the presence of 3.2 μM etomidate to control GABA EC50. Etomidate modulation was also quantified as changes in Gibbs free energy, using the fitted GABA EC50values in the absence and presence of etomidate (eq. 3):

Maximal direct activation by etomidate was assessed as response to 300 μM etomidate, normalized to maximal GABA response. Higher etomidate concentrations produce a second, inhibitory effect on GABAAreceptors.

Results are reported as mean ± SD unless otherwise noted. Standard errors for fitted log(EC50) values were used to calculate 95% CIs for EC50s. Comparisons of concentration-responses in the presence and absence of etomidate and for different receptors were performed using the comparison function in the nonlinear fitting module of Graphpad Prism. Statistical comparison of etomidate-induced shifts for different types of channels were performed by calculating the z-statistic from log(EC50) differences. Other statistical analyses were performed in Graphpad Prism or Microsoft Excel (Redmond, WA) using one-way ANOVA with Tukey post hoc  test. For pairwise comparisons (e.g. , GABA concentration-responses in the presence vs.  absence of etomidate) we used P < 0.05 to establish significance. For multiple comparisons of each receptor type to three others, we applied Bonferroni correction, and used P < 0.015 to establish significance.

GABA Concentration-responses and EC50s

All four messenger RNA combinations of dimers (Dwtor DαM236W) and trimers (Twtor TαM236W) produced functional GABA-responsive receptors in oocytes, with maximal currents typically over 2 μA at a clamping potential of −50 mV (figs. 2A–D). Oocytes injected with only dimer or only trimer messenger RNA produced maximal currents in response to GABA that were at most 10-fold smaller than those from oocytes coexpressing both dimers and trimers. The wild-type dimer and trimer combination, DwtTwt, displayed a GABA EC50of 36 μM (fig. 2E; table 1). Receptors with mutations in both etomidate sites, DαM236WTαM236W, showed a much lower GABA EC50of 1.0 μM, (fig. 2F; table 1; P < 0.0001 vs.  DwtTwt). The two single-site mutant receptors, DαM236WTwtand DwtTαM236W, were characterized by indistinguishable GABA EC50values: 5.1 and 5.5 μM, respectively (figs. 2G and 2H; table 1; P = 0.53). These values differed significantly from GABA EC50s for both DwtTwtand DαM236WTαM236W(P < 0.0001 for all pairwise comparisons). Plotting all four GABA concentration responses on a single semilogarithmic plot reveals that the midpoints (EC50) for both single-site mutant channels are approximately halfway between those for DwtTwtand DαM236WTαM236Wchannels (fig. 3A). Calculating the Gibbs free energy for GABA EC50shifts associated with the different mutated channels relative to DwtTwt(eq. 2) reveals that the single-site mutants, DαM236WTwtand DwtTαM236W, contribute −4.9 ± 0.48 and −4.7 ± 0.48 kJ/mol, respectively, each about half of the energy shift (−8.9 ± 0.52 kJ/mol) calculated for DαM236WTαM236Wchannels (fig. 3B).

Etomidate Modulation of GABA Responses

To assess etomidate modulation of GABAAreceptor activation, we measured GABA-dependent responses in the presence of 3.2 μM etomidate (figs. 2E–H, open symbols) and for each channel type calculated the ratios of GABA EC50s measured in the absence and presence of anesthetic (Left Shift Ratios, table 1). In DwtTwtreceptors, etomidate lowered the GABA EC5019-fold, from 36 to 1.9 μM (P < 0.0001), whereas DαM236WTαM236WGABA EC50was reduced only 1.5-fold, from 1.0 to 0.69 μM (P = 0.0065). The EC50ratios in DwtTwtversus  DαM236WTαM236Wwere significantly different (P < 0.0001). Etomidate shifted GABA EC50s 3.9-fold, from 5.1 to 1.3 μM, in DαM236WTwtreceptors (P < 0.0001), and 4.5-fold, from 5.5 to 1.2 μM, in DwtTαM236Wreceptors (P < 0.0001). The two single-site mutant left-shift ratios were not significantly different from each other (P = 0.21), but they differed significantly from values for both DwtTwt(P < 0.0001) and DαM236WTαM236W(P < 0.0001). Figure 4shows etomidate left-shifts expressed as free energy changes, calculated with eq. 3. This display reveals that adding the gating free energy change associated with etomidate modulation in DαM236WTwtand DwtTαM236Wreceptors (−3.4 ± 0.49 and −3.7 ± 0.38 kJ/mol, respectively) sum to a value (−7.1 ± 0.62 kJ/mol) that is not significantly different (P = 0.11) from the sum of free energy changes for DwtTwtplus DαM236WTαM236W(−8.3 ± 0.75 kJ/mol).

Etomidate Direct Activation

In the absence of GABA, etomidate directly activated all four receptors (fig. 5; table 1). Maximal etomidate currents in DwtTwtchannels, elicited with 100–300 μM anesthetic, were 16% of currents stimulated with maximal GABA, and the fitted half-maximal activation (EC50) etomidate concentration was 61 μM. DαM236WTαM236Wreceptors displayed greater sensitivity to etomidate direct activation, with half-maximal activation at 20 μM (P < 0.0001 vs.  DwtTwt) and maximal efficacy relative to GABA of 53% (P < 0.0001 vs.  DwtTwt). Etomidate activation studies in DαM236WTwtand DwtTαM236Wreceptors revealed similar etomidate EC50s (52 vs.  41 μM; P = 0.38) and similar maximal etomidate efficacies (27 vs.  31%; P = 0.16). The maximal etomidate activation efficacies of the DαM236WTwtand DwtTαM236Wchannels were both significantly higher than that of DwtTwtand significantly lower than that of DαM236WTαM236W(P < than 0.01 for all pairs by one-way ANOVA). Etomidate EC50s for DαM236WTwtand DwtTαM236Wchannels were similar to that for DwtTwt(P = 0.84 and 0.21, respectively), but significantly higher than that for DαM236WTαM236W(P < 0.001).

Receptor Spontaneous Activity and Maximal GABA Efficacy

Spontaneous activity in GABAAreceptors was assessed using the noncompetitive antagonist picrotoxin. When present, spontaneously open channels produce a resting-state current “leak,” which is inhibited by picrotoxin, resulting in an apparently outward current.7,13,15None of the concatenated GABAAdimer and trimer combinations displayed measurable picrotoxin-induced outward currents. Given a maximal GABA signal-to-noise ratio of about 1,000 in these experiments, these results indicate that spontaneous activity is less than 0.1% of maximal GABA response (table 1).

The maximal GABA efficacy (table 1) represents the fraction of activatable receptors that open when all agonist sites are bound to GABA. GABA efficacy in DwtTwtreceptors was 0.79, based on etomidate enhancement of 3 mM GABA responses (fig. 2E). In contrast, etomidate did not significantly enhance maximal (1 mM) GABA responses in either DαM236WTwtor DwtTαM236Wreceptors (figs. 2G and 2H), indicating maximal GABA efficacies near 1.0. In DαM236WTαM236Wreceptors, which displayed low sensitivity to etomidate, alphaxalone was a strong allosteric enhancer at low GABA, but did not significantly enhance responses to maximal (0.1 mM) GABA (not shown). Thus, we infer that the maximal GABA efficacy in DαM236WTαM236Wchannels is near 1.0. All mutant channels displayed GABA efficacies significantly higher than DwtTwt(P < 0.001 for all pairs by one-way ANOVA).

We coexpressed concatenated GABAAsubunit β2-α1 dimers and γ2-β2-α1 trimers that formed functional α1β2γ2 l receptors containing α1M236W mutations in neither, either, or both of the two etomidate sites formed between α1-M1 and β2-M3 domains. The major finding of this study is that a single α1M236W mutation in either the dimer or trimer etomidate binding site produces receptors with equivalent functional characteristics. With respect to both GABA sensitivity and etomidate modulation, each site contributes about half the effect observed in wild-type receptors with two etomidate sites.

Implicit in our experimental design is the assumption that subunit concatenation itself does not significantly alter the interaction of etomidate with GABAAreceptors. Concatenated wild-type control receptors assembled from DwtTwtdisplayed etomidate sensitivity similar to that previously reported in wild-type α1β2γ2 channels formed with free α1, β2, and γ2 subunits. Etomidate at 3.2 μM reduced GABA EC5019-fold in DwtTwtreceptors, comparable with shifts ranging from 11-fold to 23-fold previously reported for α1β2γ2 l receptors.7,13,16Etomidate direct agonism in DwtTwtwas characterized by EC50and efficacy parameters similar to those reported for α1β2γ2 channels by Rüsch et al.  7 

The effects of two α1M236W mutations in the concatenated receptors (DαM236WTαM236W) also paralleled effects previously reported in α1M236Wβ2γ2 l GABAAreceptors formed from free subunits.13In DαM236WTαM236Wreceptors, GABA EC50was reduced 35-fold relative to wild-type (DwtTwt), and the GABA EC50ratio was 1.5-fold at 3.2 μM etomidate. In receptors formed from free subunits, α1M236W mutations reduced GABA EC5020-fold, and GABA EC50ratio with 3.2 μM etomidate was 1.7-fold.13Another similarity to studies in GABAAreceptors with free subunits is that etomidate direct activation in DαM236WTαM236Wchannels was more potent and efficacious than in DwtTwt. These effects are consistent with previous analysis7,13suggesting that α1M236W mutations produce a loss-of-function with respect to etomidate efficacy, combined with increased basal gating. The former explains the reduced etomidate left shift, whereas the latter underlies the increased sensitivity to orthosteric agonism by GABA and allosteric agonism by etomidate.13In brief, tryptophan mimics the presence of etomidate, increasing GABA sensitivity and efficacy, while also reducing etomidate's effectiveness as a modulator. Although photolabeling indicates that αM236 is near bound etomidate, we do not know precisely how the tryptophan mutation alters etomidate binding interactions. Based on the relative size of methionine and tryptophan sidechains, a steric component is postulated.

Calculating the gating free energy changes associated with one versus  two α1M236W mutations reveals that each mutation contributes equally and additively. Both single-mutant receptors (DαM236WTwtand DwtTαM236W) displayed indistinguishable GABA EC50s and each mutation accounted for approximately half of the GABA gating energy change calculated for DαM236WTαM236W(fig. 3; table 1). Similarly, DαM236WTwtand DwtTαM236Wdisplayed indistinguishable etomidate modulation, with each site contributing approximately half of the total energy change associated with etomidate modulation in DwtTwtreceptors (fig. 4; table 1). These experiments also demonstrate that a single etomidate site is sufficient for receptor modulation to occur.

In addition to the results discussed above, we found that etomidate direct activation, spontaneous activation, and maximal GABA efficacy were all indistinguishable in receptors with one α1M236W mutation in either the dimer or trimer (fig. 5; table 1). Thus, the two sites have indistinguishable structures and interactions with etomidate molecules, and the γ2 subunit does not produce significant asymmetrical effects on the etomidate sites.

We can infer from our free energy calculations whether cooperativity between the two etomidate sites is present. If positive cooperativity between two equivalent sites exists, each single-site mutant (with one intact site) would display significantly less than half of the etomidate-induced energy shift observed in wild-type receptors, whereas negative cooperativity would produce converse results. The energy additivity found for both GABA gating and etomidate shift effects indicates that cooperativity between etomidate sites is negligible.

Previous studies using GABAAsubunit concatemers and loss-of-function mutations for various ligands provide additional evidence for symmetry in transmembrane modulator sites, whereas studies of the extracellular agonist sites reveal some asymmetrical effects.17,18Neuroactive steroids potentiate GABA currents and directly activate receptors at high concentrations, effects that are reduced by mutations at α1Q241.19In concatenated γ2-β2-α1 trimers and β2-α1 dimers, single α1Q241 mutations produced similar functional effects when introduced into either peptide.17Orthosteric agonists and competitive antagonists bind at two extracellular α/β interfacial sites.1,18Single βY205S GABA-site mutations, which produce profound loss of agonist function, produced asymmetrical effects on GABA and muscimol agonist EC50s, and symmetrical effects on bicuculline IC50.18These studies also showed that for efficient channel activation, both GABA sites must be occupied, whereas antagonist binding to either GABA site effectively blocks activation.

Some of our studies characterizing DαM236WTαM236Wreceptor function revealed differences from α1M236Wβ2γ2 GABAAreceptors formed from free subunits, which displayed both spontaneous activity and full agonism by etomidate.13Several factors may contribute to these differences. First, our concatenated GABAAreceptors contain rat subunits, whereas previous studies on α1M236W mutations were performed using human GABAAsubunits with slightly different amino acid sequences. Second, receptors formed from concatemers are constrained to include a γ subunit, whereas evidence suggests that γ subunits are not consistently incorporated into receptors when free subunits are coexpressed in Xenopus  oocytes.20,21Finally, GABA EC50s in concatenated GABAAreceptors have been reported to be higher than receptors formed from identical free subunits, suggesting that concatenation itself may reduce basal gating.12,22 

In studies using concatenated channel subunit assemblies it is important to verify that linked subunits are not enzymatically cleaved into free subunits, and in the present case, to demonstrate that expression of dimers alone or trimers alone does not lead to significant formation of alternative channel assemblies that might influence results.22Our control experiments showed that expression of dimers alone or trimers alone produced small GABA-responsive currents, with amplitudes less than 10% of those recorded when dimers and trimers were coexpressed. Thus, alternative concatemer channel assemblies are at most a minor contributor to our electrophysiological measurements. Concatemer cleavage leading to assembly of receptors from free subunits also appears to be insignificant, based on these observations. The high Hill slopes observed in GABA concentration responses for DαM236WTwtand DwtTαM236Wreceptors (figs. 2, 3) is further evidence against concatemer breakdown. If free subunits were produced, mixtures of receptors containing zero, one, or two α1M236W mutations would form, resulting in shallow (low Hill slope) GABA concentration-response curves reflecting the dose-dependent gating effects of the mutation.11 

The experiments reported here did not address whether a single etomidate site is capable of directly activating wild-type GABAAreceptors. We observed etomidate direct activation in DαM236WTwtand DwtTαM236Wreceptors, but this is likely because of the combined effects of one intact etomidate site together with basal gating enhancement associated with one α1M236W mutation, which contributes gating energy similar to partial etomidate occupancy. A related study using concatemers combining etomidate-sensitive β2 and etomidate-insensitive β1 subunits in the same GABAAreceptors suggests that a single β2 subunit is not sufficient for direct activation with etomidate.23This question could be further addressed with concatemer studies using point mutations that selectively and profoundly reduce etomidate sensitivity.

In conclusion, our results show that etomidate interactions at each of its two GABAAreceptor sites produce equal and additive gating modulation effects. These results support functional models wherein the two etomidate sites in α1β2γ2 l receptors are equivalent and noncooperative.7In terms of clinical relevance, these studies add important details to our understanding of anesthetic mechanisms at their molecular targets, a key step toward developing improved anesthetic drugs.24Of more direct relevance, heteromeric GABAAreceptors containing both β1 and β2/3 subunits are postulated to exist in the nervous system.23In the presence of etomidate, our results predict that such receptors will experience approximately half the gating energy effect of receptors containing two β2/3 subunits. Future studies using concatenated GABAAsubunit assemblies will be useful in investigating the linkages between the etomidate and GABA sites on the same subunit, and assessing the mechanisms of other anesthetics, which also likely bind to multiple sites.25 

The authors thank Erwin Sigel, Ph.D., Professor, University of Bern, Bern, Switzerland, for plasmids encoding concatenated GABAAsubunits, and Aiping Liu, M.S., Senior Laboratory Technologist, Massachusetts General Hospital, Boston, Massachusetts, for technical support on this project.

Sieghart W: Structure, pharmacology, and function of GABAAreceptor subtypes. Adv Pharmacol 2006; 54:231–63
Olsen RW, Sieghart W: GABAAreceptors: Subtypes provide diversity of function and pharmacology. Neuropharmacology 2009; 56:141–8
Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U: General anesthetic actions in vivo  strongly attenuated by a point mutation in the GABAAreceptor beta3 subunit. FASEB J 2003; 17:250–2
Forman SA: Clinical and molecular pharmacology of etomidate. ANESTHESIOLOGY 2011; 114:695–707
Belelli D, Muntoni AL, Merrywest SD, Gentet LJ, Casula A, Callachan H, Madau P, Gemmell DK, Hamilton NM, Lambert JJ, Sillar KT, Peters JA: The in vitro  and in vivo  enantioselectivity of etomidate implicates the GABAAreceptor in general anaesthesia. Neuropharmacology 2003; 45:57–71
Yang J, Uchida I: Mechanisms of etomidate potentiation of GABAAreceptor-gated currents in cultured postnatal hippocampal neurons. Neuroscience 1996; 73:69–78
Rüsch D, Zhong H, Forman SA: Gating allosterism at a single class of etomidate sites on α1β2γ2L GABAAreceptors accounts for both direct activation and agonist modulation. J Biol Chem 2004; 279:20982–92
Belelli D, Lambert JJ, Peters JA, Wafford K, Whiting PJ: The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid. Proc Natl Acad Sci U S A 1997; 94:11031–6
Li GD, Chiara DC, Sawyer GW, Husain SS, Olsen RW, Cohen JB: Identification of a GABAAreceptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. J Neurosci 2006; 26:11599–605
Bali M, Jansen M, Akabas MH: GABA-induced intersubunit conformational movement in the GABAAreceptor α1M1-β2M3 transmembrane subunit interface: Experimental basis for homology modeling of an intravenous anesthetic binding site. J Neurosci 2009; 29:3083–92
Chang Y, Wang R, Barot S, Weiss DS: Stoichiometry of a recombinant GABAA receptor. J Neurosci 1996; 16:5415–24
Baumann SW, Baur R, Sigel E: Subunit arrangement of gamma-aminobutyric acid type A receptors. J Biol Chem 2001; 276:36275–80
Stewart D, Desai R, Cheng Q, Liu A, Forman SA: Tryptophan mutations at azi-etomidate photo-incorporation sites on α1 or β2 subunits enhance GABAAreceptor gating and reduce etomidate modulation. Mol Pharmacol 2008; 74:1687–95
Baumann SW, Baur R, Sigel E: Forced subunit assembly in α1β2γ2 GABAAreceptors. Insight into the absolute arrangement. J Biol Chem 2002; 277:46020–5
Chang Y, Weiss DS: Allosteric activation mechanism of the α1β2γ2 γ-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine. Biophys J 1999; 77:2542–51
Desai R, Ruesch D, Forman SA: Gamma-amino butyric acid type A receptor mutations at β2N265 alter etomidate efficacy while preserving basal and agonist-dependent activity. ANESTHESIOLOGY 2009; 111:774–84
Bracamontes JR, Steinbach JH: Steroid interaction with a single potentiating site is sufficient to modulate GABAAreceptor function. Mol Pharmacol 2009; 75:973–81
Baumann SW, Baur R, Sigel E: Individual properties of the two functional agonist sites in GABAAreceptors. J Neurosci 2003; 23:11158–66
Hosie AM, Wilkins ME, da Silva HM, Smart TG: Endogenous neurosteroids regulate GABAAreceptors through two discrete transmembrane sites. Nature 2006; 444:486–9
Boileau AJ, Baur R, Sharkey LM, Sigel E, Czajkowski C: The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAAreceptors expressed in Xenopus  oocytes. Neuropharmacology 2002; 43:695–700
Boileau AJ, Li T, Benkwitz C, Czajkowski C, Pearce RA: Effect of γ2S subunit incorporation on GABAAreceptor macroscopic kinetics. Neuropharmacology 2003; 44:1003–12
Steinbach JH, Akk G: Use of concatemers of ligand-gated ion channel subunits to study mechanisms of steroid potentiation. ANESTHESIOLOGY 2011; 115:1328–37
Boulineau N, Baur R, Minier F, Sigel E: Consequence of the presence of two different beta subunit isoforms in a GABA(A) receptor. J Neurochem 2005; 95:1724–31
Forman SA: Molecular approaches to improving general anesthetics. Anesthesiol Clin 2010; 28:761–71
Olsen RW, Li GD: GABA(A) receptors as molecular targets of general anesthetics: Identification of binding sites provides clues to allosteric modulation. Can J Anaesth 2011; 58:206–15