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J. Biol. Chem., Vol. 279, Issue 20, 20982-20992, May 14, 2004

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Gating Allosterism at a Single Class of Etomidate Sites on _122L GABA_A Receptors Accounts for Both Direct Activation and Agonist Modulation^*

Dirk Rüsch, Huijun Zhong, and Stuart A. Forman¶||

From the Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston, Massachusetts 02114, the ^¶Department of Anesthesia, Harvard Medical School, Boston, Massachusetts 02115, and the Department of Anesthesia and Critical Care, University Hospital Schleswig-Holstein, 24105 Kiel, Germany

Received for publication, January 15, 2004 , and in revised form, March 11, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
At clinical concentrations, the potent intravenous general anesthetic etomidate enhances -aminobutyric acid, type A (GABA_A) receptor activity elicited with low -aminobutyric acid (GABA) concentrations, whereas much higher etomidate concentrations activate receptors in the absence of GABA. Therefore, GABA_A receptors may possess two types of etomidate sites: high affinity GABA-modulating sites and low affinity channel-activating sites. However, GABA modulation and direct activation share stereoselectivity for the (R)(+)-etomidate isomer and display parallel dependence on GABA_A subunit isoforms, suggesting that these two actions may be mediated by a single class of etomidate site(s) that exert one or more molecular effects. In this study, we assessed GABA modulation by etomidate using leftward shifts of electrophysiological GABA concentration responses in cells expressing human _122L receptors. Etomidate at up to 100 µM reduced GABA EC₅₀ values by over 100-fold but without apparent saturation, indicating the absence of high affinity etomidate sites. In experiments using a partial agonist, P4S, etomidate both reduced EC₅₀ and increased maximal efficacy, demonstrating that etomidate shifts the GABA_A receptor gating equilibrium toward open states. Results were quantitatively analyzed using equilibrium receptor gating models, wherein a postulated class of equivalent etomidate sites both directly activates receptors and enhances agonist gating. A Monod-Wyman-Changeux co-agonist mechanism with two equivalent etomidate sites that allosterically enhance GABA_A receptor gating independently of agonist binding most simply accounts for direct activation and agonist modulation. This model also correctly predicts the actions of etomidate on GABA_A receptors containing a point mutation that increases constitutive gating activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The major mediator of synaptic inhibition in the brain is the GABA_A¹ receptor-chloride channel complex, which is formed from five homologous subunits arranged around a gated transmembrane pore. Multiple GABA_A receptor subunit classes (, , , , , , and ) and isoforms have been identified, and most native receptors in brain contain a combination of , , and subunits (1, 2). GABA_A receptors are modulated by sedatives, anxiolytics, anticonvulsants, and many general anesthetics, including neurosteroids (3–5), barbiturates (6), propofol (7, 8), etomidate (9, 10), alcohols (11, 12), and halogenated volatile inhalants (12–14).

Clinical concentrations of these anesthetics enhance GABA_A receptor activity (15), which is observed electrophysiologically as increased multichannel responses to low GABA concentrations (e.g. EC₅ or EC₁₀). Responses to maximally activating GABA concentrations are either not or weakly enhanced by general anesthetics. As a result, general anesthetics shift GABA concentration-response relationships leftward (reduce EC₅₀), which could be caused by either enhanced GABA binding to agonist sites or to enhanced gating of ligand-bound receptors (16, 17).

Supra-clinical concentrations of general anesthetics can also directly activate GABA_A receptors (agonism or GABA-mimetic activity) (18–20). The widely different concentrations of general anesthetics that induce GABA modulation versus direct activation suggest that there may be two distinct types of sites on GABA_A receptors as follows: high affinity sites that enhance apparent GABA sensitivity, and low affinity sites that directly activate receptors. Alternative mechanisms, postulating a single class of sites that change affinity for general anesthetic depending on the receptor state, can also account for multiple actions. These unified mechanisms fall into two generic classes: orthosteric partial agonism (where anesthetics act via the GABA sites) and allosteric co-agonism (where anesthetics act via sites distinct from GABA sites). In one electrophysiological study, three effects of n-octanol on _122S GABA_A receptors, GABA modulation, direct activation, and inhibition, were explained by partial agonism (21). In a study of the effects of pentobarbital on brain synaptosomal GABA_A channels, Cash and Subbarao (22) considered an allosteric co-agonist mechanism where distinct pentobarbital and GABA-binding sites couple independently to the receptor open-closed equilibrium via Monod-Wyman-Changeux (MWC) allosterism (preferential binding to the open state).

These different mechanisms predict different effects of general anesthetic on experimental parameters that reflect the equilibria between closed and open receptors. If two distinct classes of saturable sites have sufficiently different affinities for an anesthetic, then reliable measures of GABA modulation should display maximal effects at anesthetic concentrations below those that induce maximal direct activation. Partial agonist and co-agonist mechanisms also predict different relationships between GABA EC₅₀ and anesthetic concentration. Orthosteric partial agonism predicts that as general anesthetic concentration rises, GABA EC₅₀ will at first decrease (positive modulation) but then increase again as competition for occupation of GABA-binding sites develops. In contrast, allosteric co-agonism predicts that GABA EC₅₀ will only decrease, approaching a minimum in the same anesthetic concentration range where direct activation reaches a maximum. Furthermore, models that postulate a single class of sites mediating multiple anesthetic actions predict that structurally similar anesthetics (e.g. stereoisomers) will likely display similar relative potencies for their multiple actions.

The intravenous general anesthetic etomidate (ethyl-1-(-methylbenzyl)imidazole-5-carboxylate) presents several advantages for distinguishing between possible mechanisms of GABA_A receptor modulation. It is a very potent clinical anesthetic, causing loss of righting responses in tadpoles at about 3 µM (19, 20) and loss of responsiveness in humans at about 2 µM (23). Modulation of apparent GABA affinity for GABA_A receptors (i.e. K_d or EC₅₀) is seen with etomidate concentrations as low as 0.1 µM (9, 24). Etomidate alone at concentrations above 10 µM directly activates GABA_A receptors, exhibiting maximal efficacy up to 50% relative to GABA, depending on subunit combination (9, 25). Moreover, etomidate contains a chiral center. Both direct activation of GABA_A receptors and modulation of GABA responses by (R)(+)-etomidate is seen at about 20 times lower concentrations than with (S)(-)-etomidate, closely paralleling the relative anesthetic potency of these enantiomers in animals (19, 20, 26). Together with recent transgenic animal experiments (27, 28), stereoselectivity tightly links etomidate anesthesia to GABA_A receptors containing ₂ and ₃ subunits. Stereoselectivity also constitutes the strongest evidence for etomidate-binding sites within the GABA_A receptor-ionophore complex (26).

In this study, we examined the relationship between etomidate-induced direct activation of GABA_A receptors and GABA modulation assessed with EC₅₀ shifts. We also used a partial agonist, P4S, to investigate whether etomidate-induced leftward shift is due to enhancement of agonist-receptor binding or channel gating. Activity was measured electrophysiologically in voltage-clamped Xenopus oocytes or HEK293 cells expressing human _122L GABA_A receptors. Results obtained over a wide range of etomidate concentrations were comparatively analyzed using equilibrium partial agonist and co-agonist models that account for multiple etomidate effects via a single class of GABA_A receptor-binding sites. Finally, we tested whether an MWC co-agonist model predicts etomidate effects on mutant GABA_A receptors characterized by a high degree of spontaneous activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Recombinant _122L GABA_A Receptors in Xenopus laevis Oocytes and HEK293 Cells—cDNAs encoding human ₁, ₂, and _2L GABA_A receptor subunits in pCDM8 vectors (Invitrogen) were gifts from Dr. Paul Whiting (Merck). The ₁L264T mutation was introduced as described previously (29). Subunit mRNAs were mixed in a w/w ratio of 1:1:4 and injected into oocytes as described previously (19). Injected oocytes were incubated at 17 °C for 48–144 h before electrophysiological experiments.

Maintenance of HEK293 cells (ATCC; Manassas, VA) and methods for transient transfection of GABA_A receptors have been described (29). Cells were transfected with GABA_A subunit cDNA mixtures at a ratio of 1:1:5, along with a marker plasmid encoding green fluorescent protein.

Chemicals—All chemicals were from Sigma unless otherwise indicated. GABA was stored at -80 °C as an aqueous 1 M stock solution, and aliquots were thawed and diluted into electrophysiology solution each day. P4S was freshly prepared each day as a 1 M stock. (R)(+)-etomidate (Bedford Laboratories, Bedford, OH) solutions were also prepared daily.

Two Microelectrode Oocyte Electrophysiology—Oocyte electrophysiological measurements were carried out at 21–23 °C in ND-96 solution (in mM: NaCl 96, KCl 2, MgCl₂ 0.8, CaCl₂ 1.8, HEPES 5, pH 7.5) as described previously (19). Oocytes were placed in a custom-built low volume (20 µl) flow chamber, and currents were elicited with ND-96 solutions containing etomidate, GABA (± etomidate), or P4S (± etomidate), delivered by gravity from glass reservoirs via polytetrafluoroethylene (PTFE) tubing and computer-actuated solenoid valves.

Half-maximal agonist concentrations (EC₅₀ values) were assessed both in the absence and presence of (R)(+)-etomidate, in individual oocytes. Every other recording used a maximal activating GABA concentration (1–10 mM) for normalization of oocyte responses, which can change over time. Oocytes were washed for at least 5 min in ND-96 between experiments. After completion of a control agonist concentration-response study, ND-96 was replaced with ND-96 plus (R)(+)-etomidate. Etomidate at up to 32 µM induced minimal run-down of oocyte currents and was continuously present during the agonist concentration-response experiments. Oocyte currents were elicited with agonist plus etomidate alternating with 1 mM GABA plus etomidate for normalization. Maximal responses in the absence and presence of etomidate were also compared, enabling all currents from a single oocyte to be normalized to the same standard response in 1–10 mM GABA alone. In the continuous presence of 100 µM etomidate, significant run-down of oocyte current responses was observed, which we supposed was due to GABA_A receptor desensitization. Therefore, oocytes were bathed in ND-96, and 100 µM etomidate was applied for 30 s prior to GABA plus 100 µM etomidate exposure. Oocytes were washed for 15 min in ND-96 before subsequent experiments.

HEK293 Patch Clamp Electrophysiology—Experiments were performed at 21–23 °C as described previously (29). Extracellular solution contained (in mM) 135 NaCl, 5.4 KCl, 1 MgCl₂, 1.8 CaCl₂, 5 HEPES, pH 7.2, and patch pipettes were filled with intracellular solution (in mM: 140 KCl, 2 MgCl₂, 10 HEPES, 1 EGTA, pH 7.3). Open pipette tip resistance ranged from 2 to 5 megohms. Transfected cells were identified by green fluorescent protein fluorescence. Membrane patches with greater than 1 gigohm seal resistance were excised in the outside-out configuration. For whole-cell recordings, small cells (<10 µm diameter) were lifted from coverslips, and pipette capacitance and series resistance compensation were applied. Patches and cells were voltage-clamped at -50 mV during current recordings.

Rapid application of solutions was achieved using a piezo-driven quad (2 x 2) capillary tube with up to four flowing solutions (30). One channel of the quad tube was reserved for external solution, and an adjacent channel contained external solution with 1 mM GABA as a normalization standard. The other two channels, each fed by multiple upstream reservoirs via selector valves, contained extracellular solutions with GABA, etomidate, or combinations of the two. When responses to GABA plus etomidate were studied, the patch or cell was first exposed to etomidate alone for 0.1–0.5 s before subsequent exposure to the combination. To both normalize responses among cells and to correct for run-down, every second or third trace was a control current elicited with 1 mM GABA. Cells and patches were washed in external buffer for 10–60 s between experiments. Solution exchange times were under 1 ms, measured by open pipette junction currents. Whole-cell solution exchange times, estimated from currents elicited with 0.1–1 mM GABA, were 2–5 ms.

Analysis of GABA Concentration Responses—Data analysis was performed offline. For responses elicited with GABA alone, peak currents were corrected by subtracting the base-line leak current, which was usually below 0.1 µA in oocytes. Corrected peak currents were normalized to the average of pre- and post-control maximal GABA responses measured at 1–10 mM, . Direct activation by etomidate was estimated by subtracting the average leak current from sweeps where there was no etomidate from the average leak when etomidate was present in the same oocyte. Oocyte current responses elicited with etomidate plus GABA were corrected by subtracting only the average leak in buffer (i.e. the directly activated component of the current was not subtracted). Leak-corrected responses were normalized to the average of pre- and post-control responses to etomidate plus maximal activating GABA (1–10 mM), . Scaling of responses in the presence versus absence of etomidate was achieved by multiplying by the ratio of currents elicited with maximal GABA plus etomidate and with maximal GABA alone, /. At etomidate concentrations below 100 µM, inhibition by etomidate was minimal, and was larger than in most oocytes.

In the absence of etomidate, _122L GABA_A receptors display negligible spontaneous activity. Therefore, normalized data from individual oocytes were fitted by non-linear least squares with Equation 1 (Hill or logistic), where is the half-maximal activating GABA concentration, and n is the Hill coefficient,

(Eq. 1)

Normalized GABA concentration responses recorded in the presence of etomidate were fitted with a similar logistic equation (Equation 2), where the minimum was constrained to equal the normalized directly activated current, ,

(Eq. 2)

The ratios of EC₅₀ values measured in the presence and absence of etomidate (left-shift ratios), /, were calculated for data recorded in individual oocytes.

Determination of Model Parameters—By using equations defining equilibrium P_open as functions of GABA and etomidate concentrations, parameters for the partial agonist model (Fig. 5) and allosteric co-agonist models (Fig. 6) were independently fitted to pooled concentration-response data from oocytes. Data for these fits were converted to estimated P_open by re-normalizing to a maximal open probability () of 0.85 in the presence of GABA alone. was based both on published single channel estimates (31–33) and on the degree of current enhancement observed in the presence of high GABA plus etomidate. Because values varied among individual oocytes, we fitted GABA parameters for the models to a restricted set of GABA concentration responses from 10 oocytes characterized by values between 32 and 40 µM. Etomidate direct activation parameters were fitted to the oocyte data shown in Fig. 1 (right panel).

View larger version (32K): [in this window] [in a new window]   FIG. 5. A partial agonist mechanism for etomidate modulation of GABAA receptors. A, the mechanism is based on Kurata et al. (21) and has seven free parameters. Two equivalent binding sites can be occupied by either GABA (G) or etomidate (E), with binding dissociation constants KG and KE, respectively. Binding allosterism alters the second site affinity by factors a when GABA is bound or b when etomidate is bound. Only doubly occupied receptors open, with efficacy factors G, E, and GE, depending on bound ligands. B, optimal parameters fitted to oocyte data were KG = 34.6 µM, a = 1.7, G = 5, KE = 121 µM, b = 0.17, G = 0.23, and GE = 100. Model-simulated GABA concentration responses are shown for etomidate concentrations between 0 and 1000 µM (compare with Fig. 3, top). C, model-predictions (lines) are overdrawn on re-normalized oocyte data for both EC5 enhancement by etomidate (squares; data from Husain et al. (19)) and direct activation (circles; data from Fig. 1). D, model predictions for EC50 ratios (lines) are overdrawn on oocyte data from Fig. 3. Different lines represent models with different GE values: GE = 80 (dashed line); GE = 100 (solid line); GE = 120 (dotted line). E, modeling the effects of etomidate on P4S-activated currents. Data from Fig. 4, bottom, were re-normalized assuming GABA (dashed line). The line overlaying control data (squares) represents a fit to Equation 4 from Kurata et al. (21) (substituting KP for KG and P for G), fitted KP = 12.6 µM, a = 3.9, and P = 0.50. The other lines overlaying the data obtained with 3.2 µM etomidate (circles) were calculated using Equation 6 from Kurata et al. (21) using fitted P4S and etomidate agonist parameters reported above. Lines are labeled with various PE values used in the model, between 10 and 50.

View larger version (30K): [in this window] [in a new window]   FIG. 6. An MWC co-agonist model for etomidate modulation of GABAA receptor gating. A, this model is based on Cash and Subbarao (22) and depicts allosteric co-agonism with two equivalent GABA sites and two equivalent etomidate sites. Related models with different numbers of etomidate sites (nE) would be depicted with nE + 1 vertically stacked layers. All models have five free parameters. Basal equilibrium between unliganded closed (R) and open (O) states is described by L0 = [R]/[O]. GABA binding (horizontal arrows) to closed states is described by microscopic dissociation constant KG, and efficacy per GABA is c. Etomidate binding (vertical arrows) to closed states is described by microscopic dissociation constant KE, and efficacy per etomidate is d. Open states are stabilized by GABA and/or etomidate binding, because c < 1 and d < 1. Closed-open transitions (diagonal arrows) between states in which GABA and/or etomidate sites are occupied are labeled with equilibrium constants. B, GABA EC50 shows an inverse square root dependence on direct activation at high etomidate, as predicted by the co-agonist model. Points represent results from individual oocytes exposed to directly activating etomidate concentrations: 10 µM (squares; n = 10), 32 µM (circles; n = 5), and 100 µM (triangles; n = 5). The slope of a linear least squares fit to the log transformed data is -0.48 ± 0.072 (R = -0.84; p < 0.0001). Back extrapolation to the average control EC50 (36 µM) results in an estimate of basal receptor activation: Po = 1.4 x 10-5. C, optimal parameters fitted to oocyte data for the nE = 2 model are reported in Table I. Model-simulated GABA concentration responses are shown for etomidate concentrations between 0 and 1000 µM (compare with Fig. 3, top). D, model predictions (lines) are overdrawn on re-normalized oocyte data for both EC5 enhancement by etomidate (squares; data from Husain et al. (19)) and direct activation (circles; data from Fig. 1). Different lines represent models with different nE values: 1, long dash; 2, solid; 3, short dash; 4, dot-dash; 5, dotted. E, model predictions for EC50 ratios (lines representing models with different nE values are as in D) are overdrawn on oocyte data from Fig. 3, bottom. F, error analysis comparing the predictions of models with different nE values to oocyte results. Sum-of-squares deviations are plotted for EC5 enhancement (squares), direct activation (circles), and EC50 ratios (triangles). G, modeling the effects of etomidate on P4S-activated currents. Data from Fig. 4, bottom, were re-normalized assuming GABA (dashed line). The line overlaying control data (squares) represents a fit to Equation 3 (substituting KP for KG), with L0 = 70,000 and nE = 2. Fitted KP = 44 ± 10 µM and c = 0.0055 ± 0.0017. The line overlaying the data with 3.2 µM etomidate (circles) was calculated using Equation 5 with L0 = 70,000, KP = 44 µM, c = 0.0055, KE = 35 µM, d = 0.0077, and nE = 2.

View larger version (17K): [in this window] [in a new window]   FIG. 1. Direct activation of 122L GABAA receptor-mediated currents by etomidate. Left, traces show currents recorded from an HEK cell patch. Three traces were activated using etomidate (concentrations labeled in µM). For normalization, a trace activated with 1 mM GABA is also shown. A notable difference between etomidate-activated currents from oocytes and HEK293 membrane patches is that patch currents displayed larger "surge" currents, evident after discontinuing etomidate above 100 µM. This indicates the presence of an inhibitory etomidate action that rapidly reverses before deactivation is complete. Right, etomidate direct activation responses from HEK293 cells and patches (open circles) and oocytes (solid squares) are plotted. Oocyte data include some previously published data (19) combined with new results. Points represent mean ± S.D. of at least four measurements. Peak responses to etomidate (surge current peak was used, if present) were normalized to maximal GABA currents (1 mM).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Etomidate effects on P4S-activated currents. Top, currents recorded from an oocyte expressing 122L GABAA receptors. The left trace shows a current activated with 10 mM P4S, whereas the right trace shows a current activated with 3.2 µM etomidate plus 10 mM P4S. For comparison, a current elicited with 1 mM GABA is shown in the middle trace. Addition of etomidate more than doubled the maximal P4S current, and etomidate plus P4S was more efficacious than GABA alone. Bottom, effect of etomidate on P4S concentration response. Solid squares depict average P4S concentration responses (n = 3) for oocytes, normalized to 1 mM GABA response (dashed line). The overlaying line is a fit to Equation 1 (substituting P4S for GABA): /; ; nH = 1.0. Open circles are normalized responses to P4S in the presence of 3.2 µM etomidate (n = 3), and the overdrawn line is a fit with Equation 2: /; /; ; nH = 1.0.

(Eq. 3)

Parameters for etomidate binding and efficacy (K_E and d, defined analogously to K_G and c, above) in models with different numbers of etomidate sites were derived from the direct activation estimated P_open versus [etomidate] data set by non-linear least squares fits to a similar Equation 4, with L₀ constrained to 70,000, and the number of etomidate sites (n_E) constrained to integers between 1 and 5,

(Eq. 4)

Simulated P_open values for the MWC co-agonist models containing different numbers of etomidate sites were generated by using Equation 5, using L₀ = 70,000, K_G, and c from the fit to Equation 3, and K_E, d, and n_E from the appropriate fit to Equation 4,

(Eq. 5)

Model calculations were performed using spreadsheet software (Excel, Microsoft Corp., Redmond, WA), and the results were transferred to Origin worksheets for display and analysis. Statistical analyses were performed with Excel. Non-linear least squares fits were performed using Origin software (version 6.1, Microcal Software, Inc., Northampton, MA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Direct Activation and GABA Modulation by (R)-Etomidate in Human _122L GABA_A Receptors Expressed in Xenopus Oocytes and HEK293 Cells—We and others have previously reported (19, 20, 25) electrophysiological data from Xenopus oocytes expressing _122L GABA_A receptors that compared direct activation by etomidate with maximal activation by GABA. Our results showed that etomidate is a moderately efficacious agonist, eliciting about 20% of the maximal GABA-activated current in oocytes, with an EC₅₀ of 64 µM and Hill coefficient of 1.5 (19).

A potential limitation in electrophysiological studies of ligand-gated channels using Xenopus oocytes, because of their size, is slow agonist concentration-jump rates. If the current activation rate is slower than receptor desensitization, then peak currents can be significantly underestimated, leading to underestimated EC₅₀ values and Hill slopes. In our low volume flow chamber for oocytes (20 µl) using superfusion rates of 5 ml/min, we achieve 4 volume exchanges/s. This is almost 20 times faster than maximal desensitization rates of 0.25 s^-1 recently reported by Boileau et al. (36) for HEK293-expressed ₁₂₂ receptors. Based on the observation that overexpression of ₂ minimizes fast desensitization (36), we used 4–5-fold excess ₂ relative to ₁ and ₂ mRNA or cDNA. We then compared normalized peak responses of the oocyte-expressed _122L GABA_A receptors with currents from small HEK293 cells and excised membrane patches, elicited using a device that exchanges solutions in less than 1 ms (Figs. 1 and 2). When peak HEK293 cell and patch currents elicited with etomidate were normalized to maximal GABA-activated responses, the apparent potency and efficacy of etomidate direct activation was similar to that observed in oocyte currents (Fig. 1, right). In addition, maximal GABA-induced desensitization rates in the currents we recorded from HEK293 cells and patches (n = 20) averaged 0.7 s^-1. Simulations using a solution exchange rate of 4 s^-1 (an underestimate of activation rate at high GABA) and desensitization rates of 0.25 and 0.7 s^-1 suggest that oocyte peak currents at high GABA may be underestimated by about 20–30%. In concentration-response analyses, this degree of error would cause underestimation of EC₅₀ values and Hill slopes by less than 30%.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Enhancement of GABA-activated currents in HEK293 cells and Xenopus oocytes by 3.2 µM etomidate. Top, traces show four currents recorded from an HEK293 cell expressing 122L GABAA receptors. Labels indicate GABA and etomidate concentrations. Cells were exposed to etomidate alone for 500 ms prior to activation with GABA plus etomidate (3.2 µM etomidate alone did not elicit an observable current). Bottom left, an EC50 shift study from a single Xenopus oocyte. Solid boxes represent the control GABA concentration response (; nH = 1.4 ± 0.27), and open circles represent the GABA concentration response during continuous exposure to 3.2 µM etomidate (; nH = 1.2 ± 0.18). /. All currents were normalized to control responses in 10 mM GABA. Note that etomidate increases maximal response at 10 mM GABA by about 10%. Bottom right, combined results, normalized to 1 mM GABA responses, from HEK293 cells, and patches are plotted as mean ± S.D. Control GABA concentration response (solid squares; n 5) was fitted with and nH = 1.2 ± 0.13. In the presence of 3.2 µM etomidate (open circles; n 4), and nH = 1.3 ± 0.16. /. In the presence of etomidate, maximal GABA responses were enhanced by 7%.

View larger version (24K): [in this window] [in a new window]   FIG. 3. High concentrations of etomidate induce both direct activation of GABAA receptors and leftward shifts in GABA concentration-response relationships. Top, examples of data from four individual oocytes in which GABA concentration responses were measured in the absence and presence of etomidate. All currents were normalized to the maximal GABA-induced current in the same oocyte. For display, GABA concentrations were also normalized to values for each individual oocyte. Solid squares, combined control GABA concentration responses; min = 0 and max = 1.0 , nH = 1.4. Open triangles, 3.2 µM etomidate; /; /; /; nH = 1.0. Open triangles, 10 µM etomidate; /; /; /; nH = 1.2. Open circles, 32 µM etomidate; /; /; /; nH = 1.7. Solid circles, 100 µM etomidate; /; /; /; nH = 0.8. Bottom, average (± S.D.; n 5) EC50 ratios for all oocytes (solid triangles) plotted against etomidate concentration. The EC50 ratio determined in HEK293 cells at 3.2 µM (Fig. 2) is plotted as an open triangle. The small open triangle at 100 µM etomidate represents an EC50 estimate from HEK293 currents measured at 0.32 µM, 1 µM, and 1 mM GABA (three cells).

In the presence of 0.32 µM etomidate, GABA was 2–3-fold lower than (/ ratio = 0.4 ± 0.15) and dropped steeply as etomidate concentration increased to 10 µM. ratios determined at 10, 32, and 100 µM etomidate (Fig. 3) averaged 0.019, 0.014, and 0.010, respectively, and pairwise comparisons of shifts at these three etomidate concentrations were all significantly different (p 0.03 using two-tailed Student's t tests). Fig. 3, bottom, shows that does not reach a minimum at etomidate concentrations up to 100 µM, although the / versus [etomidate] relationship appears to flatten somewhat above 32 µM.

Attempts to quantify values while exposing oocytes to etomidate concentrations above 100 µM were unsuccessful, because of rapid and irreversible rundown of current responses, which was apparently caused by both desensitization of GABA_A receptors and very slow washout of etomidate from oocytes. By using voltage-clamped HEK293 cells, where etomidate washout is much faster, experiments comparing responses to sub-micromolar GABA and 1 mM GABA in the presence of 100 µM etomidate (data not shown) indicated a GABA near 0.4 µM, corresponding to an / ratio of 0.009, which closely matched the value derived from oocyte experiments (Fig. 3, bottom).

Etomidate Effects on Currents Elicited with P4S—Reduced GABA EC₅₀ values in the presence of etomidate could be due to either enhanced GABA binding or to enhanced gating efficacy of GABA-bound receptors (16, 17). It is not possible to distinguish unambiguously these possibilities from macrocurrents stimulated with GABA, because high concentrations of GABA alone activate nearly all receptors (). To test whether etomidate enhances GABA_A receptor gating, we used a partial agonist, P4S, that acts at the GABA sites (Fig. 4). In whole oocytes, 70 ± 24 µM P4S induced half-maximal activation of GABA_A receptors, and P4S concentrations up to 10 mM (140 x EC₅₀) elicited 40 ± 3.2% (n = 3) of the maximal GABA-activated current. Thus, P4S is a partial agonist in _122L receptors. In the presence of 3.2 µM etomidate, the P4S EC₅₀ was reduced to 6.3 ± 0.79 µM (/), and the apparent efficacy of 1–10 mM P4S increased dramatically, eliciting currents that averaged 10% higher than those elicited with 1 mM GABA (Fig. 4).

Analyses of GABA and Etomidate Data Based on Partial Agonist and Co-agonist Models—We limited our analysis to two quantitative equilibrium models that minimized the number of free parameters by assuming two equivalent GABA sites and equivalent etomidate sites as follows: a partial agonist model (21) and an MWC allosteric co-agonist model (22). After independently fitting portions of the models to estimated P_open versus [GABA] or [etomidate] data, model-generated P_open values in the presence of both GABA and etomidate were compared with experimental results (Figs. 5 and 6).

Partial Agonism—The partial agonist model is depicted in Fig. 5A. Six out of its seven free equilibrium parameters (K_G, K_E, a, b, _G, and _E) were directly fitted and are reported in the legend to Fig. 5. The remaining parameter, _GE, was optimized to a value of 100, to account for the large etomidate-induced leftward shifts in GABA concentration responses (Fig. 5D). Independent of _GE, the fitted partial agonist model predicts a biphasic relationship between etomidate concentration and GABA EC₅₀ with a minimum at about 30 µM etomidate. At etomidate concentrations near or above the fitted K_E value of 120 µM, is predicted to rise because of competitive occupation of agonist sites (Fig. 5, B and D).

We tried to model our P4S results (Fig. 4) using the partial agonist mechanism for etomidate. Estimated P_open versus [P4S] data were used to derive K_P, a, and _P values (reported in the legend to Fig. 5), and _PE was set 20-fold higher than _P, (i.e. equal to the optimal _GE/_G ratio). When these parameters were combined with fitted K_E, b, and _E values, the model predicted a large leftward shift but no significant increase in the maximum efficacy of P4S (Fig. 5E; _PE = 10). Further increasing _PE produced larger leftward shifts and increased maximal efficacy, but the competitive interaction between P4S and etomidate in the model kept the predicted efficacy of 1–10 mM P4S plus etomidate well below that of maximal GABA.

MWC Co-agonism—Unlike the partial agonist model, MWC co-agonism provides no a priori constraint on the number of etomidate sites. Fig. 6A depicts the model with two equivalent etomidate sites. Each MWC model is defined by the number of equivalent etomidate sites, n_E, and only five additional free parameters, L₀, K_G, c, K_E, and d. Only K_E and d are dependent on the number of etomidate sites in the model. All these models specify two equivalent GABA sites and therefore predict that GABA should show an inverse square root dependence on etomidate direct activation, / (16, 29, 35). Fig. 6B demonstrates that for oocytes exposed to 10, 32, or 100 µM etomidate, where both direct activation and were measured, an inverse square root relationship is indeed present (log-log slope = -0.48). Analysis using / instead of resulted in a similar fitted slope of -0.51 ± 0.083 but did not improve the correlation coefficient of the line (R = -0.82). Extrapolation of the fitted line in Fig. 6B to the average GABA value of 36 µM provided an estimate of GABA_A receptor spontaneous activation probability in the absence of both etomidate and GABA (the R O transition in Fig. 6A). The basal P_open estimate (P_o) was 1.4 x 10^-5, corresponding to an L₀ value of 70,000 that was used in fitting the four additional free model parameters (see "Experimental Procedures").

Fitting Equation 3 to pooled re-normalized GABA concentration-response data resulted in estimates for K_G = 57 µM and c = 1.5 x 10^-3. With n_E constrained to integers between 1 and 5, a set of K_E and d values (Table I) were fitted to etomidate direct activation data (Equation 4). The fit for the single etomidate site model was inferior to the two-site model fit (3-fold reduction in ²), but there was no appreciable improvement or degradation in the fits to models with more than two etomidate sites (Fig. 6F, circles). When model predictions for EC₅ enhancement (Fig. 6D) and EC₅₀ ratios (Fig. 6E) were compared with experimental results, it was apparent that the n_E = 2 model provided a much better fit than models with fewer or more etomidate sites (Fig. 6F, squares and triangles). The n_E = 2 model fit gave K_E = 35 µM and d = 7.7 x 10^-3.

A Test of MWC Co-agonism Using a Receptor Mutation That Alters Gating—An important feature of MWC gating models is their ability to account for the behavior of receptors containing mutations that display "constitutive" or "spontaneous" activation in the absence of agonist, by varying the basal equilibrium between closed and open channels (L₀). Indeed, Chang and Weiss (35) showed that an MWC allosteric gating mechanism with one fixed set of equilibrium GABA binding and efficacy values (K_G and c) could elegantly account for the apparent GABA sensitivities (EC₅₀ values) of oocyte-expressed wild-type ₁₂₂ GABA_A receptors and multiple mutant receptors containing substitutions at the highly conserved leucines of M2 domains (L9s). This was achieved by noting that as experimental estimates of the spontaneous opening probability (P_o = (1 + L₀)^-1) of mutant channels increased, their GABA EC₅₀ values decreased. Analogously, the MWC co-agonist model predicts that etomidate will appear to be a more potent and efficacious agonist when mutations that induce spontaneous gating are introduced into GABA_A receptors.

Oocytes expressing ₁(L264T)_22L receptors displayed large picrotoxin-sensitive "leak" currents in the absence of GABA, which are associated with spontaneously open GABA_A channels. The portion of the leak current associated with channel activity was estimated using picrotoxin (2 mM), which produced an apparent outward current (actually a reduced leak current) that averaged 10 ± 3.2% of the maximal GABA-activated current (Fig. 7, top). In oocytes expressing ₁(L264T)_22L receptors, etomidate concentrations as low as 10 nM directly elicited inward currents, and etomidate concentrations above 10 µM produced current amplitudes comparable with those elicited with high GABA (100 µM).

View larger version (31K): [in this window] [in a new window]   FIG. 7. Co-agonism accounts for etomidate activation of constitutively active mutant GABAA receptors. Top, current traces recorded from a single oocyte expressing 1L264T22L GABAA receptors are labeled with the conditions used for current activation. Picrotoxin (PTX) (2 mM) elicited an apparent outward current, due to inhibition of constitutively active channels. Inward currents from the same cell elicited with etomidate alone or with 1 mM GABA are also shown. Based on the average ratio of outward PTX currents and inward GABA currents in oocytes, we estimated L0 = 10 for 1L264T22L GABAA receptors. Bottom left, the MWC nE = 2 model was used to predict the interaction of GABA and etomidate in mutant channels. Parameters were those given in Table I for nE = 2, except for L0 = 10. Equation 5 was used to generate GABA concentration responses at various etomidate concentrations. The model predicts that very little enhancement of GABA currents can be observed in the presence of 3.2 µM etomidate, because etomidate alone activates over 90% of mutant receptors. Bottom right, etomidate direct activation of 1L264T22L GABAA receptors in oocytes. Points represent mean ± S.D. from at least five oocytes. The line represents the predictions of the wild-type nE = 2 MWC model with L0 = 10.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The major goals of this study were to examine both the concentration dependence and the mechanism of etomidate-induced modulation of GABA_A receptor activity. Voltage clamp electrophysiology data were mostly obtained from oocytes, providing rough estimates of receptor open probabilities. These were analyzed using binding-gating models that are characterized only by microscopic equilibrium constants between adjacent states. Based on comparison to parallel experiments in rapidly superfused HEK293 cells and patches (Figs. 1, 2, 3), our oocyte studies appear quite adequate for comparative evaluation of equilibrium mechanistic models that do not incorporate transition rates or receptor desensitization.

GABA Modulation Is Not Mediated by a High Affinity Site for Etomidate—Our first aim was to determine whether high affinity (K_d 10 µM) etomidate sites on GABA receptors mediate enhancement of GABA responses. The presence of these sites was suggested by previous studies of GABA modulation, which typically show half-maximal enhancement at 2–5 µM etomidate and maximal enhancement at 10–20 µM (e.g. Fig. 6D, squares) (19, 20, 25). In contrast, GABA EC₅₀ ratios (Fig. 3) do not reach a minimum at etomidate concentrations below 100 µM. These data suggest that GABA modulation saturates in the same etomidate concentration range (near 300 µM) where direct activation reaches its maximum (Fig. 1).

Many studies of GABA modulation by general anesthetics have used a single low GABA concentration, for instance, one that activates 5% of maximal responses (EC₅). Based on single channel estimates of at high [GABA] near 85% (31–33), the greatest observable anesthetic enhancement at GABA EC₅ would be 24-fold (100/0.05/0.85), and this measurement will underestimate modulation by compounds that enhance receptor activity more than 24-fold. Etomidate is clearly an example where this is the case. EC₅₀ ratios also reduce errors in measuring positive modulation caused by combined inhibiting and enhancing drug effects (37).

Etomidate Enhances GABA_A Receptor Gating—The observation that etomidate increases maximal P4S agonist efficacy (Fig. 4) indicates that etomidate shifts the equilibrium between agonist-bound closed and open GABA_A receptors toward the open state. The partial agonist P4S has been used previously to demonstrate enhanced gating of GABA_A receptors by propofol and volatile anesthetics (17, 38). This result is also consistent with single channel evidence for stabilization of GABA_A receptor open states in the presence of etomidate (24). Based on P4S modulation alone, we cannot rule out the possibility that etomidate also enhances P4S binding to agonist sites, but quantitative modeling suggests that a gating effect alone can account for reduced GABA and P4S EC₅₀ values (see below).

Single Site Equilibrium Models for Etomidate Actions on GABA_A Receptors—Our evidence that both GABA modulation and direct activation are maximized at similar etomidate concentrations adds quantitative support to the hypothesis that identical sites mediate both actions. Previous studies showed that both GABA modulation and direct activation display similar stereoselectivity for (R)(+)- versus (S)(-)-etomidate (19, 20, 26) and similar dependence on subunit isoform (9, 25). One amino acid at the 15' position of subunit M2 domains (Ser in ₁ and Asn in _2,3) was shown to influence both direct activation and GABA modulation in parallel (10, 27, 39, 40). The remarkable correspondence of affinity, stereoselectivity, and subunit dependence makes it highly unlikely that distinct etomidate sites mediate direct activation and GABA modulation. Previously, there has been both speculation about (40–42) and modeling of (21, 22) single site mechanisms to explain multiple general anesthetic effects on GABA_A receptors. For etomidate, the data presented above represent the first critical test of this hypothesis and comparison of the proposed mechanisms.

The partial agonist mechanism of Kurata et al. (21) suggests that two equivalent GABA activation sites (Fig. 5) display binding allosterism and also bind general anesthetics, resulting in direct activation, enhanced activation at low GABA concentrations, and inhibition at high GABA concentrations. The model enabled fitting of most free parameters to estimated P_open versus [GABA] or P_open versus [etomidate] data. To account for the large GABA EC₅₀ decreases induced by etomidate, this model required that the RGE state (Fig. 5A; receptors with one GABA and one etomidate bound) gate 20 times more efficaciously (_GE = 100) than the RG₂ state (_G = 5). In essence, this model explains multiple actions using a single type of etomidate site with a single binding parameter, K_E, but each action is mediated through distinct underlying mechanisms that are associated with different model parameters. Thus, direct activation is described by the gating efficacy parameter, _E. GABA enhancement under selected conditions is partially due to positive binding allosterism between agonist sites in the model (b <1), whereas most of the GABA enhancement in the model is driven by positive gating allosterism (_GE > _G). The optimized partial agonist model does a reasonable job predicting estimated P_open when both etomidate and GABA are present (Fig. 5, C and D).

However, there are significant problems with this and other partial agonist models. Our fitted model incorrectly predicts that GABA EC₅₀ reaches a minimum near 30 µM etomidate and rises at higher concentrations (Fig. 5D). The fitted etomidate parameters that adequately describe interactions with GABA clearly cannot simulate the interaction between etomidate and P4S, even when we postulate 100-fold gating enhancement when both P4S and etomidate are bound (Fig. 5E). Partial agonist models predict that all the actions of etomidate should display similar stereoselectivity, but the apparent inhibitory potencies of (R)(+)- versus (S)(-)-etomidate do not display the same stereoselectivity seen for direct activation and GABA modulation (19). Also, structure-function studies using subunit chimeras and point mutations suggest that etomidate (40) and other intravenous general anesthetics (41–43) activate GABA_A receptors via sites that are distinct from the GABA agonist sites. Thus, there are more reasons to reject than to accept partial agonist models for etomidate.

The MWC co-agonist mechanism (Fig. 6) is a simple model that incorporates symmetric allosteric channel gating by both GABA and etomidate at distinct and independent sets of sites. It is highly constrained and defined by only five equilibrium parameters, yet it predicts with remarkable accuracy the combined effects of GABA and etomidate on GABA_A receptors. Moreover, a single underlying mechanism (positive gating modulation) mediated by a single class of equivalent etomidate-binding sites accounts for both GABA modulation and direct activation in the model. Most important, the same etomidate binding and efficacy parameters that describe GABA modulation also correctly predict both the leftward shift and the increase in apparent efficacy of P4S in the presence of etomidate (Fig. 6G). Because agonist modulation (EC₅₀ ratio) is similar with either GABA or P4S, our results support an implicit feature of the MWC model, that the impact of etomidate on channel gating is independent of agonist site occupancy and agonist efficacy. Furthermore, the co-agonist model is consistent with both etomidate stereoselectivity studies and structure-function studies of GABA versus etomidate agonism.

In contrast to sequential binding-gating models of receptor activation, MWC models (16, 44) incorporate the assumption that unliganded receptors can spontaneously activate with some low probability P₀ = (1 + L₀)^-1. The L₀ value derived from the model-predicted relationship between etomidate direct activation and GABA EC₅₀ (Fig. 6B) has nearly 10-fold uncertainty. Nonetheless, it is within 2-fold of previous L₀ estimates for _122L receptors in oocytes (35) and HEK293 cells (29). Binding and efficacy parameters were derived by fitting independent estimated P_open versus [GABA] or [etomidate] data sets. Based on direct activation, EC₅ enhancement and EC₅₀ ratios, the n_E = 2 model best described experimental results (Fig. 6E). This outcome fits nicely with subunit substitution and site mutation studies that have located determinants of etomidate actions on GABA_A receptor subunits (9, 25) and others indicating the presence of two ₂ subunits per ₁₂₂ channel (45, 46).

Fitted efficacy parameters for the n_E = 2 model (Table I) suggest that etomidate binds about 130-fold (d^-1) more tightly to open versus closed receptors and that occupation of both sites therefore shifts the open-closed equilibrium 130² = 16,900-fold toward the open state, corresponding to a stabilization energy of -5.8 kcal/mol. The model also implies that GABA binds 670-fold (c^-1) more tightly to open versus closed receptor sites. Two GABAs shift the equilibrium 450,000-fold toward open states (-7.7 kcal/mol). The value for the model for GABA is 0.86, and adding etomidate increases toward 1.0, because the open state stabilization energies of etomidate and GABA are additive. Experimentally, we observed that etomidate enhances maximal GABA ac