Photomotor Responses in Zebrafish and Electrophysiology Reveal Varying Interactions of Anesthetics Targeting Distinct Sites on Gamma-Aminobutyric Acid Type A Receptors

Abstract

Background:

Etomidate, barbiturates, alphaxalone, and propofol are anesthetics that allosterically modulate GABA_A receptors via distinct sets of molecular binding sites. Two-state concerted co-agonist models account for anesthetic effects and predict supra-additive interactions between drug pairs acting at distinct sites. Some behavioral and molecular studies support these predictions, while other findings suggest potentially complex anesthetic interactions. We therefore evaluated interactions among four anesthetics in both animals and GABA_A receptors.

Methods:

We used video assessment of photomotor responses in zebrafish larvae and isobolography to evaluate hypnotic drug pair interactions. Voltage-clamp electrophysiology and allosteric shift analysis evaluated co-agonist interactions in α1β3γ2L receptors activated by GABA vs. anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA.

Results:

In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-mTFD-MPAB (mean ± SD α = 1.0 ± 0.07 and 0.96 ± 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean α range 0.76 to 0.89). Electrophysiologic allosteric shifts revealed that both propofol and R-mTFD-MPAB modulated etomidate-activated receptors much less than GABA-activated receptors [log(d, AN):log(d, GABA) ratios = 0.09 ± 0.021 and 0.38 ± 0.024, respectively], while alphaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alphaxalone was supra-additive (n = 6; P = 0.023 by paired t-test), but etomidate plus R-mTFD-MPAB or propofol was not.

Conclusion:

In both zebrafish and GABA_A receptors, anesthetic drug pairs interacted variably, ranging from additivity to synergy. Pairs including etomidate displayed corresponding interactions in animals and receptors. Some of these results challenge simple two-state co-agonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.

INTRODUCTION

Etomidate, barbiturates, alphaxalone, and propofol are general anesthetics that enhance GABA_A receptor activity, contributing to neuronal circuit effects underlying sedation and hypnosis.¹ GABA_A receptors are pentameric ligand-gated chloride ion channels.² In typical synaptic α1β3γ2L receptors, subunits are pseudo-symmetrically arranged β-α-β-α-γ counterclockwise from an extracellular perspective. Mutant function, photolabeling, substituted cysteine modification-protection, and structural imaging studies indicate that general anesthetics bind in multiple GABA_A receptor sites formed at transmembrane subunit interfaces.^3–5 Although each approach has limitations, the majority of data indicate that etomidate selectively binds in outer transmembrane β+/α- interfaces; the potent barbiturate R-mTFD-MPAB selectively binds in outer transmembrane α+/β- and γ+/β- interfaces; propofol binds in all four etomidate and R-mTFD-MPAB sites; and alphaxalone selectively binds in inner transmembrane β+/α- interfaces (Fig. 1).

Figure 1: Multiple Distinct Sets of Anesthetic Binding Sites on Synaptic GABA_A Receptors.

Structural features of αβγ synaptic GABA_A receptors are depicted schematically. Subunits are arranged pseudo-symmetrically around the chloride ion channel (grey oval), labeled and color-coded α (yellow), β (blue), and γ (green). Each subunit’s extracellular domain is shown as an oval and the transmembrane domain as a rhomboid solid. The locations of transmembrane helical elements (M1 through M4) also are shown within each subunit’s transmembrane domain, and visible “+” (adjacent to M3) and “–” (adjacent to M1) inter-subunit faces are labeled. Anesthetics in their binding pockets are also depicted as follows: etomidate in outer transmembrane β+/α- pockets (red circles); alphaxalone in inner transmembrane β+/α- pockets (blue ovals); and R-mTFD-MPAB in outer transmembrane α+/β- and γ+/β-- pockets (green boxes). Propofol binding sites overlap with those for both etomidate and R-mTFD-MPAB.

Concerted two-state co-agonist models account for the functional effects of etomidate, propofol, and pentobarbital on synaptic GABA_A receptors.^6–8 These anesthetics allosterically potentiate receptor activation by GABA and at high concentrations activate receptors without GABA.⁹ This mechanism predicts independent and additive receptor-activating energy contributions from anesthetics binding at distinct co-agonist sites, resulting in synergistic (supra-additive) interactions among drug pairs.^9,10 Alternatively, additive interactions are predicted for drug pairs competing for shared sites.^10,11 These predictions are supported by electrophysiologic studies of several anesthetic combinations in GABA_A receptors.^12,13. Studies using Xenopus tadpole loss-of-righting reflexes and isobolographic analysis to assess interactions among hypnotic drugs targeting several distinct sets of co-agonist sites on GABA_A receptors also reported results consistent with concerted model predictions, including synergy between propofol + etomidate and propofol + R-mTFD-MPAB pairs where mixed interactions might be anticipated.¹⁴

Other functional and structural evidence suggests potentially complex interactions between GABA_A receptor sites targeted by different anesthetics. Quantitative electrophysiologic data analyzed using allosteric models revealed that the energetic effects of anesthetic site mutations were not additive.¹⁵ Bulky mutations reduced the efficacies of abutting drugs as expected, but also weakened anesthetic effects at distant sites. Furthermore, the concept that receptors adopt only a few shared conformations is challenged by cryo-electron microscopic structures of α1β2γ2L GABA_A receptors bound to GABA, etomidate, propofol, and phenobarbital.⁵ While the locations of anesthetic binding sites in cryo-electron microscopy structures largely agree with other studies, they also indicate that etomidate and propofol occupation of β+/α- sites is associated with different configurations of the α+/β- and γ+/β- barbiturate sites. Thus, anesthetic interactions in GABA_A receptors might vary depending on crosstalk between sites.

In this study, we evaluated pair-wise interactions of etomidate, R-mTFD-MPAB, propofol and alphaxalone in both animals and GABA_A receptors. Hypnosis in large groups of zebrafish larvae was tested using automated video quantification of motor responses¹⁶ to inverted photic stimuli (“dark flashes”), suitable for isobolographic analysis. Electrophysiology in human α1β3γ2L GABA_A receptors was used to compare anesthetic interactions with other anesthetics versus GABA using quantitative allosteric shift analysis.

METHODS

Animals:

Zebrafish (Danio rerio, Tubingen AB strain) were used with approval from the Massachusetts General Hospital Institutional Animal Care and Use Committee (protocol #2014N000031) and in accordance with ARRIVE guidelines. Adult zebrafish were mated to produce embryos and larvae as needed. Embryos and larvae were maintained in Petri dishes (140 mm diameter, less than 100 per dish) filled with E3 medium (in mM: 5.0 sodium chloride, 0.17 potassium chloride, 0.33 calcium chloride, 0.33 magnesium sulfate, 2 HEPES, pH 7.4) in a 28.5°C incubator under a 14/10 h light/dark cycle. The light intensity inside the incubator ranged from 3000–4000 lux (measured with a calibrated light meter from Thermo Fischer, Waltham, MA). Experiments were performed on larvae at 7 days post-fertilization. After use in experiments or at 8 days post-fertilization, larvae were euthanized.

Xenopus laevis oocytes were used in electrophysiology experiments. Female frogs were housed in a veterinarian supervised facility and used with approval from the Massachusetts General Hospital Institutional Animal Care and Use Committee (protocol #2005N000051). Oocytes were harvested via mini-laparotomy from frogs anesthetized by immersion in 0.2% Tricaine, in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Materials:

Propofol (2,6 di-isopropyl phenol), GABA and salts were purchased from Sigma-Aldrich (St. Louis, MO). R-etomidate (a gift from Prof. Douglas Raines (Department of Anesthesia Critical Care & Pain Medicine, Massachusetts General Hospital, Boston, MA) was synthesized by Bachem America (Torrance, CA). R-mTFD-MPAB (R-5-allyl-1-methyl m-trifluoromethyl mephobarbital) was synthesized by KareBay Biochem Inc. (Monmouth Junction, NJ). Alphaxalone was purchased from Steraloids Inc. (Newport, RI). Anesthetic stock solutions in dimethylsulfoxide were stored at −20ºC and diluted to final concentration in experimental buffers on the day of use. Coding DNAs (cDNA) for human GABA_A receptor wild-type α1, β3, and γ2L subunits and mutants (α1S270I and γ2LS280W) were cloned in pCDNA3.1 vectors.^15,17 Capped messenger RNAs were synthesized on linearized cDNA templates using mMessage mMachine kits (Sigma- Aldrich) and stored in nuclease-free water at −80°C.

Overview of Study Design

Binarized zebrafish larvae motor responses to photic stimuli were measured in quadruplicate for each animal. Development of the inverted photomotor response assay used approximately 2000 zebrafish larvae. We next established hypnotic drug IC50s by varying anesthetic concentrations (n = 752 to 1680 larvae per drug, for 4 anesthetics). Drug pair experiments were then performed with one drug fixed at a fraction of its IC50 and the other varied. Each such drug pair study was used to calculate a pairwise interactive parameter, α (n = 240 to 480 larvae per α determination). Six or seven such α-values under different fixed drug conditions were combined for isobolographic analysis of each drug pair (n = 1440 to 1920 larvae per isobologram). Six anesthetic pairs were studied.

Drug interactions in α1β3γ2L GABA_A receptors were studied using voltage-clamp electrophysiology in Xenopus oocytes. We compared anesthetic modulation of receptors activated by GABA vs. another anesthetic as primary agonist, based on fitted log(d) values from allosteric co-agonist shift analyses. The ratio of log(d) values indicated whether anesthetic pairs interacted in accord with co-agonist models. For these experiments, estimated open probability was assessed at 9 to 11 primary agonist concentrations in 5 oocytes per concentration-response curve. Concentration-dependent activation by 4 primary agonists (GABA, etomidate, propofol, and R-mTFD-MPAB) and 11 studies of anesthetic modulation (4 with GABA, 3 with etomidate, 3 with propofol, and one with R-mTFD-MPAB) were assessed (n = 75 oocytes in total). Another set of experiments assessed anesthetic additivity in receptors activated by low GABA, using an isobolographic approach (3 drug pairs; n = 6 oocytes per anesthetic pair).

Activity tracking of zebrafish larvae:

Single zebrafish larvae (7 days post-fertilization, sex indeterminate) were randomly selected and placed into wells of standard 96-well plates containing 150 µL E3 buffer. Anesthetic stocks were diluted in E3 buffer to four times final concentrations and 50 µL of 4x solution was transferred to each well using a multi-pipetter, bringing the final well volumes to 200 µL. Drug transfers took less than 5 minutes per plate. All control and final drug solutions contained 0.1% dimethylsulfoxide. Each 96-well plate was placed in a Zebrabox (Viewpoint Life Sciences, Montreal, Canada) and maintained at 28°C in a thermostatically controlled water bath. The duration of acclimation to the Zebrabox environment was varied during assay development and set at 30 min for experiments. During experiments, Zebralab v5.15 software (Viewpoint Life Sciences) coordinated stimuli while recording and integrating the motor activity of individual larvae during programmed epochs, using infrared video data as previously described.¹⁸

Inverted photomotor responses:

To assess the hypnotic effects of anesthetics singly and in pairs for isobolographic analyses, we used an inverted photomotor response assay that provided a baseline response probability near 100%. Larvae in groups of 8 were placed in E3 buffer containing no drug or a specified drug solution. Larvae were acclimated in a Zebrabox illuminated with white light at 4000 lux for 30 min while spontaneous activity stabilized. During inverted photomotor response trials, baseline activity was measured for 10 s followed by a 1 s exposure to darkness (< 15 lux) followed by renewed exposure to bright white light. Trials were repeated four times (up to ten times during assay development) with 3 min recovery periods between trials. Activity was integrated over 0.5 s epochs during baseline periods and 0.1 s epochs during each “dark flash”. For analysis, activity values were normalized to epoch duration. To establish binary outcomes, the means and standard deviations for normalized activity during pre-stimulus basal periods (80 total epochs) were calculated for individual larvae. Binary responses for each trial were scored as positive (1) if normalized activity in any of the ten 0.1-s epochs during a dark flash exceeded the upper 99% CI (mean + 2.8 × SD, using a Bonferroni adjustment for four comparisons) for normalized basal activity. Otherwise, the photomotor response response was scored as negative (0). Cumulative response probability (P_Resp) for each larva was calculated by averaging its four binary trial results. For statistical analyses, results from all larvae in each exposure group were pooled.

Drug concentration-response studies in zebrafish larvae:

P_Resp results for all larvae within exposure groups (mean ± 95% CI; n ≥ 24 per condition; 240 to 1680 larvae per concentration-response study) were plotted against log[drug]. The data were fitted to three-variable logistic functions (Eq. 1) using nonlinear least-squares in GraphPad Prism version 8 (GraphPad Software, San Diego, CA).

$${\text{P}}_{\text{Resp}}=\frac{{\text{P}}_{\text{max}}}{1+{10}^{\left(\log \left[\text{Drug}\right]-\log {\text{IC}}_{50}\right)\times \text{nH}}}$$ Eq. 1

Logistic fits calculated mean and standard errors values for maximum response probability at 0 drug (P_max), log half-maximal inhibitory concentration (log[IC₅₀]), and Hill slope (nH). The software also calculated the log drug concentration at which P_Resp = 0.5 (log[IC50]), and 95% confidence intervals for IC₅₀ and IC50 and other parameters. Note that IC₅₀ and IC50 (drug concentration resulting in 50% response probability) are identical when P_max = 1.0 but diverge when P_max < 1.0.

Isobolographic analyses:

We adopted the approach used by Kent et al.¹⁴ IC50 values for photomotor response inhibition by single drugs were determined multiple times. An initial set of single-drug IC50s was established to guide subsequent experiments on drug pairs. In drug pair experiments, one drug was held at a fixed fraction (0.25, 0.5 or 0.75) of its IC50 while the other drug was varied. Each pair was studied with both drugs fixed at 3 or more values and the other varied, resulting in 6 or more separate concentration-response studies per combination. The IC50 for the varied drug (at P_Resp = 0.5) was calculated as described (n = 240 to 480 larvae for each concentration-response; n = 1440 to 1920 per combination). Single-drug experiments with the varied drug were performed at least once on the same day as drug pair experiments, using larvae from the same batches. These single-drug data were then combined with earlier results, and an updated overall single-drug IC50 was calculated (final n = 752 to 1680 larvae per drug). After all pairs were studied, fixed drug concentrations were recalculated as fractions of final IC50s.

Two-dimensional isobolograms display plotted pairs of drug concentrations, each normalized to its single-drug IC50, that produce equivalent effects, namely P_Resp = 0.5. The sum of normalized drug concentrations is an interaction index, α (Eq. 2). If α is 1.0, the drug interaction is additive and if α is less than 1.0, the interaction is supra-additive (synergistic).¹⁹

$$\alpha =\frac{\left[\text{A}\right]}{\text{IC}{50}_{\text{A}}}+\frac{\left[\text{B}\right]}{\text{IC}{50}_{\text{B}}}$$ Eq. 2

GABA_A Receptor Expression in Xenopus Oocytes:

Harvested Xenopus oocytes were treated with collagenase and defolliculated as previously described,¹⁵ then microinjected with 0.5 to 1.0 ng of mRNA mixtures in ratio 1α:1β:5γ. Before use in electrophysiologic experiments, injected oocytes were maintained for up to 48 hours at 18°C in ND-96 (in mM: 96 NaCl, 2 KCL, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, pH 7.4) supplemented with 0.05 mg/ml gentamicin and 0.01 mg/ml ciprofloxacin.

Oocyte Electrophysiology:

Two microelectrode voltage-clamp electrophysiology was performed at room temperature (21–23°C) as previously described.¹⁵ Oocytes in a 0.2 ml flow chamber were impaled with glass microelectrodes filled with 3M KCl (tip resistance 0.5–2 MΩ) and clamped at −50 mV (model OC-725C; Warner Instruments). Gravity-driven solutions in ND-96 were delivered at 4 ml/min from glass syringes via PTFE tubing and a micro-manifold. LabScribe 2 software and an RA834 interface (both from iWorx, Dover, NH) coordinated timing of solutions through valves and digitized currents at 200 Hz. Current data were digitally filtered with a 10-Hz low-pass Bessel function and analyzed off-line using Clampfit8 software (Molecular Devices, San Jose, CA).

GABA_A receptor agonist concentration-responses:

Voltage-clamp electrophysiology in Xenopus oocytes expressing α1β3γ2L GABA_A receptors was used to compare anesthetic modulation of GABA-activated receptors and receptors activated with another anesthetic as primary agonist (etomidate, propofol or R-mTFD-MPAB). Modulation of primary agonist responses was tested in the presence of 3.2 µM etomidate, 5 µM propofol, 2.5 µM alphaxalone or 8 µM R-mTFD-MPAB (n ≥ 5 oocytes per condition), which similarly modulate GABA-activated GABA_A receptors.²⁰ Voltage-clamped oocytes expressing GABA_A receptors were exposed to solutions of variable primary agonist concentrations alternating with maximal agonist controls (3 mM GABA, 1 mM etomidate, 300 µM propofol, or 150 µM R-mTFD-MPAB) with at least 5 minutes of ND-96 wash between recordings. After baseline correction, peak currents were normalized to the average of preceding and following control responses.

For modulation experiments, oocytes were pre-exposed to the modulating drug for 10 sec before activation with primary agonist + modulator. Peak currents were normalized to the average of preceding and following responses to maximal agonist + modulator.

To explore whether the two R-mTFD-MPAB binding sites (α+/β- and γ+/β- transmembrane interfaces) differentially contributed to etomidate-R-mTFD-MPAB interactions, we studied etomidate-dependent activation in the absence and presence of 8 µM R-mTFD-MPAB in oocytes expressing α1S270Iβ3γ2L and α1β3γ2LS280W GABA_A receptors, using the same approach used for wild-type receptors.

Estimated Open Probability Calculations:

Estimated open probability P_open was calculated as previously described.¹⁵ This calculation explicitly adds spontaneous basal channel activity and re-normalizes experimental peak currents to account for maximal agonist efficacy, based on anesthetic-enhanced maximal GABA-induced currents that are assumed to represent 100% activated channels (Eq. 3).

$${\text{P}}_{\text{open}}=\frac{\frac{\text{I}}{{\text{I}}_{\text{GABA}}^{\max }}+\frac{{\text{I}}_{\text{PTX}}}{{\text{I}}_{\text{GABA}}^{\max }}}{\frac{{\text{I}}_{\text{GABA}+\text{Mod}}^{\text{max}}}{{\text{I}}_{\text{GABA}}^{\max }}+\frac{{\text{I}}_{\text{PTX}}}{{\text{I}}_{\text{GABA}}^{\max }}}$$ Eq. 3

Spontaneous channel activation was assessed in wild-type and mutant GABA_A receptors by measuring outward currents from oocyte-expressed receptors exposed to 2 mM picrotoxin, which inhibits channel activity. Picrotoxin-sensitive currents were normalized to maximal inward currents elicited with 3 mM GABA ($\frac{{\text{I}}_{\text{PTX}}}{{\text{I}}_{\text{GABA}}^{\max }}$; n = 5 oocytes per receptor type). Maximal agonist efficacies in wild-type receptors were assessed by activating oocyte currents with maximally activating concentrations (see above) and normalizing to currents elicited with 3 mM GABA + a modulator ($\frac{{\text{I}}_{\text{GABA}+\text{Mod}}^{\text{max}}}{{\text{I}}_{\text{GABA}}^{\max }}$; n = 5 oocytes per condition). Alphaxalone or etomidate-enhanced GABA responses were used to assess GABA and etomidate maximal efficacy in α1S270Iβ3γ2L and α1β3γ2LS280W mutant receptors.

Analysis of Electrophysiologic Concentration-Responses:

P_open data (mean ± 95% CI; n ≥ 5 per condition) was plotted against log([Agonist]) and parametrically analyzed by fitting to four-variable logistic equations (Eq. 4) using non-linear least squares (GraphPad Prism). Fitted parameters reported from these fits include log(EC₅₀), which was used to calculate 95% confidence intervals for EC₅₀, nH, P_min (non-zero in some mutant receptors) and P_max. Pairwise parameter comparisons between fits to different data sets were performed using F-tests with P = 95% as a significance threshold.

$${\text{P}}_{\text{open}}=\frac{{\text{P}}_{\max }-{\text{P}}_{\min }}{1+{10}^{\left(\log \text{EC}50-\log \left[\text{Agonist}\right]\right)*\text{nH}}}+{\text{P}}_{\min }$$ Eq. 4

To compare allosteric gating energy shifts caused by modulating anesthetics with different primary agonists, we utilized Monod-Wyman-Changeux (MWC) log(d) shift analysis as described previously.¹⁵ This approach simultaneously fits agonist-dependent P_open results in the absence and presence of a specified modulator concentration to a simplified two-state (resting and active) co-agonist model (Eq. 5).¹⁵

$${\text{P}}_{\text{open}}=\frac{1}{1+{\text{L}}_0\times {\left(\frac{1+\left[\text{Agonist}\right]/{\text{K}}_{\text{Ag}}}{1+\left[\text{Agonist}\right]/{\text{cK}}_{\text{Ag}}}\right)}^2\left(\frac{1+\left[\text{Mod}\right]/{10}^{-6}}{1+\left[\text{Mod}\right]/{10}^{\left[\log \left(\text{d}\right)-6\right]}}\right)}$$ Eq. 5

L₀ is the equilibrium constant for resting:active receptors in the absence of ligands. K_Ag is the dissociation constant for primary agonist binding to resting receptors and c is the ratio of dissociation constants in active vs. resting receptors, reflecting agonist efficacy. Log(d) is a fitted model parameter that is proportional to the allosteric gating free energy contributed by the modulating drug at its specified concentration. Modulator-induced EC₅₀ shifts and changes in both P_min and P_max are incorporated into the single log(d) parameter, making it suitable for quantitative comparison of modulator effects on receptors activated with different primary agonists or in different receptor types.^15,21 For analyses of results in wild-type, we compared log(d, GABA) for a modulator when GABA was the primary agonist to log(d, AN) when another anesthetic was the primary agonist. Fits to allosteric shift models designated the modulator as a binary factor (0 if absent, 1 if present) and were performed using non-linear least squares in Origin 6.1 (OriginLab, Northampton, MA). The models were constrained to two primary agonist (GABA or anesthetic) sites and a single modulatory (co-agonist) site. L₀ was constrained to a value of 25,000 for wild-type receptors²¹ and for mutant receptors was calculated based on spontaneous P_open estimates (L₀ = [1-P₀]/P₀).

All the anesthetics we studied act as co-agonists with GABA in α1β3γ2L GABA_A receptors, resulting in supra-additive activation. Two-state co-agonist models predict that a modulator at a selected concentration should result in the same log(d) value with any primary agonist. Thus a ratio of log(d, AN):log(d, GABA) near 1.0 is indicative of strict energy additivity and activation synergy between the co-agonist modulator and the anesthetic primary agonist. In calculating log(d) ratios, standard errors were propagated as described by Bevington and Robinson (2002).²² To estimate log(d) values for additive (competitive) drug interactions, we used the primary agonist logistic model for etomidate and calculated the effect of adding 3.2 µM etomidate as a modulator to the varied etomidate agonist concentrations. The etomidate agonist model combined with its “concentration-enhanced” model (20 points each) was then fitted with Eq. 5. The resulting log(d) value (−0.008 ± 0.010) was not significantly different from zero. Thus, a log(d, AN) near zero is indicative of a strictly additive interaction between the modulator and the primary agonist anesthetic, consistent with competition for shared agonist sites.

Interactions of Hypnotic Range Drug Combinations in GABA-Activated α1β3γ2L GABA_A Receptors:

We also electrophysiologically assessed whether etomidate combined with R-mTFD-MPAB, propofol, or alphaxalone produced supra-additive modulation of low GABA responses in Xenopus oocytes expressing α1β3γ2L receptors. These experiments used approximately equi-effective drug concentrations that were close to the hypnotic IC50s observed in zebrafish larvae. We first compared the effects of 10 µM GABA (∼EC8) alone and combined with each drug individually at its zebrafish IC50 in independent sets of 3 oocytes. We then adjusted anesthetic concentrations to produce responses to 10 µM GABA + anesthetic near 40% of maximal GABA activation (3 mM) in additional sets of oocytes. This process identified 0.50 µM etomidate, 1.70 µM propofol 1.03 µM alphaxalone and 1.85 µM R-mTFD-MPAB as approximately equally modulatory, based on one-way ANOVA and Tukey’s multiple comparisons (n = 5 each).

We next tested whether drug pairs were supra-additive in separate sets of 6 oocytes for each combination. To illustrate, in one set of oocytes we measured currents elicited with 10 µM GABA + 0.5 µM etomidate, 10 µM GABA + 1.03 µM alphaxalone, and 10 µM GABA + 0.25 µM etomidate + 0.51 µM alphaxalone (a 1:1 mixture of the first two solutions). The other drug combinations were similarly tested in separate groups of six oocytes. Anesthetics were pre-applied to oocytes for 10 s before activation using combinations of GABA + anesthetics, and normalizing responses to 3 mM GABA were recorded both before and after each GABA + anesthetic response. To control for the order of drug applications, each oocyte in a set of six was studied using a different permutation of the 3 drug-containing solutions.

EC1 Enhancement Ratios:

We used data from electrophysiologic concentration-responses in the presence and absence of modulators to estimate EC1 enhancement ratios. Primary agonist concentration responses were examined to identify concentrations that produced activation with P_open in the range 0.5% to 1%. P_open data from studies with modulator plus the same primary agonist concentration were then identified. Each data set was used to calculate mean ± sd and the EC1 enhancement ratio was calculated as P_open with modulator:P_open without modulator. Standard deviations for ratios were propagated according to Bevington & Robinson.²²

Statistical Methods and Scientific Rigor:

Hypnotic concentration-response studies in zebrafish larvae were performed in multiple groups of randomly selected larvae (sex indeterminate) from at least 3 separate breeding clutches, and included 9 to 11 concentrations (n = 24 to 48 larvae per experimental condition; n = 240 to 480 per study). No a priori statistical power calculations were conducted. The number of zebrafish larvae studied was based on our prior experience with this approach. Photomotor response outcomes in individual zebrafish larvae were binarized based on quantitative and automated motion analysis, eliminating observer bias. Investigator blinding was not used in performance of experiments or analyses. Each animal was tested four times. No corrections were made for larvae that may have died during experiments. For isobolographic analyses of each drug pair, six or seven conditions from separate sets of trials were tested, with one drug fixed and the other varied. Controls with no drugs were also included in every 96 well plate to confirm high photomotor response probability. Between 1440 and 1920 zebrafish larvae (5760 to 7680 photomotor response trials) were studied to generate multiple independent α-values for each of the 6 drug pairs, from which averages and variances were calculated. Each set of α-values was compared to the additivity model (α = 1.0) using a single sample Students t-test. Supra-additivity was inferred for a drug pair when the upper 95% confidence interval for mean α was less than 1.0. This also corresponded with the majority of confidence intervals for individual α-values in a drug pair set below 1.0.

In electrophysiologic experiments, the number of oocytes studied for each condition (n ≥ 5) was based on our extensive prior experience with this method. Oocytes were randomly assigned to groups and tested for adequate and stable receptor expression before experiments. Results were normalized to frequently measured within-oocyte controls to account for variations in functional receptor expression levels. Non-linear least squares fits to normalized electrophysiologic results (Eqs. 4 and 5) were performed in GraphPad Prism v8 or MicroCal Origin 6.1. Statistical comparisons based on ANOVA, F-tests, and t-tests, were performed in GraphPad Prism. The threshold for statistical significance was P < 0.05.

To determine whether anesthetic pairs at low concentrations produced supra-additive effects on GABA_A receptors, we used a variation of isobolography. In sets of 6 oocytes, we compared within-oocyte averaged normalized peak responses recorded with GABA + single anesthetics to the normalized response elicited by the 1:1 mixture of GABA + anesthetics (i.e. GABA + two anesthetics, each at half concentration), using paired Students t-tests (GraphPad Prism 8). Supra-additivity was inferred when the combination of two drugs at half concentrations was significantly (p < 0.05) larger than the average responses to individual drugs alone at approximately equally modulating concentrations.

Correlations between zebrafish α values and metrics of receptor modulation ([og(d) and EC1 enhancement] were calculated as Pearson r values in GraphPad Prism 8.

RESULTS

Inversion of Zebrafish Larvae Photomotor Response Assay Increases Response Probability and Affects Sedative-Hypnotic Potencies

Binarized photomotor responses in our prior studies of hypnotic drugs in zebrafish larvae were based on movement of dark-adapted animals following brief exposures to bright white light.¹⁶ Using that approach, control response probabilities were under 90% and diminished with repeated trials. However, consistent control response rates near 100% are desirable for studying pharmacodynamic interactions between sedative-hypnotic drugs using isobolographic analyses. It is known that zebrafish larvae are more active in lighted than dark environments and reliably respond to sudden darkness with vigorous movements.^23,24 We therefore tested response probabilities using an inverted photomotor response assay, with larvae maintained in a lighted environment and exposed to brief periods of darkness (dark flashes). Initial trials showed that inverted photomotor response probabilities were consistently near 95% for zebrafish adapted to white light intensities ranging from 100 to 5000 lux. We chose to use 4000 lux white background light to approximate the conditions used in cultivating zebrafish embryos and larvae. In comparison to traditional photomotor response, inverted photomotor response resulted in consistently higher initial probabilities of movement and a significantly smaller drop in cumulative response probability with repeated trials at 3-minute intervals (Fig 2A). Varying the period of environmental adaptation before testing motor responses to photic stimuli revealed unchanged “dark flash” response probability with longer adaptation periods in light but diminishing light flash response probability with longer dark adaptation periods (Fig 2B), as previously reported.¹⁶ This suggested that exposure to a dark environment alone caused zebrafish larvae to become sedated and less responsive to photic stimuli, possibly through onset of natural sleep. Consistent with this idea, we found that anesthetic IC50s for sedation (reduced spontaneous activity) in a lighted environment (Table 1) were much higher than when measured in dark-adapted larvae¹⁸ and also higher than IC50s for hypnosis (reduced responses to photic stimuli) using either approach (Fig 2C; Table 1).

Figure 2: Inverted Photomotor Responses in Zebrafish Larvae.

Panel A compares the cumulative response probabilities for our previous photomotor response (open squares) assay and the inverted photomotor response (filled circles) assay adopted for isobolographic analyses. Each point represents the cumulative mean and 95% CI (n = 96 each) for zebrafish larvae subjected to 10 inverted or traditional photomotor response trials with 3-minute recovery periods between trials. Lines are linear regression fits. Inverted photomotor response is characterized by a high response probability (intercept = 0.98 ± 0.010) that diminishes little with repetitive trials (slope = −0.0046 ± 0.0016). Traditional photomotor response response probability is lower (intercept = 0.74 ± 0.021) and diminishes more with repetitive trials (slope = -0.016 ± 0.0033). Panel B shows the effect of varying pre-trial adaptation to either a lighted environment (filled circles) or a dark environment (open squares). Each point represents the cumulative response probability (mean and 95% CI) for 96 zebrafish larvae subjected to 4 trials with 3-minute recovery periods between trials. Lines are linear regression fits. Again, inverted photomotor response is characterized by a high response probability (intercept = 0.91 ± 0.022) and negligible change with increased adaptation time (slope = 0.0003 ± 0.0006 min⁻¹). Traditional photomotor response is characterized by a lower initial response probability (intercept = 0.70 ± 0.036) that diminishes with increased adaptation time (slope = −0.0071 ± 0.00093 min⁻¹). Symbols without error bars indicate small variances. Panel C shows etomidate-dependent inhibition of inverted photomotor responses (solid red circles) and spontaneous activity (open red squares). Inverted photomotor response data represents the average probability (mean and 95% CI) for 4 trials per animal in 96 to 128 animals per concentration. Spontaneous Activity data represents normalized averaged activity integration from 80 0.5-s baseline epochs in the same zebrafish, using the zero etomidate group for normalization. Lines through data are non-linear least squares fits to Eq. 1 (Methods) and fitted parameters are reported in Table 1.

Table 1:

Hypnotic and Sedative Population IC50s* in Zebrafish Larvae Based on Inverted Photomotor Responses

Anesthetic Drug	Inverted Photomotor Response IC50 (µM) [95% CI]	Spontaneous Activity IC50 (µM) [95% CI]	# animals
Alphaxalone	1.02 [0.97 to 1.06]	2.2 [1.9 to 2.7]	1680
Etomidate	0.34 [0.33 to 0.36]	0.67 [0.52 to 0.86]	1336
R-mTFD-MPAB	1.8 [1.6 to 1.9]	2.8 [2.0 to 4.3]	1464
Propofol	0.55 [0.51 to 0.59]	1.3 [1.0 to 1.7]	752

IC50 values are defined as the drug concentration resulting in 50% response. Note that when the control probability of response (P_max) is below 1.0, IC50 values are lower than IC₅₀ values that represent half-maximal effect concentrations. IC₅₀ values were calculated in non-linear least squares fits to 3-parameter logistic equations (Eq. 1 in Methods) with P_max, IC₅₀, and Hill slope (nH) as free parameters. Population IC50 values and 95% confidence intervals were calculated from these fits. Each animal was studied in a single condition with 4 repeated binary outcomes, resulting in 5 possible response probabilities (0, 0.25, 0.5, 0.75, 1.0).

Isobolographic Analyses of Hypnotic Combinations in Zebrafish Larvae Reveal both Supra-Additive and Additive Interactions

We assessed the interactions of etomidate, alphaxalone, R-mTFD-MPAB, and propofol alone and in paired combinations in 7 days post-fertilization zebrafish larvae using inverted photomotor response hypnosis assays. Multiple single-drug concentration-responses, including on days when two drugs were studied in combination, were performed to define IC50 values (Table 1). Multiple drug pair interaction studies, with one drug fixed at a fraction of its IC50 and the other drug varied were performed. IC50s fitted to the varied drug were used to calculate α values (Eq. 2).

Figure 3 illustrates experimental data for etomidate as the varied drug alone (red circles) or combined with 0.43 µM R-mTFD-MPAB (∼0.24 x IC50; green squares) or 0.3 µM alphaxalone (∼0.29 x IC50; blue triangles). For larvae studied with etomidate alone in these trials, the control (no drug) response probability was 94% [95% CI = 92 to 97%]. The fitted etomidate IC₅₀ = 0.36 µM [95% CI = 0.33 to 0.38] results in a 47% (half-maximal) response probability, while the IC50 eliciting 50% response probability (dotted line) is 0.35 µM [95% CI = 0.32 to 0.37]. Unsurprisingly, response rates at 0 etomidate are slightly lower in the presence of R-mTFD-MPAB or alphaxalone. The IC50 for etomidate combined with 0.43 µM R-mTFD-MPAB is 0.23 µM [95% CI = 0.21 to 0.26] and combined with 0.3 µM alphaxalone is 0.15 µM [95% CI = 0.13 to 0.17]. The calculated α value from these data for R-mTFD-MPAB + etomidate is 0.93 and for alphaxalone + etomidate is 0.74. Six or more such drug pairs with different drugs held constant were studied to construct isobolograms (Fig 4) and calculate mean ± sd α values for each set of experiments for a drug pair (Table 2).

Figure 3: Inverted Photomotor Response Data with Drug Combinations for Isobolographic Analysis.

The figure shows etomidate dependent response probabilities assessed with etomidate alone (red circles; n = 40 larvae per concentration) or combined with either alphaxalone (blue triangles; n = 24 larvae per concentration) or R-mTFD-MPAB (green squares; n = 30 larvae per concentration) at about 25% of their respective IC50s. Data points represent mean and 95% CI for the average of 4 trials at each condition (etomidate data is a subset of that shown in Fig 2). Lines through data represent non-linear least squares fits to Eq. 1. Shaded areas around lines represent 95% confidence intervals to fits. The dotted line at 50% response probability identifies the isobolographic condition (IC50) calculated from fits. Fitted parameters [95% CI] were: for etomidate alone: P_max = 0.94 [0.92 to 0.97], IC₅₀ = 0.36 µM [0.33 to 0.38] and IC50 = 0.35 µM [0.32 to 0.37]; for etomidate + 0.43 µM R-mTFD-MPAB: P_max = 0.93 [0.88 to 0.99], IC₅₀ = 0.25 µM [0.22 to 0.29] and IC50 = 0.23 µM [0.21 to 0.26]; for etomidate + 0.30 µM alphaxalone: P_max = 0.89 [0.84 to 0.95], IC₅₀ = 0.18 µM [0.15 to 0.22] and IC50 = 0.15 µM [0.13 to 0.20].

Figure 4: Isobolograms Summarizing Hypnotic Drug Combinations Tested in Zebrafish.

Each panel depicts results from inverted photomotor response studies in zebrafish for one of six pairs of hypnotic drugs. Symbols represent mean and 95% CI data normalized to each drug’s IC50, with the experimentally varied drug indicated by shape, color, and error bars: etomidate (red circles), alphaxalone (blue triangles), R-mTFD-MPAB (green squares) and propofol (open hexagons). Calculated α-values (Eq. 2 in Methods) for each experimental condition and α-value means with 95% CI for each drug pair are reported in Table 2.

Table 2:

Fixed Concentrations, IC50s and α Values for Anesthetic Combinations in Larval Zebrafish

Alphaxalone [95% CI] (µM)	Etomidate [95% CI] (µM)	R-mTFD-MPAB [95% CI] (µM)	Propofol [95% CI] (µM)	α [95% CI]	Average α ± SD P-value
0.24	0.22 [0.18 to 0.25]	---	---	0.87 [0.78 to 0.95]
0.3	0.15 [0.13 to 0.17]	---	---	0.74 [0.68 to 0.80]
0.56	0.11 [0.083 to 0.14]	---	---	0.86 [0.79 to 0.94]
0.7	0.019 [0 to 0.05]	---	---	0.73 [0.59 to 0.83]
0.58 [0.50 to 0.66]	0.1	---	---	0.85 [0.78 to 0.93]
0.24 [0.17 to 0.33]	0.175	---	---	0.74 [0.67 to 0.83]	0.83 ± 0.096
0.24 [0.17 to 0.34]	0.26	---	---	1.00 [0.92 to 1.09]	P = 0.003
0.25	---	0.88 [0.72 to 1.05]	---	0.75 [0.66 to 0.85]
0.5	---	0.68 [0.51 to 0.87]	---	0.88 [0.78 to 0.98]
0.75	---	0.072 [ 0 to 0.16]	---	0.77 [0.73 to 0.82]
0.35 [0.30 to 0.40]	---	0.43	---	0.58 [0.53 to 0.64]
0.29 [0.21 to 0.38]	---	0.86	---	0.77 [0.70 to 0.87]	0.76 ± 0.096
0.048 [0 to 0.082]	---	1.29	---	0.79 [0.72 to 0.82]	P = 0.0015
0.28	---	---	0.44 [0.38 to 0.50]	1.05 [0.95 to 1.16]
0.56	---	---	0.13 [0.089 to 0.17]	0.77 [0.70 to 0.86]
0.83	---	---	0.006 [0 to 0.032]	0.81 [0.77 to 0.86]
0.49 [0.39 to 0.60]	---	---	0.17	0.77 [0.67 to 0.88]
0.29 [0.21 to 0.40]	---	---	0.24	0.71 [0.63 to 0.82]	0.82 ± 0.12
0.09 [0.02 to 0.12]	---	---	0.42	0.83 [0.74 to 0.90]	P = 0.015
---	0.075	1.02 [0.84 to 1.23]	---	0.81 [0.70 to 0.93]
---	0.15	1.03 [0.81 to 1.26]	---	1.03 [0.91 to 1.16]
---	0.225	0.40 [0.30 to 0.51]	---	0.89 [0.83 to 0.95]
---	0.23 [0.21 to 0.26]	0.43	---	0.93 [0.86 to 1.01]
---	0.22 [0.19 to 0.25]	0.86	---	1.13 [1.04 to 1.21]	0.96 ± 0.112
---	0.083 [0.062 to 0.107]	1.29	---	0.99 [0.92 to 1.05]	P = 0.44
---	0.1	---	0.35 [0.30 to 0.40]	0.92 [0.84 to 1.02]
---	0.175	---	0.26 [0.22 to 0.30]	0.98 [0.91 to 1.06]
---	0.3	---	0.049 [0 to 0.10]	0.97 [0.88 to 1.06]
---	0.23 [0.19 to 0.26]	---	0.175	0.98 [0.89 to 1.09]
---	0.15 [0.12 to 0.18]	---	0.3	0.99 [0.90 to 1.06]	1.0 ± 0.073
---	0.043 [0 to 0.093]	---	0.50	1.03 [0.88 to 1.18]	P = 0.88
---	---	0.3	0.48 [0.42 to 0.55]	1.05 [0.92 to 1.17]
---	---	1	0.25 [0.20 to 0.31]	1.03 [0.93 to 1.13]
---	---	1.3	0.080 [0.034 to 0.13]	0.89 [0.81 to 0.98]
---	---	0.97 [0.84 to 1.10]	0.14	0.81 [0.73 to 0.89]
---	---	0.93 [0.69 to 1.18]	0.18	0.85 [0.71 to 0.99]
---	---	0.24 [0.16 to 0.35]	0.35	0.77 [0.72 to 0.83]	0.89 ± 0.110
---	---	0.09 [0.04 to 0.15]	0.42	0.81 [0.78 to 0.84]	P = 0.033

Each anesthetic concentration pair includes one fixed concentration and a calculated value for the paired anesthetic IC50 with 95% confidence interval, representing a combination that suppresses inverted photomotor responses in 50% of zebrafish larvae. The α values and confidence intervals were calculated (Eq. 2) after normalizing each drug to its IC50 in the absence of other drugs (Table 1). Individual α values were based on studies using 240 to 480 zebrafish larvae. Averages and standard deviations of collected α values for each drug pair are reported in the right-most column (n = 1440 to 1920 larvae per drug combination). P-values were derived using a single parameter t-test against a theoretical value of 1.0, representing an additive drug interaction.

Consistent with previous studies in tadpoles¹⁴ and rodents,¹³ combining the neurosteroid alphaxalone with etomidate, R-mTFD-MPAB, or propofol resulted in synergistic hypnotic interactions with mean α values significantly less than 1.0 (Table 2; Fig 4). Combinations of R-mTFD-MPAB + propofol also resulted in α-values indicative of weak synergy (mean ± sd = 0.89 ± 0.110; P = 0.033). However, contrasting with results in tadpoles,¹⁴ our results in zebrafish larvae for combinations of etomidate + propofol (mean α = 1.0 ± 0.07; P = 0.88) and etomidate + R-mTFD-MPAB (mean α = 0.96 ± 0.11; P = 0.44) were inconsistent with synergy, and instead consistent with additive interactions.

Modulation of GABA-Activated vs. Anesthetic-Activated α1β3γ2L GABA_A Receptors

To test whether our zebrafish results reflect molecular effects of hypnotic combinations in synaptic α1β3γ2L GABA_A receptors, we used two-microelectrode voltage-clamp electrophysiology to compare anesthetic modulation of currents activated by GABA (an orthosteric agonist) vs. another anesthetic (allosteric agonist). Two-state co-agonist models predict that anesthetic modulation of GABA_A receptor activation will be independent of the agonist applied as long as agonist and modulator sites are structurally distinct.^14,25,26 Thus, log(d, AN):log(d, GABA) ratios near 1.0 are predicted for anesthetic pairs acting through distinct sites and ratios near 0 are consistent with competition for shared sites. Etomidate at 3.2 µM, propofol at 5 µM, R-mTFD-MPAB at 8μM and alphaxalone at 2.5μM were previously shown to produce similar modulation of GABA-dependent receptor activation.^15,20 Figure 5A shows GABA-dependent receptor activation (estimated P_open) in the absence (black circles) and presence of anesthetics. Spontaneous activation of α1β3γ2L receptors was undetectable, indicating basal P_open < 0.0005. GABA EC₅₀ and maximal efficacy values, derived from non-linear least-squares fits to logistic functions (Eq. 4 in Methods), are reported in Table 3. The shifts in GABA responses produced by R-mTFD-MPAB (green squares) or etomidate (red diamonds) appeared larger than those produced by propofol (hexagons) or alphaxalone (blue triangles). At the concentrations used, R-mTFD-MPAB and etomidate resulted in lower GABA EC₅₀s than alphaxalone or propofol (P < 0.01 by F-tests), while all four drugs similarly increased maximal GABA responses. Figure 5B shows the same data subjected to allosteric shift analysis (Eq. 5 in Methods), which calculates log(d) values proportional to the gating energy attributable to modulating drugs. The fitted parameters from these analyses are summarized in Table 4. Of note, the fitted values for K and c, reflecting GABA binding affinity and efficacy, are similar in all four shift models, The quality of fits, reflected in R² values (Tables 3 and 4) are slightly better for logistic fits than for MWC models, likely because of the variable Hill slope.

Figure 5: Anesthetic Modulation of GABA-Activated and Etomidate-Activated α1β3γ2L GABA_A Receptors.

Panel A summarizes results for GABA-activated receptors in the absence (black circles) or presence of 3.2 µM etomidate (red diamonds), 2.5 µM alphaxalone (blue triangles), 5 µM propofol (open hexagons) and 8 µM R-mTFD-MPAB (green squares). Data are mean and 95% CI (n = 5 each) for estimated P_open (Eq. 3 in Methods). Lines are fits to logistic functions (Eq. 4 in Methods). Fitted parameters are reported in Table 3. Panel B shows the same data from Panel A with lines representing fitted allosteric log(d) shift models (Eq. 5 in Methods). For clarity, a single fit is shown for GABA alone. Fitted parameters are reported in Table 4. Panel C summarizes results for etomidate-activated receptors in the absence (red diamonds) and presence of propofol (open hexagons), R-mTFD-MPAB (green squares) and alphaxalone (blue triangles). Data are estimated P_open (mean and 95% CI; n = 5) and lines represent fits to logistic functions. Fitted parameters are reported in Table 3. Panel D shows the same data from Panel C with lines representing fitted allosteric log(d) shift models. For clarity, a single fit is shown for etomidate alone. Fitted parameters are reported in Table 4. Panel E shows current sweeps recorded from oocytes expressing α1β3γ2L receptors during exposure to etomidate (100 µM) alone or combined with alphaxalone (2.5 µM), R-mTFD-MPAB (8 µM) or propofol (5 µM). Bars above sweeps represent drug applications. Currents were recorded from separate oocytes and normalized so that within-cell etomidate controls are of equal height.

Table 3:

Fitted Logistic Parameters^* for Anesthetic Modulation of α1β3γ2L GABA_A Receptors Activated by GABA or Another Anesthetic

		Agonist EC50	Maximal Efficacy	#		EC1
Agonist	Modulator	[95% CI] (µM)	[95% CI]	oocytes	R2	Ratio (± SD)
GABA	--	77 [68 to 88]	0.84 [0.81 to 0.88]	10	0.98	--
GABA	Etomidate	5.4 [4.7 to 6.1]	1.0 [0.99 to 1.03]	5	0.99	53 ± 11
GABA	Propofol	9.9 [8.5 to 11.6]	0.95 [0.93 to 0.98]	5	0.99	28 ± 5.5
GABA	Alphaxalone	8.5 [6.4 to 11.3]	0.99, [0.95 to 1.05]	5	0.97	35 ± 10
GABA	R-mTFD-MPAB	4.6 [3.4 to 6.0]	0.96, [0.92 to 1.00]	5	0.97	55 ± 14
Etomidate	--	76 [58 to 98]	0.20, [0.18 to 0.22]	9	0.86	--
Etomidate	Alphaxalone	9.6 [6.2 to 14]	0.75, [0.69 to 0.81]	5	0.90	36 ± 19
Etomidate	R-mTFD-MPAB	19 [12 to 31]	0.48 [0.43 to 0.54]	5	0.84	14 ± 8.3
Etomidate	Propofol	83 [61 to 130]	0.27 [0.24 to 0.32]	5	0.95	3 ± 1.4
Propofol	--	60 [44 to 86]	0.33 [0.29 to 0.39]	5	0.83	--
Propofol	R-mTFD-MPAB	10 [6.2 to 18]	0.35 [0.32 to 0.40]	5	0.85	21 ± 13
Propofol	Alphaxalone	8.2 [5.6 to 12]	0.35 [0.32 to 0.38]	5	0.90	20 ± 11
Propofol	Etomidate	49 [28 To 86]	0.29 [0.25 to 0.33]	5	0.89	1.1 ± 0.73
R-mTFD-MPAB	--	73 [21 to ???]	0.08 [0.05 to ???]	5	0.93	--
R-mTFD-MPAB	Alphaxalone	2.2 [1.4 to 3.4]	0.18 [0.16 to 0.20]	5	0.79	20 ± 9.2

^*Parameters derived from P_open vs. log[Agonist] data sets (Fig 5A and C, S1 A and C) using non-linear least squares fits to a 4-variable logistic equation (Eq. 4 in Methods). The relative quality of the fits is reflected in the R² values (i.e. closer to 1.0 is better). The number of data points used in fits was 9 or 10 times the number of oocytes. Modulator concentrations were: 3.2 µM etomidate, 5 µM propofol, 2.5 µM alphaxalone, and 8 µM R-mTFD-MPAB. Fitted 95% confidence intervals for baseline P_open values were all consistent with 0. Fitted Hill slopes varied from 0.9 to 2.0.

EC1 Enhancement Ratios were calculated from peak current data (mean ± sd) at 3 µM GABA, 10 µM etomidate, 10 µM propofol or 3 µM R-mTFD-MPAB with and without modulators at the concentrations indicated above. Standard deviations were propagated according to Bevington & Robinson.²²

Table 4.

Fitted MWC Log(d) Shift Parameters^* for Anesthetic Modulation of α1β3γ2L GABA_A Receptors Activated by GABA or Another Anesthetic

Agonist	Modulator	K ± se (µM)	c ± se	Log(d) ± se	Log(d) Ratio ± se	R2
GABA	Etomidate	108 ± 10	0.0030 ± 0.00012	−1.91 ± 0.064	NA	0.97
GABA	Propofol	98 ± 9.2	0.0031 ± 0.00012	−1.33 ± 0.061	NA	0.98
GABA	Alphaxalone	100 ± 11	0.0030 ± 0.00014	−1.47 ± 0.072	NA	0.96
GABA	R-mTFD-MPAB	106 ± 13	0.0030 ± 0.00015	−1.91 ± 0.081	NA	0.96
Etomidate	Alphaxalone	17 ± 2.3	0.0132 ± 0.00056	−1.28 ± 0.053	0.87 ± 0.056	0.93
Etomidate	R-mTFD-MPAB	20 ± 2.4	0.013 ± 0.00041	−0.73 ± 0.035	0.38 ± 0.024	0.90
Etomidate	Propofol	43 ± 5.3	0.012 ± 0.00031	−0.11 ± 0.028	0.086 ± 0.021	0.87
Propofol	R-mTFD-MPAB	14 ± 3.0	0.0096 ± 0.00049	−0.18 ± 0.051	0.096 ± 0.027	0.78
Propofol	Alphaxalone	11 ± 2.3	0.0099 ± 0.00049	−0.21 ± 0.050	0.14 ± 0.035	0.79
Propofol	Etomidate	38 ± 7.0	0.0082 ± 0.00036	0.10 ± 0.044	−0.075 ± 0.033	0.84
R-mTFD-MPAB	Alphaxalone	1.1 ± 0.24	0.033 ± 0.0036	−0.82 ± 0.095	0.55 ± 0.070	0.84

^*Parameters were derived from P_open vs. [Agonist] data sets in the absence and presence of a Modulator (Figs 5B and D, S1 B and D) using non-linear least squares fits to Eq. 5 in Methods. The relative quality of the fits is reflected in the R² values (i.e. closer to 1.0 is better). The number of data points in fits is 9 or 10 times the number of total combined oocytes for agonist with and without modulator (reported in Table 3). Modulator concentrations were: 3.2 µM etomidate, 5 µM propofol, 2.5 µM alphaxalone, and 8 µM R-mTFD-MPAB. L₀ (basal gating parameter) was fixed at a value of 25,000; K is the dissociation constant for agonist (GABA or etomidate) interactions with inactive receptors; c is agonist efficacy, defined as the ratio of dissociation constants in active vs. inactive receptors; Log(d) is proportional to the gating energy shift in the presence of Modulator. Log(d, AN):log(d, GABA) ratios are calculated for modulator effects on receptors activated with another anesthetic vs. GABA as primary agonist. MWC models assumed 2 equivalent agonist sites and 1 anesthetic site.

Of particular interest was whether interactions between etomidate and other anesthetics in synaptic GABA_A receptors corresponded to the varying interactions observed in zebrafish experiments (Fig. 4) or with predictions of two-state co-agonism. Figure 5C shows etomidate-dependent receptor activation in the absence (red diamonds) and presence of alphaxalone (blue triangles), propofol (open hexagons) or R-mTFD-MPAB (green squares) at the same concentrations co-applied with GABA. Etomidate EC₅₀s and maximal efficacy values from logistic fits are reported in Table 3. In contrast to results in GABA-activated receptors, alphaxalone produced larger leftward shifts in etomidate responses than R-mTFD-MPAB or propofol. The etomidate EC₅₀ in the presence of alphaxalone was lower than in the presence of R-mTFD-MPAB (9.6 vs. 19 µM; P = 0.036 by F-test) or propofol (9.6 vs, 83 µM; P < 0.0001 by F-test) and the maximum efficacy of etomidate + alphaxalone was higher than that of etomidate + R-mTFD-MPAB or etomidate + propofol (0.75 vs. 0.48 or 0.27; both P < 0.0001 by F-test). The allosteric shift model fits for etomidate-activated receptor currents are shown in Figure 5D and the fitted parameters are reported in Table 4. Log(d, AN):log(d, GABA) derived from shift analysis (Table 4) indicated that alphaxalone interacts with etomidate with 87 ± 5.6% of the energy observed with alphaxalone + GABA. In stark contrast, the interaction between propofol and etomidate resulted in a log(d) value of −0.11 ± 0.028, which was only 8.6 ± 2.1% of the log(d) calculated for propofol modulation of GABA responses. We also assessed the effect of 3.2 µM etomidate on propofol-activated receptor currents and observed a similarly low log(d) value (Fig S1; Table 4). The R-mTFD-MPAB log(d) for etomidate responses was 38 ± 2.4% of the value for GABA responses, intermediate between alphaxalone and propofol.

We also studied the effects of R-mTFD-MPAB and alphaxalone on propofol-activated GABA_A receptors and the effects of alphaxalone on R-mTFD-MPAB-activated receptors. Results are displayed in Fig S1. Logistic fit parameters and results of allosteric shift calculations are reported in Tables 3 and 4, respectively. The effects of R-mTFD-MPAB and alphaxalone on propofol-activated currents were similar, producing a 6- to 7-fold reduction in propofol EC₅₀, but without increasing maximal responses at high [propofol]. These results were poorly fit with MWC allosteric shift models, reflected in low R² values (Table 4). R-mTFD-MPAB was a weak agonist of α1β3γ2L GABA_A receptors and at up to 30 µM R-mTFD-MPAB-activated currents were enhanced up to 20-fold by alphaxalone. At higher R-mTFD-MPAB concentrations in the presence of alphaxalone, inhibition in the form of surge currents was observed. The alphaxalone log(d) for R-mTFD-MPAB-activated receptors was 55 ± 7.0% of the value for GABA-activated receptors.

For all six drug pairs tested, correlation between zebrafish α values and log(d) ratios resulted in a Pearson r = −0.46 (P = 0.35).

Figure 5E displays representative current traces recorded from oocytes used in experiments shown in Figs. 5C and 5D. These revealed additional details about the interactions of etomidate with R-mTFD-MPAB, propofol and alphaxalone in α1β3γ2L receptors. Traces elicited with high etomidate (100 µM) alone showed channel activation and deactivation only. With the addition of 2.5 µM alphaxalone, peak currents were amplified about 4-fold and very small surge currents were evident during drug washout. In contrast, addition of 8 µM R-mTFD-MPAB to 100 µM etomidate increased peak current about 2-fold and large surge currents were evident after drug exposure ended. Addition of 5 µM propofol to 100 µM etomidate increased peak currents by only ∼15% without inducing surge currents.

Zebrafish Hypnotic-Range Anesthetic Additivity Studies in α1β3γ2L GABA_A Receptors

The receptor-activating anesthetic concentrations used in co-agonist shift experiments were mostly higher than those present in zebrafish hypnosis experiments. We assessed anesthetic interactions at lower concentrations in two ways. EC1 enhancement ratios (Table 3) were calculated from subsets of data generated for allosteric shift analysis, specifically modulation of anesthetic concentrations activating approximately 1% of maximal GABA currents. EC1 enhancement ratios were largest for etomidate + alphaxalone, and lower but similar for R-mTFD-MPAB + alphaxalone, propofol + R-mTFD-MPAB and propofol + alphaxalone. The two drug pairs that appeared additive in zebrafish hypnosis assays (etomidate + R-mTFD-MPAB and etomidate + propofol) produced the two lowest EC1 enhancement ratios.

We also assessed interactions between etomidate + R-mTFD-MPAB, etomidate + alphaxalone and etomidate + propofol in α1β3γ2L receptors activated with low GABA (10μM ∼ EC8) using concentrations close to zebrafish hypnosis IC50s. We found that 10 µM GABA combined with 0.5 µM etomidate, 1.03 µM alphaxalone 1.85 µM R-mTFD-MPAB, or 1.70 µM propofol all elicited peak currents that were ∼40% of maximal GABA (3 mM) responses. To test anesthetic interactions, we compared the average of two currents elicited with GABA + single anesthetic solutions to the current elicited with GABA + both anesthetics at half concentrations (n = 6 oocytes per drug pair). Fig 6A shows results for alphaxalone and etomidate. The normalized currents elicited with GABA + ½alphaxalone + ½etomidate (mean ± sd = 0.45 ± 0.079) were consistently higher than the within-oocyte averages of normalized currents elicited with GABA + alphaxalone and GABA + etomidate (0.35 ± 0.062; P = 0.023 by paired Student’s t-test), indicating a supra-additive interaction. Based on the within-oocyte ratios of currents with ½alphaxalone + ½etomidate to the averages of single-anesthetic responses (1.28 ± 0.25), the degree of synergy was not large. Fig 6B shows results for R-mTFD-MPAB and etomidate in another group of oocytes. Within-oocyte normalized currents elicited with GABA + ½R-mTFD-MPAB + ½etomidate (0.43 ± 0.043) were mostly higher than averaged results elicited with GABA + R-mTFD-MPAB and GABA + etomidate (mean ± sd = 0.38 ± 0.038; ratio = 1.15 ± 0.17; n = 6) but the results did not reach statistical significance (P = 0.064 by paired Student’s t-test). Fig 6C shows results for propofol and etomidate. The normalized currents elicited with GABA + ½propofol + ½etomidate (0.27 ± 0.035) were very similar to average within-oocyte currents elicited with GABA + propofol and GABA + etomidate (mean ± sd = 0.26 ± 0.050; ratio = 1.0 ± 0.14; n = 6; P = 0.86 by paired Student’s t-test). These results are consistent with an additive drug interaction. In summary, our studies of anesthetic interactions in GABA-activated α1β3γ2L receptors echoed findings in zebrafish larvae at comparable drug concentrations. Combining alphaxalone and etomidate produced modest but significant supra-additive effects, while R-mTFD-MPAB + etomidate or propofol + etomidate interacted additively.

Figure 6: Hypnotic Concentration Anesthetic Interactions in GABA_A Receptors Activated with Low GABA.

Panel A shows normalized (to 3 mM GABA responses) peak current results from six oocytes exposed to 10 µM GABA in the presence of 0.5 µM etomidate (red), 1.03 µM alphaxalone (blue), or 0.25 µM etomidate + 0.51 µM alphaxalone (filled purple), each oocyte exposed to a different permutation of the 3 solutions. Pairs of symbols on the right compare the within-oocyte averages of responses with etomidate and alphaxalone alone (open circles) with both combined at half concentrations (filled circles). Panel B shows results for similar experiments using 0.5 µM etomidate and 1.85 µM R-mTFD-MPAB (green circles) and Panel C shows results for similar experiments using 0.5 µM etomidate and 1.7 µM propofol (white hexagons). P-values were calculated using paired Student’s t-tests (n = 6 oocytes per drug pair). Mixtures of half-drug concentrations producing significantly larger responses than the within-oocyte average of single drugs was interpreted as a supra-additive drug interaction.

R-mTFD-MPAB Subsite Mutations to Probe Contributions to Modulation of Etomidate-Activated Receptors

The appearance of surge currents following co-application of etomidate + R-mTFD-MPAB (Fig 5E) indicated that both activation and inhibition were induced in α1β3γ2L GABA_A receptors. We considered whether the two distinct R-mTFD-MPAB subsites might differentially contribute to these opposing effects, as observed for diazepam by McGrath et al.²⁷ We therefore tested the effects of 8 µM R-mTFD-MPAB on etomidate-activated GABA_A receptors containing a mutation in either the α+/β- or γ+/β- site. Results are shown in Fig 7.

Figure 7: R-mTFD-MPAB Site Mutations on α1 and γ2L Subunits Both Obliterate R-mTFD-MPAB Modulation of Etomidate Activation.

Data points represent mean and 95% CI (n = 5 per condition) P_open data derived from electrophysiologic recordings (Eq. 3 in Methods) of etomidate-activated α1S270Iβ3γ2L (open symbols) and α1β3γ2LS280W (solid symbols) receptors in the absence (red circles) or presence (green squares) of 8 µM R-mTFD-MPAB. Lines through data points represent logistic fits (Eq. 4 in Methods). Data were also fitted with Eq. 5 to derive log(d) allosteric shifts induced by R-mTFD-MPAB. Parameters from fits to Eq. 5 for α1β3γ2LS280W were: L₀ = 50, K_ETO = 130 + 23 µM, c_ETO = 0.18 ± 0.008, and log(d) = 0.20 ± 0.038. Parameters from fits to Eq. 5 for α1S270Iβ3γ2L were: L₀ = 50, K_ETO = 33 + 7.7 µM, c_ETO = 0.043 ± 0.0061, and log(d) = −0.36 ± 0.71.

Both α1S270Iβ3γ2L and α1β3γS280W receptors displayed spontaneous activation of ∼2%, consistent with prior results.¹⁵ Based on logistic fits, etomidate as an agonist in α1S270Iβ3γ2L receptors (Fig 7, open red circles) is characterized by EC₅₀ = 19 µM and high efficacy (P_max ≈ 1.13), while in α1β3γS280W receptors (solid red circles) etomidate activates with lower potency (EC₅₀ = 200 µM) and lower efficacy (P_max ≈ 0.40). In α1S270Iβ3γ2L receptors, addition of 8 µM R-mTFD-MPAB (open green squares) did not significantly alter peak currents elicited by etomidate at any tested concentration and etomidate EC₅₀ was not significantly changed (P = 0.45 by F-test). In α1β3γ2LS280W receptors, addition of 8 µM R-mTFD-MPAB (solid green squares) moderately reduced currents elicited by etomidate (P_max ≈ 0.28; P = 0.15 by F-test) without altering apparent potency (EC₅₀ = 160 µM; P = 0.73 by F-test). Shift analysis was also applied to quantify the allosteric effects of 8 µM R-mTFD-MPAB in the two mutant GABA_A receptors. In α1S270Iβ3γ2L receptors, 8 µM R-mTFD-MPAB resulted in log(d) = −0.3 ± 0.71, indicating no significant overall gating effect. In α1β3γ2LS280W receptors, 8 µM R-mTFD-MPAB resulted in log(d) = 0.20 ± 0.038, indicating a small but significant inhibitory effect.

We also tested whether R-mTFD-MPAB directly activated the mutant GABA_A receptors (data not shown). At concentrations up to 300 μM, R-mTFD-MPAB activated less than 1% of maximal GABA currents in α1β3γ2S280W receptors. R-mTFD-MPAB at concentrations above 10 µM activated up to 18% of α1S270Iβ3γ2L receptors.

DISCUSSION

We tested the hypothesis, based on two-state allosteric co-agonist models, that pairs of four anesthetics (etomidate, propofol, R-mTFD-MPAB and alphaxalone) acting through distinct sets of GABA_A receptor sites would interact synergistically in both zebrafish larvae hypnosis assays and in electrophysiologic measures of α1β3γ2L receptor activation.^11,14

Hypnotic drug interactions in zebrafish larvae were not consistently synergistic. All three anesthetic pairs containing alphaxalone displayed synergy in zebrafish, agreeing with prior studies of similar neurosteroids in tadpoles and rodents.^13,14 Propofol + R-mTFD-MPAB also displayed synergy in zebrafish larvae. However, etomidate + R-mTFD-MPAB and etomidate + propofol pairs were additive (not synergistic) in zebrafish, contrasting with tadpole results.¹⁴ When observed, anesthetic synergy in zebrafish was also generally weaker than that reported in Xenopus tadpole righting reflex tests, reflected in higher α-values (Table 2).¹⁴ This difference probably reflects lower drug IC50 values for zebrafish inverted photomotor responses vs. tadpole righting reflexes along with differences in species and neurological development. These factors may also have contributed to the contrasting results in zebrafish and tadpoles for etomidate + R-mTFD-MPAB and etomidate + propofol.

Based on allosteric shift analysis of α1β3γ2L receptor activation assessed electrophysiologically (Table 4), etomidate + alphaxalone interacted in close accord with predictions of allosteric co-agonism [log(d) ratio near 1.0], but the other 5 drug pairs did not. Notably, etomidate + propofol interacted with log(d) near 0, consistent with competition for shared sites. Thus, most of our log(d) results appear to conflict with prior electrophysiologic studies reporting drug interactions fully in accord with allosteric co-agonism. However, most of the drug interactions that we studied in α1β3γ2L receptors have not previously been reported. Additionally, electrophysiologic experiments by others (Akk and colleagues) used α1β2γ2L receptors formed from concatenated subunit assemblies and low concentrations of agonists and co-agonists.^11,12 For all 6 drug pairs in our study, no significant correlation was found between log(d, AN):log(d, GABA) ratios and zebrafish α-values. EC1 enhancement ratios (Table 3), based on results at relatively low anesthetic concentrations, correlated better than log(d) ratios with zebrafish α-values (Spearman r = −0.66; P = 0.15).

We also examined the 3 drug pairs that included etomidate in receptors activated with low GABA and low anesthetic concentrations near zebrafish hypnotic IC50s. Under these conditions, alphaxalone + etomidate interacted supra-additively, while R-mTFD-MPAB + etomidate and etomidate + propofol were additive (Fig 6). Thus, these 3 low concentration drug pair interactions in synaptic receptors correlate well with zebrafish photomotor response results.

Oocyte current recordings (Fig 5E) provided evidence that different hypnotic pair interactions in GABA_A receptors induce different functional state mixtures. Currents elicited with etomidate alone or combined with alphaxalone or propofol showed primarily activation and deactivation phases, consistent with two dominant functional states: resting and active. In contrast, during etomidate + R-mTFD-MPAB washout we observed large surge currents indicating reactivation from additional non-conductive states. Channel blockade by anesthetics is a possible explanation but neither etomidate nor R-mTFD-MPAB alone inhibit receptors at the relevant concentrations. Another explanation is receptor desensitization. GABA_A receptors can re-open during recovery from desensitization, prolonging deactivation and generating surge currents.²⁸ We speculate that R-mTFD-MPAB enhanced both activation and desensitization by high etomidate concentrations.⁶ Nonetheless, under non-desensitizing conditions, low concentrations of R-mTFD-MPAB and etomidate also cooperated more weakly than alphaxalone and etomidate (Fig 6). We also explored the possibility that the α+/β- and γ+/β- binding sites might mediate opposing R-mTFD-MPAB effects, finding that both α+ (S270I) and γ+ (S280W) mutations essentially eliminated R-mTFD-MPAB modulation (Fig 7) of etomidate-activated receptors. The log(d) shifts for etomidate + R-mTFD-MPAB in mutant receptors were similar to those for GABA + R-mTFD-MPAB,¹⁵ suggesting that R-mTFD-MPAB binding at both β- sites cooperatively enhances activation without selectively affecting inhibition.

Overall, our results indicate that anesthetic interactions in GABA_A receptors were not consistently explained by two-state allosteric co-agonism. Qualitatively, these models correctly predicted synergistic interactions in both zebrafish and synaptic GABA_A receptors for four hypnotic drug pairs at low concentrations: alphaxalone + etomidate, alphaxalone + propofol, alphaxalone + R-mTFD-MPAB and propofol + R-mTFD-MPAB. However, for higher drug concentrations, this mechanism quantitatively approximated only the interaction of alphaxalone + etomidate. Indeed, our findings suggest that allosteric co-agonist models best describe the interactions of agonists and co-agonists that selectively bind in β+/α- subunit interfaces: GABA, etomidate, and alphaxalone. The interactions of etomidate + R-mTFD-MPAB and etomidate + propofol in both zebrafish and GABA_A receptors clearly disagreed with co-agonism. Receptor structure-function¹⁵ and cryo-electron microscopy⁵ studies provide alternative frameworks for interpreting these drug pair interactions.

The weak interaction of etomidate + R-mTFD-MPAB is explained by cryo-electron microscopy images of GABA_A receptors with etomidate occupying β+/α- sites, showing collapse of α+/β- and γ+/β- cavities where R-mTFD-MPAB binds.⁵ The cryo-electron microscopy structures are thought to represent desensitized-inactive receptors but likely maintain open-active state features, because activation and desensitization involve rearrangement of different ion channel regions.^17,29 Allosteric shift analyses of mutant function also indicate that etomidate binding may weaken R-mTFD-MPAB interactions with GABA_A receptors. Mutations in etomidate sites weaken R-mTFD-MPAB modulation and mutations in R-mTFD-MPAB sites weaken etomidate modulation.¹⁵ This negative cross-talk echoes our current findings that cooperativity is much weaker between these drugs than between either drug and GABA.

Etomidate and propofol interacted additively in both zebrafish and α1β3γ2L receptors, consistent with competition for the same co-agonist sites. Indeed, evidence from photolabeling, mutant analyses, substituted cysteine modification and protection, and cryo-electron microscopy all indicate that propofol and etomidate bind at overlapping sites in transmembrane β+/α- inter-subunit pockets.^5,20,30–33 However, most studies also locate propofol binding in β- sites overlapping those for R-mTFD-MPAB. Assuming that propofol binds at the two R-mTFD-MPAB (β-) and the two etomidate (β+) sites and that all four sites contribute equal gating energy, one might expect a log(d) ratio near 0.5, reflecting co-agonism between etomidate and propofol at the β- sites. In contrast, cryo-electron microscopy shows propofol binding only in β+/α- pockets.⁵ Also, mutations in β+/α- sites nearly obliterate modulation by both etomidate and propofol, while α+/β- and γ+/β- mutations produce smaller effects.¹⁵ Thus, the β+/α- sites may mediate nearly all of the allosteric gating effects of propofol and our initial assertion that propofol and etomidate act via different sets of sites needs reconsideration.

Given that propofol and etomidate both act through β+/α- sites in GABA_A receptors, one might also expect that the combination of propofol + R-mTFD-MPAB would interact similarly to etomidate + R-mTFD-MPAB (i.e. weakly). However, the EC1 enhancement ratio for propofol + R-mTFD-MPAB was larger than that for etomidate + R-mTFD-MPAB, and similar to that for propofol + alphaxalone (Fig S1 & Table 3). Both propofol + R-mTFD-MPAB and propofol + alphaxalone pairs also displayed synergy in zebrafish. Cryo-electron microscopy suggests why the propofol + R-mTFD-MPAB interaction is more cooperative than the etomidate + R-mTFD-MPAB interaction. In contrast to etomidate, propofol bound in the outer β+/α- sites is associated with accessible α+/β- and γ+/β- pockets where R-mTFD-MPAB binds,⁵ suggesting that propofol and R-mTFD-MPAB can simultaneously and cooperatively occupy their distinct binding sites.

This study has several limitations that influence comparison of results in zebrafish versus tadpoles and GABA_A receptors. The nervous systems of larval zebrafish are not fully developed, and generalizability of our findings needs testing in older animals and in other species. Our prior photomotor response studies in larval zebrafish showed strong correlations with tadpole results.¹⁶ However, our α-values for drug interactions based on inverted photomotor response results differ quantitatively from results in Xenopus tadpoles.¹⁴ For example, etomidate and R-mTFD-MPAB were weakly cooperative in GABA_A receptors and additive in zebrafish, but synergized in tadpoles. There are multiple advantages of zebrafish over tadpoles for hypnotic drug assays,¹⁶ while one disadvantage is the slightly lower control response probability. Inverted photomotor response assays also resulted in different hypnotic IC50 values than our earlier photomotor response assays in zebrafish larvae,¹⁸ while greatly increasing IC50s for spontaneous activity. Thus, zebrafish inverted photomotor response results may not correlate as well with tadpole results.

We found that allosteric shift models are of limited relevance for analyzing anesthetic interactions at concentrations affecting zebrafish, because direct GABA_A receptor agonism requires high anesthetic concentrations. Studies in GABA-activated receptors and EC1 enhancement values using lower anesthetic concentrations were alternatives that also better correlated with our zebrafish results.

Finally, our analysis is framed by the assumption that most of the behavioral effects of the studied drugs are mediated by GABA_A receptors with pharmacology similar to α1β3γ2L. However, non-synaptic GABA_A receptors and other molecular targets may also contribute to the actions of anesthetics we studied.³⁴

In conclusion, our findings in both zebrafish larvae and α1β3γ2L GABA_A receptors correlate modestly, while challenging the general validity of simple two-state allosteric co-agonism when more than one anesthetic is present. Our results indicate that cross-talk between allosteric co-agonist sites on GABA_A receptors varies, probably influencing drug interactions in animals. They also support the functional and pharmacological relevance of distinct cryo-electron microscopy structures of GABA_A receptors bound to different anesthetics, despite the non-physiological conditions used in these studies.

Supplementary Material

Figure S1. Figure S1: Anesthetic Modulation of Propofol-Activated and R-mTFD-MPAB-Activated α1β3γ2L GABA_A Receptors.

Panel A summarizes results for propofol-activated receptors in the absence (open hexagons) or presence of 2.5 µM alphaxalone (blue triangles) 8 µM R-mTFD-MPAB (green squares), or 3.2 µM etomidate (red diamonds). Data are mean and 95% CI (n = 5 per condition) for estimated P_open (Eq. 3 in Methods). Lines are fits to logistic functions (Eq. 4 in Methods). Fitted parameters are reported in Table 3. Panel B shows the same data from Panel A with fitted lines representing allosteric log(d) shift models (Eq. 5 in Methods). For clarity, a single fit is shown for propofol alone. Fitted parameters are reported in Table 4. Panel C summarizes results for R-mTFD-MPAB-activated receptors in the absence and presence of alphaxalone. Data are estimated P_open (mean and 95% CI; n = 5) and lines represent fits to logistic functions. Fitted parameters are reported in Table 3. Panel D shows the same data from Panel C with lines representing the fitted allosteric log(d) shift model. Fitted parameters are reported in Table 4.

Abbreviations:

IC₅₀

concentration resulting in half-maximal inhibition

IC50

concentration resulting in 50% response probability

EC1

concentration eliciting 1% of maximal response

GABA

gamma-aminobutyric acid

GABA_A

gamma-aminobutyric acid type A

R-mTFD-MPAB

R-5-allyl-1-methyl m-trifluoromethyl mephobarbital