It is widely accepted that many anesthetic drugs generate their effects by acting on γ-aminobutyric acid type A receptors (GABAARs).1–3 GABAARs are the most abundant inhibitory receptor in the central nervous system (CNS). They are members of the Cys-loop ligand–gated ion channel superfamily that consists of 5 subunits arranged pseudosymmetrically around a central ion pore. There are 16 different but highly homologous GABAAR subunits (α1–6, β1–3, γ1–3, δ, ε, θ, and π).4 Although all pentameric configurations that occur naturally are not thoroughly established, the most common arrangement reading anticlockwise around the central ion pore, as viewed from the extracellular side, is thought to be β–α–β–α–x, where x might be any subunit except possibly α. In practice, the most common fifth subunit in GABAARs in the CNS is β, γ, and δ.
R-etomidate is a general anesthetic that acts on GABAARs with high affinity and enantioselectivity. Importantly, its potency is subunit dependent; GABAARs containing a β1 subunit are insensitive to R-etomidate, whereas those with β2 or β3 subunits are sensitive.5 These observations show that general anesthetics can act selectively on certain subtypes of GABAARs. Furthermore, it was discovered that mutating a single residue in both β2 and β3 subunits (N256S and N265M, respectively) could render GABAARs that contained them insensitive to etomidate.6 Introduction of each of these mutations into mice (knock-in mice) has provided a tool for examining the contributions of β2- and β3-containing GABAARs to the many components underlying the state of general anesthesia.7,8 Subsequently, photolabeling with etomidate derivatives in heterologously expressed GABAARs has identified the binding site for R-etomidate within the transmembrane domain at the interface between the β3 and α1 subunits.9 On the basis of homology models of the GABAAR and the structure of a β3 homopentameric GABAAR,10 the photolabeled residues and β3 N265 site all reside within the β+-α− subunit interface and within approximately 10 Å of each other (Figure 1), suggesting that they constitute the etomidate-binding site.
It was shown in 2013, however, that the photoactivatable barbiturate R–mTFD-MPAB also acts on GABAARs but primarily by binding at the γ-β interface, with a 60-fold preference over R-etomidate’s site at the β+-α− interface.11 Thus, R–mTFD-MPAB differs in vitro from etomidate in interacting exclusively with GABAARs that contain a γ subunit and by acting at a single subunit interface that is homologous to, but separate from, the etomidate site. Given this difference in drug-binding sites established by the in vitro experiments, we hypothesized that in experiments in vivo, the β3-N265M–mutant mice would not be resistant to the anesthetic effects of R–mTFD-MPAB, whereas the same mutant mice would be resistant to the anesthetic effects of R-etomidate, as shown in previous work.7,13
All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011) and were approved by the University of Wisconsin Institutional Animal Care and Use Committee, Madison, Wisconsin. All efforts were made to minimize the animal suffering and reduce the number of animals used.
Male and female offspring of heterozygous breeding pairs homozygous for the asparagine-to-methionine point mutation at GABAAR β3 subunit position 265 (β3-N265M) as well as wild-type (WT) controls were used for this study. Mice were housed in an animal care facility with continuous access to standard mouse chow and water. Twelve-hour light-dark cycles were maintained. Mice were genotyped by the use of DNA from tail tips, which was amplified by polymerase chain reaction.
Etomidate (Tocris Bioscience, Bristol, United Kingdom) was dissolved in sterile saline to 5 mg/mL stock solution. R–mTFD-MPAB14 was dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) to 5 mg/mL stock solution. These stock solutions were further diluted with additional saline or DMSO on the day of injections to working solutions of a concentration such that an injection volume of 2.5 mL/kg would deliver the desired dose of drug while keeping the volume of DMSO as low as possible and consistent among mice while injecting R-mTFD-MPAB.
Injections were performed on mice aged 38 to 120 days. Drug was prepared on the day of injections as described previously. The individual performing the injections was blinded to the genotype of the mice being injected. Mice were weighed, and an appropriate volume of drug was drawn up in a 1-mL syringe (BD Biosciences, San Jose, CA). Mice were restrained and injected in a lateral tail vein using a 30-gauge half-inch needle (BD Biosciences). Mice were removed immediately from the restraint device and placed on their backs to check for righting reflex. Once a mouse lost righting reflex (which was generally immediately), a timer was started and the mouse was placed in an empty housing container and observed continuously. Stimulation was provided every 30 seconds in the form of a gentle nudge. The time at which the mouse regained its righting reflex, that is, was able to maintain an upright posture on all 4 paws, was recorded and is referred to as the time (in seconds) until “return of righting reflex” (RORR). Some mice made an initial attempt to regain an upright stance but instead fell to the opposite side, usually repeatedly, resulting in the appearance of a “rolling motion.” If this rolling occurred, the time at which a mouse began to undertake these attempts at righting was also recorded and is referred to as the “return of attempted righting” (ROAR). The “duration of anesthesia” was defined as the time until RORR or time until ROAR.
Doses used for etomidate injections were 2.5, 5, and 10 mg/kg. Doses used for R–mTFD-MPAB injections were 5, 7.5, and 10 mg/kg. Five additional mice (3 WT and 2 mutant) were injected with 2.5 mL/kg of plain DMSO and observed for loss of righting reflex (LORR), which did not occur. Repeat injections were performed on mice only if tail veins appeared viable after initial injection. Any repeat injection was performed at a minimum of 4 days after the previous injection.
No a priori statistical power calculation was conducted to guide sample size. Instead, the sample size was based on our previous experience with these methods. For the experiments, 76 animals were observed in 98 conditions with 20 animals used in 2 conditions and 1 animal used in 3 conditions. For the analyses, the responses from the animals were treated as independent of one another (ie, that an animal’s response at one dose was independent of that in another dose). To examine the differences between mutant and WT animals at each dose, a Mann-Whitney U test was applied with a Bonferroni correction applied to adjust for the fact that 3 comparisons were made for each drug. Medians (interquartile range [IQR]) were used to report the data, with median differences and bootstrapped 95% confidence interval (CI) of these differences used to index the degree of difference between each group. All analyses were conducted with R statistical software (R Foundation for Statistical Computing, Vienna, Austria). Where appropriate, all analyses were 2 tailed, and P value <0.05 was used to interpret the statistical significance.
IV injection of etomidate (2.5–10 mg/kg) to WT mice produced a dose-dependent LORR, with larger doses leading to a greater duration of anesthesia (Figure 2A). Similarly, injection of MPAB (5–10 mg/kg) to WT mice produced a dose-dependent LORR (Figure 2B). Because the greatest dose of MPAB (10 mg/kg) caused a very prolonged duration of anesthesia and, in many cases, death upon injection, it was administered only in a limited number of times, and these data were not included in the statistical analysis.
IV injection of etomidate (2.5–10 mg/kg) to mutant mice also produced a dose-dependent LORR. These mice, however, displayed unique behavior on emergence from anesthesia. Before being able to maintain all 4 paws on the ground, these mice would attempt to right themselves, but, lacking the apparent coordination to do so, would fall to the opposite side in what was deemed a “rolling” behavior. WT mice did not display this behavior, nor did either genotype after injection with MPAB. This behavior was deemed ROAR as opposed to the classic RORR in which mice are able to maintain all 4 paws in contact with the ground.
Mutant mice receiving etomidate had significantly shorter duration of anesthesia, defined as the time until ROAR or RORR, than their WT counterparts (Figure 2A). At the 2.5 mg/kg dose, 7 of 8 mutant animals did not lose righting reflex, while 8 of 8 WT animals were observed to sleep for a median [IQR] duration of 175  seconds. The median difference (95% CI) between the 2 groups was 175 (119, 344), P = 0.015. At the 5.0 mg/kg dose, the mutant animals slept for 46  seconds, while the WT slept for 357.5 [84.25] seconds, median difference: 311.5 (95% CI: 273, 381), P = 0.0021. At the 10.0 mg/kg dose, the mutant animals slept for 195  seconds, 10 while the WT slept for 1189 [1086.5] seconds, median difference: 994 (95% CI: 586, 2464), P = 0.017.
Although the duration of anesthesia was shorter for mutant than for WT mice, there was no statistical difference between genotypes in time to recovery of balance and coordination. The 2.5, 5, and 10 mg/kg doses yielded median RORR times of [137.75], 348 , and 926  seconds, respectively. Compared with WT mice, the median difference for the 2.5 mg/kg dose was 61.5 (95% CI, –93.5 to 218.4), P = 0.226. The median difference for 5 mg/kg was 9.5 (95% CI, −141 to 163), P = 0.768, and for the 10 mg/kg dose was 263 (95% CI, −101 to 1221), P = 0.202.
For mice receiving MPAB at the 5 mg/kg dose, mutant mice had a median [IQR] RORR time of 106 [85.25] seconds, whereas WT mice had a median RORR time of 44  seconds (Figure 2B). The median difference was −62 (95% CI, −114.6 to 2.87), P = 0.023. A single mouse died immediately after injection of the 5 mg/kg dose. For the 7.5 mg/kg dose, mutant mice had a median RORR time of 257 [203.25] seconds, whereas WT mice had a median RORR time of 318 [427.25] seconds. The median difference of 61 (95% CI, −160.5 to 257.5) was not statistically significantly different (P = 0.999). There were 3 deaths associated with the 7.5 mg/kg dose of MPAB. Two of these occurred immediately after injection, whereas the other occurred the night after injection.
Five (3 mutant and 2 WT) of the first 9 mice injected with 10 mg/kg of MPAB died either shortly after injection or later on the day of injection, and thus injections of this dose were discontinued. No data from any mouse that died in conjunction with injection of MPAB were used in the data analysis. Because there were only 2 mice for each genotype injected with the 10 mg/kg dose, these data also were not included in the analysis.
Our results clearly show that the LORR caused by IV injection of R–mTFD-MPAB is insensitive to the β3-N265M mutation, whereas the potency of R-etomidate is dramatically shifted in these mutant animals. This finding indicates that R–mTFD-MPAB does not cause general anesthesia by acting on the etomidate site in the β+-α− interfaces of GABAARs.
All functional combinations of GABAAR subunits are thought to contain β+-α− interfaces because the GABA-binding site is located at this interface in the extracellular domain, approximately 50 Å from the etomidate site in the transmembrane domain.15 The action of R-etomidate is dependent on the subtype of β subunit. Receptors with β1 subunits, which constitute a minority of GABAARs in the CNS,4 are relatively insensitive, whereas those with β2 and β3 subunits are equally sensitive.5 The type of α subunit is less important for affinity, although the magnitude of current enhancement may vary.5 Thus, β1-containing receptors appear to play little if any role in etomidate-induced anesthesia.
Observations from this study do not provide any additional information about how exactly R–mTFD-MPAB causes anesthesia, but 4 lines of evidence support the hypothesis that it causes anesthesia by acting on GABAARs that contain γ-subunits.11,14 First, in an equilibrium LORR study of tadpoles immersed in R–mTFD-MPAB solutions, the 50% effective concentration was found to be 3.7 μM. Second, this concentration is comparable with that which enhances currents induced by low concentrations of GABA in α1β3γ2 GABAARs in oocytes (50% effective concentration is 2.1 μM). Third, the enantiomer, S–mTFD-MPAB, is much less potent both in tadpoles and in modulation of recombinant GABAARs.14 Fourth, R–mTFD-MPAB photolabels α1β3γ2 GABAARs in the γ2+-β3− interfaces at the level of the transmembrane domain at a site that is homologous to the etomidate site in the β+-α− interfaces.11
All synaptic GABAA receptors are thought to contain γ2 subunits because of their critical role in targeting receptors to the synapse, and GABAA receptors lacking γ2 are thought to be exclusively extrasynaptic; however, not all γ2-containing GABAARs are synaptic. Thus, R–mTFD-MPAB and etomidate will both act on native synaptic receptors, but they will only act together on a subset of extrasynaptic GABAA receptors that contain γ subunits. Thus, we might expect the physiologic processes underlying the global anesthetic state to differ between the 2 agents. For example, δ-subunit–containing extrasynaptic receptors have been implicated in the action of etomidate16 but are unlikely to be important in the action of R–mTFD-MPAB because γ2 Ser-301 in the R–mTFD-MPAB–binding site is homologous with δ Trp-299, a residue that is large enough to sterically hinder binding. Indeed, site-directed mutagenesis to a tryptophan is commonly used for this purpose.17,18
An unexpected observation during these experiments was the unique behavior of mutant mice receiving etomidate. To our knowledge, this rolling behavior has not been reported previously. Reported studies in which β3-N265M mice received etomidate7,13 indeed showed decreased times until RORR in mutants, but little detail was given as to what the authors defined as RORR or any subjective differences in behavior during experiments. Previous studies of genetically modified mice that lack specific GABAAR subunits, or that carry mutations rendering specific subunits insensitive to anesthetics, have shown that specific end points depend upon certain subsets of GABAARs.1,16 For example, immobility (impaired hindlimb withdrawal reflex) produced by etomidate and propofol is mediated by GABAARs that incorporate β3 subunits.7 By contrast, sedation (reduced spontaneous motor activity) is mediated by GABAARs that incorporate β2 subunits.8 Similarly, memory impairment produced by a low dose of etomidate (reduced fear conditioning to context) depends upon modulation of GABAARs that incorporate β2 subunits together with α5 subunits.19–21 Our present finding that LORR is shorter in mice that carry the β3-N265M mutation is consistent with previous studies showing that hypnosis depends in part on GABAARs that incorporate β3 subunits.7,13 Although N265M mice attempted to right themselves sooner than WT mice, our novel observation that β3-N265M mice did not recover the ability to maintain an upright posture on all 4 paws until the same time as WT mice indicates that impaired coordination or balance induced by etomidate does not depend upon β3 subunits, so presumably is produced by modulation of β2 subunits because GABAA receptors that incorporate β1 subunits are relatively insensitive to etomidate.
During experimentation, an attempt was made to blind the injector/observer to prevent bias as much as possible. After each round of injections, however, the observer was unblinded for data tabulation. The unique behavior of mutants receiving etomidate thus diminished the blinding process during later rounds of injections.
A weakness of the present study is the apparently limited therapeutic range for R–mTFD-MPAB. Injections of the 10 mg/kg dose were halted because of a large proportion of postinjection deaths from the drug, and 12.5% of animals died after successful injection with the next greatest dose (7.5 mg/kg). Previous experiments (data not shown) indicated limited efficacy with doses as low as 2.5 to 3 mg/kg, making the therapeutic window for R–mTFD-MPAB in mice quite narrow. This was not an issue for our purposes because we sought only to compare 2 genotypes receiving similar doses and not to establish a dose-response curve.
The primary aim of this study was to further test the hypothesis that R–mTFD-MPAB binds to the GABAAR at a site remote from the site of etomidate binding. The data we obtained largely support the hypothesis because mice with the β3-N265M mutation were not resistant to the anesthetic effects of R–mTFD-MPAB. For reasons unclear at this time, mutant mice receiving 5 mg/kg R–mTFD-MPAB had in fact a significantly longer (2-fold) time to RORR. In addition, the observed differences in emergence behaviors in mutant versus WT mice receiving etomidate raises further questions as to the location, composition, and function of the GABAAR. Further research is needed on the subunit dependence of the action of R–mTFD-MPAB on GABAARs because it also photolabeled to a lesser degree the α1+-β3− subunit interface.11 The ability to compare the in vivo actions of R-etomidate and R–mTFD-MPAB, however, clearly holds promise both for finding the common pathways by which they both exert anesthesia and for teasing apart GABAAR-mediated contributions to the different components of anesthetic action. Such an endeavor could eventually lead to the development of agents that act more selectively on the CNS to the benefit of patients.
Name: Corey A. Amlong, MD, MS.
Contribution: This author helped to design and conduct the experiments, analyze, create the figures, and write and edit the manuscript.
Name: Mark G. Perkins, BS.
Contribution: This author helped with the animal husbandry and in conducting the experiments.
Name: Timothy T. Houle, PhD.
Contribution: This author performed the statistical analysis.
Name: Keith W. Miller, DPhil.
Contribution: This author helped concept the data, create the figures, and write and edit the manuscript.
Name: Robert A. Pearce, MD, PhD.
Contribution: This author helped design the experiment, analyze, and write and edit the manuscript.
This manuscript was handled by: Gregory J. Crosby, MD.
The authors thank Professor Karol S. Bruzik and Dr. Pavel Y. Savechenkov of the Department of Medicinal Chemistry and Pharmacology, University of Illinois, Chicago, Illinois, for providing the R–mTFD-MPAB. They also thank Drs. Rachel Jurd and Uwe Rudolph for providing the genetically modified mice studied. Figure 1 was produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health [NIH] P41 RR-01081)22 with a homology model kindly supplied by David C. Chiara.
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