Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors.

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1ß2/3γ2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near ßM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1ß3M286Cγ2L receptors to estimate the distance from etomidate to ß3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cß3γ2L receptors; (ii) studies in α1L232Wß3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to ß3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cß3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wß3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1ß3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to ß3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between ß3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane ß+/α- inter-subunit pockets. Comparing results in α1ß3M286Cγ2L and α1L232Wß3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward ß3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.

Competitive Interactions Between Propofol and Diazepam: Studies in GABAA Receptors and Zebrafish.

Although propofol is among the most commonly administered general anesthetics, its mechanism of action is not fully understood. It has been hypothesized that propofol acts via a similar mechanism as (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate) by binding within the GABAA receptor transmembrane receptor domain at the two ß +/α - subunit interfaces with resultant positive allosteric modulation. To test this hypothesis, we leveraged the ability of diazepam to bind to those sites and act as a competitive antagonist. We used oocyte-expressed α 1 ß 3 γ 2L GABAA receptors to define the actions of diazepam (± flumazenil) on currents activated or potentiated by propofol and a zebrafish activity assay to define the impact of diazepam and flumazenil on propofol-induced anesthesia. We found that diazepam increased the amplitudes of GABAA receptor-mediated currents at nanomolar concentrations but reduced them at micromolar concentrations. The current amplitude changes produced by nanomolar diazepam concentrations were inhibited by flumazenil whereas those produced by micromolar diazepam concentrations were not. Studies of agonist potentiation showed that the micromolar inhibitory action of diazepam was surmountable by high concentrations of propofol and produced a rightward shift in the propofol concentration-response curve characterized by a Schild slope not statistically significantly different from 1, consistent with competition between diazepam and propofol. Although micromolar concentrations of diazepam (plus flumazenil) similarly reduced GABAA receptor currents modulated by propofol and etomidate, it only reduced the anesthetic actions of etomidate. We conclude that while both propofol and etomidate can modulate GABAA receptors by binding to the ß +/α - subunit interfacial sites, propofol-induced anesthesia likely involves additional target sites. SIGNIFICANCE STATEMENT: Although the drug combination of diazepam and flumazenil reverses the GABAA receptor positive modulatory actions of both propofol and (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate), it only reverses the in vivo anesthetic actions of etomidate. These results strongly suggest that distinct mechanisms of action account for the anesthetic actions of these two commonly administered anesthetic agents.

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

BACKGROUND: Etomidate, barbiturates, alfaxalone, and propofol are anesthetics that allosterically modulate γ-aminobutyric acid type A (GABAA) receptors via distinct sets of molecular binding sites. Two-state concerted coagonist 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 GABAA receptors. METHODS: The authors 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 coagonist interactions in α1ß3γ2L receptors activated by γ-aminobutyric acid (GABA) versus 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-5-allyl-1-methyl m-trifluoromethyl mephobarbital (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 alfaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alfaxalone was supra-additive (n = 6; P = 0.023 by paired t test), but etomidate plus R-mTFD-MPAB or propofol was not. CONCLUSIONS: In both zebrafish and GABAA 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 coagonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.

Selective actions of benzodiazepines at the transmembrane anaesthetic binding sites of the GABAA receptor: In vitro and in vivo studies.

BACKGROUND AND PURPOSE: In addition to binding to the classical high-affinity extracellular benzodiazepine binding site of the GABAA receptor, some benzodiazepines occupy transmembrane inter-subunit anaesthetic sites that bind etomidate (ß+ /α- sites) or the barbiturate derivative R-mTFD-MPAB (α+ /ß- and γ+ /ß- sites). We aimed to define the functional effects of these interactions on GABAA receptor activity and animal behaviour. EXPERIMENTAL APPROACH: With flumazenil blocking classical high-affinity extracellular benzodiazepine site effects, modulation of GABA-activated currents by diazepam, midazolam and flurazepam was measured electrophysiologically in wildtype and M2-15' mutant α1 ß3 γ2L GABAA receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil. KEY RESULTS: In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABAA receptor currents. ß3 N265M (M2-15' in the ß+ /α- sites) and α1 S270I (M2-15' in the α+ /ß- site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2 S280W mutation (M2-15' in the γ+ /ß- site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2-15' mutations. In the presence of flumazenil, zebrafish locomotion was enhanced by diazepam at concentrations up to 30 µM and suppressed at 100 µM, suppressed by midazolam and enhanced by flurazepam. CONCLUSIONS AND IMPLICATIONS: Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.

Substituted Cysteine Modification and Protection with n-Alkyl- Methanethiosulfonate Reagents Yields a Precise Estimate of the Distance between Etomidate and a Residue in Activated GABA Type A Receptors.

The anesthetic etomidate modulates synaptic α1ß2/3γ2 GABAA receptors via binding sites located in transmembrane ß+/α- interfaces. Various approaches indicate that etomidate binds near ß2/3M286 side chains, including recent cryogenic electron microscopy images in α1ß2γ2L receptors under nonphysiologic conditions with ∼3.5-Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized n-alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and ß3M286 side chains in activated functional receptors. Using voltage-clamp electrophysiology in Xenopus oocytes expressing α1ß3M286Cγ2L GABAA receptors, we measured functional changes after exposing GABA-activated receptors to n-alkyl-MTS reagents, from methyl-MTS to n-decyl-MTS. Based on previous studies using a large sulfhydryl reagent, we anticipated that cysteine modifications large enough to overlap etomidate sites would cause persistently increased GABA sensitivity and decreased etomidate modulation and that etomidate would hinder these modifications, reducing effects. Based on altered GABA or etomidate sensitivity, ethyl-MTS and larger n-alkyl-MTS reagents modified GABA-activated α1ß3M286Cγ2L GABAA receptors. Receptor modification by n-propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked ß3M286C modification by n-propyl-MTS, n-butyl-MTS, and n-hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, and ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and n-propyl-MTS, from which we infer that -S-(n-propyl) is the smallest ß3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and -S-ethyl and -S-(n-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the ß3M286 side chain. SIGNIFICANCE STATEMENT: Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. This study combined electrophysiology, a cysteine substitution, and n-alkyl-methanethiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1ß3γ2L GABA type A receptor residue and etomidate bound in the transmembrane ß+/α- interface.

Competitive Antagonism of Etomidate Action by Diazepam: In Vitro GABAA Receptor and In Vivo Zebrafish Studies.

BACKGROUND: Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the α1ß3γ2L γ-aminobutyric acid type A (GABAA) receptor, diazepam also binds to etomidate binding sites located in the transmembrane receptor domain. Because such binding is characterized by low modulatory efficacy, the authors hypothesized that diazepam would act in vitro and in vivo as a competitive etomidate antagonist. METHODS: The concentration-dependent actions of diazepam on 20 µM etomidate-activated and 6 µM GABA-activated currents were defined (in the absence and presence of flumazenil) in oocyte-expressed α1ß3γ2L GABAA receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified α1ß3γ2L GABAA receptors by [H]azietomidate was assessed in photoaffinity labeling protection studies. The impact of diazepam (in the absence and presence of flumazenil) on the anesthetic potencies of etomidate and ketamine was compared in a zebrafish model. RESULTS: At nanomolar concentrations, diazepam comparably potentiated etomidate-activated and GABA-activated GABAA receptor peak current amplitudes in a flumazenil-reversible manner. The half-maximal potentiating concentrations were 39 nM (95% CI, 27 to 55 nM) and 26 nM (95% CI, 16 to 41 nM), respectively. However, at micromolar concentrations, diazepam reduced etomidate-activated, but not GABA-activated, GABAA receptor peak current amplitudes in a concentration-dependent manner with a half-maximal inhibitory concentration of 9.6 µM (95% CI, 7.6 to 12 µM). Diazepam (12.5 to 50 µM) also right-shifted the etomidate-concentration response curve for direct activation without reducing the maximal response and inhibited receptor photoaffinity labeling by [H]azietomidate. When administered with flumazenil, 50 µM diazepam shifted the etomidate (but not the ketamine) concentration-response curve for anesthesia rightward, increasing the etomidate EC50 by 18-fold. CONCLUSIONS: At micromolar concentrations and in the presence of flumazenil to inhibit allosteric modulation via the classical benzodiazepine binding site of the GABAA receptor, diazepam acts as an in vitro and in vivo competitive etomidate antagonist.

A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors.

The pentameric γ-aminobutyric acid type A receptors (GABAARs) are the major inhibitory ligand-gated ion channels in the central nervous system. They mediate diverse physiological functions, mutations in them are associated with mental disorders and they are the target of many drugs such as general anesthetics, anxiolytics and anti-convulsants. The five subunits of synaptic GABAARs are arranged around a central pore in the order ß-α-ß-α-γ. In the outer third of the transmembrane domain (TMD) drugs may bind to five homologous intersubunit binding sites. Etomidate binds between the pair of ß - α subunit interfaces (designated as ß+/α-) and R-mTFD-MPAB binds to an α+/ß- and an γ+/ß- subunit interface (a ß- selective ligand). Ligands that bind selectively to other homologous sites have not been characterized. We have synthesized a novel photolabel, (2,6-diisopropyl-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl)methanol or pTFD-di-iPr-BnOH). It is a potent general anesthetic that positively modulates agonist and benzodiazepine binding. It enhances GABA-induced currents, shifting the GABA concentration-response curve to lower concentrations. Photolabeling-protection studies show that it has negligible affinity for the etomidate sites and high affinity for only one of the two R-mTFD-MPAB sites. Exploratory site-directed mutagenesis studies confirm the latter conclusions and hint that pTFD-di-iPr-BnOH may bind between the α+/ß- and α+/γ- subunits in the TMD, making it an α+ ligand. The latter α+/γ- site has not previously been implicated in ligand binding. Thus, pTFD-di-iPr-BnOH is a promising new photolabel that may open up a new pharmacology for synaptic GABAARs.

Effects of Anticholinesterase Reversal Under General Anesthesia on Postoperative Cardiovascular Complications: A Retrospective Cohort Study.

BACKGROUND: The anticholinesterase neostigmine and the muscarinic inhibitor glycopyrrolate are frequently coadministered for the reversal of neuromuscular blockade. This practice can precipitate severe bradycardia or tachycardia, but whether it affects the incidence of cardiovascular complications remains unclear. We hypothesized that anticholinesterase reversal with neostigmine and glycopyrrolate versus no anticholinesterase reversal increases the risk of postoperative cardiovascular complications among adult patients undergoing noncardiac surgery with general anesthesia. METHODS: We conducted a prespecified retrospective analysis of hospital registry data from a major health care network for patients undergoing surgery with general anesthesia from January 2007 to December 2015. The primary outcome was a composite of cardiac dysrhythmia, acute heart failure, transient ischemic attack, ischemic stroke, and acute myocardial infarction within 30 days after surgery. We performed sensitivity analyses in subgroups and propensity score adjustment and explored the association between exposure and outcome in subgroups of patients with high risk of cardiovascular complications. RESULTS: Of the 98,147 cases receiving neuromuscular blockade, 73,181 (74.6%) received neostigmine and glycopyrrolate, while 24,966 (25.4%) did not. A total of 5612 patients (7.7%) in the anticholinesterase reversal group and 1651 (6.6%) in the control group (P < .001) experienced the primary outcome. After adjustment for clinical covariates, neostigmine and glycopyrrolate exposure was significantly associated in a dose-dependent fashion (P for trend <.001, respectively) with tachycardia (adjusted odds ratio = 2.1 [95% CI, 1.97-2.23]; P < .001) and bradycardia (adjusted odds ratio = 2.84 [95% CI, 2.49-3.24]; P < .001) but not with postoperative cardiovascular complications (adjusted odds ratio = 1.03 [95% CI, 0.97-1.1]; P = .33). We identified a significant effect modification of anticholinesterase reversal by high age, high-risk surgery, and history of atrial fibrillation (P for interaction = .002, .001, and .02, respectively). By using linear combinations of main effect and exposure-risk interaction terms, we detected significant associations between anticholinesterase reversal and cardiovascular complications toward a higher vulnerability in these patient subgroups. CONCLUSIONS: Neuromuscular blockade reversal with neostigmine and glycopyrrolate was associated with an increased incidence of intraoperative tachycardia and bradycardia but not with 30-day postoperative cardiovascular complications. Exploratory analyses suggest that a high postoperative cardiovascular complication risk profile may modify the effects of anticholinesterase reversal toward clinical relevance.

Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor ß3 Subunits.

BACKGROUND: Transgenic mouse studies suggest that γ-aminobutyric acid type A (GABAA) receptors containing ß3 subunits mediate important effects of etomidate, propofol, and pentobarbital. Zebrafish, recently introduced for rapid discovery and characterization of sedative-hypnotics, could also accelerate pharmacogenetic studies if their transgenic phenotypes reflect those of mammals. The authors hypothesized that, relative to wild-type, GABAA-ß3 functional knock-out (ß3) zebrafish would show anesthetic sensitivity changes similar to those of ß3 mice. METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mutagenesis was used to create a ß3 zebrafish line. Wild-type and ß3 zebrafish were compared for fertility, growth, and craniofacial development. Sedative and hypnotic effects of etomidate, propofol, pentobarbital, alphaxalone, ketamine, tricaine, dexmedetomidine, butanol, and ethanol, along with overall activity and thigmotaxis were quantified in 7-day postfertilization larvae using video motion analysis of up to 96 animals simultaneously. RESULTS: Xenopus oocyte electrophysiology showed that the wild-type zebrafish ß3 gene encodes ion channels activated by propofol and etomidate, while the ß3 zebrafish transgene does not. Compared to wild-type, ß3 zebrafish showed similar morphology and growth, but more rapid swimming. Hypnotic EC50s (mean [95% CI]) were significantly higher for ß3 versus wild-type larvae with etomidate (1.3 [1.0 to 1.6] vs. 0.6 [0.5 to 0.7] µM; P < 0.0001), propofol (1.1 [1.0 to 1.4] vs. 0.7 [0.6 to 0.8] µM; P = 0.0005), and pentobarbital (220 [190 to 240] vs. 130 [94 to 179] µM; P = 0.0009), but lower with ethanol (150 [106 to 213] vs. 380 [340 to 420] mM; P < 0.0001) and equivalent with other tested drugs. Comparing ß3 versus wild-type sedative EC50s revealed a pattern similar to hypnosis. CONCLUSIONS: Global ß3 zebrafish are selectively insensitive to the same few sedative-hypnotics previously reported in ß3 transgenic mice, indicating phylogenetic conservation of ß3-containing GABAA receptors as anesthetic targets. Transgenic zebrafish are potentially valuable models for sedative-hypnotic mechanisms research.

Monod-Wyman-Changeux Allosteric Shift Analysis in Mutant α1ß3γ2L GABAA Receptors Indicates Selectivity and Crosstalk among Intersubunit Transmembrane Anesthetic Sites.

Propofol, etomidate, and barbiturate anesthetics are allosteric coagonists at pentameric α1ß3γ2 GABAA receptors, modulating channel activation via four biochemically established intersubunit transmembrane pockets. Etomidate selectively occupies the two ß +/α - pockets, the barbiturate photolabel R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) occupies homologous α +/ß - and γ +/ß - pockets, and propofol occupies all four. Functional studies of mutations at M2-15' or M3-36' loci abutting these pockets provide conflicting results regarding their relative contributions to propofol modulation. We electrophysiologically measured GABA-dependent channel activation in α1ß3γ2L or receptors with single M2-15' (α1S270I, ß3N265M, and γ2S280W) or M3-36' (α1A291W, ß3M286W, and γ2S301W) mutations, in the absence and presence of equipotent clinical range concentrations of etomidate, R-mTFD-MPAB, and propofol. Estimated open probabilities were calculated and analyzed using global two-state Monod-Wyman-Changeux models to derive log(d) parameters proportional to anesthetic-induced channel modulating energies (where d is the allosteric anesthetic shift factor). All mutations reduced the log(d) values for anesthetics occupying both abutting and nonabutting pockets. The Δlog(d) values [log(d, mutant) - log(d, wild type)] for M2-15' mutations abutting an anesthetic's biochemically established binding sites were consistently larger than the Δlog(d) values for nonabutting mutations, although this was not true for the M3-36' mutant Δlog(d) values. The sums of the anesthetic-associated Δlog(d) values for sets of M2-15' or M3-36' mutations were all much larger than the wild-type log(d) values. Mutant Δlog(d) values qualitatively reflect anesthetic site occupancy patterns. However, the lack of Δlog(d) additivity undermines quantitative comparisons of distinct site contributions to anesthetic modulation because the mutations impaired both abutting anesthetic binding effects and positive cooperativity between anesthetic binding sites.

Etomidate Effects on Desensitization and Deactivation of α4ß3δ GABAA Receptors Inducibly Expressed in HEK293 TetR Cells.

Central α4ßδ receptors are the most abundant isoform of δ subunit-containing extrasynaptic GABAA receptors that mediate tonic inhibition. Although the amplitude of GABA-activated currents through α4ßδ receptors is modulated by multiple general anesthetics, the effects of general anesthetics on desensitization and deactivation of α4ßδ receptors remain unknown. In the current study, we investigated the effect of etomidate, a potent general anesthetic, on the kinetics and the pseudo steady-state current amplitude of α4ß3δ receptors inducibly expressed in human embryonic kidney 293 TetR cells. Etomidate directly activates α4ß3δ receptors in a concentration-dependent manner. Etomidate at a clinically relevant concentration (3.2 µM) enhances maximal response without altering the EC50 of GABA concentration response. Etomidate also increases the extent of desensitization and prolongs the deactivation of α4ß3δ receptors in the presence of maximally activating concentrations of GABA (1 mM). To mimic the modulatory effect of etomidate on tonic currents, long pulses (30-60 seconds) of a low GABA concentration (1 µM) were applied to activate α4ß3δ receptors in the absence and presence of etomidate. Although etomidate increases the desensitization of α4ß3δ receptors, the pseudo steady-state current amplitude at 1 µM GABA is augmented by etomidate. Our data demonstrate that etomidate enhances the pseudo steady-state current of α4ß3δ receptors evoked by a GABA concentration comparable to an ambient GABA level, suggesting that α4ß3δ receptors may mediate etomidate's anesthetic effect in the brain.

Inhibitable photolabeling by neurosteroid diazirine analog in the ß3-Subunit of human hetereopentameric type A GABA receptors.

Pregnanolone and allopregnanolone-type ligands exert general anesthetic, anticonvulsant and anxiolytic effects due to their positive modulatory interactions with the GABAA receptors in the brain. Binding sites for these neurosteroids have been recently identified at subunit interfaces in the transmembrane domain (TMD) of homomeric ß3 GABAA receptors using photoaffinity labeling techniques, and in homomeric chimeric receptors containing GABAA receptor α subunit TMDs by crystallography. Steroid binding sites have yet to be determined in human, heteromeric, functionally reconstituted, full-length, glycosylated GABAA receptors. Here, we report on the synthesis and pharmacological characterization of several photoaffinity analogs of pregnanolone and allopregnanolone, of which 21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone (21-pTFDBzox-AP) was the most potent ligand. It is a partial positive modulator of the human α1ß3 and α1ß3γ2L GABAA receptors at sub-micromolar concentrations. [3H]21-pTFDBzox-AP photoincorporated in a pharmacologically specific manner into the α and ß subunits of those receptors, with the ß3 subunit photolabeled most efficiently. Importantly, photolabeling by [3H]21-pTFDBzox-AP was inhibited by the positive steroid modulators alphaxalone, pregnanolone and allopregnanolone, but not by inhibitory neurosteroid pregnenolone sulfate or by two potent general anesthetics and GABAAR positive allosteric modulators, etomidate and an anesthetic barbiturate. The latter two ligands bind to sites at subunit interfaces in the GABAAR that are different from those interacting with neurosteroids. 21-pTFDBzox-AP's potency and pharmacological specificity of photolabeling indicate its suitability for characterizing neurosteroid binding sites in native GABAA receptors.

High-throughput Screening in Larval Zebrafish Identifies Novel Potent Sedative-hypnotics.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. METHODS: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. RESULTS: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. CONCLUSIONS: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.

Combining Mutations and Electrophysiology to Map Anesthetic Sites on Ligand-Gated Ion Channels.

General anesthetics are known to act in part by binding to and altering the function of pentameric ligand-gated ion channels such as nicotinic acetylcholine and γ-aminobutyric acid type A receptors. Combining heterologous expression of the subunits that assemble to form these ion channels, mutagenesis techniques and voltage-clamp electrophysiology have enabled a variety of "structure-function" approaches to questions of where anesthetic binds to these ion channels and how they enhance or inhibit channel function. Here, we review the evolution of concepts and experimental strategies during the last three decades, since molecular biological and electrophysiological tools became widely used. Topics covered include: (1) structural models as interpretive frameworks, (2) various electrophysiological approaches and their limitations, (3) Monod-Wyman-Changeux allosteric models as functional frameworks, (4) structural strategies including chimeras and point mutations, and (5) methods based on cysteine substitution and covalent modification. We discuss in particular depth the experimental design considerations for substituted cysteine modification-protection studies.

Comparison of αßδ and αßγ GABAA receptors: Allosteric modulation and identification of subunit arrangement by site-selective general anesthetics.

GABAA receptors play a dominant role in mediating inhibition in the mature mammalian brain, and defects of GABAergic neurotransmission contribute to the pathogenesis of a variety of neurological and psychiatric disorders. Two types of GABAergic inhibition have been described: αßγ receptors mediate phasic inhibition in response to transient high-concentrations of synaptic GABA release, and αßδ receptors produce tonic inhibitory currents activated by low-concentration extrasynaptic GABA. Both αßδ and αßγ receptors are important targets for general anesthetics, which induce apparently different changes both in GABA-dependent receptor activation and in desensitization in currents mediated by αßγ vs. αßδ receptors. Many of these differences are explained by correcting for the high agonist efficacy of GABA at most αßγ receptors vs. much lower efficacy at αßδ receptors. The stoichiometry and subunit arrangement of recombinant αßγ receptors are well established as ß-α-γ-ß-α, while those of αßδ receptors remain controversial. Importantly, some potent general anesthetics selectively bind in transmembrane inter-subunit pockets of αßγ receptors: etomidate acts at ß+/α- interfaces, and the barbiturate R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (R-mTFD-MPAB) acts at α+/ß- and γ+/ß- interfaces. Thus, these drugs are useful as structural probes in αßδ receptors formed from free subunits or concatenated subunit assemblies designed to constrain subunit arrangement. Although a definite conclusion cannot be drawn, studies using etomidate and R-mTFD-MPAB support the idea that recombinant α1ß3δ receptors may share stoichiometry and subunit arrangement with α1ß3γ2 receptors.

Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABAA Receptors.

GABAA receptors can be directly activated and potentiated by the intravenous anesthetic propofol. Previous photolabeling, modeling, and functional data have identified two binding domains through which propofol acts on the GABAA receptor. These domains are defined by the ß(M286) residue at the ß"+"-α"-" interface in the transmembrane region and the ß(Y143) residue near the ß"-" surface in the junction between the extracellular and transmembrane domains. In the ternary receptor, there are predicted to be two copies of each class of sites, for a total of four sites per receptor. We used ß2α1γ2L and ß2α1 concatemeric constructs to determine the functional effects of the ß(Y143W) and ß(M286W) mutations to gain insight into the number of functional binding sites for propofol and the energetic contributions stemming from propofol binding to the individual sites. A mutation of each of the four sites affected the response to propofol, indicating that each of the four sites is functional in the wild-type receptor. The mutations mainly impaired stabilization of the open state by propofol, i.e., reduced gating efficacy. The effects were similar for mutations at either site and were largely additive and independent of the presence of other Y143W or M286W mutations in the receptor. The two classes of sites appeared to differ in affinity for propofol, with the site affected by M286W having about a 2-fold higher affinity. Our analysis indicates there may be one or two additional functionally equivalent binding sites for propofol, other than those modified by substitutions at ß(Y143) and ß(M286).

Alphaxalone Binds in Inner Transmembrane ß+-α- Interfaces of α1ß3γ2 γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αßγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane ß-α interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in ß-α interfaces. METHODS: The authors created 12 single-residue ß3 cysteine mutations: ß3T262C and ß3T266C in ß3-M2; and ß3M283C, ß3Y284C, ß3M286C, ß3G287C, ß3F289C, ß3V290C, ß3F293C, ß3L297C, ß3E298C, and ß3F301C in ß3-M3 helices. The authors coexpressed α1 and γ2L with each mutant ß3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. RESULTS: Eleven mutant ß3 subunits, when coexpressed with α1 and γ2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with ß3V290C, ß3F293C, ß3L297C, or ß3F301C mutations. Both etomidate and propofol protected receptors with ß3M286C or ß3V290C mutations. Etomidate also protected ß3F289C. In α1ß3γ2L structural homology models, all these protected residues are located in transmembrane ß-α interfaces. CONCLUSIONS: Alphaxalone binds in transmembrane ß-α pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.