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.

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.

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.