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Mutations at beta N265 in ?-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

ABSTRACT: Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane ?+/?- inter-subunit interfaces. Early experiments in heteromeric receptors identified ?N265 (M2-15') on ?2 and ?3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the ?1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the ?N265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between ?N265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous ?1?2N265C?2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the ?-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on ?1?2N265C?2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive ?N265M mutants, we applied indirect structure-function strategies, exploiting second mutations in ?1?2/3?2L GABAA receptors. Using ?1M236C as a modifiable and anesthetic-protectable site occupancy reporter in ?+/?- interfaces, we found that ?N265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. ?N265M also impaired the transduction of gating effects associated with ?1M236W, a mutation that mimics ?+/?- anesthetic site occupancy. Our results show that ?N265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in ?+/?- sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for ?N265 in anesthetic binding within the ?+/?- transmembrane sites.

SUBMITTER: Stewart DS

PROVIDER: S-EPMC4210246 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Mutations at beta N265 in γ-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

Stewart Deirdre S DS Pierce David W DW Hotta Mayo M Stern Alex T AT Forman Stuart A SA

PloS one 20141027 10

Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α- inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15') on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate se ...[more]

PMID: 25347186

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Project description:?-Aminobutyric acid type A (GABAA) receptors mediate important effects of intravenous general anesthetics. Photolabel derivatives of etomidate, propofol, barbiturates, and a neurosteroid get incorporated in GABAA receptor transmembrane helices M1 and M3 adjacent to intersubunit pockets. However, photolabels have not been consistently targeted at heteromeric ??? receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors.Two mutation-based strategies, substituted tryptophan sensitivity and substituted cysteine modification-protection, combined with voltage-clamp electrophysiology in Xenopus oocytes, were used to evaluate interactions between four intravenous anesthetics and six amino acids in M1 helices of ?1, ?3, and ?2L GABAA receptor subunits: two photolabeled residues, ?1M236 and ?3M227, and their homologs.Tryptophan substitutions at ?1M236 and positional homologs ?3L231 and ?2L246 all caused spontaneous channel gating and reduced ?-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at ?1L232C and ?1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at ?3M227C and ?3L231C, and propofol protection at ?1M236C and ?3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected ?2I242C or ?2L246C.All five intersubunit transmembrane pockets of GABAA receptors display similar allosteric linkage to ion channel gating. Substituted cysteine modification and protection results were fully concordant with anesthetic photolabeling at ?1M236 and ?3M227 and revealed overlapping noncongruent sites for etomidate and propofol in ?-? interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in ?-? and ?-? interfaces. The authors' results identify the ?-? transmembrane interface as a potentially unique orphan modulator site.

Project description:BackgroundEtomidate, 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.MethodsThe 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 α1β3γ2L 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.ResultsIn 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.ConclusionsIn 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.Editor’s perspective

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Mutations at beta N265 in ?-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

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Mutations at beta N265 in γ-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.

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