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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.

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

Publications

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

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