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Cysteine substitutions define etomidate binding and gating linkages in the ?-M1 domain of ?-aminobutyric acid type A (GABAA) receptors.

ABSTRACT: Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled ?Met-236 in transmembrane domain M1, which structural models locate in the ?+/?- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human ?1?2?2L GABAA receptors, we applied the substituted cysteine accessibility method to ?1-M1 domain residues extending from ?1Gln-229 to ?1Gln-242. We used electrophysiology to characterize each mutant's sensitivity to GABA and etomidate. We also measured rates of sulfhydryl modification by p-chloromercuribenzenesulfonate (pCMBS) with and without GABA and tested if etomidate blocks modification of pCMBS-accessible cysteines. Cys substitutions in the outer ?1-M1 domain impaired GABA activation and variably affected etomidate sensitivity. In seven of eight residues where pCMBS modification was evident, rates of modification were accelerated by GABA co-application, indicating that channel activation increases water and/or pCMBS access. Etomidate reduced the rate of modification for cysteine substitutions at ?1Met-236, ?1Leu-232 and ?1Thr-237. We infer that these residues, predicted to face ?2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer ?1-M1 helix within a subdomain that undergoes significant structural rearrangement during channel gating. Our results are consistent with in silico docking calculations in a homology model that orient the long axis of etomidate approximately orthogonal to the transmembrane axis.

SUBMITTER: Stewart DS

PROVIDER: S-EPMC3798502 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

Stewart Deirdre S DS Hotta Mayo M Li Guo-Dong GD Desai Rooma R Chiara David C DC Olsen Richard W RW Forman Stuart A SA

The Journal of biological chemistry 20130905 42

Etomidate is a potent general anesthetic that acts as an allosteric co-agonist at GABAA receptors. Photoreactive etomidate derivatives labeled αMet-236 in transmembrane domain M1, which structural models locate in the β+/α- subunit interface. Other nearby residues may also contribute to etomidate binding and/or transduction through rearrangement of the site. In human α1β2γ2L GABAA receptors, we applied the substituted cysteine accessibility method to α1-M1 domain residues extending from α1Gln-22 ...[more]

PMID: 24009076

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Project description:Backgroundγ-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 αβγ receptors and do not form adducts with all contact residues. Complementary approaches may further define anesthetic sites in typical GABAA receptors.MethodsTwo 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 α1, β3, and γ2L GABAA receptor subunits: two photolabeled residues, α1M236 and β3M227, and their homologs.ResultsTryptophan substitutions at α1M236 and positional homologs β3L231 and γ2L246 all caused spontaneous channel gating and reduced γ-aminobutyric acid EC50. Substituted cysteine modification experiments indicated etomidate protection at α1L232C and α1M236C, R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid protection at β3M227C and β3L231C, and propofol protection at α1M236C and β3M227C. No alphaxalone protection was evident at the residues the authors explored, and none of the tested anesthetics protected γ2I242C or γ2L246C.ConclusionsAll 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 α1M236 and β3M227 and revealed overlapping noncongruent sites for etomidate and propofol in β-α interfaces and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid and propofol in α-β and γ-β interfaces. The authors' results identify the α-γ transmembrane interface as a potentially unique orphan modulator site.

Dataset Information

Cysteine substitutions define etomidate binding and gating linkages in the ?-M1 domain of ?-aminobutyric acid type A (GABAA) receptors.

Publications

Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-aminobutyric acid type A (GABAA) receptors.

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