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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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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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