title: Cysteine substitutions define etomidate binding and gating linkages in the α-M1 domain of γ-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 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-229 to α1Gln-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 α1-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 α1Met-236, α1Leu-232 and α1Thr-237. We infer that these residues, predicted to face β2-M3 or M2 domains, contribute to etomidate binding. Thus, etomidate interacts with a short segment of the outer α1-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.
authors.affiliation: From the Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114,; the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and.
authors.forename: Deirdre S
authors.initials: DS
authors.lastname: Stewart
authors.affiliation: From the Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
authors.forename: Mayo
authors.initials: M
authors.lastname: Hotta
authors.affiliation: the Departments of Molecular and Medical Pharmacology and; Anesthesiology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095.
authors.forename: Guo-Dong
authors.initials: GD
authors.lastname: Li
authors.affiliation: From the Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114.
authors.forename: Rooma
authors.initials: R
authors.lastname: Desai
authors.affiliation: the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and.
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.affiliation: the Departments of Molecular and Medical Pharmacology and.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Olsen
authors.affiliation: From the Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114,. Electronic address: saforman@partners.org.
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Forman
mesh_terms: D002731:4-Chloromercuribenzenesulfonate / Q000737:chemistry* / Q000494:pharmacology; D019943:Amino Acid Substitution; D018686:Anesthetics, Intravenous / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005045:Etomidate / Q000737:chemistry* / Q000494:pharmacology; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D062105:Molecular Docking Simulation; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D018686:Anesthetics, Intravenous; D004791:Enzyme Inhibitors; D011963:Receptors, GABA-A; D002731:4-Chloromercuribenzenesulfonate; D005045:Etomidate
keywords: Allosteric Regulation; Anesthetics; GABA Receptors; Ion Channels; Nicotinic Acetylcholine Receptors; Sulfhydryl