The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

Belelli, Delia; Lambert, Jeremy J.; Peters, John A.; Wafford, Keith A.; Whiting, Paul J.

doi:10.1073/pnas.94.20.11031

Cited by 361 publications

(342 citation statements)

References 27 publications

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“…In addition, aliphatic carbocation intermediates are known to be highly reactive with water (t1 ⁄ 2 Ͻ1 ns (25)), further limiting the possibility of diffusional encounters with reactive side chains distant from the binding site. That the photolabeling of ␣1Met-236 and ␤Met-286 was potentiated in the presence of GABA, fully inhibitable by etomidate (12), and inhibitable by propofol 6 provides further evidence that [ 3 H]azietomidate is acting as a specific affinity label and that the photolabeling results provide a direct identification of two amino acids contributing to the etomidate binding site.…”

Section: Photoaffinity Labeling With [mentioning

confidence: 70%

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Chiara

Cohen

et al. 2009

Journal of Biological Chemistry

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) J. Neurosci. 26, 11599 -11605) azietomidate photolabeling of GABA A R ␣1Met-236 and ␤Met-286 (in ␣M1 and ␤M3). Positioning these two photolabeled amino acids in a single type of binding site at the interface of ␤ and ␣ subunits (two copies per pentamer) is consistent with a GABA A R homology model based upon the structure of the nicotinic acetylcholine receptor and with recent ␣M1 to ␤M3 cross-linking data. Biologically active neurosteroids enhance rather than inhibit azietomidate photolabeling, as assayed at the level of GABA A R subunits on analytical SDS-PAGE, and protein microsequencing establishes that the GABA A R-modulating neurosteroids do not inhibit photolabeling of GABA A R ␣1Met-236 or ␤Met-286 but enhance labeling of ␣1Met-236. Thus modulatory steroids do not bind at the same site as etomidate, and neither of the amino acids identified as neurosteroid activation determinants (Hosie, A. M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature 444, 486 -489) are located at the subunit interface defined by our etomidate site model. GABA A3 receptors (GABA A R) are major mediators of brain inhibitory neurotransmission and participate in most circuits and behavioral pathways relevant to normal and pathological function (1). GABA A R are subject to modulation by endogenous neurosteroids, as well as myriad clinically important central nervous system drugs including general anesthetics, benzodiazepines, and possibly ethanol (1, 2). The mechanism of GABA A R modulation by these different classes of drugs is of major interest, including identification of the receptor amino acid residues involved in binding and action of the drugs.In the absence of high resolution crystal structures of drugreceptor complexes, the locations of anesthetic binding sites in GABA A Rs have been predicted based upon analyses of functional properties of point mutant receptors, which identified residues in the ␣ and ␤ subunit M1-M4 transmembrane helices important for modulation by volatile anesthetics (primarily ␣ subunit) and by intravenous agents, including etomidate and propofol (␤ subunit) (3-5). Position ␤M2-15, numbered relative to the N terminus of the helix, functions as a major determinant of etomidate and propofol potency as GABA modulators in vitro and in vivo (6 -8). By contrast, this residue is not implicated for modulation by the neurosteroids, potent endogenous modulators of GABA A R (9).Photoaffinity labeling, which allows the identification of residues in proximity to drug binding sites (10, 11), has been used to identify two GABA A R amino acids covalently modified by the etomidate analog [ 3 H]azietomidate (12): ␣1Met-236 within ␣M1 and ␤Met-286 within ␤M3. Photolabeling of these residues was inhibited equally by nonradioactive etomidate and enhanced proportionately by GABA present in the assay, consistent with the presence of these two residues in a common drug binding pocket that would be located at the interface between the ␤ and ␣ subunits in the transmembrane domain (12). Mutational analyses identi...

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Section: Photoaffinity Labeling With [mentioning

confidence: 70%

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Chiara

Cohen

et al. 2009

Journal of Biological Chemistry

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“…GABA A receptor residues that are photolabeled by etomidate derivatives (13,14), in both ␣-M1 and ␤-M3, are highlighted in pink. We also illustrate ␤2Asn-265 on ␤-M2, a residue where mutations influence etomidate sensitivity (37,38).…”

Section: Chemicals-r-(ϩ)-etomidate Was Obtained From Bedford Laboratomentioning

confidence: 99%

“…In the 100 lowest energy docking orientations in the binding sites at GABA A receptor ␤2-M3/␣1-M1 interfaces, the etomidate molecule is oriented with the benzene ring located near ␤2-M2. In the lowest energy poise, the non-branching nitrogen in the imidazole group is located 3.0 Å from the amide nitrogen of ␤2N265 (M2-15Ј), a residue where mutations affect etomidate sensitivity (37,38). The ester leaving group (ethanol) projects outward from the ion channel toward the lipid-protein interface between ␣1-M1 and ␤2-M3.…”

Section: Etomidate Docking To a Molecular Structure Homologymentioning

confidence: 99%

Cysteine Substitutions Define Etomidate Binding and Gating Linkages in the α-M1 Domain of γ-Aminobutyric Acid Type A (GABAA) Receptors

Stewart

Hotta

et al. 2013

Journal of Biological Chemistry

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Background: Etomidate induces anesthesia via intersubunit transmembrane sites in GABA A receptors. Results: In receptors engineered with ␣-M1 domain cysteines, GABA accelerates modification. Etomidate inhibits modification at three positions. Conclusion: Etomidate contacts a subdomain of ␣-M1 linked to channel gating, consistent with in silico model docking. Significance: We identify new structures that both bind anesthetic and modulate channel gating through rearrangement.

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“…This patient showed a decreased sensitivity to etomidate and propofol. In studies on recombinant receptors, mutations at amino acid 265 in the second and amino acid 286 in the third transmembrane region of the GABA A receptor b subunits b1, b2, and b3 have been demonstrated to render GABA A receptors largely insensitive to etomidate and/or propofol action in vitro (Belelli et al, 1997;Krasowski et al, 1998;Siegwart et al, 2002). We therefore sequenced these regions in all three b subunit genes, but were unable to detect a mutation that would cause an amino-acid change.…”

Section: Discussionmentioning

confidence: 99%

“…Propofol potentiation of GABA-induced currents was abolished by a point mutation in the GABA A receptor b1 subunit (Krasowski et al, 1998). Etomidate has also been shown to influence the function of recombinant human GABA A receptors in vitro (Belelli et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Diminished GABAA Receptor-Binding Capacity and a DNA Base Substitution in a Patient with Treatment-Resistant Depression and Anxiety

Kosel¹,

Rudolph

Wielepp

et al. 2003

Neuropsychopharmacol

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In this report, we describe the case of a caucasion male patient, aged 42 years, suffering from severe treatment-resistant generalized anxiety disorder with panic attacks and from major depression for which he was treated with a course of electroconvulsive therapy. During electroconvulsive treatment, anesthesia was difficult to induce with etomidate and, once, propofol. Bispectral indices recordings (assessing the depth of anesthesia) revealed a much shorter duration of loss of responsiveness compared to a control patient receiving also a course of electroconvulsive therapy. Since GABA A receptors with 123 I-iomazenil SPECT and found a clearly diminished binding of the radiotracer in the right frontal and orbitotemporal regions compared to the recordings in a 38-year-old healthy male control. Genetic analysis of the exons 7 and 8 of the GABRB1-3 genes coding for the1-3-subunits of the GABA A receptors revealed a silent G to A substitution in the third position of amino acid 257 of the b1-subunit. To our knowledge, this is the first report of a link between insensivity to anesthetic agents and altered GABA A receptor function in a clinical case. Whereas reduced GABA A receptor-binding capacity has been investigated in anxiety disorders, this has not been the case in depressive disorders. This case illustrates how clinical observations in psychiatry can prompt investigations by modern techniques and potentially link clinics and basic sciences. No conclusion can, however, be made about casual links in this single case.

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The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

Cited by 361 publications

References 27 publications

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

Cysteine Substitutions Define Etomidate Binding and Gating Linkages in the α-M1 Domain of γ-Aminobutyric Acid Type A (GABAA) Receptors

Diminished GABAA Receptor-Binding Capacity and a DNA Base Substitution in a Patient with Treatment-Resistant Depression and Anxiety

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