3 research outputs found

    Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [<sup>3</sup>H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

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    The γ-aminobutyric acid type A receptor (GABA<sub>A</sub>R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA<sub>A</sub>Rs purified from bovine brain, [<sup>3</sup>H]­azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA<sub>A</sub>R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) <i>J. Neurosci. 26</i>, 11599–11605]. To further define GABA<sub>A</sub>R etomidate binding sites, we now use [<sup>3</sup>H]­TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA<sub>A</sub>Rs and more extensively identify photolabeled GABA<sub>A</sub>R amino acids. [<sup>3</sup>H]­TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [<sup>3</sup>H]­azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA<sub>A</sub>R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [<sup>3</sup>H]­TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA<sub>A</sub>R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [<sup>3</sup>H]­TDBzl-etomidate and [<sup>3</sup>H]­azietomidate also provides evidence of a homologous etomidate binding site at the β3−β3 subunit interface in the α1β3 GABA<sub>A</sub>R

    Photoaffinity Labeling the Propofol Binding Site in GLIC

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    Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABA<sub>A</sub>R), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus. In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) <i>Nature 469</i>, 428–431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3<i>H</i>-diazirin-3-yl]­phenol or AziP<i>m</i>) that acts as an anesthetic in vivo and potentiates GABA<sub>A</sub>R in vitro. For GLIC expressed in <i>Xenopus</i> oocytes, propofol and AziP<i>m</i> inhibited current responses at pH 5.5 (EC<sub>20</sub>) with IC<sub>50</sub> values of 20 and 50 μM, respectively. When [<sup>3</sup>H]­AziP<i>m</i> (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziP<i>m</i> bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket

    Allyl <i>m</i>-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic

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    We synthesized 5-allyl-1-methyl-5-(<i>m</i>-trifluoromethyl-diazirynylphenyl)­barbituric acid (<b>14</b>), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as <i>S</i> by X-ray crystallography. Additionally, we obtained the <sup>3</sup>H-labeled ligand with high specific radioactivity. <i>R</i>-(−)-<b>14</b> is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC<sub>50</sub> approaches those for propofol and etomidate, whereas <i>S</i>-(+)-<b>14</b> is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, <i>R</i>-(−)-<b>14</b> both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1β2/3γ2L GABA<sub>A</sub> receptors. Finally, <i>R</i>-(−)-<b>14</b> was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and β3 subunits of human α1β3 GABA<sub>A</sub> receptors. These results indicate <i>R</i>-(−)-<b>14</b> is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors
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