title: Photoaffinity labeling the propofol binding site in GLIC.
abstract: Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), 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) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 μM, respectively. When [(3)H]AziPm (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 AziPm 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.
authors.affiliation: Department of Neurobiology, Harvard Medical School , Boston, Massachusetts 02115, United States.
authors.forename: David C
authors.initials: DC
authors.lastname: Chiara
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Gill
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Tommy
authors.initials: T
authors.lastname: Tillman
authors.forename: William P
authors.initials: WP
authors.lastname: Dailey
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Pei
authors.initials: P
authors.lastname: Tang
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Cohen
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001665:Binding Sites; D003978:Diazomethane / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry*; D058446:Ligand-Gated Ion Channels; D008958:Models, Molecular; D015742:Propofol / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000378:metabolism
chemical_list: C551949:2-isopropyl-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol; D000345:Affinity Labels; D001426:Bacterial Proteins; D007473:Ion Channels; D058446:Ligand-Gated Ion Channels; D011963:Receptors, GABA-A; D003978:Diazomethane; D015742:Propofol