title: Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.
abstract: BACKGROUND
  Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined.
  
  
  METHODS
  We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations.
  
  
  RESULTS
  Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC.
  
  
  CONCLUSIONS
  The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.
authors.affiliation: From the Departments of Anesthesiology (M.N.K., Q.C., Y.X., P.T.), Pharmacology and Chemical Biology (Y.X., P.T.), Structural Biology (Y.X.), and Computational and Systems Biology (P.T.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (W.B., R.G.E.).
authors.forename: Monica N
authors.initials: MN
authors.lastname: Kinde
authors.forename: Weiming
authors.initials: W
authors.lastname: Bu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
authors.forename: Pei
authors.initials: P
authors.lastname: Tang
mesh_terms: D000777:Anesthetics / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016974:Dickeya chrysanthemi; D005260:Female; D058446:Ligand-Gated Ion Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D014982:Xenopus laevis
chemical_list: D000777:Anesthetics; D058446:Ligand-Gated Ion Channels