title: Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.
abstract: BACKGROUND
  Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC.
  
  
  METHODS
  We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes.
  
  
  RESULTS
  EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels.
  
  
  CONCLUSIONS
  Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas.
authors.forename: Suzzane
authors.initials: S
authors.lastname: Horani
authors.affiliation: Chemistry Department , Skidmore College, Saratoga Springs, New York.
authors.forename: Evan P
authors.initials: EP
authors.lastname: Stater
authors.affiliation: Channel-Receptor Research Group , Pasteur Institute, Bâtiment Fernbach, Paris, France.
authors.forename: Pierre-Jean
authors.initials: PJ
authors.lastname: Corringer
authors.affiliation: Department of Anesthesia , Stanford University School of Medicine, Stanford, California.
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas.
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas.|Chemistry Department , Skidmore College, Saratoga Springs, New York.
authors.forename: Rebecca J
authors.identifier: http://orcid.org/0000-0003-2049-3378
authors.initials: RJ
authors.lastname: Howard
mesh_terms: D000818:Animals; D000458:Cyanobacteria; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D058446:Ligand-Gated Ion Channels / Q000737:chemistry / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects*; D009865:Oocytes; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000737:chemistry* / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D058446:Ligand-Gated Ion Channels; D018009:Receptors, Glycine; D000431:Ethanol
keywords: Chimera; Glycine; Ligand-Gated Ion Channel; Picrotoxin