title: Identification of an Inhibitory Alcohol Binding Site in GABAA ρ1 Receptors.
abstract: Alcohols inhibit γ-aminobutyric acid type A ρ1 receptor function. After introducing mutations in several positions of the second transmembrane helix in ρ1, we studied the effects of ethanol and hexanol on GABA responses using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. The 6' mutations produced the following effects on ethanol and hexanol responses: small increase or no change (T6'M), increased inhibition (T6'V), and small potentiation (T6'Y and T6'F). The 5' mutations produced mainly increases in hexanol inhibition. Other mutations produced small (3' and 9') or no changes (2' and L277 in the first transmembrane domain) in alcohol effects. These results suggest an inhibitory alcohol binding site near the 6' position. Homology models of ρ1 receptors based on the X-ray structure of GluCl showed that the 2', 5', 6', and 9' residues were easily accessible from the ion pore, with 5' and 6' residues from neighboring subunits facing each other; L3' and L277 also faced the neighboring subunit. We tested ethanol through octanol on single and double mutated ρ1 receptors [ρ1(I15'S), ρ1(T6'Y), and ρ1(T6'Y,I15'S)] to further characterize the inhibitory alcohol pocket in the wild-type ρ1 receptor. The pocket can only bind relatively short-chain alcohols and is eliminated by introducing Y in the 6' position. Replacing the bulky 15' residue with a smaller side chain introduced a potentiating binding site, more sensitive to long-chain than to short-chain alcohols. In conclusion, the net alcohol effect on the ρ1 receptor is determined by the sum of its actions on inhibitory and potentiating sites.
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Borghese
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Carlos I
authors.initials: CI
authors.lastname: Ruiz
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Ui S
authors.initials: US
authors.lastname: Lee
authors.affiliation: Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Madeline A
authors.initials: MA
authors.lastname: Cullins
authors.affiliation: Department of Anesthesia & Beckman Program for Molecular and Genetic Medicine, Stanford University , Palo Alto, California 94305, United States.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Bertaccini
authors.affiliation: Department of Anesthesia & Beckman Program for Molecular and Genetic Medicine, Stanford University , Palo Alto, California 94305, United States.
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 78712, United States.
authors.forename: R Adron
authors.initials: RA
authors.lastname: Harris
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D018682:GABA Agents / Q000494:pharmacology; D000441:Hexanols / Q000494:pharmacology; D008845:Microinjections; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000187:drug effects* / Q000235:genetics; D017433:Protein Structure, Secondary; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D002492:Central Nervous System Depressants; D018682:GABA Agents; D000441:Hexanols; D011963:Receptors, GABA-A; D000431:Ethanol; D005680:gamma-Aminobutyric Acid
keywords: 6′ position; Ligand-gated ion channel; homology model; oocyte; pore; transmembrane