title: Evidence for α-helices in the large intracellular domain mediating modulation of the α1-glycine receptor by ethanol and Gβγ.
abstract: The α1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gβγ actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the α1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and Gβγ. The data strongly suggest the existence of an α-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the γ2-subunit of the GABAA receptor predicted to have little helical conformation. This region of γ2 was able to bind Gβγ and form a functional channel when combined with α1-GlyR, but it was not sensitive to ethanol. Mutations in the N- and C-terminal regions introduced to replace corresponding amino acids of the α1-GlyR sequence restored the ability to be modulated by ethanol and Gβγ. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to α1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Burgos
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
authors.forename: Patricio A
authors.initials: PA
authors.lastname: Castro
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
authors.forename: Trinidad
authors.initials: T
authors.lastname: Mariqueo
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
authors.forename: Marta
authors.initials: M
authors.lastname: Bunster
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Guzmán
authors.affiliation: Laboratory of Neurophysiology, Department of Physiology (C.F.B., .P.A.C., T.M., L.G.A.), Laboratory of Molecular Neurobiology, Department of Physiology (L.G.), Laboratory of Molecular Biophysics, Department of Biochemistry and Molecular Biology (M.B.), and Ph.D. program in Pharmacology (T.M.), University of Concepción, Concepción, Chile laguayo@udec.cl.
authors.forename: Luis G
authors.initials: LG
authors.lastname: Aguayo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism / Q000494:pharmacology*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism; D018009:Receptors, Glycine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; D000431:Ethanol