title: Mutagenesis and computational docking studies support the existence of a histamine binding site at the extracellular β3+β3- interface of homooligomeric β3 GABAA receptors.
abstract: Histamine is an important neurotransmitter that exerts its physiological actions through H1-4 metabotropic receptors in mammals. It also directly activates ionotropic GABAA receptor (GABAAR) β3 homooligomers and potentiates GABA responses in αβ heterooligomers in vitro, but the respective histamine binding sites in GABAARs are unknown. We hypothesized that histamine binds at the extracellular β+β- interface at a position homologous to the GABA binding site of heterooligomeric GABAARs. To test this, we individually mutated several residues at the putative ligand binding minus side of a rat GABAAR β3 wild type subunit and of a β3 subunit that was made insensitive to trace Zn(2+) inhibition [β3(H267A); called (Z)β3]. (Z)β3, (Z)β3(Y62L), (Z)β3(Q64A), (Z)β3(Q64E), α1(Z)β3, or α1(Z)β3(Y62L) receptors were studied in HEK293T cells using whole cell voltage clamp recording. β3, β3(Y62C), β3(Q64C), β3(N41C), β3(D43C), β3(A45C) or β3(M115C) receptors were examined in Xenopus oocytes using two-electrode voltage clamp. Histamine directly activated (Z)β3 and β3 homooligomers and potentiated GABA actions in α1(Z)β3 heterooligomers. Receptors containing (Z)β3(Y62L), β3(Y62C) and β3(D43C) showed markedly reduced histamine potency, but homo- and heterooligomers with (Z)β3(Q64E) exhibited increased potency. The GABAAR αβ(γ) competitive antagonist bicuculline elicited sub-maximal agonist currents through (Z)β3 homooligomers, the potency of which was strongly decreased by (Z)β3(Y62L). Mutations β3(N41C), β3(A45C) and β3(M115C) disturbed receptor expression or assembly. Computational docking into the crystal structure of homooligomeric β3 receptors resulted in a histamine pose highly consistent with the experimental findings, suggesting that histamine activates β3 receptors via a site homologous to the GABA site in αβγ receptors.
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY, 12208, USA. Electronic address: Hoerbelt@stanford.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Hoerbelt
authors.affiliation: Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090, Vienna, Austria.
authors.forename: Joachim
authors.initials: J
authors.lastname: Ramerstorfer
authors.affiliation: Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090, Vienna, Austria.
authors.forename: Margot
authors.initials: M
authors.lastname: Ernst
authors.affiliation: Department of Molecular Neurosciences, Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090, Vienna, Austria.
authors.forename: Werner
authors.initials: W
authors.lastname: Sieghart
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY, 12208, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Thomson
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY, 12208, USA.
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Hough
authors.affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY, 12208, USA.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Fleck
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005260:Female; D057809:HEK293 Cells; D006632:Histamine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D062105:Molecular Docking Simulation / Q000379:methods*; D016296:Mutagenesis / Q000502:physiology*; D017433:Protein Structure, Secondary; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C546276:Gabrb3 protein, rat; D011963:Receptors, GABA-A; D006632:Histamine
keywords: (−)-bicuculline methobromide (PubChem CID: 171729); Bicuculline; Docking; GABA (PubChem CID: 119); GABA(A) receptor; Histamine; Histamine dihydrochloride (PubChem CID: 5818); Ligand-gated ion channel; Mutagenesis; Pentobarbital sodium (PubChem CID: 23676152)