title: Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.
abstract: OBJECTIVE
  β2/3-subunit-selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors.
  
  
  METHODS
  Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed.
  
  
  RESULTS
  A binding pocket for VA at the β(+) /α(-) interface encompassing amino acid β3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on β3M286W channels as well as significantly decreased efficacy and potency of VA on β3N265S and β3F289S receptors. In addition, reduced efficacy of VA-induced IGABA enhancement was also observed for α1M235W, β3R269A and β3M286A constructs.
  
  
  CONCLUSIONS
  Our data suggest that amino acid residues β3N265, β3F289, β3M286, β3R269 in the β3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands.
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Luger
authors.affiliation: Department of Pharmacy, University of Pisa, Pisa, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Poli
authors.affiliation: Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Wieder
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Stadler
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Ke
authors.affiliation: Department of Molecular Neurosciences, Center of Brain Research, Medical University of Vienna, Vienna, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Ernst
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Hohaus
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Linder
authors.affiliation: Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Seidel
authors.affiliation: Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Langer
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Khom
authors.affiliation: Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Hering
mesh_terms: D000818:Animals; D001665:Binding Sites; D005260:Female; D007192:Indenes / Q000494:pharmacology*; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology*; D014982:Xenopus laevis
chemical_list: D007192:Indenes; D011963:Receptors, GABA-A; D012717:Sesquiterpenes; C037133:valerenic acid