Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Br J Pharmacol
2015 Nov 01;17222:5403-13. doi: 10.1111/bph.13329.
Show Gene links
Show Anatomy links
Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.
Luger D
,
Poli G
,
Wieder M
,
Stadler M
,
Ke S
,
Ernst M
,
Hohaus A
,
Linder T
,
Seidel T
,
Langer T
,
Khom S
,
Hering S
.
???displayArticle.abstract???
BACKGROUND AND PURPOSE: 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.
EXPERIMENTAL APPROACH: 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.
KEY 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 AND IMPLICATIONS: 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.
Figure 1. Putative binding pocket(s) of VA located at the β+/αâ interface on GABAA receptors and twoâdimensional representations of VA are shown. The α1 subunit is coloured in brown, and the respective β subunits (β3 in (A, B) and β1 in (D, E)) are shown in green. VA and interacting amino acid side chains are shown in stick rendering, colour coded as to atom type: red, oxygen; dark blue, nitrogen; yellow, sulfur. Top row: VA poses derived from docking: TM residues β1/3T262, β3N265/β1S265, β1/3T266, β1/3R269, β1/3M286 and β1/3F289 and α1I227, α1L231, α1P232, α1M235 and α1L268 are pocketâdefining or very close to the pocket. Energetically, most favourable orientations of VA obtained from docking into (A) α1β3γ2S and (D) α1β1γ2S GABAA receptor homology models are illustrated. Dashed lines indicate distances between VA's carboxylate group and putative Hâbond interaction partners on β3N265 (A) and β1S265 (D) respectively. Middle row: Refined poses and resulting putative pharmacophore: (B, E) Optimized docking poses of VA (marked in red/ purple) in the β3+/α1â and β1+/α1â binding pockets are shown. All surrounding amino acid side chains were kept flexible. Strong changes in rotamers compared with the docking results shown in the top row can be observed for β1/3R269 and β1/3F289. The structureâbased pharmacophore features three lipophilic contacts (yellow spheres), two putative Hâbond acceptor interactions (red arrows) and one putative ionic interaction (red star). All amino acids that interact with the ligand are highlighted in a stick display style. Bottom row: Twoâdimensional rendering of the structureâbased pharmacophores: (C, F) Schematic twoâdimensional representations of the structureâbased pharmacophores of VA in the proposed binding pockets are shown. The carboxyl group forms an H bond with the âNH2 group of β3N265, or the âOH group of β1S265S. Additionally, the guanidinium group of β1/3R269 could form ionic or Hâbonding interactions with the carboxylate group. Hydrophobic interactions occur between VA's three methyl groups and the side chains of α1I227, α1M235, α1L268, β1/3T262, β1/3M286 and β1/3F289, which form the lipophilic part of the binding pocket surface.
Figure 2. GABA concentrationâresponse curves for wildâtype α1β3γ2S and the mutant GABAA channels indicated are compared. Panel (A) illustrates the effect of mutations of the α1 subunit (coâexpressed with β3 and γ2S subunits) on GABA sensitivity compared with wildâtype α1β3γ2S channels, while in panel (B), the impact of the β3 mutations on the GABAâconcentration response relation is shown (wild type illustrated as dashed line). Responses at indicated concentrations in each cell were normalized to the maximum GABAâevoked peak current. Each data point represents the mean ± SEM of â¥5 oocytes from at least two batches.
Figure 3. Potentiation of submaximal GABA responses (EC3â7) by 1âμM diazepam of mutant receptors (black bars) is compared with wildâtype α1β3γ2S receptors (white bar). Bars represent means ± SEM (n = 3 for α1L231Aβ3γ2S, α1M235Aβ3γ2S α1L268Aβ3γ2S, α1β3T262Aγ2S, α1β3R269Aγ2S and α1M286Aβ3γ2S; n = 4 for α1I227Aβ3γ2S; n = 5 for α1M235Wβ3γ2S and α1β3T266Aγ2S; n = 6 for α1β3γ2S, α1β3T262Sγ2S and α1β3F289Sγ2S; n = 7 for α1β3N265Sγ2S and α1β3286Wγ2S; cells were taken from at least two different oocyte batches).
Figure 4. Effects of mutating residues β3N265, β3R269, β3F289, β3T262, and β3T266 on efficacy and potency of I
GABA enhancement by VA are shown. Concentrationâresponse curves for VAâinduced I
GABA enhancement on (A) α1β3γ2S, α1β3N265Sγ2S, (B) α1β3R269Aγ2S, α1β3F289Sγ2S, (C) α1β3T262Aγ2S, α1β3T262Sγ2S and α1β3T266Aγ2S receptors are illustrated. Responses in each cell were normalized to a submaximal GABA EC3â7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of â¥5 oocytes from at least two batches. Error bars smaller than the symbol are not shown. Grey symbols are excluded from the fit. (D) Representative current traces in the presence of 20âs application of a GABA EC3â7 concentration (single bar) or coâapplication of GABA EC3â7 and 100âμM VA recorded from Xenopus laevis oocytes voltageâclamped at â70âmV expressing the indicated receptor subtype.
Figure 5. Effects of mutating residues β3M286 and α1M235 on efficacy and potency of I
GABA enhancement by VA are illustrated. Concentrationâresponse curves for VAâinduced I
GABA enhancement on (A) α1β3M286Wγ2S and α1β3M286Aγ2S and (B) α1M235Wβ3γ2S and α1M235Aβ3γ2S receptors are shown. Dashed line in (A) represents I
GABA enhancement by VA on wildâtype channels. Responses in each cell were normalized to a submaximal GABA EC3â7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of â¥6 oocytes from at least two batches. Error bars smaller than the symbol are not shown. Grey symbols are excluded from the fit. (C) Representative current traces in the presence of 20âs application of a GABA EC3â7 concentration (single bar) or coâapplication of GABA EC3â7 and 100âμM VA recorded from Xenopus laevis oocytes voltageâclamped at â70âmV expressing the indicated receptor subtype.
Figure 6. Concentrationâresponse curves for VAâinduced I
GABA enhancement on α1I227Aβ3γ2S, α1L231Aβ3γ2S and α1L268β3γ2S receptors. Dashed line represents I
GABA enhancement by VA on wildâtype channels. Responses in each cell were normalized to a submaximal GABA EC3â7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of â¥5 oocytes from at least two batches. Error bars smaller than the symbol are not shown. Grey symbols are excluded from the fit.
Alexander,
The Concise Guide to PHARMACOLOGY 2013/14: ligand-gated ion channels.
2013, Pubmed
Alexander,
The Concise Guide to PHARMACOLOGY 2013/14: ligand-gated ion channels.
2013,
Pubmed
Baburin,
Automated fast perfusion of Xenopus oocytes for drug screening.
2006,
Pubmed
,
Xenbase
Baumann,
Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement.
2002,
Pubmed
,
Xenbase
Baur,
A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits.
2006,
Pubmed
,
Xenbase
Belelli,
The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid.
1997,
Pubmed
,
Xenbase
Benke,
GABA A receptors as in vivo substrate for the anxiolytic action of valerenic acid, a major constituent of valerian root extracts.
2009,
Pubmed
Bianchi,
Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.
2003,
Pubmed
Boileau,
The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes.
2002,
Pubmed
,
Xenbase
Chen,
Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor.
2012,
Pubmed
Chiara,
Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor.
2013,
Pubmed
Crow,
Insomnia: chasing the dream.
2013,
Pubmed
Groves,
The role of GABAbeta2 subunit-containing receptors in mediating the anticonvulsant and sedative effects of loreclezole.
2006,
Pubmed
Halliwell,
Subunit-selective modulation of GABAA receptors by the non-steroidal anti-inflammatory agent, mefenamic acid.
1999,
Pubmed
,
Xenbase
Hibbs,
Principles of activation and permeation in an anion-selective Cys-loop receptor.
2011,
Pubmed
Hintersteiner,
Esters of valerenic acid as potential prodrugs.
2014,
Pubmed
,
Xenbase
Hosie,
Neurosteroid binding sites on GABA(A) receptors.
2007,
Pubmed
Hosie,
Conserved site for neurosteroid modulation of GABA A receptors.
2009,
Pubmed
Hosie,
Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
2006,
Pubmed
Jayakar,
Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.
2014,
Pubmed
Jurd,
General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.
2003,
Pubmed
Khom,
Pharmacological properties of GABAA receptors containing gamma1 subunits.
2006,
Pubmed
,
Xenbase
Khom,
Valerenic acid potentiates and inhibits GABA(A) receptors: molecular mechanism and subunit specificity.
2007,
Pubmed
,
Xenbase
Kilkenny,
Animal research: reporting in vivo experiments: the ARRIVE guidelines.
2010,
Pubmed
Li,
Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog.
2006,
Pubmed
Luger,
Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.
2016,
Pubmed
,
Xenbase
McGrath,
Guidelines for reporting experiments involving animals: the ARRIVE guidelines.
2010,
Pubmed
Miller,
Crystal structure of a human GABAA receptor.
2014,
Pubmed
Möhler,
The GABA system in anxiety and depression and its therapeutic potential.
2012,
Pubmed
Möhler,
GABAA receptors in central nervous system disease: anxiety, epilepsy, and insomnia.
2006,
Pubmed
Morris,
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility.
2009,
Pubmed
Olsen,
GABA(A) receptors as molecular targets of general anesthetics: identification of binding sites provides clues to allosteric modulation.
2011,
Pubmed
Olsen,
International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.
2008,
Pubmed
Olsen,
GABA A receptors: subtypes provide diversity of function and pharmacology.
2009,
Pubmed
Olsen,
Molecular biology of GABAA receptors.
1990,
Pubmed
Pawson,
The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands.
2014,
Pubmed
Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed
Sigel,
Impact of subunit positioning on GABAA receptor function.
2006,
Pubmed
Sigel,
Structure, function, and modulation of GABA(A) receptors.
2012,
Pubmed
Simon,
Analysis of the set of GABA(A) receptor genes in the human genome.
2004,
Pubmed
Stewart,
Mutations at beta N265 in γ-aminobutyric acid type A receptors alter both binding affinity and efficacy of potent anesthetics.
2014,
Pubmed
,
Xenbase
Stewart,
Tryptophan mutations at azi-etomidate photo-incorporation sites on alpha1 or beta2 subunits enhance GABAA receptor gating and reduce etomidate modulation.
2008,
Pubmed
,
Xenbase
Tretter,
Stoichiometry and assembly of a recombinant GABAA receptor subtype.
1997,
Pubmed
Wafford,
A novel allosteric modulatory site on the GABAA receptor beta subunit.
1994,
Pubmed
,
Xenbase
Wallace,
LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions.
1995,
Pubmed
Wingrove,
The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.
1994,
Pubmed
,
Xenbase
Wolber,
LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters.
2005,
Pubmed
