Luger D et al. (2015), Identification of the putative binding pocket o...

XB-ART-51310

Br J Pharmacol 2015 Nov 01;17222:5403-13. doi: 10.1111/bph.13329.

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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 .

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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.

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Species referenced: Xenopus laevis
Genes referenced: gabarap

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	Figure 1. Putative binding pocket(s) of VA located at the β+/α− interface on GABAA receptors and two‐dimensional 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 pocket‐defining 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 H‐bond 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+/α1− and β1+/α1− 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 structure‐based pharmacophore features three lipophilic contacts (yellow spheres), two putative H‐bond 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: Two‐dimensional rendering of the structure‐based pharmacophores: (C, F) Schematic two‐dimensional representations of the structure‐based pharmacophores of VA in the proposed binding pockets are shown. The carboxyl group forms an H bond with the –NH2 group of β3N265, or the –OH group of β1S265S. Additionally, the guanidinium group of β1/3R269 could form ionic or H‐bonding 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 concentration‐response curves for wild‐type α1β3γ2S and the mutant GABAA channels indicated are compared. Panel (A) illustrates the effect of mutations of the α1 subunit (co‐expressed with β3 and γ2S subunits) on GABA sensitivity compared with wild‐type α1β3γ2S channels, while in panel (B), the impact of the β3 mutations on the GABA‐concentration response relation is shown (wild type illustrated as dashed line). Responses at indicated concentrations in each cell were normalized to the maximum GABA‐evoked peak current. Each data point represents the mean ± SEM of ≥5 oocytes from at least two batches.
	Figure 3. Potentiation of submaximal GABA responses (EC3–7) by 1 μM diazepam of mutant receptors (black bars) is compared with wild‐type 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. Concentration‐response curves for VA‐induced 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 EC3–7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of ≥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 20 s application of a GABA EC3–7 concentration (single bar) or co‐application of GABA EC3–7 and 100 μM VA recorded from Xenopus laevis oocytes voltage‐clamped at −70 mV 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. Concentration‐response curves for VA‐induced 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 wild‐type channels. Responses in each cell were normalized to a submaximal GABA EC3–7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of ≥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 20 s application of a GABA EC3–7 concentration (single bar) or co‐application of GABA EC3–7 and 100 μM VA recorded from Xenopus laevis oocytes voltage‐clamped at −70 mV expressing the indicated receptor subtype.
	Figure 6. Concentration‐response curves for VA‐induced I GABA enhancement on α1I227Aβ3γ2S, α1L231Aβ3γ2S and α1L268β3γ2S receptors. Dashed line represents I GABA enhancement by VA on wild‐type channels. Responses in each cell were normalized to a submaximal GABA EC3–7 concentration determined at the beginning of each experiment. Data points represent the mean ± SEM of ≥5 oocytes from at least two batches. Error bars smaller than the symbol are not shown. Grey symbols are excluded from the fit.

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