22 research outputs found

    The gating properties of α4β3N–Flag–δ GABA<sub>A</sub>Rs.

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    <p>(<b>A</b>) GABA concentration-response curve for α4β3N–Flag–δ receptor mediated currents. Currents were elicited by application of varying concentrations of GABA (0.1–10 mM). Peak current amplitudes in each cell were normalized to that obtained with 10 mM GABA. (<b>B</b>) Representative current trace obtained by application of an 8.5 ms pulse of 100 μM GABA to measure the deactivation rate. (<b>C</b>) α4β3N–Flag–δ receptors are spontaneously open. (Left panel) Representative trace of outward currents observed by application of 2 mM Picrotoxin to inhibit the spontaneously open receptors. (Right panel) The estimate of the maximum inward currents obtained by co-application of 10 mM GABA with 10 μM Etomidate to gate all available receptors. The gray lines are drawn by eye to represent the baseline. At least three cells per concentration were used throughout experiments.</p

    Stability of the α4-subunit.

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    <p>(<b>A</b>) Western blot depicting fragmentation of α4-subunit seen as two bands in N-Flag-α4β3 and α4β3N-Flag-δ receptors reconstituted into CHAPS/asolectin micelles is presented. Both α4 bands are identified by polyclonal anti-α4 and monoclonal anti-Flag antibodies in the former receptor, confirming identity of the band. Numbers on the side indicate the position of the molecular weight markers (kDa). (<b>B</b>) Cells induced to express indicated GABA<sub>A</sub>Rs were prepared for Western blotting by either 1. suspending cells in suspension buffer; 2. suspending and sonicating; 3. leaving in a monolayer (untreated); or 4. lysing directly in the well with suspension buffer supplemented with 10 mM DDM; as indicated, prior to lysing cells with a 4x Laemmli sample buffer with 10% β-mercaptoethanol. Suspension buffer was supplemented with Protease Inhibitor Cocktail (Sigma) at 1:100 dilution. (<b>C</b>) Representative Western blot of samples obtained during α4β3N-Flag-δ receptor purification, as described in the materials and methods section. Numbers under each lane indicate the fraction the lower band comprises of the higher band, expressed as percentile points. (<b>D</b>) Membrane fraction from the α4β3N-Flag-δ was incubated for 1 hour at indicated temperatures prior to analysis by Western blotting. All blots are presented as grayscale and were uniformly adjusted for brightness and contrast to facilitate analysis. Full immunoblots used to make panels A-D are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s004" target="_blank">S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191583#pone.0191583.s007" target="_blank">S7</a> Figs.</p

    All subunits of the α4β3N-Flag-δ GABA<sub>A</sub> receptor are glycosylated.

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    <p>Purified receptors were resolved by Western blotting with antibodies for α4- and β3-subunits or Flag, as shown on the left, middle and right panels, respectively. In each of the three panels, reading left to right the lanes are: purified receptor; purified receptor after deglycosylation with PNGase F, and PNGase F alone. The band below the 43 kDa marker was nonspecific and associated with PNGase F. Numbers on the left side indicate MW in kDa. The α4 and β3 antibodies are polyclonal. Brightness and contrast were uniformly adjusted for each panel.</p

    DS2 enhances agonist binding in δ-subunit containing receptors.

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    <p>The δ-subunit specific modulator, DS2, modulates [<sup>3</sup>H]muscimol binding (2 nM) in α4β3N-Flag-δ GABA<sub>A</sub>Rs. For the membranes, the data are the mean and standard deviation of two experiments, and for micelle reconstituted receptors for a single experiment in triplicate. The curves were fitted by nonlinear least squares to the Hill equation. The EC<sub>50</sub> (μM), Hill coefficient and maximum modulation were: for membranes, 2.0 ± 0.7 μM, 0.9 ± 0.2, 139 ± 4%; for reconstituted 2.3 ± 0.8 μM, 1.2 ± 0.5, 132 ± 4%.</p

    The binding isotherm of the agonist [<sup>3</sup>H]muscimol to the α4β3N-Flag-δ GABA<sub>A</sub> receptor in native membranes and reconstituted into CHAPS/lipid micelles.

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    <p>Binding curves of [<sup>3</sup>H]muscimol to α4β3N–Flag–δ GABA<sub>Α</sub>Rs, both, in cell membranes (pmol/mg membrane protein) and after purification and reconstitution into micelles of 5 mM CHAPS and 200 μM DOPC:DOPS:Cholesterol in mole ratio 52:15:33 (pmol/mL). Displaceable binding was determined as the difference between binding in the presence and absence of 1 mM GABA using a filtration assay in triplicate. The displaceable binding and its standard deviation was determined by subtracting these two values and propagating errors at each total muscimol concentration. The curves were fitted by nonlinear least squares with weighting by standard deviation. These yielded apparent dissociation constants of 9.2 ± 0.6 and 35 ± 12 nM, respectively. The B<sub>max</sub> of the membranes was 22.6 ± 0.5 pmol/mg and for reconstituted receptors in micelles was 19 ± 3 pmol/mL. The Hill coefficients differed little from one (1.07 ± 0.3 and 0.90 ± 0.05 respectively).</p

    Table_1_Histone Deacetylase Inhibitor Alleviates the Neurodegenerative Phenotypes and Histone Dysregulation in Presenilins-Deficient Mice.pdf

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    <p>Histone acetylation has been shown to play a crucial role in memory formation, and histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) has been demonstrated to improve memory performance and rescue the neurodegeneration of several Alzheimer’s Disease (AD) mouse models. The forebrain presenilin-1 and presenilin-2 conditional double knockout (cDKO) mice showed memory impairment, forebrain degeneration, tau hyperphosphorylation and inflammation that closely mimics AD-like phenotypes. In this article, we have investigated the effects of systemic administration of NaB on neurodegenerative phenotypes in cDKO mice. We found that chronic NaB treatment significantly restored contextual memory but did not alter cued memory in cDKO mice while such an effect was not permanent after treatment withdrawal. We further revealed that NaB treatment did not rescue reduced synaptic numbers and cortical shrinkage in cDKO mice, but significantly increased the neurogenesis in subgranular zone of dentate gyrus (DG). We also observed that tau hyperphosphorylation and inflammation related protein glial fibrillary acidic protein (GFAP) level were decreased in cDKO mice by NaB. Furthermore, GO and pathway analysis for the RNA-Seq data demonstrated that NaB treatment induced enrichment of transcripts associated with inflammation/immune processes and cytokine-cytokine receptor interactions. RT-PCR confirmed that NaB treatment inhibited the expression of inflammation related genes such as S100a9 and Ccl4 found upregulated in the brain of cDKO mice. Surprisingly, the level of brain histone acetylation in cDKO mice was dramatically increased and was decreased by the administration of NaB, which may reflect dysregulation of histone acetylation underlying memory impairment in cDKO mice. These results shed some lights on the possible molecular mechanisms of HDAC inhibitor in alleviating the neurodegenerative phenotypes of cDKO mice and provide a promising target for treating AD.</p

    The δ-subunit is expressed in the α4β3N–Flag–δ GABA<sub>A</sub>R stable cell line.

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    <p>Representative current traces show the effect of DS2 (<b>A</b>) and Etomidate (<b>B</b>) on 10 mM GABA–elicited currents on α4β3N–Flag–δ (upper panel) compared to N–Flag–α4β3 (lower panel). Currents were elicited in a notch protocol by an eight second pulse of GABA, during which drug was co-applied for 4 seconds 1 second after the GABA perfusion started. Concentrations are indicated in the figure.</p

    Mapping General Anesthetic Binding Site(s) in Human α1β3 γ-Aminobutyric Acid Type A Receptors with [<sup>3</sup>H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue

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    The γ-aminobutyric acid type A receptor (GABA<sub>A</sub>R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA<sub>A</sub>Rs purified from bovine brain, [<sup>3</sup>H]­azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA<sub>A</sub>R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) <i>J. Neurosci. 26</i>, 11599–11605]. To further define GABA<sub>A</sub>R etomidate binding sites, we now use [<sup>3</sup>H]­TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA<sub>A</sub>Rs and more extensively identify photolabeled GABA<sub>A</sub>R amino acids. [<sup>3</sup>H]­TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [<sup>3</sup>H]­azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA<sub>A</sub>R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [<sup>3</sup>H]­TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA<sub>A</sub>R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [<sup>3</sup>H]­TDBzl-etomidate and [<sup>3</sup>H]­azietomidate also provides evidence of a homologous etomidate binding site at the β3−β3 subunit interface in the α1β3 GABA<sub>A</sub>R

    Image_1_Histone Deacetylase Inhibitor Alleviates the Neurodegenerative Phenotypes and Histone Dysregulation in Presenilins-Deficient Mice.pdf

    No full text
    <p>Histone acetylation has been shown to play a crucial role in memory formation, and histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) has been demonstrated to improve memory performance and rescue the neurodegeneration of several Alzheimer’s Disease (AD) mouse models. The forebrain presenilin-1 and presenilin-2 conditional double knockout (cDKO) mice showed memory impairment, forebrain degeneration, tau hyperphosphorylation and inflammation that closely mimics AD-like phenotypes. In this article, we have investigated the effects of systemic administration of NaB on neurodegenerative phenotypes in cDKO mice. We found that chronic NaB treatment significantly restored contextual memory but did not alter cued memory in cDKO mice while such an effect was not permanent after treatment withdrawal. We further revealed that NaB treatment did not rescue reduced synaptic numbers and cortical shrinkage in cDKO mice, but significantly increased the neurogenesis in subgranular zone of dentate gyrus (DG). We also observed that tau hyperphosphorylation and inflammation related protein glial fibrillary acidic protein (GFAP) level were decreased in cDKO mice by NaB. Furthermore, GO and pathway analysis for the RNA-Seq data demonstrated that NaB treatment induced enrichment of transcripts associated with inflammation/immune processes and cytokine-cytokine receptor interactions. RT-PCR confirmed that NaB treatment inhibited the expression of inflammation related genes such as S100a9 and Ccl4 found upregulated in the brain of cDKO mice. Surprisingly, the level of brain histone acetylation in cDKO mice was dramatically increased and was decreased by the administration of NaB, which may reflect dysregulation of histone acetylation underlying memory impairment in cDKO mice. These results shed some lights on the possible molecular mechanisms of HDAC inhibitor in alleviating the neurodegenerative phenotypes of cDKO mice and provide a promising target for treating AD.</p
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