The gating properties of α4β3N–Flag–δ GABA_ARs.

<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

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

<p>Binding curves of [³H]muscimol to α4β3N–Flag–δ GABA_ΑRs, 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_max 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

Stability of the α4-subunit.

<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_ARs 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

DS2 enhances agonist binding in δ-subunit containing receptors.

<p>The δ-subunit specific modulator, DS2, modulates [³H]muscimol binding (2 nM) in α4β3N-Flag-δ GABA_ARs. 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 EC₅₀ (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

Representative yield of α4β3N-Flag-δ GABA_AR purification by anti-Flag affinity chromatography.

<p>Representative yield of α4β3N-Flag-δ GABA_AR purification by anti-Flag affinity chromatography.</p

All subunits of the α4β3N-Flag-δ GABA_A receptor are glycosylated.

<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

The δ-subunit is expressed in the α4β3N–Flag–δ GABA_AR stable cell line.

<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

Primers sequences.

<p>Primers sequences.</p

Impaired neuronal differentiation of ebiNSc expressing IDH1^R132H.

<p><b>(A)</b> Immunocytochemical characterization of MAP2 expression after 0, 7 and 14 days of differentiation in ebiNSc cultures expressing IDH1^R132H compared to control cultures (magnification 200x, scale bars mark 100μm). Fraction of MAP2-positive cells is present before the differentiation. The early clusters of MAP2-positive cells are visible after 7 days in ebiNSc, ebiNSc^empty and ebiNSc^IDH1wt controls. No MAP2-positive clusters and a low fraction of MAP2-positive cells are visible in ebiNSc^IDH1R132H after either 7 or 14 days. <b>(B)</b> Graph demonstrating the percentage of MAP2-positive cells with the elongated morphology after 7 days of differentiation in ebiNSc expressing IDH1^R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Kruskal-Wallis with Dunn’s multiple comparison test. ***, p<0.005. <b>(C)</b> Graph demonstrating the quantitative analysis of <i>MAP2</i> expression at the mRNA level after 0, 7 and 14 days of differentiation (d0, d7, d14) in ebiNSc expressing IDH1^R132H and control cultures. Error bars indicate SEM. Two-way ANOVA analysis did not reveal any statistical significance between samples, however, the trend can be appreciated.</p

Impaired astrocytic differentiation of ebiNSc expressing IDH1^R132H.

<p><b>(A)</b> Immunocytochemical characterization of GFAP expression after 0, 7 and 14 days of differentiation in ebiNSc cultures expressing IDH1^R132H compared to control cultures (magnification 200x, scale bars mark 100μm). GFAP-positive cells are present after 7 days in ebiNSc, ebiNSc^empty and ebiNSc^IDH1wt cultures. No GFAP-positive cells are visible in ebiNSc^IDH1R132H after either 7 or 14 days. <b>(B)</b> Graph demonstrating the percentage of GFAP-positive cells after 7 days of differentiation in ebiNSc expressing IDH1^R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Kruskal-Wallis with Dunn’s multiple comparison test. ***, p<0.005. <b>(C</b>) Graph demonstrating the quantitative analysis of <i>GFAP</i> expression at the mRNA level after 0, 7 and 14 days of differentiation (d0, d7, d14) in ebiNSc expressing IDH1^R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Two-way ANOVA with Bonferroni’s post-comparison test. *, p<0.05; **, p<0.01; ***, p<0.005; ns, not significant.</p