Journal: eLife

Article Title: A motogenic GABAergic system of mononuclear phagocytes facilitates dissemination of coccidian parasites

doi: 10.7554/eLife.60528

Figure Lengend Snippet: ( A, B ) Relative mRNA expression (2 -ΔCt ) of cation chloride transporters (CCCs) in unchallenged ( A ) mBMDCs and ( B ) hMoDCs (n = 3 independent experiments). ( C, D ) Heat map depicts (%) transcriptional expression changes of CCCs in ( C ) mBMDCs and ( D ) hMoDCs challenged with T. gondii (PRU-RFP) relative to unchallenged cells at indicated time points. ( X ) indicates no amplification (n = 3 independent experiments). ( E ) Immunostaining of mBMDCs challenged with T. gondii (ME49/PTG-GFP) stained with NKCC1/2 monoclonal antibody (Alexa Flour 594-conjugated anti-mouse secondary antibody) and DAPI (nuclei). Scale bars: 10 μm. ( F ) Representative Western blot of lysates from T. gondii -challenged mBMDCs for indicated time, immunoblotted with phospho-NKCC1 and total NKCC1/2 antibodies. GAPDH was used as loading reference. (n = 4 independent experiments). ( G, H ) Representative motility plots of unchallenged and T. gondii -infected ( G ) mBMDCs and ( H ) hMoDCs treated with NKCC1 inhibitor (bumetanide). X- and y-axes indicate distances in μm. ( I, J ) Box-and-whisker dot plots show, for each indicated condition, median velocities (μm/min) of unchallenged and T. gondii -infected ( I ) mBMDCs and ( J ) hMoDCs as in ( G, H ) (n = 3 independent experiments). ( K, L ) The mRNA expression (2 -ΔCt ) of control shLuc- and shNKCC1-treated unchallenged ( K ) mBMDCs and ( L ) hMoDCs related (%) to mock-treated cells (n = 7 independent experiments for mBMDCs and n = 4 for hMoDCs). Bar graphs show mean + SEM. ( M, N ) Histograms show accumulated distances migrated (μm) by control shLuc-treated and shNKCC1-treated ( M ) mBMDCs and ( N ) hMoDCs, respectively, challenged with T. gondii (PRU-RFP). Dotted lines indicate median values (n = 3 independent experiments). ( O ) Velocities of unchallenged and T. gondii -infected mBMDCs treated with GABA or muscimol in presence of bumetanide. ( P ) Representative motility plots of T. gondii -infected shGAD67- and shNKCC1-treated hMoDCs in presence of GABA. ( Q ) Velocities of unchallenged and T. gondii -infected shLuc-, shGAD67- and shNKCC1-treated hMoDCs with or without GABA. Statistical significance was tested by ordinary one-way ANOVA with Dunnett’s multiple comparison test for (I, J, K, L, O, Q) and by Mann-Whitney test for (M, N), *p<0.05, **p<0.01, ***p<0.001, ns p≥0.05.

Article Snippet: To probe GABA-A R subunits, cells were incubated with rabbit anti-GABA-A R α3 polyclonal antibody, rabbit anti-GABA-A R α5 polyclonal antibody, rabbit anti-GABA-A R ρ1 polyclonal antibody (all from Alomone labs, Jerusalem, Israel), mouse anti-GABA-A R β3 monoclonal antibody (NeuroMab, UC Davis, CA, US) and for NKCC, with mouse anti-NKCC1/2 monoclonal antibody (clone T4, Developmental Studies Hybridoma Bank, DSHB, Iowa, USA) ON at 4°C.

Techniques: Expressing, Amplification, Immunostaining, Staining, Western Blot, Infection, Whisker Assay, Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Kinase activity simultaneously determines the constitutive and the orthosteric gating in α 4 β 1/3 δ GABA A receptors in hippocampal granule cells

doi: 10.1101/318543

Figure Lengend Snippet: The effect of two GABA agonists, THIP (δ-subunit preferring) and Thio-THIP (α 4 β 1/3 δ selective) on the TCD is dependent on temperature, intracellular Ca 2+ -EGTA chelation and PKC activity. A : Full recording traces of voltage clamped (−70 mV) DGGCs upon bath application of 100 μM Thio-THIP followed by PTX. Thio-THIP induces a ~4 x larger TCD in condition a , c and d than condition b . Note the presence of a positive endogenous TCD in condition b only. B : Traces of mIPSCs display the Thio-THIP induced reduction of the average mIPSC in all recording conditions. C : Summary barplot of TCD values induced by Thio-THIP (n=12-14, *** P<0.001 ANOVA/Bonferroni). D-E : Summary of peak and decay time of the average mIPSCs (** P<0.01 paired t-test). F-G : Representative traces and barplot showing the effect of THIP in recording condition a through d (n=8-10, ** P<0.01 ANOVA/Bonferroni). H : The TCD induced by Thio-THIP in adult δ +/+ and δ -/- mice and the effect on the average mIPSC peak in the same recordings (n =, *** P<0.001, ** P<0.01 paired t-test for same-cell measurements, unpaired for different cells).

Article Snippet: Prof. Dr. Petra Scholze, Medical University of Vienna, Center for Brain Research), anti-δ (#868A-GDN, Phosphosolutions, USA), anti-γ 2 (#NB300-190, Novus Biologicals, Littleton, USA), anti-β3 (#NBP1-47613, Novus Biologicals), anti-GAPDH (# NB600-502, Novus Biologicals, Littleton, USA) and anti-Na + /K + -ATPase (#ab76020.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Kinase activity simultaneously determines the constitutive and the orthosteric gating in α 4 β 1/3 δ GABA A receptors in hippocampal granule cells

doi: 10.1101/318543

Figure Lengend Snippet: GBZ induces a δ-subunit dependent TCD in adult rat DGGCs under recording conditions phenomenologically characterized by the absence of a constitutive current. A : Recording traces displaying the effect of GBZ and PTX in recording conditions a - d . GBZ quickly blocks mIPSCs, but also induces a TCD in condition a , c and d , which is reversed by PTX. B : Shutting of the constitutive current by BIC (20 μM) in recording condition b is reversed by GBZ (10 μM). C-D : Concentration-response relation of GBZ in blocking mIPSCs, and inducing a TCD in recording condition d . E : Summary of effects of GBZ to induce a TCD in recording condition a - d , incl. recording condition b in which the constitutive current is shut by BIC. F-G : The δ-subunit selective positive modulator DS2 potentiates the effect of GBZ in producing a PTX-sensitive tonic current. H-I : Recordings of DGGCs from adult δ +/+ and δ -/- mice demonstrate that the effect of GBZ inducing a TCD is dependent on expression of the δ-subunit. ** P<0.01, t-test (figure I), *** P<0.001, ANOVA/Bonferroni.

Article Snippet: Prof. Dr. Petra Scholze, Medical University of Vienna, Center for Brain Research), anti-δ (#868A-GDN, Phosphosolutions, USA), anti-γ 2 (#NB300-190, Novus Biologicals, Littleton, USA), anti-β3 (#NBP1-47613, Novus Biologicals), anti-GAPDH (# NB600-502, Novus Biologicals, Littleton, USA) and anti-Na + /K + -ATPase (#ab76020.

Techniques: Concentration Assay, Blocking Assay, Expressing

Journal: bioRxiv

Article Title: Kinase activity simultaneously determines the constitutive and the orthosteric gating in α 4 β 1/3 δ GABA A receptors in hippocampal granule cells

doi: 10.1101/318543

Figure Lengend Snippet: GBZ does not antagonize the effect of Thio-THIP. A : Thio-THIP, GBZ and PTX on DGGCs in recording condition b in adult rats (n=8). B : Same experiment as A, recording condition d . Note that 10 μM GBZ neither blocks, nor increases the TCD already induced by Thio-THIP (right panel, n=13). C-D : Thio-THIP and GBZ recorded in condition d in adult β +/+ and β -/- mice (n=18/22). E : The noise (SD) induced by Thio-THIP and GBZ in β +/+ and β -/- mice. The noise in Thio-THIP + GBZ is significantly lower in β -/- than β +/+ mice. F-G : The average mIPSC peak is similarly reduced by Thio-THIP in β +/+ and β -/- mice. H : Western blots of whole hippocampal homogenate from β +/+ and β -/- mice, displaying absence of β subunit immunoreactivity in β -/- mice (left) and no significant compensatory changes in α , α 4 , β 2 , β 3 , δ and γ 2 subunits in β -/- mice compared to β +/+ (right, see extended data - for individual WBs).

Article Snippet: Prof. Dr. Petra Scholze, Medical University of Vienna, Center for Brain Research), anti-δ (#868A-GDN, Phosphosolutions, USA), anti-γ 2 (#NB300-190, Novus Biologicals, Littleton, USA), anti-β3 (#NBP1-47613, Novus Biologicals), anti-GAPDH (# NB600-502, Novus Biologicals, Littleton, USA) and anti-Na + /K + -ATPase (#ab76020.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Kinase activity simultaneously determines the constitutive and the orthosteric gating in α 4 β 1/3 δ GABA A receptors in hippocampal granule cells

doi: 10.1101/318543

Figure Lengend Snippet: GBZ antagonizes effect of GABA but does not induce a current alone in constitutively silent α 4 β 1 δ and α 4 β 3 δ receptors expressed in HEK-293 Flp-In cells. A, D : GABA ( ) activates human α 4 β 1 δ (A) and α 4 β 3 δ (D) receptors with a similar EC 50 of 3.5E-7 M. B, E : GBZ ( ) concentration-dependently blocks the response to an EC 80 concentration of GABA with IC 50 = 4.2E-7 M at α 4 β 1 δ receptors (B) and 5E-7 M at α 4 β 3 δ receptors (E). C, F : GBZ does not induce a current alone in any receptor type. All datapoints in graph A-F are means of triplicate measurements ±SD. FU, fluorescence unit. Symbols below belong to both figures in the three vertical panels.

Article Snippet: Prof. Dr. Petra Scholze, Medical University of Vienna, Center for Brain Research), anti-δ (#868A-GDN, Phosphosolutions, USA), anti-γ 2 (#NB300-190, Novus Biologicals, Littleton, USA), anti-β3 (#NBP1-47613, Novus Biologicals), anti-GAPDH (# NB600-502, Novus Biologicals, Littleton, USA) and anti-Na + /K + -ATPase (#ab76020.

Techniques: Concentration Assay, Fluorescence

Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 3. GABRB3 mutations altered the stability of GABAA receptors. (A) Cryo-EM structure of the pentameric α1β3γ2 GABAA receptor (6HUP) viewed from the side, with the β subunits in red, the α subunits in blue, and the γ subunit in gray. GABRB3 mutations are mapped onto the structure and represented as green sticks and numbered according to the panels in (B). Network of interatomic interactions predicted by wild-type (wt, left panels) and β3 mutant (mut, right panels) 6HUP receptors. The panels are numbered according to the labels in panel (A), where the mutations are mapped. wt and mut amino acids are colored in light green, and the neighborhood residues are in CPK representation. All residues are represented as sticks. In brackets, the residues are numbered based on the 6HUP structure. For correspondence with the GABRB3 protein sequence deposited in UniProt-P28472, 25 must be added to the number indicated on the panel. Structural domains involved in the network of interactions are labeled, and residues are equally colored. ∆∆G values are shown on the corresponding panels.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Sequencing, Labeling

Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 5. GABRB3 mutations altered the flexibility and function of GABAA receptors. (A) Structural representation of the GABAA receptor flexible conformation based on the vibrational entropy energy change (∆∆SVib ENCoM) predicted between the wild-type and the mutant β3 subunit in the 6HUP structure. Mutant β3 subunits are colored according to the vibrational entropy perturbation upon mutation. Shades of blue represent a loss of flexibility (rigidification of the structure), and shades of red mean an increase in structural flexibility. ∆∆SVib ENCoM values are shown at the bottom of the corresponding mutation. In brackets, the variants are numbered based on the 6HUP structure. (B) In the upper panel, concentration–response curves of wild-type and mutant α1β3γ2 receptors are shown. GABA-evoked currents were normalized to the maximal response to 1 mM GABA. Lower panel, Pearson correlation coefficients obtained from the ∆∆G of the mutant β3 structures were plotted against surface (left graph) and total (right graph) expression levels of α1, β3, and γ2 subunits according to the data displayed in panels (C,D), respectively. Lines represent linear regression fits. (C,D) Surface and total expression levels of wild-type and mutant β3 subunits coexpressed with α1 and γ2 subunits, respectively.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Mutagenesis, Concentration Assay, Expressing