Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 2. Whole-cell patch-clamp recording of selected eight GABAA receptor α1 subunit DAVs. a HEK293T cells were transiently transfected with α1 (wild type or the indicated variants), β2, and γ2 cDNAs of GABAA receptors. Forty-eight hours post transfection, whole-cell currents were recorded using the IonFlux Mercury 16 ensemble plates at a holding voltage of −60 mV. Representative whole-cell voltage-clamp recording traces are shown. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (Imax) is shown on the right (n = 4–10). nA: nano Ampere. b Dose-response curves of GABA are shown for the calculation of EC50 values for wild type and the indicated α1 DAVs (n = 3–4). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. Statistical significance was labelled for the comparison to the WT group. ***, p < 0.001.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Patch Clamp, Transfection, Comparison

Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 4. Hispidulin and TP003 promote the functional surface expression of α1 DAVs. a Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on the surface protein expression of the α1 variants in HEK293T cells expressing α1(A322D)β2γ2, α1(D219N)β2γ2, α1(P260L)β2γ2, α1(R214C)β2γ2, α1(S76R)β2γ2, α1(G251D)β2γ2, α1 (M263T)β2γ2, or α1(T289P)β2γ2 GABAA receptors according to surface biotinylation analysis. Na+/K+ ATPase serves as a plasma membrane protein loading control. Quantification of the surface α1 band intensities was shown on the bottom panels (n = 3). b Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on GABA- induced peak current amplitudes in HEK293T cells expressing α1(A322D)β2γ2, α1(D219N)β2γ2, α1(P260L)β2γ2, α1(R214C)β2γ2, α1(S76R)β2γ2, α1(G251D)β2γ2, α1(M263T)β2γ2, or α1(T289P)β2γ2 GABAA receptors. Representative whole-cell voltage-clamp recording traces are shown. The holding voltage was set at −60 mV. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (Imax) are shown on the bottom panels (n = 4–11). pA: pico Ampere. c Comparison of the peak current amplitudes of compound (DMSO, Hispidulin, or TP003)-treated α1 variants with those in wild type (WT) receptors. Data was taken from Fig. 2a and Fig. 4b. Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. Statistical significance was labelled for the comparison to the DMSO group ((a, b) or WT group (a, b) or WT group (c). NS, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Also see Supplementary Fig. S5.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Functional Assay, Expressing, Clinical Proteomics, Membrane, Control, Comparison

Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 8. Proposed mechanism of action of Hispidulin and TP003 on rescuing α1 DAV trafficking. Hispidulin and TP003 act as pharmacological chaperones to bind GABAA receptor variants to stabilize them in the ER. Pharmacological chaperone treatment enhances the assembly of the variants and their forward trafficking to the Golgi and plasma membrane. The effect of Hispidulin and TP003 is also reflected from the enhanced folding and reduced degradation of the variants. Consistently, the interactions between the variants and pro-folding chaperones (BiP and calnexin) and a trafficking factor (LMAN1) are enhanced, whereas the interactions be tween the variants and ERAD factors (Grp94 and VCP) are reduced by pharmacological chaperone treatment.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: A therapeutic small molecule lead enhances γ-oscillations and improves cognition/memory in Alzheimer’s disease model mice

doi: 10.1101/2023.12.04.569994

Figure Lengend Snippet: a. Many PV+INs in CA3 express the α1 subunit (examples at arrows). Their distribution closely resembles that of δ subunit-expressing PV+INs in CA3 (described previously ). b. PV+INs along the base of the granule cell layer also exhibit strong α1 subunit labeling along their cell surface (arrow). c. In Gabrd +/+ animals, α1 subunit labeling is similarly expressed on the surface of these interneurons, with lighter, diffuse labeling within the cytoplasm. In contrast, in Gabrd -/- animals, many α1-labeled interneurons have reduced surface labeling and exhibit increased punctate labeling within the cytoplasm. This suggests that loss of the δ subunit leads to altered localization of the α1 subunit in these interneurons, consistent with a favored partnership of the δ and α1 subunits. Scale bars: a = 50 µm; b,c = 10 µm.

Article Snippet: For double immunofluorescence labeling, free-floating sections (30 μm) were incubated for 72 h at room temperature in the following combinations of primary antisera: 1) rabbit anti-parvalbumin (1:5,000; KG Baimbridge, R301) and either guinea pig anti-α1 subunit (1:30,000; J-M Fritschy) or guinea pig anti-α1 subunit (1:2,000; Alomone, AGA-001-GP); 2) rabbit anti-δ subunit (1:3,000; W. Sieghart) and either mouse anti-parvalbumin (1:2,000; Millipore, MAB1572) or guinea pig anti-α1 subunit (J-M Fritschy, as above); 3) guinea pig anti-α1 subunit (Alomone, as above) and either rabbit anti-β2 subunit (1:100; PhosphoSolutions, 862-GB2C) or anti-β3 subunit (1:250; Millipore, AB5563).

Techniques: Expressing, Labeling, Immunofluorescence

Journal: Animal Models and Experimental Medicine

Article Title: 1,2‐propanediol reformulation improves tribromoethanol safety and reveals pontine GABRA1 enrichment as a candidate mechanistic correlate of anesthesia

doi: 10.1002/ame2.70265

Figure Lengend Snippet: Molecular docking identifies GABRA1 as a high‐affinity binding target for TBE, with pontine enrichment. (A–D) Predicted binding poses of TBE within the active sites of GABRA1, NMDA, TREK‐1, and α₂ A ‐adrenergic receptors. (E) Western blot analysis of GABRA1 expression across nine brain regions.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against GABRA1 (ABclonal, Cat# A8299, RRID:AB_2769558; 1:750) and GAPDH (Abcam, Cat# 2251‐1, RRID:AB_1267174; 1:10 000).

Techniques: Binding Assay, Western Blot, Expressing