Fig. 4

Both AA147 and AA263 promote the surface expression of a variety of trafficking-deficient mutant GABA_A receptors. AA147 (5 µM, 24 h) and AA263 (5 µM, 24 h) increase the surface protein expression of the variant γ2 subunits in HEK293T cells stably expressing α1β2γ2(R177G) (A) and α1β2γ2(R82Q) (B) GABA_A receptors. AA147 (2.5 µM, 24 h) and AA263 (2.5 µM, 24 h) increase the surface protein expression of the variant γ2 subunit in neuronal SH-SY5Y cells expressing α1β2γ2(R177G) (C) and α1β2γ2(R82Q) (D) GABA_A receptors according to surface biotinylation analysis. Na⁺/K⁺-ATPase serves as membrane protein loading control. Quantification of the band intensities is shown on the bottom panels (n = 3). E HEK293T cells stably expressing α1β2γ2(R177G) receptors were treated with DMSO vehicle control, AA147 (5 µM, 24 h) or AA263 (5 µM, 24 h). Surface γ2 staining was in green (column 1), and plasma membrane marker Na⁺/K⁺-ATPase staining in red (column 2). Merge of these two signals and nucleus staining by DAPI in blue was shown in column 3. Scale bar = 20 μm. Quantification of the fluorescence intensity of the surface subunits from 35–45 cells per condition is shown on the right. F Human-induced pluripotent stem cells (hiPS)-derived cortical neurons carrying the γ2(R82Q) variant were treated with DMSO vehicle control, AA147 (2.5 µM, 24 h) or AA263 (2.5 µM, 24 h). Surface γ2 staining was in green and nucleus staining by DAPI was in blue. Quantification of the fluorescence intensity of the surface subunits from 20–30 cells per condition is shown on the right. Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001