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: Molecular characterization of selected disease-associated variants (DAVs) of GABA A receptor α1 subunits. a Pathogenic probability of 149 clinical variants of the GABA A receptor α1 subunit is predicted using Rhapsody and plotted against its primary protein sequence. A schematic of the mature α1 sequence (residue 28–456) showing the N-terminal domain (NTD) and the transmembrane domain (M1-M4) is displayed on the bottom. The variants are classified into three categories: mild (pathogenic probability < 0.30), moderate (0.30 ≤ pathogenic probability < 0.60), and severe (pathogenic probability ≥ 0.60). Eight variants are selected for further characterization and colored in green. b The spatial distribution of the positions of α1 variants is illustrated in cryo-EM structure of α1β2γ2 GABA A receptors (6X3S.pdb), rendered using PyMOL. The C β of the residues (C α in the case of glycine) are shown as spheres. Mild variants are colored in blue, moderate variants in yellow, and severe variants in red. Eight selected variants are labelled. c HEK293T cells were transfected with α1 (wild type or the indicated variants), β2, and γ2 at a ratio of 1:1:1. Forty-eight hours post transfection, cells were lysed with a lysis buffer containing 2 mM DDM. The total proteins were subjected to SDS-PAGE and Western blot analysis. β-actin serves as a total protein loading control. Quantification of the normalized α1 band intensity is shown on the bottom (n = 3). d Surface biotinylation assay was used to quantify the surface α1 protein level. Na + /K + -ATPase serves as a plasma membrane protein loading control. Quantification of the normalized surface α1 band intensity is shown on the bottom (n = 3). e Insoluble α1 fraction was generated from removing soluble α1 fraction as shown in c by extracting proteins with a lysis buffer containing 2 mM DDM; residual insoluble α1 was re-suspended with Laemmli sample buffer containing 2 % SDS and subjected to Western blot analysis. The band intensity was quantified by including the entire lane as indicated within the two arrows. Quantification of the ratio of insoluble over soluble α1 as a measure of aggregation propensity is shown on the bottom (n = 3). f Non-reducing protein gel was used to determine the oligomerization of α1 subunits. Total proteins were extracted and resolved through non-reducing SDS-PAGE in the absence of a reducing reagent. Quantification of oligomeric α1, as indicated within the two arrows, is shown on the bottom (n = 3). 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. NS, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Also see , , and .

Article Snippet: Briefly, intact cells were washed twice with ice-cold DPBS and incubated with the membrane-impermeable biotinylation reagent Sulfo-NHS SS-Biotin (0.5 mg / mL; APExBIO, #A8005) in DPBS containing 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS+CM) for 30 min at 4 °C to label surface membrane proteins.

Techniques: Sequencing, Residue, Cryo-EM Sample Prep, Transfection, Lysis, SDS Page, Western Blot, Control, Surface Biotinylation Assay, Clinical Proteomics, Membrane, Generated, 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: 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 GABA A 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 GABA A 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 (I max ) 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 and . 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 .

Article Snippet: Briefly, intact cells were washed twice with ice-cold DPBS and incubated with the membrane-impermeable biotinylation reagent Sulfo-NHS SS-Biotin (0.5 mg / mL; APExBIO, #A8005) in DPBS containing 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS+CM) for 30 min at 4 °C to label surface membrane proteins.

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