Improving Proteostasis of Trafficking-Deficient GABA_A Receptor Variants by Activating IRE1

Abstract

Gamma-aminobutyric acid type A receptors (GABA_ARs) are essential for maintaining the excitation–inhibition balance in the central nervous system. Genetic variations of GABA_ARs result in a variety of neurological disorders, such as epilepsy. A key pathogenic mechanism involves protein misfolding and defective assembly of GABA_ARs in the endoplasmic reticulum (ER), resulting in impaired surface expression and loss of function. Here, we investigated three trafficking-deficient variants of the GABA_AR α1 subunit (GABRA1), including D219N (ClinVar Variation ID: 127232), G251D (Variation ID: 419523), and P260L. We demonstrated that selective pharmacological activation of the IRE1/XBP1s signaling arm of the unfolded protein response using IXA62, IXA554, and IXA105 increases total and surface protein levels of all three α1 variants without affecting wild-type receptor protein levels in HEK293T cells. Patch-clamping recordings further showed that treatment with IXA62, IXA554, and IXA105 increases the peak GABA-evoked current amplitudes in HEK293T cells expressing α1­(D219N) and α1­(G251D). Mechanistic analyses revealed that IXA62 and IXA554 remodel the GABA_AR-associated proteostasis network by promoting folding and anterograde trafficking while inhibiting degradation in HEK293T cells expressing α1­(D219N) variant and human iPSC-derived neurons carrying α1­(G251D) variant. These results suggest that selective IRE1/XBP1s activation pharmacologically can be further developed to provide a potential therapeutic avenue for genetic epilepsies caused by GABA_AR trafficking defects.

Introduction

The endoplasmic reticulum (ER) plays an essential role in the folding and maturation of proteins destined to environments through the secretory pathway, including the plasma membrane. Perturbation of ER homeostasis, often caused by excessive protein misfolding inside the ER, results in ER stress. In response, cells activate a series of adaptive signaling pathways, known as the unfolded protein response (UPR), to restore proteostasis (protein homeostasis) in the ER. In mammalian cells, the UPR comprises three primary signaling pathways that are initiated by distinct ER membrane sensor proteins: inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6). , The UPR alleviates ER stress by reducing protein translation and promoting the transcriptional remodeling of ER proteostasis pathways involved in protein folding, trafficking, and degradation. , Under prolonged or severe ER stress, the UPR induces a pro-apoptotic response to remove the damaged cells.

IRE1 is the most conserved arm of the UPR and is found in all organisms, from yeast to mammals. Upon ER stress, IRE1 is activated through a mechanism involving oligomerization and autophosphorylation that allosterically activates the cytosolic IRE1 RNase domain involved in the unconventional splicing of X-box binding protein 1 (XBP1) mRNA to generate spliced XBP1 (XBP1s), the active transcription factor that transactivates a cluster of IRE1/XBP1s target genes, including chaperones and ER-associated degradation (ERAD) factors. This triggers a cascade of events aimed at enhancing the ER’s protein folding capacity through the adaptive remodeling of ER proteostasis pathways. However, prolonged or high levels of IRE1 activation can mediate cell death through regulated IRE1-dependent decay (RIDD) and activate the JNK and NF-κB pathways. Thus, moderately activating the IRE1 pathway pharmacologically offers a unique and promising opportunity to alleviate ER stress and correct imbalanced ER proteostasis in diverse diseases.

Gamma-aminobutyric acid type A receptors (GABA_ARs) mediate the majority of fast inhibitory neurotransmission in the mammalian brain. Functional GABA_ARs are pentamers assembled from a combination of 19 subunits (α1–6, β1–3, γ1–3, δ, ε, π, θ, and ρ1–3). The most common GABA_AR subtype in the human synaptic sites is composed of two α1 subunits, two β2 subunits, and one γ2 subunit (Figure S1A). Over 1000 variations in GABA_AR subunits are involved in different types of genetic epilepsies, including childhood absence epilepsy (CAE), febrile seizures (FS), Dravet syndrome (DS), and West syndrome (WS). − Proteostasis maintenance of GABA_ARs is essential for their normal physiological functions, and proteostasis defects of GABA_ARs cause various neurological diseases. Emerging lines of evidence indicate a close association between the dysfunction of GABA_AR variants and reduced surface expression of receptors, particularly at synaptic sites. , The α1 subunits are the requisite subunits as they form the ligand binding pockets for the neurotransmitter GABA, and play a key role in channel inhibitory function and early brain development. Many missense variations in α1 subunits critically affect the receptor folding, assembly, trafficking, and expression on the cell surface. , Here, we focus on three trafficking-deficient α1 variants, including D219N (Variation ID: 127232), G251D (Variation ID: 419523), and P260L. These variants led to significantly reduced receptor surface expression and GABA-induced current amplitudes; in addition, these variants displayed channel gating defects on the plasma membrane. , We chose these variants based on the following two criteria. First, these variants cover a broad phenotypic spectrum: G251D and P260L cause severe epileptic encephalopathy with developmental delay, , whereas D219N is associated with milder idiopathic generalized epilepsy (IGE) or febrile seizures. , Second, these variants cover diverse spatial distribution in the α1 subunit: G251D and P260L are located in the transmembrane helix 1 (TM1), whereas D219N is located in the ER lumen (or extracellular) domain (Figure S1A). High-resolution cryo-electron microscopy (cryo-EM) structures of pentameric GABA_A receptors have been reported, providing valuable insights about how pathogenic variants may impact receptor folding, assembly, and function. The D219 residue resides in the β9 strand, showing contact with K247 in the β10 strand, and is adjacent to TM1 (Figure S1B); therefore, D219 could regulate the transduction of ligand binding to channel gating. The D219N variation removes the side-chain negative charge, which could induce local protein misfolding and impair coupling between ligand binding and channel gating. The G251 residue is located in the initial segment of TM1 (Figure S1C), and the G251D variation introduces a negative charge to the lipid bilayer and likely destabilizes TM1, leading to the subunit misfolding and aggregation. The P260 residue, which is located in the middle segment of TM1 (Figure S1C), is critical for helix packing. The P260L variation likely destabilizes TM1 substantially, leading to subunit misfolding and degradation. In addition, many pathogenic GABA_AR variants cause drug-resistant epilepsy since current antiseizure drugs only work on the cell surface receptors while such variants have substantially reduced surface trafficking capability. , Therefore, the development of novel therapeutic strategies that aim to restore the receptor variant surface expression and thus function is urgently needed.

In this study, we focused on investigating the effect of pharmacological activation of IRE1/XBP1s on proteostasis maintenance of epilepsy-associated trafficking-deficient GABA_AR α1 subunit variants. We identified three stress-independent IRE1/XBP1s activators (IXA62, IXA105, and IXA554) as effective reagents that enhance the folding, assembly, and trafficking of pathologic GABA_AR variants. In addition, molecular mechanism studies demonstrated that these three compounds promote the folding and trafficking as well as attenuating the ERAD of GABA_ARs. Our findings provide new insights into the development of targeted therapies for GABA_AR variation-associated neurological disorders.

Results

IXA62, IXA554, and IXA105 Stimulate IRE1/XBP1s Signaling in HEK293T Cells Carrying Pathogenic α1­(D219N)­β2γ2 GABA_ARs

Previous high-throughput screening of 646,251 compounds from the Scripps Drug Discovery Library identified top compounds with high XBP1 splicing activity. Such stress-independent IRE1 activators were reported to correct pathologic imbalances in ER proteostasis in cellular and mouse models of numerous diseases, such as Alzheimer’s disease and obesity. , Here, we assessed the potential of IRE1 activators in promoting the function of pathogenic GABA_AR variants using a well-established trafficking-deficient α1­(D219N) variant , as the starting point. Two compounds, IXA62 and IXA554, emerged as promising candidates that increased the total protein levels of the α1­(D219N) variant in HEK293T cells stably expressing α1­(D219N)­β2γ2 receptors (Figure A). In addition, IXA105 (an IXA62 analog) (Figure A) activated the IRE1/XBP1s pathway according to luciferase-based XBP1 splicing reporter assay (Figure S1D) and increased α1­(D219N) protein levels. Therefore, these three IRE1/XBP1s activators, namely IXA62, IXA554, and IXA105, were selected for further studies.

1.

IRE1/XBP1s activators IXA62, IXA554, and IXA105 selectively induce the IRE1 arm of UPR in HEK293T cells expressing a pathogenic GABA_AR variant. (A) Chemical structures of three prioritized IRE1/XBP1s activators. (B) RT-qPCR measurements of GABRA1 and IRE1 targeting gene sets in HEK293T cells expressing α1­(D219N)­β2γ2 GABA_ARs after treatment with the indicated compound (10 μM) for 12 h (n = 3 independent experiments). (C) HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs were treated with IXA62 (10 μM), IXA554 (10 μM), IXA105 (10 μM), or DMSO control for 24 h and then subjected to Western blot analysis (n = 3 independent experiments). XBP1s is the protein marker for the activation of the IRE1 pathway. (D–F) HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs were treated with DMSO vehicle control, identified compounds [IXA62 (10 μM) (D); IXA554 (10 μM) (E); and IXA105 (10 μM) (F)], 4μ8C (32 μM), identified compound (10 μM) with 4μ8C (32 μM) cotreatment, or thapsigargin (500 nM) for 24 h. 4μ8C is an inhibitor of the IRE1 pathway, whereas thapsigargin (Tg), a pan UPR activator, serves as a positive control to induce XBP1s expression. Quantification of the band intensities is shown on the bottom panels (n = 5 independent experiments). Each data point is reported as mean ± SEM. One-way ANOVA followed by posthoc Tukey test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001. Also see Figure S1 and Table S1.

To further define the effects of IXA62, IXA554, and IXA105 on the activation of the IRE1/XBP1s signaling pathway in HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs, we determined how drug treatments influenced the mRNA expression level of known transcriptional target genes of IRE1/XBP1s, including XBP1s, Sec24D, ERdj4, Creb3L2, and HerpUD1. All IRE1/XBP1s targeted genes were significantly induced after drug treatments for 12 h (Figure B). In addition, Western blot analysis showed that all three compounds (24-h treatment) increased the protein levels of XBP1s (Figure C). These results confirmed that all three compounds activated the IRE1/XBP1s pathway. In the meantime, we did not observe any significant changes in protein levels of CHOP after 24-h drug treatments, indicating that the PERK pathway was not activated (Figure S1E). Such results are consistent with our recent report showing that IXA62 and IXA554 are selective IRE1/XBP1s activators in HEK293 cells.

We next determined the drug effects using 10 μM of each compound to treat HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs. IXA62, IXA554, and IXA105 increased the expression of α1­(D219N) proteins by 2.4-fold, 1.6-fold, and 2.8-fold, respectively (Figure D–F). Combined with the unchanged GABRA1 mRNA level after drug treatment (Figure B), our data indicated that these compounds increased the expression of α1­(D219N) protein level in a post-transcriptional manner. Co-treatment of an IRE1 RNase active site inhibitor, 4μ8C, attenuated the compound-induced increases in the α1­(D219N) steady-state protein levels and blocked compound-stimulated XBP1 splicing level (Figure D–F, cf. lane 4 to 2), indicating that these compounds stimulated the α1­(D219N) protein expression primarily through an IRE1-dependent mechanism. Collectively, these observations demonstrated that IXA62, IXA554, and IXA105 selectively activated the IRE1/XBP1s signaling pathway to adjust the ER proteostasis network to enhance the protein expression of a pathogenic GABA_AR variant.

IRE1/XBP1s Activators IXA62, IXA554, and IXA105 Promote the Functional Surface Protein Expression of Trafficking-Deficient α1­(D219N)­β2γ2 GABA_ARs

We next determined the optimal concentration and treatment time for the application of IXA62, IXA554, and IXA105. We treated HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs with a range of concentrations of IXA62 from 0.1 μM to 25 μM for 24 h. The concentrations of IXA554 and IXA105 were similarly tested in the range of 0.1 μM to 10 μM due to the drug toxicity to cells at 25 μM (Figure A). Dose–response analysis showed that half maximal effective concentration (EC₅₀) values are 7.57 ± 3.07 μM for IXA62, 1.35 ± 0.76 μM for IXA554, and 4.25 ± 1.06 μM for IXA105 (Figure B). In addition, dose–response analysis in HEK293T cells expressing wild-type GABA_ARs showed that IXA62 and IXA554 treatment did not increase the wild-type α1 protein levels (Figure S2A,B), and IXA105 treatment only had a minimal effect on wild-type α1 proteins (Figure S2C), indicating the selectivity of these IRE1 activators in the misfolding-prone pathogenic GABA_AR variants. The time-course analysis showed that a single application of IXA62, IXA554, or IXA105 reached the optimal effects at 48, 24, or 48 h, respectively (Figure C–F). Thus, in subsequent experiments, we chose 10 μM, 24-h drug treatment to maximize their effects on increasing α1 variant protein levels.

2.

IRE1/XBP1s activators IXA62, IXA554, and IXA105 enhance the functional surface expression of epilepsy-associated GABA_ARs harboring the α1­(D219N) variant. (A, B) Dose–response analysis of IXA62, IXA554, and IXA105 treatments (24 h) on the total α1­(D219N) protein levels in HEK293T cells stably expressing α1­(D219N)­β2γ2 receptors. Western blot images are shown in (A), and dose–response curve fittings are shown in (B) (n = 3 independent experiments). (C–F) Time-course study of IXA62, IXA554, and IXA105 treatments (10 μM) on the total α1­(D219N) protein levels in HEK293T cells stably expressing α1­(D219N)­β2γ2 receptors. Western blot images are shown in (C). Quantifications of the α1 band intensities are shown in (D) for IXA62, (E) for IXA554, and (F) for IXA105 (n = 5 independent experiments). (G, H) Effect of IXA62, IXA554, and IXA105 (10 μM, 24 h) treatments on the surface α1­(D219N) protein expression according to surface biotinylation assay. Western blot images are shown in (G) and quantification of surface α1 intensities are shown in (H) (n = 3 independent experiments). Na⁺/K⁺-ATPase serves as membrane protein loading control. (I) Representative images of surface staining of HEK293T cells stably expressing α1­(D219N)­β2γ2 receptors. Cells were treated with DMSO vehicle control, IXA62, IXA554, or IXA105 (10 μM, 24 h). Surface α1 staining was in green, surface marker Na⁺/K⁺-ATPase was in red, and nucleus staining by Hoechst 33342 was in blue. Scale bar: 25 μm. Arrows indicate surface stainings. (J) Quantification of the single-cell fluorescence intensity of the α1 surface expression from (I) (n = 40–50 cells). (K) Diagram of whole-cell auto patching recording traces of 100 μM GABA-induced chloride currents in HEK293T cells stably expressing α1­(D219N)­β2γ2 receptors in the DMSO vehicle control, IXA62 treatment (10 μM, 24 h), IXA554 treatment (10 μM, 24 h), or IXA105 treatment (10 μM, 24 h). (L) Quantification of the peak currents (I _max) from (K) (n = 8 independent experiments). pA, picoampere. (M) Dose–response analysis using HEK293T cells expressing α1­(D219N)­β2γ2 receptors (n = 4 independent experiments). Each data point is reported as mean ± SEM. One-way ANOVA followed by posthoc Tukey test was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. Also see Figure S2.

GABA_ARs must be transported from the ER to the cell surface for function, and many missense variations in epilepsy-associated GABA_AR subunits critically affect the expression of the receptors on the cell surface, thus suppressing the overall inhibitory input and predisposing the brain to hyperexcitability. We performed cell surface biotinylation assays and showed that IXA62, IXA554, and IXA105 increased the surface protein levels of α1­(D219N) by 1.8-fold, 2.5-fold, and 1.8-fold, respectively (Figure G,H). In addition, we carried out confocal microscopy from immunofluorescence surface staining experiments and demonstrated that all three compounds significantly increased the fluorescence intensities of the surface α1­(D219N) proteins (Figure I,J). Akin to what was observed for total protein levels (Figure S2A–C), these three IRE1 activators also did not significantly alter the surface expression level of wild-type α1 subunit in HEK293T cells stably expressing wild-type GABA_ARs (Figure S2D).

To determine the effects of these three IRE1 activators on the function of this pathogenic GABA_AR variant, we carried out whole-cell patch-clamping electrophysiological recording experiments using an automated patch-clamping system with HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs. The 100 μM GABA-induced peak currents were significantly increased to 2.2-fold by IXA62 treatment, to 3.0-fold by IXA554 treatment, and to 2.4-fold by IXA105 treatment (Figure K,L), indicating that the promoted receptor trafficking from ER to cell surface led to enhanced function. Since it was previously reported that the D219N variation reduced the peak current amplitude to 60.3% of wild-type receptors, these three IRE1 activators enhanced the peak current amplitudes to a level that was comparable to wild-type receptors, suggesting the clinical potential of this treatment strategy. EC₅₀ values of GABA-induced currents are similar between untreated and compound-treated groups: 2.82 ± 0.71 μM for DMSO vehicle control, 2.09 ± 0.30 μM for IXA62 treatment, 2.35 ± 0.73 μM for IXA554 treatment, and 1.96 ± 0.24 μM for IXA105 treatment (Figure M). These findings indicated that these IRE1 activators stimulated adaptive remodeling of pathogenic GABA_AR expression and function, providing a potential small molecule therapeutic strategy for genetic epilepsies. Since IXA105, which is an IXA62 analog (Figure A), had very similar effects as IXA62 on both protein expression and functional performance levels, only IXA62 and IXA554 were selected for the following mechanistic studies.

IXA62 and IXA554 Promote Folding and Forward Trafficking of α1­(D219N) Subunit

To investigate whether IXA62 and IXA554 promoted the folding and trafficking of mutant GABA_ARs, we carried out a series of assays to evaluate the folded and unfolded populations of α1­(D219N) proteins in HEK293T cells expressing α1­(D219N)­β2γ2 GABA_ARs. The n-Dodecyl-β-d-maltoside (DDM) detergent solubility assay was used to quantify the relative folding degree of α1­(D219N) through the ratio of soluble/pellet fraction. Both compound treatments enhanced the protein folding of α1­(D219N) (Figure A). In addition, each folded α1 subunit forms a signature disulfide bond in the ER lumen, which is disrupted during the misfolding process. Therefore, we developed a PEGylation assay to monitor the free sulfhydryls in unpaired cysteines using a gel mobility shift assay (Figure B). Free sulfhydryls in unpaired cysteines, but not paired disulfides in correctly folded α1­(D219N) subunits, can be covalently attached to methoxypolyethylene glycol maleimide (Mal-PEG), a 5 kDa modifier. The PEGylation assay showed that IXA62 and IXA554 significantly increased the intensity of lower molecular weight α1 bands at 60 kDa, which correspond to less PEGylated, folded α1­(D219N) subunits (Figure B), indicating the selected IRE1/XBP1s activators enhanced the protein folding of this variant. Furthermore, we used nonreducing SDS-PAGE to evaluate the oligomerization states of GABA_ARs. Both IXA62 and IXA554 significantly increased α1­(D219N) protein dimers (Figure C), suggesting that drug treatments increased the population of the assembly intermediates. Overall, these results indicated that IXA62 and IXA554 improved the folding and assembly ability of the epilepsy-associated α1­(D219N) variant.

3.

IRE1/XBP1s activators IXA62 and IXA554 enhance the folding and trafficking of trafficking-deficient α1­(D219N) subunit. (A) Effect of IXA62 and IXA554 treatment (10 μM, 24 h) on the folding degree of α1­(D219N) subunits according to detergent solubility assay. Two mM n-Dodecyl-β-D-maltoside (DDM) is used as the solubilization detergent. Quantification of the detergent soluble/insoluble fractions is shown on the bottom panel (n = 4 independent experiments). (B) PEGylation assay shows the effect of IXA62 and IXA554 (10 μM, 24 h) on the folding degree of α1­(D219N) subunits. The unfolded and folded bands are indicated on the gel. Quantification of the folded α1/unfolded α1fractions is shown on the bottom panel (n = 5 independent experiments). (C) Immunoblot shows the assembly intermediates of α1­(D219N)­β2γ2 GABA_ARs after IXA62 and IXA554 treatments (10 μM, 24 h) in nonreducing SDS-PAGE conditions. Fold change of α1 dimers is quantified in the absence of β-mercaptoethanol (βME) and shown on the right panel (n = 3 independent experiments). (D) Deglycosylation assay shows that IXA62 and IXA554 (10 μM, 24 h) treatments increase the endo H-resistant post-ER glycoform of the α1­(D219N) subunit. PNGase F treatment indicates the unglycosylated α1 subunit. Quantification of the endo H-resistant/total α1 band intensity is shown on the bottom panel (n = 4 independent experiments). (E) Immunoblot analysis of steady-state protein levels of BiP, calnexin, HSP47, and LMAN1 after DMSO control, IXA62, or IXA554 (10 μM, 24 h) treatments in HEK293T cells stably expressing α1­(D219N)­β2γ2 GABA_ARs. Quantifications of the normalized band intensities are shown on the right (n = 3 independent experiments). (F) Effects of IXA62 and IXA554 (10 μM, 24 h) treatment on the interactions between α1­(D219N) subunits and BiP, calnexin, HSP47, and LMAN1. Apyrase (10 units/mL), which hydrolyzes ATP, was added during the coimmunoprecipitation experiments to enhance the detection of the interactions between BiP and α1­(D219N). Quantification of the ratio of the indicated proteins and α1­(D219N) post immunoprecipitation (IP) is shown on the right panel (n = 3 independent experiments). Each data point is reported as mean ± SEM. One-way ANOVA followed by posthoc Tukey test was used for statistical analysis. *, p < 0.05, ***, p < 0.001.

Properly folded and assembled α1­(D219N) subunits must exit the ER for forward trafficking. An endoglycosidase H (Endo H) enzyme digestion assay was used to test whether IXA62 and IXA554 enhanced the receptor trafficking. Endo H treatment increased the upper two endo H-resistant α1­(D219N) bands (Figure D, cf. lanes 5 and 7 with lane 3), corresponding to post-ER, mature glycoforms that trafficked at least to the Golgi. The bottom endo H sensitive α1­(D219N) bands were retained in the ER. Thus, the increased ratio of endo H-resistant/total α1­(D219N) by IXA62 and IXA554 treatments indicated that both compound treatments enhanced the ER-to-Golgi trafficking efficiency of α1­(D219N) (Figure D).

Subsequently, we investigated how IXA62 and IXA554 adapted the ER proteostasis network. The folding and assembly of GABA_ARs in the ER is facilitated by ER luminal chaperones, including BiP and HSP47, two heat shock protein family members, and calnexin, a lectin chaperone. , In the meantime, upon activation of the IRE1 pathway, IRE1α has been found to interact with HSP47. Thus, we evaluated how IXA62 and IXA554 influenced these chaperones. Both compound treatments had no apparent effect on calnexin and HSP47 protein levels, whereas IXA62 slightly increased the BiP protein level (Figure E). In addition, a coimmunoprecipitation assay showed that IXA62 enhanced the interaction between BiP and α1­(D219N) subunits (Figure F). Previously, we demonstrated that LMAN1 (also known as ERGIC53), a lectin ER Golgi intermediate compartment 53 kDa protein, plays an important role in GABA_AR trafficking. We showed that both compound treatments did not alter the protein level of LMAN1 (Figure E), but significantly increased the interaction between LMAN1 and α1­(D219N) subunits (Figure F). These findings suggested that selected IRE1/XBP1s activators enhanced the α1­(D219N) subunits forward trafficking by increasing the interaction between LMAN1 and α1­(D219N) subunits, and IXA62 upregulated the BiP level as well as the interaction between BiP and α1­(D219N) to facilitate protein folding of α1­(D219N) subunits.

IXA62 and IXA554 Reduce the Degradation of the α1­(D219N) Variant

Since the ERAD pathway is critical in regulating the degradation of misfolded GABA_ARs, , we sought to determine how the selected IRE1/XBP1s activators affect the GABA_AR-associated ERAD network. To determine how drug treatments affected the degradation kinetics of mutant GABA_ARs, we applied cycloheximide, a potent inhibitor of protein biosynthesis, to cell culture media. The cycloheximide-chase assay showed that the degradation of α1­(D219N) proteins was significantly delayed after IXA62 and IXA554 treatments (Figure A,B). We next assessed how IXA62 and IXA554 regulated ERAD factors to attenuate the degradation of α1­(D219N) subunits. Previously, we demonstrated that GABA_ARs interact with multiple ERAD factors, including Grp94, Hrd1-Sel1L, and VCP. , Grp94 is a 90 kDa heat shock protein (HSP90) chaperone in the ER lumen that recognizes the non-native states of α1 subunits and directs those misfolded α1 subunits to the ERAD pathway. Hrd1, a major ubiquitin E3 ligase, together with its obligate partner sel-1 suppressor of lin-12-like (Sel1L), coordinates the substrate ubiquitination process. Then valosin-containing protein (VCP), an AAA-ATPase, facilitates substrate dislocation and extraction. The HRD1-Sel1L-VCP pathway represents the most conserved ERAD machinery. Both IXA62 and IXA554 treatments did not alter the protein levels of Grp94, Sel1L, Hrd1, or VCP (Figure C,D), but they significantly decreased the interaction between α1­(D219N) subunits with these ERAD factors (Figure E,F). We further evaluated some other important ERAD factors, including Edem1, Erlin2, HSPA13, HUWE1 and HerpUD1, which are known UPR targets. None of these ERAD factor protein levels were altered by drug treatments, except that IXA62 modestly increased HerpUD1 protein level (Figure S3). Collectively, these results demonstrated that IXA62 and IXA554 inhibited the ERAD pathway of misfolded α1­(D219N) subunits by attenuating the interaction between α1­(D219N) and known GABA_A-associated ERAD factors, including Grp94, Hrd1-Sel1L, and VCP.

4.

Both IXA62 and IXA554 inhibit the ERAD of misfolding-prone α1­(D219N) subunit. (A, B) Cycloheximide (CHX)-chase assay to detect the effect of IXA62 and IXA554 treatment (10 μM, 24 h) on the degradation of the α1­(D219N) subunit. n = 6 independent experiments. (C, D) Protein level changes of the indicated ERAD factors after IXA62 and IXA554 treatments (10 μM, 24 h) (n = 3 independent experiments). (E, F) Effect of IXA62 and IXA554 treatments (10 μM, 24 h) on the interactions between α1­(D219N) subunits and selected ERAD factors. Quantification of the ratio of the target proteins and α1­(D219N) post immunoprecipitation (IP) is on (F) (n = 3 independent experiments). (G) Dendra2 assay to quantify the stability of α1­(D219N) on the cell surface. The representative time trace TIRF images of the Dendra2 labeled α1­(D219N) subunit with DMSO vehicle control, IXA62 treatment (10 μM, 24 h), and IXA554 treatment (10 μM, 24 h) showing the fluorescence intensity reduced over time. Normalized fluorescence decay curves of α1­(D219N) subunit are shown on the right panels (n = 8 cells). Each data point is reported as mean ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001. Also see Figure S3.

In addition, we tagged the receptors with a photoconvertible fluorescent protein, Dendra2, and used Total Internal Reflection Fluorescence (TIRF) microscopy to determine the stability of GABA_ARs on the plasma membrane. Dendra2 can be irreversibly converted from green to red emission upon exposure to 405 nm light. The TIRF orientation limits the photoconversion and detection range to a narrow region, which means that only α1­(D219N) subunits tagged with Dendra2 located near the cell surface would be converted to red emitting species and detected with 561 nm TIRF excitation light. Therefore, the stability of Dendra2-fused α1­(D219N) protein on the plasma membrane can be quantified by measuring the intensity of red emission over time. Figure G shows representative time-lapse TIRF images of the red emission during the 8-h observation period. The quantified fluorescence intensity versus time plots were used to show the decay of surface α1­(D219N) subunits. The decay of α1­(D219N) subunits was significantly reduced by both drug treatments (Figure G).

IRE1/XBP1s Activators Enhance the Surface Expression of Three Trafficking-Deficient GABA_AR Variants

The above results demonstrated the effectiveness of selected IRE1/XBP1s activators in enhancing the functional surface expression of epilepsy-associated α1­(D219N)­β2γ2 GABA_ARs. We continued to explore whether IXA62, IXA554, and IXA105 could mitigate multiple trafficking-deficient GABA_AR variants. We tested the drug effects in two additional pathogenic α1 variants: α1­(G251D) and α1­(P260L). IXA62, IXA554, and IXA105 increased total protein levels of α1­(G251D) by 2.6-fold, 1.8-fold, and 3.1-fold (Figure A,B) and total protein levels of α1­(P260L) by 2.3-fold, 1.9-fold, and 2.6-fold (Figure S4A,B), respectively. Furthermore, cell surface biotinylation assays demonstrated that IXA62, IXA554, and IXA105 increased the surface protein levels of α1­(G251D) by 3.0-fold, 3.1-fold, and 2.3-fold (Figure A,C), and surface protein levels of α1­(P260L) by 1.4-fold, 1.9-fold, and 1.5-fold, respectively (Figure S4A,C). In addition, we confirmed the drug effects on surface expression enhancement using confocal immunofluorescence imaging assay. Through indirect surface staining experiments using Na⁺/K⁺-ATPase as a surface membrane control, we showed that all three compounds significantly increased the fluorescence intensities of the surface α1­(G251D) staining (Figure D,E). Alternative surface staining strategies include: (I) the utilization of a GFP tag that was fused to the N-terminus of α1 subunit, as was reported to robustly visualize surface GABA_A receptors; (II) dual-color staining using α1 antibody that has an extracellular epitope to label surface receptors without membrane permeabilization and then using the same antibody conjugated to a different fluorophore to label total receptors after membrane permeabilization, which enables direct comparison of surface versus total receptor levels within the same cells. These alternative surface staining strategies merit future investigation to enhance the efficiency and clarity of surface receptor labeling. Moreover, the whole-cell patch-clamping electrophysiological recording results demonstrated that IXA62, IXA554, and IXA105 significantly increased GABA-induced peak current amplitudes by 2.1-fold, 2.6-fold, and 1.6-fold, respectively (Figure F,G), indicating that these compounds promoted functional GABA_ARs on the cell surface. Since it was previously reported that the G251D variation reduced the peak current amplitude to 48.4% of wild-type receptors, these three IRE1/XBP1s activators increased the peak current amplitudes to a level that was comparable to wild-type receptors, suggesting the clinical potential of this strategy. Once reaching the plasma membrane, Dendra2-based time-lapse TIRF measurements demonstrated that IXA62 and IXA554 treatments enhanced the residence time of α1­(G251D) on the cell surface (Figure H,I).

5.

Three IRE1/XBP1s activators promote the functional surface expression of a variety of trafficking-deficient GABA_AR variants. (A–C) Effect of IXA62, IXA554, or IXA105 (10 μM, 24 h) treatments on the total (B) (n = 3 independent experiments) and surface (C) (n = 4 independent experiments) protein expression of the α1­(G251D) subunits in HEK293T cells stably expressing α1­(G251D)­β2γ2 GABA_A receptors. β-actin serves as total protein loading control and Na⁺/K⁺-ATPase serves as membrane protein loading control according to surface biotinylation analysis. (D) Representative images of surface staining of HEK293T cells stably expressing α1­(G251D)­β2γ2 receptors, which were treated with DMSO vehicle control, compound IXA62, IXA554, or IXA105 (10 μM, 24 h). Surface α1 staining was in green, surface marker Na⁺/K⁺-ATPase was in red, and nucleus staining by Hoechst was in blue. Scale bar: 25 μm. Arrows indicate surface stainings. (E) Quantification of the single-cell fluorescence intensity of the surface subunits from (D) (n = 40–50 cells). (F, G) Treatments of IXA62, IXA554, or IXA105 increase the peak amplitude of 1 mM GABA-induced chloride currents in HEK293T cells stably expressing α1­(G251D)­β2γ2 receptors. Representative whole-cell automatic patch-clamping recording traces are shown in (F). Quantification of the peak currents (I _max) are shown in (G) (n = 8 independent experiments). pA, picoampere. (H) Dendra2 assay to quantify the stability of α1­(G251D) on the cell surface. The representative time trace TIRF images of the Dendra2 labeled α1­(G251D) subunit with DMSO vehicle control, IXA62 treatment (10 μM, 24 h), and IXA554 treatment (10 μM, 24 h) showing the fluorescence intensity reduced over time. (I) Normalized fluorescence decay curves of α1­(G251D) subunit (n = 12 cells). Each data point is reported as mean ± SEM * p < 0.05, ** p < 0.01, *** p < 0.001. Also see Figure S4.

IRE1/XBP1s Activators Promoted the Surface Trafficking of Pathogenic GABA_ARs in Human-Induced Pluripotent Stem Cell (iPSC)-Derived Neurons Carrying α1­(G251D) Variant

To mimic the native neuronal environment of the pathogenic GABA_ARs, we developed a human iPSC model carrying the pathogenic α1­(G251D) variant. We then differentiated these iPSCs into GABAergic neurons by the expression of transcription factors ASCL1 and DXL2 using lentivirus transduction. Neurons were further matured for additional three to 4 weeks. Surface staining and confocal microscopy experiments showed that IXA62 and IXA554 treatment (10 μM, 24 h) significantly increased the fluorescence intensities of the major endogenous subunits, including α1­(G251D), β2/β3, and γ2 of GABA_ARs (Figure A).

6.

IRE1/XBP1s activators adapt the proteostasis network to enhance the surface trafficking of α1­(G251D) in human iPSC-derived neurons. (A) Effect of IXA62 (10 μM, 24 h) and IXA554 (10 μM, 24 h) on the surface protein levels of endogenous GABA_AR subunits in human iPSC-induced GABAergic neurons carrying the α1­(G251D) variant. Surface GABA_A receptors were stained using anti-α1 subunit, anti-β2/β3 subunit, or anti-γ2 subunit antibodies without membrane permeabilization. 19–35 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. Quantification of the fluorescence intensity of the surface GABA_A receptor subunits after background correction is shown on the right panels. (B) Effect of IXA62 (10 μM, 24 h) and IXA554 (10 μM, 24 h) on the interactions between α1­(G251D) and selected ER proteostasis network components and γ2 subunits according to proximity ligation assay (PLA). 22–36 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. Quantification of the PLA puncta number per cell was achieved using the ImageJ Analyze Particles plug-in and shown on the right panels. Each data point is reported as mean ± SEM. ANOVA followed by posthoc Tukey test was used to evaluate the statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Furthermore, to determine the mechanism of action of these IRE1/XBP1s activators in iPSC-derived neurons, we used Proximity Ligation Assay (PLA) to quantify the endogenous interactions between α1­(G251D) and selected GABA_AR-associated proteostasis network components. IXA62 and IXA554 treatments increased the interactions between α1­(G251D) and BiP, a pro-folding chaperone, and LMAN1, a trafficking receptor, whereas both compounds decreased the interaction between α1­(G251D) and Grp94, a chaperone that promotes the degradation of GABA_ARs (Figure B). In addition, IXA62 and IXA554 treatments enhanced the interaction between α1­(G251D) and γ2 subunits of GABA_ARs (Figure B, top row), indicating that these activators promoted the subunit–subunit assembly process. Therefore, consistent with the results in HEK293T cells, IXA62 and IXA554 treatment adapted the ER proteostasis network to promote the folding and trafficking of α1­(G251D) while inhibiting the ERAD in iPSC-derived neurons.

Discussion

Here we demonstrated that three IRE1/XBP1s activators, namely IXA62, IXA554, and IXA105, effectively enhanced the surface trafficking of three epilepsy-associated GABRA1 variants, including α1­(D219N), α1­(G251D), and α1­(P260L) (Figures , , and S4). Functional GABA_A receptors must be present on the plasma membrane as pentamers containing α1, β2, and γ2 subunits as the prominent subtype in the synaptic sites. We demonstrated that IXA62 and IXA554 enhanced surface trafficking of α1, β2, and γ2 subunits of GABA_A receptors in human iPSC-derived neurons carrying the α1­(G251D) variant (Figure A). In addition, we showed that IXA62, IXA554, and IXA105 increased GABA-induced peak current amplitudes in HEK293T cells expressing α1­(D219N)­β2γ2 receptors (Figure K,L) and α1­(G251D)­β2γ2 receptors (Figure F,G), indicating that these compounds enhanced the function of pentameric receptors. These results suggest that these IRE1/XBP1s activators increased the surface trafficking of α1, β2, and γ2 subunits in pathogenic GABA_A receptor variants to promote their function. Moreover, we demonstrated that these IRE1/XBP1s activators increased peak current amplitudes for α1­(D219N) ((Figure K,L) and α1­(G251D) (Figure F,G) to levels that are comparable to wild-type receptors in HEK293T cells, suggesting that these compounds merit further development to achieve optimal potency, efficacy, and drug properties for their translational application. One limitation of our current evaluation is that we did not use native wild-type receptors side-by-side for comparison with variant receptors with or without drug treatments, which would provide direct interpretation of the degree of rescue. Since we used stable cells expressing GABA_A receptors in our drug treatment experiments, which would have various receptor expression levels depending on the selection process, comparison between wild type and variants in stable cell lines is not viable. As a surrogate, since we have a comparison between transiently transfected wild-type and variant receptors, we used that data in our analysis to achieve a relative fold change of GABA_A receptor variants afforded by drug treatment, as an indirect way to quantify the degree of rescue. Nonetheless, future studies should aim to incorporate direct comparisons between native wild-type receptors and drug-treated variants.

The mechanism of action of these IRE1/XBP1s activators on enhancing the functional surface expression of GABA_AR variants was summarized in Figure . Activation of the IRE1/XBP1s pathway enhanced the ER folding capacity and increased the interactions between the α1 variant and pro-folding chaperones, including BiP, and trafficking machinery, including LMAN1. In the meantime, the degradation of the α1 variant was attenuated with the reduced interactions between the α1 variant and ERAD factors, including Grp94, Hrd1-Sel1L, and VCP. This would increase the folded population of the α1 variant and the properly assembled pentameric receptors in the ER membrane, leading to enhanced trafficking efficiency from the ER to the Golgi and onward to the plasma membrane. After reaching the plasma membrane, the stability of the α1 variant was also increased after drug treatments, which might result from the enhanced proteostasis network capacity that acts on the cell surface receptors.

7.

Mechanism of action of IRE1/XBP1s activators on enhancing GABA_AR variant proteostasis. Certain GABA_AR variants misfold in the ER and undergo excessive ER-associated degradation (ERAD), leading to their trafficking deficiency to the plasma membrane. IRE1/XBP1s activators enhance the ER folding capacity to promote the folding of a variant by increasing the interaction between the variant and BiP, a pro-folding chaperone, and further promote the assembly into pentameric receptors in the ER membrane. In addition, such IRE1/XBP1s activators inhibit the ERAD of the variant by decreasing the interaction between the variant and Grp94, Hrd1-Sel1L, and VCP, known ERAD factors for GABA_ARs. Consequently, IRE1/XBP1s activators enhance the anterograde trafficking of the variant to the plasma membrane by increasing the interaction between the variant and LMAN1. The receptor variant is also stabilized after reaching the plasma membrane.

Recent advances in genetic screening have identified a growing number of disease-associated variants in genes encoding GABA_AR subunits, including α1 (>300 variants), α2 (>200 variants), β1 (>180 variants), β2 (>300 variants), β3 (>300 variants), and γ2 (>300 variants) according to NIH Clinvar as of August 2025. The molecular consequences of these variants include missense, frameshift, nonsense, splice site, and Untranslated Region (UTR). GABA_AR variants are linked to a broad phenotypic spectrum including various types of epilepsies with or without neurodevelopmental delay. However, the majority of these variants have not been characterized for their potential proteostasis and functional defects, calling for substantial efforts to comprehensively evaluate such a large number of GABA_AR variants. Artificial intelligence-based modeling using AlphaMissense or Rhapsody predicted the pathogenic probability of saturating variants of α1, β2, β3, and γ2 subunits, providing an initial clue about their functional consequences. We proposed to categorize clinical variants of GABA_ARs into four classes based on their molecular defects: (I) proteostasis defects, arising from abnormalities in protein folding, assembly, trafficking, degradation, aggregation, or endocytosis; (II) gating defects, caused by impaired ligand binding or alterations in current amplitude and kinetics (activation, desensitization, and deactivation) at the plasma membrane; (III) mRNA defects, resulting from nonsense-mediated decay triggered by premature stop codons; and (IV) other defects not captured by the above categories. It is increasingly recognized that proteostasis deficiency and channel gating defects represent major disease-causing mechanisms for pathogenic GABA_AR variants. In many cases, these defects coexist, reflecting the complex and multifactorial nature of GABA_AR-related diseases. , In the present study, the three α1 variants examined, including α1­(D219N), α1­(G251D), and α1­(P260L), exhibit significantly reduced surface trafficking and peak current amplitudes, consistent with impaired proteostasis, and also display altered channel gating kinetics. , All three variants show reduced desensitization compared to wild-type receptors, , and α1­(D219N) also displays accelerated deactivation kinetics. Certain trafficking-deficient pathogenic GABA_AR variants, such as γ2­(R177G), appear to have similar macroscopic kinetic properties compared to wild-type receptors. Together, these findings highlight that GABA_AR variants frequently harbor overlapping folding, trafficking, and gating abnormalities. For severe pathogenic GABA_AR variants exhibiting both proteostasis defects and gating defects, a combination use of trafficking enhancers and gating potentiators may provide the greatest functional rescue for potential disease treatment. This principle is exemplified by the cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy for cystic fibrosis, sold under the brand name of Trikafta, which contains two CFTR correctors (elexacaftor and tezacaftor) to improve channel surface trafficking and one CFTR potentiator (ivacaftor) to enhance surface channel gating.

Since the stress-independent activation of the IRE1/XBP1s pathway pharmacologically promotes the ER folding capacity to enhance proteostasis of GABA_AR variants, the advantage of IRE1/XBP1s activators is that they are expected to be effective on numerous trafficking-deficient variants. It is also important to note that these IRE1/XBP1s activators can achieve specificity toward the destabilized α1 variants over wild-type proteins (Figures and S2). IRE1/XBP1s activators have beneficial effects toward the functional rescue of trafficking-deficient variants by increasing the number of surface receptors per se, which also enhances the pool available for modulation by direct GABA_ΑR-targeting compounds. Because many GABA_AR variants are expected to exhibit both proteostasis and gating defects, an effective therapeutic strategy would involve combining a proteostasis regulator, to restore surface trafficking, with a direct GABA_AR regulator, to correct gating abnormalities. Therefore, stress-independent IRE1/XBP1s activators have the promise to be further developed for the treatment of genetic epilepsy resulting from trafficking deficiency of GABA_ARs.

One technical limitation of this study is that most experiments were performed in HEK239T cells expressing exogenous GABA_ARs. HEK293T cells are commonly used as a heterologous expression system for GABA_ARs because they allow precise control over receptor subunit composition. Here, to reduce the impact of plasmid overexpression on the endogenous proteostasis environment, we minimized the amount of plasmid DNA used for transfection. Nonetheless, human iPSC-derived neurons carrying disease-associated variants, which closely recapitulate the native neuronal context, represent a valuable cellular system for modeling GABA_AR variant-related diseases to elucidate disease-causing mechanisms and develop therapeutic strategies. Indeed, such endogenous models for GABA_AR variants have begun to emerge in the literature, underscoring the importance of further development in this direction.

Recently, we demonstrated that small molecule pharmacological chaperones, such as hispidulin and TP003, directly bind GABA_ARs to stabilize them, leading to enhanced functional surface expression of multiple GABRA1 variants. These pharmacological chaperones do not induce the UPR. Therefore, IRE1/XBP1s activators and pharmacological chaperones have distinct mechanisms of action, and their coapplication has the promise to produce additive/synergistic rescue of trafficking-deficient GABA_AR variants.

GABA_ARs are subject to post-translational modifications, including glycosylation, phosphorylation, ubiquitination, and palmitoylation, which can be manipulated to regulate receptor folding, degradation, and trafficking. For example, calnexin, a lectin chaperone, plays a critical role in facilitating the folding of glycosylated GABA_ARs; overexpressing calnexin is sufficient to enhance the anterograde trafficking of α1­(D219N) variant to the plasma membrane in a glycan-dependent manner. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) can phosphorylate serine residues in β1–3 and γ2 subunits; phosphorylation of β3 at S383 by CaMKII enhances the rapid receptor insertion on the surface. Hrd1, a conserved E3 ubiquitin ligase, ubiquitinates α1 subunits, and overexpressing Hrd1 increases α1 ubiquitination, thereby expediting their degradation. Palmitoylation of γ2 subunits is mediated by GODZ (Golgi-specific DHHC zinc finger protein, ZDHHC3), which is required for their efficient insertion at synaptic sites; overexpressing GODZ increases the palmitoylation level and surface expression of trafficking-deficient γ2­(G257R) variant, which has reduced palmitoylation. Many of the GABA_AR variants are resistant to current GABA_AR-targeting drugs since they only act on the surface receptors. As such, it is urgently needed to develop proteostasis-based therapeutic strategies, including proteostasis regulators, pharmacological chaperones, and manipulation of post-translational modifications of GABA_ARs, to rescue the surface trafficking and function of GABA_AR variants to treat such devastating drug-resistant epilepsy.

Methods

Reagents

IXA62 (#Z15671089, Enamine) (CAS #: 956783-34-9), IXA554 (#F2506-1107, Life Chemicals) (CAS #: 894053-81-7), and IXA105 (#Z14109985, Enamine) were purchased from commercial vendors. 4μ8C (#412512) was obtained from EMD Millipore. Thapsigargin (#50-464-295) was obtained from Fisher Scientific. Apyrase (#A6237) was obtained from Sigma. Cycloheximide (CHX) (#3342C067, armesco) was obtained from VWR.

Antibodies

GABA_A receptor α1 subunit antibody (#MAB339, 1:2000) and GABA_A receptor β2/β3 subunit antibody (#05-474, 1:500) were obtained from Millipore. The rabbit polyclonal anti-GABA_A α1 subunit antibody (catalog #: 224203, 1:250) and rabbit polyclonal anti-GABA_A γ2 antibody (catalog #: 224003, 1:250) were obtained from Synaptic systems. The fluorescent anti-β-actin antibody Rhodamine came from Biorad (catalog #: 12004163, 1:10000). Calnexin (#ADI-SPA-860-F, 1:1000) and Grp94 (#ADI-SPA-850-F, 1:1000) antibodies were purchased from Enzo Life Sciences. Sel1L antibody (#PA5–18943, 1:1000) was obtained from ThermoFisher Scientific. HRD1 antibody (#AP2184e, 1:1000), BiP antibody (#AP5041C, 1:1000) were obtained from Abgent. VCP (#ab109240, 1:1000), LMAN1 (#ab125006, 1:1000), Hsp47 (# ab109117, 1:1000), and Na/K-ATPase (#ab76020, 1:2000), and BiP (#ab21685, 1:250) antibodies were from Abcam. XBP1s (#12782S, 1:1000) and CHOP (#2895S, 1:1000) antibodies were purchased from Cell Signaling. The rabbit polyclonal anti-Grp94 antibody (#14700-1-AP, 1:250) was obtained from Proteintech.

Plasmids

The pCMV6 plasmids encoding human GABA_A receptor α1 (Uniprot no. P14867–1), β2 (isoform 2, Uniprot no. P47870-1), γ2 (isoform 2, Uniprot no. P18507-2) subunits, and pCMV6 Entry Vector plasmid (pCMV6-EV) were obtained from Origene. The FLAG tag was inserted between Leu31 and Gln32 in the α1 subunit for coimmunoprecipitation experiments, which do not influence trafficking function. The α1 subunit missense mutations D219N, G251D, and P260L were constructed using QuikChange II site-directed mutagenesis Kit (Agilent Genomics, #20052489). The fluorescent protein Dendra2 was incorporated into the C-terminus of each α1 construct for TIRF imaging. All cDNA sequences were confirmed by DNA sequencing.

Cell Culture and Transfection

HEK293T cells were obtained from ATCC (#CRL-3216) or Abgent (#CL1032). HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Fisher Scientific, catalog #: 10-013-CV) with 10% heat-inactivated fetal bovine serum (FBS) (Fisher Scientific, catalog #: SH30396.03HI) and 1% Penicillin–Streptomycin (Fisher Scientific, catalog #: SV30010) at 37 °C in 5% CO₂. Cells were grown in 6-well plates or 10 cm dishes and allowed to reach 50–70% confluency before transient transfection using TransIT-2020 (Mirus Bio, catalog #: MIR 5400) according to the manufacturer’s instruction. Briefly, cells were transfected with α1:β2:γ2 (0.25 μg:0.25 μg:0.25 μg), α1­(D219N):β2:γ2 (0.25 μg:0.25 μg:0.25 μg), α1­(G251D):β2:γ2 (0.25 μg:0.25 μg:0.25 μg), or α1­(P260L):β2:γ2 (0.25 μg:0.25 μg:0.25 μg) plasmids for each well of 6-well plates, or α1 WT/variant:β2:γ2 (0.8 μg:0.8 μg:0.8 μg) plasmids for each 10 cm dish. To achieve optimal transfection efficiency without apparent cell toxicity, 2.2 μL of TransIT-2020 reagent per 1 μg of DNA was used. For stable cell line generation, forty-8 h post transfection, cells were selected with G-418 (Enzo Life Sciences, #ALX-380–013, 1 mg/mL) for 10 days. Cells were then maintained in DMEM supplemented with 0.4 mg/mL G418. The G418 resistant polyclonal cells expressing α1, β2, and γ2 subunits of GABA_A receptors, which were verified by Western blot analysis, were used for experiments.

Quantitative Reverse Transcription (RT)-PCR

Cells were treated with compounds for the indicated time before total RNA was extracted using RNeasy Mini Kit (Qiagen #74104). 500 ng of total RNA was used to synthesize cDNA using QuantiTect Reverse Transcription Kit (Qiagen #205311). Quantitative RT-PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems #A25776) with corresponding primers in the QuantStudio 3 Real-Time PCR System (Applied Biosystems) and analyzed using QuantStudio software (Applied Biosystems). Three replicates were performed for each biological sample, and the mRNA expression values were normalized against RPLP2 (as housekeeping gene control). The forward and reverse primers are listed in Table S1.

Western Blot Analysis

Cells were lysed with total lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 2 mM DDM (n-Dodecyl-β-d-maltoside, GoldBio #DDM5)) supplemented with complete protease inhibitor cocktail (Roche #04693116001) and centrifuged at 21,000 g for 10 min at 4 °C to yield supernatant. Protein concentrations were measured by MicroBCA assay (ThermoFisher Pierce #23235). Protein was resuspended in 4× Laemmli buffer (Biorad #1610747) with 10% 2-Mercaptoethanol (Sigma, #M3148), loaded on SDS-PAGE gels, and transferred onto nitrocellulose membranes. Western blot analysis was performed using appropriate antibodies, followed by visualization using Azure Biosystems C600. Band intensity was quantified using ImageJ software from the NIH.

IRE1 Reporter Assay

The IRE1 reporter assay was performed as previously described. Briefly, HEK293^TREX cells stably expressing the XBP1-Rluc splicing reporter were treated with the indicated compounds (10 μM) in the presence or absence of the IRE1 active site inhibitor 4 μ8C (32 μM) for 18 h. Luminescence was measured as a relative XBP 1s amount for the indication of the IRE1 activation.

Coimmunoprecipitation

Cells were lysed with total lysis buffer and incubated with 2.0 μg of mouse anti-α1 antibody (Millipore #MAB339) overnight at 4 °C, and then with 30 μL of protein A/G plus agarose beads (Santa Cruz Biotechnology #sc-2003) overnight at 4 °C. Immunoprecipitates were washed three times with cell lysate buffer and then eluted by incubation with 30 μL of SDS loading buffer in the presence of dithiothreitol (DTT). The immunopurified eluents were loaded in SDS-PAGE gel and analyzed by Western blot analysis.

Biotinylation of Cell Surface Proteins

To label surface membrane protein with biotin, cells were incubated with Sulfo-NHS SS-Biotin (1 mg/mL; APExBIO, #A8005) in ice-cold PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS+CM) for 30 min at 4 °C. Next, to quench the reaction, cells were incubated with 10 mM glycine in ice-cold PBS+CM twice for 5 min at 4 °C, followed by 5 nM N-ethylmaleimide (NEM) incubation for 15 min at room temperature. Cells were solubilized for 6 h at 4 °C in total lysis buffer supplemented with protease inhibitor and 5 mM NEM. The lysates were centrifuged at 21,000 g for 10 min at 4 °C to remove insoluble pellet debris. To isolate biotinylated surface proteins from cell lysate, 500 μg of the above supernatant were purified by incubating with 40 μL of neutravidin-conjugated agarose bead (ThermoFisher Pierce #29201) and rotated for 2 h at 4 °C. The beads were washed 3 times with lysate buffer. Surface proteins were eluted from beads by vortexing for 15 min with 2× Laemmli sample buffer (Biorad #1610737) supplemented with 100 mM DTT and 6 M urea. The biotinylated surface protein was loaded in SDS-PAGE gel and analyzed by Western blot analysis.

PEGylation Assay

Prior to the PEGylation reaction, cells were treated with 5 mM DTT at 37 °C for 10 min. Then cells were lysed in 2x sarcosyl sample buffer (4% sarcosyl (Sigma #L5125), 125 mM Tris, 20% glycerol, 0.01% bromophenol blue, pH 6.8) supplemented with 5 mM methoxy PEG maleimide (Sigma #63187) (molecular weights 5 kDa) at room temperature for 2 h. Then, 100 mM DTT was used to quench the reaction. Immediately after sonication, pegylated cell lysate samples were loaded to SDS-PAGE gel and analyzed by Western blot.

Cycloheximide-Chase Assay

To evaluate the stability of the mutant α1­(D219N) subunit of α1­(D219N)­β2γ2 GABA_A receptors, cells were incubated with the indicated compound for 24 h before cycloheximide-chase assay. Protein synthesis was inhibited by 100 μg/mL cycloheximide treatments (Enzo Life Sciences # ALX-380-269). At five different time points (after 0, 0.5, 1, 2, and 4 h), cells were harvested and lysed for protein analysis.

Endoglycosidase H (Endo H) Enzyme Digestion Assay

Digestions were conducted directly on total cell lysates using Endo H enzyme (NEBiolab #P0702L) with G5 reaction buffer at 37 °C for 1 h, which removed asparaginyl-N-acetyl-d-glucosamine in the N-linked glycans incorporated on the α1 subunit in the ER. Control digests (for unglycosylated α1 subunit) were using Peptide-N-Glycosidase F (PNGase F) (NEBiolab, #P0704S) enzyme to treat total cell lysate. The digests were stopped by 2× Laemmli sample buffer then subjected to Western blot analysis.

iPSC Generation and Differentiation

The heterozygous knockin of the G251D GABRA1 variation (NM_001127644.2­(GABRA1):c.752G > A (p.Gly251Asp); single nucleotide variant: rs1064793933) into apparently healthy male iPSCs (Applied StemCell, catalog #: ASE-9211) was described previously and confirmed by genotyping. The iPSCs were differentiated into GABAergic neurons by the expression of transcription factors ASCL1 and DXL2 using lentivirus transduction according to published procedure. , Three to 4 weeks postdifferentiation, GABAergic neurons were subjected to immunofluorescence staining or in situ Proximity Ligation Assay (PLA) for confocal microscopy.

Confocal Immunofluorescence

Cells were cultured on glass coverslips and fixed for 5 min with 2% formaldehyde on ice for surface staining. After blocking with 10% goat serum at room temperature for 1 h, cells were treated with mouse monoclonal anti-α1 antibody (Millipore #MAB339, 1:500) and rabbit monoclonal anti-Na/K-ATPase antibody (Abcam, #ab76020, 1:500). Cells were then incubated with Alexa 488, or Alexa 568-conjugated secondary antibody (1:1000). Afterward, cells were incubated with Hoechst 33342 (ThermoFisher, #62249) (1 μg/mL) for 3 min to stain the nucleus. The coverslips were then mounted and sealed.

In situ Proximity Ligation Assay (PLA) was performed using Duolink In Situ Detection Reagents Red kit (Sigma, catalog #: DUO92008) according to manufacturer’s instructions, as described previously. The PLA puncta was quantified using the ImageJ software from the NIH with the built-in macro Analyze Particles. Images were smoothed; a threshold was selected manually to account for PLA puncta from background fluorescence, and such a threshold was applied to all images in the sample set. Objects larger than 5 μm² were excluded, such as nuclei, and the built-in macro Analyze Particles was used to count PLA puncta per cell.

An Olympus IX-81 Fluoview FV1000 confocal laser scanning system with 60X oil objective was used for confocal images. Quantification of the fluorescence intensity was done using the ImageJ software from the NIH.

Total Internal Reflection Fluorescence (TIRF) Imaging

Cells were plated in a T25 flask at day-1 and transiently transfected with α1­(D219N)-dendra2/α1­(G251D)-dendra2, β2 and γ2 subunits on the following day. After 16 h of incubation, the transfected cells were dissociated with Trypsin, and 5 × 10⁴ cells were plated on a 35 mm Matrigel-coated glass bottom dish in growth media. Cells were again incubated for an additional 8 h, and then drug treatment was performed. Dendra2 was first excited with a 488 nm laser to visualize and identify cells expressing the protein of interest. A 405 nm laser in a TIRF orientation was then used to photoconvert protein within a narrow region (∼100 nm) just above the glass substrate. A 561 nm laser focused through a high numerical aperture objective (Olympus, 60×, 1.49 NA) in a TIRF orientation was used to visualize the photoconverted population. Live cell time-course imaging was then conducted in a TIRF setup with stage incubation chamber to maintain temperature, humidity, and CO₂ level. The TIRF setup is capable of detecting plasma membrane proteins and minimizes the fluorescence background from intracellular components. Quantification of the fluorescence intensity was done using the ImageJ software from the NIH.

Autopatch

HEK293T cells expressing GABA_A receptors were recorded in whole-cell patch clamp configuration using an IonFlux 16 system (Fluxion, California, USA). Cells grown in 10 cm dishes were detached with accutase (Sigma-Aldrich, #A6964) at 37 °C for 3 min. Then cells were collected and resuspended in serum free medium HEK293 SFM II (Gibco, #11686-029) supplemented with 25 mM HEPES (Gibco, #15630-080) and 1% penicillin–streptomycin (Fisher Scientific, catalog #: SV30010). Cells were then maintained at room temperature for 30 min to 1 h with gentle shaking. Before recording, cells were resuspended in an extracellular solution containing: 142 mM NaCl, 8 mM KCl, 6 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM HEPES. Electrophysiological recordings were carried out in a 96-well Ion-Flux microfluidic plate following manufacturer suggestions. Briefly, this automated patch clamp platform would capture cells in 20 individual microchannels for cell voltage clamp in parallel. Individual cells will be sucked in each channel and cellular membrane will be broke to obtain the whole-cell configuration. GABA_A receptors were evoked by a gradient concentration of GABA at −60 mV holding potential. The intracellular solution composition for whole-cell current recordings contains 153 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES.

Quantification and Statistical Analysis

All data are presented as mean ± SEM. Statistical significance was evaluated using two-tailed Student’s t-test if two groups were compared and one-way ANOVA followed by posthoc Tukey test if more than two groups were compared. A p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.5c00227.

• Effect of IRE1/XBP1s activators IXA62, IXA554, and IXA105 on IRE1 and PERK pathways (Figure S1) and wild-type GABA_ARs (Figure S2); effect of IRE1/XBP1s activators IXA62 and IXA554 on ERAD factors (Figure S3); effect of IRE1/XBP1s activators IXA62, IXA554, and IXA105 on total and surface protein levels of pathogenic α1 variants (Figure S4); uncropped Western blot images (Figure S5); and list of qPCR primers (Table S1) (PDF)

Conceptualization, T.M.; data curation: X.F., Y.W., K.L., L.A., X.C., B.H., M.W., H.S., P.Z., and A.G.; formal analysis: X.F., Y.W., K.L., and T.M.; funding acquisition: A.S., C.R., R.L.W., J.W.K., and T.M.; supervision: R.L.W., J.W.K., and T.M.; writingoriginal draft: X.F., Y.W., K.L., R.L.W., J.W.K., and T.M.; and writingreview and editing: all.

The authors declare the following competing financial interest(s): RLW and JWK are inventors on patents describing IRE1 activator compounds. RLW and JWK are also shareholders and scientific advisory board members of Protego Biopharma who have licensed the IRE1 activators. TM is a member of the scientific advisory board of Cure GABA-A Variants Foundation, a nonprofit organization.