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Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

  • Ya-Juan Wang
    Correspondence
    For correspondence: Ting-Wei Mu; Ya-Juan Wang
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA

    Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
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  • Xiao-Jing Di
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
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  • Ting-Wei Mu
    Correspondence
    For correspondence: Ting-Wei Mu; Ya-Juan Wang
    Affiliations
    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
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Open AccessPublished:August 28, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102423
      Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

      Keywords

      Abbreviations:

      ABC (ammonium bicarbonate), ACN (acetonitrile), CANX (calnexin), CHX (cycloheximide), DAVID (Database for Annotation, Visualization and Integrated Discovery), DMEM (Dulbecco’s modified Eagle's medium), ER (endoplasmic reticulum), ERAD (ER-associated degradation), EV (empty vector), FA (formic acid), FBS (fetal bovine serum), FDR (false discovery rate), GABAA (gamma-aminobutyric acid type A), GO (Gene Ontology), HEK293T (human embryonic kidney 293T cell line), IP–MS/MS (immunoprecipitation–tandem mass spectrometry), MS (mass spectrometry), QC (quality control), RCN2 (reticulocalbin 2), SILAC (stable isotope labeling by amino acids in cell culture), SRP (signal recognition particle), TM (transmembrane), TRIM21 (tripartite motif containing-21), UGGT1 (UDP-glucose glycoprotein glucosyltransferase 1)
      Normal organismal physiology depends on the maintenance of protein homeostasis (proteostasis) in each cellular compartment (
      • Hartl F.U.
      • Bracher A.
      • Hayer-Hartl M.
      Molecular chaperones in protein folding and proteostasis.
      ,
      • Balch W.E.
      • Morimoto R.I.
      • Dillin A.
      • Kelly J.W.
      Adapting proteostasis for disease intervention.
      ,
      • Hetz C.
      Adapting the proteostasis capacity to sustain brain healthspan.
      ,
      • Powers E.T.
      • Gierasch L.M.
      The proteome folding problem and cellular proteostasis.
      ), which dictates a delicate balance between protein synthesis, folding, assembly, trafficking, and degradation while minimizing misfolding and aggregation (
      • Bukau B.
      • Weissman J.
      • Horwich A.
      Molecular chaperones and protein quality control.
      ,
      • Hetz C.
      • Zhang K.
      • Kaufman R.J.
      Mechanisms, regulation and functions of the unfolded protein response.
      ,
      • Needham P.G.
      • Guerriero C.J.
      • Brodsky J.L.
      Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.
      ). For one specific client protein, its interaction with a network of proteins, especially its proteostasis network components, in the crowded cellular environment is critical to maintain its proteostasis. However, how the proteostasis network orchestrates the biogenesis of multisubunit multispan ion channel proteins is poorly understood. The current limited knowledge about such protein quality control (QC) machinery is gained from the study of various classes of membrane proteins, including cystic fibrosis transmembrane (TM) conductance regulator (
      • Peters K.W.
      • Okiyoneda T.
      • Balch W.E.
      • Braakman I.
      • Brodsky J.L.
      • Guggino W.B.
      • et al.
      CFTR folding consortium: methods available for studies of CFTR folding and correction.
      ), T-cell receptors (
      • Feige M.J.
      • Behnke J.
      • Mittag T.
      • Hendershot L.M.
      Dimerization-dependent folding underlies assembly control of the clonotypic αβT cell receptor chains.
      ), sodium channels (
      • Buck T.M.
      • Jordahl A.S.
      • Yates M.E.
      • Preston G.M.
      • Cook E.
      • Kleyman T.R.
      • et al.
      Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.
      ), potassium channels (
      • Delaney E.
      • Khanna P.
      • Tu L.
      • Robinson J.M.
      • Deutsch C.
      Determinants of pore folding in potassium channel biogenesis.
      ), and nicotinic acetylcholine receptors (
      • Kweon H.J.
      • Gu S.
      • Witham E.
      • Dhara M.
      • Yu H.
      • Mandon E.D.
      • et al.
      NACHO engages N-glycosylation ER chaperone pathways for α7 nicotinic receptor assembly.
      ). We have been using gamma-aminobutyric acid type A (GABAA) receptors as an important membrane protein substrate to clarify its biogenesis pathway (
      • Fu Y.L.
      • Wang Y.J.
      • Mu T.W.
      Proteostasis maintenance of cys-loop receptors.
      ), which is currently understudied.
      GABAA receptors are the primary inhibitory neurotransmitter-gated ion channels in mammalian central nervous systems (
      • Macdonald R.L.
      • Olsen R.W.
      GABA(A) receptor channels.
      ) and provide most of the inhibitory tone to balance the tendency of excitatory neural circuits to induce hyperexcitability, thus maintaining the excitatory–inhibitory balance (
      • Akerman C.J.
      • Cline H.T.
      Refining the roles of GABAergic signaling during neural circuit formation.
      ). There are 19 known GABAA receptor subunits in mammals, including α1–6, β1–3, γ1–3, θ, ε, π, δ, and ρ1–3. Loss of function of GABAA receptors is a prominent cause of genetic epilepsies, and recent advances in genetics have identified a growing number of epilepsy-associated variants in over ten genes that encode the subunits of GABAA receptors, including over 150 variants in those encoding major synaptic subunits (α1, β2, and γ2 subunits) (Fig. 1A, top right figure) (
      • Fu X.
      • Wang Y.J.
      • Kang J.Q.
      • Mu T.W.
      GABAA Receptor Variants in Epilepsy.
      ,
      • Hernandez C.C.
      • Macdonald R.L.
      A structural look at GABA(A) receptor mutations linked to epilepsy syndromes.
      ,
      • Hirose S.
      Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.
      ,
      • Noebels J.L.
      The biology of epilepsy genes.
      ,
      • Steinlein O.K.
      ion channel mutations in neuronal diseases: a genetics perspective.
      ). GABAA receptors belong to the Cys-loop superfamily of ligand-gated ion channels, sharing common structural characteristics with other Cys-loop receptor members (
      • Dougherty D.A.
      Cys-loop neuroreceptors: structure to the rescue?.
      ). A functional GABAA receptor is composed of five subunits. Each subunit has a large extracellular (or the endoplasmic reticulum [ER] luminal) N terminus, four TM helices (TM1–TM4, with TM2 domain lining the interior of the pore), and a short extracellular (or the ER luminal) C terminus (Fig. 1A) (
      • Laverty D.
      • Desai R.
      • Uchański T.
      • Masiulis S.
      • Stec W.J.
      • Malinauskas T.
      • et al.
      Cryo-EM structure of the human α1β3γ2 GABA(A) receptor in a lipid bilayer.
      ,
      • Zhu S.
      • Noviello C.M.
      • Teng J.
      • Walsh Jr., R.M.
      • Kim J.J.
      • Hibbs R.E.
      Structure of a human synaptic GABAA receptor.
      ). GABA binding to GABAA receptors in the extracellular domain induces conformational changes, opens the ion pore to conduct chloride, hyperpolarizes the plasma membrane, and inhibits neuronal firing in mature neurons.
      Figure thumbnail gr1
      Figure 1Quantitative immunoprecipitation–tandem mass spectrometry (IP–MS/MS) analysis to identify GABAA receptor interactomes. A, GABAA receptor biogenesis pathways. Individual subunits of GABAA receptors fold in the endoplasmic reticulum (ER). Properly folded subunits assemble into a heteropentamer in the ER for subsequent trafficking to the plasma membrane. Unassembled and misfolded subunits are subjected to the ER-associated degradation (ERAD) pathway by the proteasome or the lysosome-related degradation. B, outline of a comparative SILAC-based quantitative proteomics approach to identify GABAA receptor interactomes in HEK293T cells. The α1(A322D) variant leads to its excessive misfolding and ERAD. C and D, HEK293T cells expressing empty vector (EV), WT, or α1(A322D)β2γ2 GABAA receptors were subjected to coimmunoprecipitation using an anti-GABAA receptor α1 subunit antibody. The immunoisolated complexes were separated by SDS-PAGE and visualized by Coomassie blue staining (C) or Western blot analysis (D). Both heavy chain and light chain bands were detected in Coomassie blue gels, whereas only light chain bands were detected in Western blot because of the usage of a light chain–specific secondary antibody for the detection. GABAA, gamma-aminobutyric acid type A; HEK293T, human embryonic kidney 293T cell line; IB, immunoblotting; IP, immunoprecipitation; SILAC, stable isotope labeling by amino acids in cell culture.
      To function properly, GABAA receptors need to fold into their native structures and assemble correctly to form a pentamer on the ER membrane and traffic efficiently through the Golgi en route to the plasma membrane (Fig. 1A). Misfolded GABAA receptors are recognized by the cellular protein QC machinery. ER-associated degradation (ERAD) is one major cellular pathway to target misfolded GABAA receptors to the cytosolic proteasome for degradation (
      • Needham P.G.
      • Guerriero C.J.
      • Brodsky J.L.
      Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.
      ,
      • Olzmann J.A.
      • Kopito R.R.
      • Christianson J.C.
      The mammalian endoplasmic reticulum-associated degradation system.
      ,
      • Di X.J.
      • Wang Y.J.
      • Han D.Y.
      • Fu Y.L.
      • Duerfeldt A.S.
      • Blagg B.S.
      • et al.
      Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.
      ,
      • Gallagher M.J.
      • Shen W.Z.
      • Song L.Y.
      • Macdonald R.L.
      Endoplasmic reticulum retention and associated degradation of a GABA(A) receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha 1 subunit.
      ,
      • Poliquin S.
      • Kang J.Q.
      Disruption of the ubiquitin-proteasome system and elevated endoplasmic reticulum stress in epilepsy.
      ). Another potential degradation pathway is to target the aggregation-prone GABAA receptors to the lysosome through autophagy, ER-phagy, or ER-to-lysosome-associated degradation (
      • Fregno I.
      • Molinari M.
      Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways.
      ,
      • Wilkinson S.
      ER-Phagy: shaping up and destressing the endoplasmic reticulum.
      ,
      • Arancibia-Cárcamo I.L.
      • Yuen E.Y.
      • Muir J.
      • Lumb M.J.
      • Michels G.
      • Saliba R.S.
      • et al.
      Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.
      ,
      • Davis K.M.
      • Sturt B.L.
      • Friedmann A.J.
      • Richmond J.E.
      • Bessereau J.L.
      • Grant B.D.
      • et al.
      Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.
      ). Maintenance of a delicate balance between GABAA receptor folding, trafficking, and degradation utilizing its interacting proteins is critical for its function. However, the GABAA receptor interactome, especially the proteostasis network that orchestrates GABAA receptor biogenesis in the ER, has not been studied systematically in the literature despite recent advances about the trafficking of GABAA receptors beyond the ER (
      • Ge Y.
      • Kang Y.
      • Cassidy R.M.
      • Moon K.M.
      • Lewis R.
      • Wong R.O.L.
      • et al.
      Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.
      ,
      • Han W.
      • Li J.
      • Pelkey K.A.
      • Pandey S.
      • Chen X.
      • Wang Y.X.
      • et al.
      Shisa7 is a GABA(A) receptor auxiliary subunit controlling benzodiazepine actions.
      ,
      • Han W.
      • Shepard R.D.
      • Lu W.
      Regulation of GABA(A)Rs by transmembrane accessory proteins.
      ,
      • Lorenz-Guertin J.M.
      • Jacob T.C.
      GABA type a receptor trafficking and the architecture of synaptic inhibition.
      ,
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ,
      • Heller E.A.
      • Zhang W.
      • Selimi F.
      • Earnheart J.C.
      • Ślimak M.A.
      • Santos-Torres J.
      • et al.
      The biochemical anatomy of cortical inhibitory synapses.
      ). Here, we used quantitative immunoprecipitation–tandem mass spectrometry (IP–MS/MS) analysis utilizing stable isotope labeling by amino acids in cell culture (SILAC) in human embryonic kidney 293T (HEK293T) cells to identify the interactomes for both WT and a misfolding-prone GABAA receptor. Endogenous interactions between selected interactors and GABAA receptors were verified in mouse brain homogenates. Furthermore, bioinformatics analysis enabled us to assemble a proteostasis network model for the cellular folding, assembly, degradation, and trafficking pathways of GABAA receptors.

      Results

      Identification of interactomes for WT and misfolding-prone GABAA receptors using comparative SILAC-based proteomics

      We employed a proteomics-based approach to identify the interactomes of GABAA receptors in HEK293T cells by carrying out a quantitative IP–MS/MS analysis utilizing SILAC (Fig. 1B) (
      • Mann M.
      Functional and quantitative proteomics using SILAC.
      ). Since HEK293T cells do not own endogenous GABAA receptors, precise control of the subtypes and variants of GABAA receptors can be achieved by exogenously expressing their subunits (
      • Thomas P.
      • Smart T.G.
      HEK293 cell line: a vehicle for the expression of recombinant proteins.
      ). Furthermore, we performed comparative proteomics by using both WT α1 subunit and a well-characterized misfolding-prone α1 subunit carrying the A322D variant as the bait proteins to determine the potential difference between them (
      • Gallagher M.J.
      • Ding L.
      • Maheshwari A.
      • Macdonald R.L.
      The GABA(A) receptor alpha 1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation.
      ). The A322D variant introduces an extra negative charge in the third TM (TM3) helix of the α1 subunit, causing its substantial misfolding and excessive degradation by ERAD (
      • Gallagher M.J.
      • Ding L.
      • Maheshwari A.
      • Macdonald R.L.
      The GABA(A) receptor alpha 1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation.
      ,
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ). HEK293T cells stably expressing either WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were labeled with heavy media, whereas HEK293T cells that were transfected with empty vector (EV) plasmids were cultured in normal light media. The same amount of light and heavy cell lysates was mixed. The α1 or α1(A322D) complexes were immunoprecipitated using a monoclonal antibody against the N-terminal region of the α1 subunit before being subjected to SDS-PAGE and in-gel digestion and tandem MS analysis. Coomassie blue–stained gels showed numerous clearly visible bands in samples expressing GABAA receptors (Fig. 1C, lanes 2 and 3), which represented potential interacting proteins for α1 subunit–containing GABAA receptors, indicating efficient coimmunoprecipitations. In addition, Western blot analysis detected α1 subunits effectively at ∼50 kDa postimmunoprecipitation in samples expressing GABAA receptors (Fig. 1D, lanes 2 and 3), whereas no α1 band was observed in the EV control sample (Fig. 1D, lane 1), supporting the efficient isolation of the α1 subunit–containing GABAA receptor complexes.
      The α1 subunit–containing GABAA receptor interactomes were identified using the SILAC ratio with arbitrary yet strict criteria to remove potential false positives. To be included as an interactor, it must (1) have a SILAC ratio of WT α1/EV or α1(A322D)/EV to be at least 1.30; (2) have a p < 0.05; and (3) have a Benjamini and Hochberg correction (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the false discovery rate - a practical and powerful approach to multiple testing.
      ) of false discovery rate (FDR) of no more than 0.10. The top right green area in Figure 2, A contains high-confidence interactors for GABAA receptors. As a result of the stringent criteria, the WT α1-containing GABAA receptor interactome contains 125 proteins, the α1(A322D)-containing GABAA receptor interactome contains 105 proteins, and 54 proteins overlap within two interactomes (Fig. 2B). These 176 interactors for GABAA receptors are used for the following bioinformatics analysis (see Table S1 for protein list).
      Figure thumbnail gr2
      Figure 2Bioinformatics analysis of GABAA receptor interactomes. A, a 2D plot showing the relationship between p value and SILAC ratio for proteins that were identified from tandem MS analysis. Proteins that are in the WT samples are colored in blue; those in α1(A322D) variant–containing GABAA receptor samples, in red. The vertical black line represents a SILAC ratio of 1.30, whereas the horizontal black line represents a p value of 0.05. B, a Venn diagram showing the protein numbers and overlap of the GABAA receptor interactomes. C, a heat map showing the protein abundance for GABAA receptor interactors in HEK293 cells and human brain tissues. D, a heat map showing the RNA abundance for GABAA receptor interactors in HEK293 cells, SH-SY5Y cells, and human brain tissues. E, analysis of cellular components for GABAA receptor interactomes. F, analysis of cellular components for GABAA receptor interactors that are integral membrane proteins. G, a pie chart showing the biological processes of the pooled 176 GABAA receptor interactors. Enriched biological processes for WT α1-containing GABAA receptor interactome (H) and α1(A322D)-containing GABAA receptor interactome (I) according to DAVID analysis. DAVID, Database for Annotation, Visualization and Integrated Discovery; FDR, false discovery rate. Fpkm, fragment per kilobase of exon per million mapped fragment; GABAA, gamma-aminobutyric acid type A; HEK293, human embryonic kidney 293 cell line; MS, mass spectrometry; SILAC, stable isotope labeling by amino acids in cell culture.

      Comparison with previous GABAA receptor proteomic analyses

      Since GABAA receptors inhibit neuronal firing in the mammalian central nervous system, we first compared the expression abundance of their interactors between HEK293 cells and the nervous system. ProteomicsDB (https://www.proteomicsdb.org/) provides an MS-based navigation of the human proteome from tissues, cell lines, and body fluids, enabling the comprehensive mapping of the protein abundance of GABAA receptor interactors (
      • Wilhelm M.
      • Schlegl J.
      • Hahne H.
      • Gholami A.M.
      • Lieberenz M.
      • Savitski M.M.
      • et al.
      Mass-spectrometry-based draft of the human proteome.
      ). Hierarchical clustering analysis showed that these interactors have comparable protein levels between HEK293 cells and human brain tissues (Fig. 2C and Table S2). For example, many molecular chaperones, such as Hsp90s (Hsp90AA1, Hsp90AB1, and Hsp90B1), Hsp70s (HspA5 and HspA8), Hsp40s (DNAJA1, DNAJA2, and DNAJB11), calnexin (CANX), and Hsp47 (SerpinH1), are abundantly expressed in both systems. In addition, tissue-based map of the human proteome based on quantitative transcriptomics (https://www.proteinatlas.org/) enabled us to compare RNA levels of GABAA receptor interactors between HEK293 cells, a human neuronal SH-SY5Y cell line, and human brain tissues (
      • Sjöstedt E.
      • Zhong W.
      • Fagerberg L.
      • Karlsson M.
      • Mitsios N.
      • Adori C.
      • et al.
      An atlas of the protein-coding genes in the human, pig, and mouse brain.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ). Hierarchical clustering analysis showed that many interactors, including molecular chaperones and ubiquitin-dependent degradation factors, such as UBA1, UBR5, UBE3C, SEL1L, and VCP, have similar RNA expression patterns between these systems (Fig. 2D and Table S2). These results are consistent with the report that most proteins are well conserved among human tissues (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ).
      Previously, proteomic analyses were carried out to identify the interacting proteins for GABAA receptor subunits using knockin mice carrying Venus (a GFP variant)-tagged α1 subunit (
      • Heller E.A.
      • Zhang W.
      • Selimi F.
      • Earnheart J.C.
      • Ślimak M.A.
      • Santos-Torres J.
      • et al.
      The biochemical anatomy of cortical inhibitory synapses.
      ), pHluorin (a pH-sensitive GFP variant)-Myc-tagged α2 subunit (
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ), or His6-FLAG-YFP-tagged γ2 subunit (
      • Ge Y.
      • Kang Y.
      • Cassidy R.M.
      • Moon K.M.
      • Lewis R.
      • Wong R.O.L.
      • et al.
      Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.
      ). GFP-affinity purification from the cerebral cortex using Venus-tagged α1 subunits as bait led to the identification of 18 proteins in the inhibitory synaptic complexes, including 11 GABAA receptor subunits, five scaffolding and adhesion proteins (gephrin [GPHN], neuroligin 2 [NLGN2], neuroligin 3 [NLGN3], collybistin [ARHGEF9], and neurexin 1 [NRXN1]), and two proteins with less known functions (neurobeachin [NBEA] and LHFPL4) (
      • Heller E.A.
      • Zhang W.
      • Selimi F.
      • Earnheart J.C.
      • Ślimak M.A.
      • Santos-Torres J.
      • et al.
      The biochemical anatomy of cortical inhibitory synapses.
      ). However, because of the use of size-exclusion chromatography during the protein complex purification process, proteins that are potentially involved in the cellular folding and trafficking of GABAA receptors were not well identified.
      GFP-trap purification from hippocampus and cortex using pHluorin-Myc-tagged α2 subunits as bait resulted in the identification of 174 proteins, including 14 GABAA receptor subunits, known GABAA receptor interactors such as gephrin, neuroligins, and collybistin, and 149 novel binding partners (
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ). The novel interactors were further categorized into five groups, including G protein–coupled receptors, ion channels, and transporters (26 interactors), factors that regulate protein trafficking, stability, and cytoskeletal anchoring (38 interactors), factors that regulate phosphorylation and GTP exchange (26 interactors), miscellaneous enzymes (27 interactors), and miscellaneous proteins (32 interactors) (
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ). Comparison between the α2 subunit–containing GABAA receptor interactome and our α1 subunit–containing GABAA receptor interactome showed overlapping interactors, including factors that regulate protein folding and degradation (DNAJA1, RPN2, SQSTM1, USP9X, and DDB1), transporters (SLC25A3, SLC25A4, and SLC25A5), and miscellaneous proteins (PHB2 and IMMT).
      Furthermore, tandem affinity purification from whole brain homogenates using His6-FLAG-YFP-tagged γ2 subunits as bait gave rise to the identification of 11 known associated proteins (GABAA receptor subunits, gephrin and neuroligin 2) and 39 novel binding partners (
      • Ge Y.
      • Kang Y.
      • Cassidy R.M.
      • Moon K.M.
      • Lewis R.
      • Wong R.O.L.
      • et al.
      Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.
      ). The novel interactors were further classified into five groups, including ion channels (two interactors), factors that regulate protein folding and trafficking (six interactors), mitochondria proteins (six interactors), miscellaneous enzymes (seven interactors), and miscellaneous proteins (18 interactors). Comparison between the γ2 subunit–containing GABAA receptor interactome and our α1 subunit–containing GABAA receptor interactome showed overlapping interactors, including a factor that regulate protein degradation (PSMC2), an enzyme (PTPLAD1), and a miscellaneous protein (EMD).
      Among the three GABAA receptor proteomic analyses using knockin mice, six synaptic proteins were recognized from at least two studies, including gephrin, neuroligin 2, neuroligin 3, collybistin, neurobeachin, and LHFPL4. However, such synaptic proteins were not identified in our α1 subunit–containing GABAA receptor interactome possibly because of their lack of expression in HEK293T cells. The only other overlapping interactor from at least two knockin mice proteomic studies is CACNA1E, the α1E subunit of R-type voltage-dependent calcium channels, which could indicate that different subunits utilize differentiating cellular interaction networks. Moreover, the limited interactome overlapping from these proteomic analyses could arise from using different purification procedures to isolate the GABAA receptor–containing complex, such as using different detergents. Nonetheless, in addition to certain GABAA receptor subunits, our α1 subunit–containing GABAA receptor interactome showed 13 overlapping interactors with one other proteomic study, providing promising candidates for future investigation.

      Gene Ontology analysis of GABAA receptor interactomes

      To annotate cellular component for GABAA receptor interactors, we used Database for Annotation, Visualization and Integrated Discovery (DAVID) to carry out Gene Ontology (GO) analysis (
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
      ,
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.
      ). Since many proteins reside in more than one cellular location, we only choose one primary subcellular location for them manually. To aid such an assignment, we also integrate subcellular location information from UniProt Database (https://www.uniprot.org/) and GeneCards: The Human Gene Database (https://www.genecards.org/). GABAA receptor interactors are distributed in various cellular locations, including the nucleus (33 interactors), ER (25 interactors), Golgi (2 interactors), proteasome (4 interactors), lysosome (1 interactor), mitochondria (21 interactors), cytoskeleton (23 interactors), cytosol (59 interactors), plasma membrane (5 interactors), and extracellular space (3 interactors) since biogenesis and function of GABAA receptors require their interactions with a network of proteins throughout the cell (Fig. 2E and Table S3). Because of the essential role of the ER in protein QC, 25 interactors (14 for WT α1-containing GABAA receptor only, three for α1(A322D) variant–containing GABAA receptor only, and eight for both) are located to this organelle. Eight overlapping interactors in the ER include molecular chaperones (CANX, HspA5, DNAJB11, Hsp90B1, and SerpinH1), factors involved in N-linked glycosylation (RPN1 and DPM1), and a Ca2+-binding protein (reticulocalbin 2 [RCN2]). RCN2 (aka ERC55) encodes RCN2 in the ER lumen; it is abundantly expressed in the brain and contains EF-hand Ca2+-binding motifs (
      • Weis K.
      • Griffiths G.
      • Lamond A.I.
      The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif.
      ). Function of RCN2 is largely unknown, although gene expression of RCN2 is upregulated in patients with idiopathic absence epilepsies (
      • Helbig I.
      • Matigian N.A.
      • Vadlamudi L.
      • Lawrence K.M.
      • Bayly M.A.
      • Bain S.M.
      • et al.
      Gene expression analysis in absence epilepsy using a monozygotic twin design.
      ). Given the critical role of GABAA receptors in the pathophysiology of epilepsy, it will be of great interest to determine how RCN2 regulates GABAA receptor biogenesis and contributes to epilepsy phenotypes in the future.
      Furthermore, since GABAA receptors are TM proteins, we extracted their interactors that are integral membrane proteins, including 13 in the ER, seven in the mitochondria (SLC25A3, SLC25A4, SLC25A5, SLC25A6, SLC25A10, IMMT, and TIMM50), two in the nucleus (EMD and LBR), and one in the plasma membrane (ATP1A1) (Fig. 2F and Table S3). ATP1A1 encodes α1 subunit of sodium/potassium-transporting ATPase in the plasma membrane. Variants in ATP1A1 cause hypomagnesemia, seizures, and mental retardation 2, an autosomal dominant disease characterized by generalized seizures in infancy and significant intellectual disability (
      • Schlingmann K.P.
      • Bandulik S.
      • Mammen C.
      • Tarailo-Graovac M.
      • Holm R.
      • Baumann M.
      • et al.
      Germline de novo mutations in ATP1A1 cause renal hypomagnesemia, refractory seizures, and intellectual disability.
      ). The 13 membrane interactors in the ER include molecular chaperones and folding enzymes (CANX, DDOST, RPN1, and RPN2), trafficking factors (SSR1 and SEC16A), ERAD factors (ERLIN2 and SEL1L), transporters (ATP2A2 and SLC39A7), enzymes involved in the lipid metabolism (TECR and PTPLAD1), and other (MTDH). Since each subunit of GABAA receptors has four TM helices, these ER membrane interactors have the potential to form intramembrane interactions with GABAA receptors to regulate their biogenesis in the ER, which needs to be explored in the future. Since the extensive dynamic ER network can form membrane contact sites with a variety of organelles, including the mitochondria (
      • Wu H.
      • Carvalho P.
      • Voeltz G.K.
      Here, there, and everywhere: the importance of ER membrane contact sites.
      ), seven mitochondria membrane interactors were identified for GABAA receptors. Interestingly, SLC25A3, SLC25A4, SLC25A5, and IMMT were also identified from a previous proteomic study using the pHluorin-Myc-tagged α2 subunit as a bait protein in the knockin mice (
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ). Although these seven mitochondria membrane interactors are annotated as inner membrane proteins, they could either utilize other adapter proteins or own cytosolic components to be involved in the ER–mitochondria interactions.
      Moreover, we used DAVID, UniProt, and GeneCards to annotate biological process for GABAA receptor interactors. The 176 interactors were annotated to the following functional categories: ion transport (4%), organic substance transport (3.4%), cytoskeleton organization (13.1%), protein complex oligomerization (1.1%), protein synthesis (16.5%), protein folding (15.3%), protein transport (8.5%), proteolysis (13.6%), signal transduction (7.4%), cellular metabolic process (6.8%), and miscellaneous (10.2%) (Fig. 2G and Table S4). Furthermore, we used DAVID to determine enriched biological process for GABAA receptor interactors. Both WT α1-containing GABAA receptor interactors and α1(A322D)-containing GABAA receptor interactors form five functional clusters (Table S5). Top enriched biological processes from those clusters are plotted in Figure 2, H and I according to FDR. Protein refolding (GO: 0042026), response to unfolded protein (GO: 0006986), and translation (GO: 0006412) are common enriched biological processes for both WT and α1(A322D) interactomes. Since the A322D variant causes extensive protein misfolding and ERAD of the α1 subunit, the α1(A322D)-containing GABAA receptor interactome enriches proteasome-mediated ubiquitin-dependent protein catabolic process (GO: 0043161) and protein polyubiquitination (GO: 0000209).

      Mapping of the proteostasis network for GABAA receptors

      We focused on identifying the proteostasis network for GABAA receptors, which regulates their folding, assembly, trafficking, and degradation. Based on GO analysis and literature knowledge, we assigned the interactors to proteostasis network categories, including protein folding (GO: 0006457), proteolysis (GO: 0006508), and protein transport (GO: 0015031) (Table S6). The proteostasis network components account for 37.4% of the total interactors, indicating their critical role in regulating GABAA receptor function. The number of interactors that belong to the proteostasis network was plotted for WT α1-containing GABAA receptor interactome, α1(A322D)-containing GABAA receptor interactome, and overlapping interactome (Fig. 3A). In addition, we visualized the proteostasis network for WT and α1(A322D) variant–containing receptors in a 2D plot showing their SILAC ratios (Fig. 3B and Table S1).
      Figure thumbnail gr3
      Figure 3Proteome profiling of proteostasis network for WT and misfolding-prone α1(A322D)-containing GABAA receptors. A, Gene Ontology analysis of the proteostasis network for GABAA receptor interactomes. Number of interactors is plotted against proteolysis, protein transport, and protein folding. B and C, a 2D plot showing SILAC ratios of the identified interactors. The x- and y-axes show log2(SILAC ratio of WT α1/EV) and log2(SILAC ratio of α1(A322D)/EV). For display purpose, if the SILAC ratio is greater than 4.0, it is displayed as 4.0; if a protein is only detected in one sample, the SILAC ratio of this protein in the other sample is artificially set to 1.0. Blue solid lines represent a cutoff SILAC ratio of 1.30 to be considered as interactors for WT α1-containing GABAA receptors or the α1(A322D) variant–containing GABAA receptor (B). Blue dashed lines represent a cutoff SILAC ratio of 1.30 to be considered as interactors that preferentially bind WT or α1(A322D) variant–containing GABAA receptor (C). Based on literature knowledge, interactors that are expected to play a role in the folding and assembly of GABAA receptors are colored in green; those in their degradation, in orange; those in their transport, in purple; and those with unknown roles in proteostasis maintenance, in black. EV, empty vector; GABAA, gamma-aminobutyric acid type A; SILAC, stable isotope labeling by amino acids in cell culture.
      Judging from the literature knowledge, interactors that are expected to regulate the folding and assembly of α1 subunit–containing GABAA receptors are indicated in green, those for protein transport in purple, and those for proteolysis in orange (Fig. 3B). Major chaperone networks and folding enzymes were identified from GABAA receptor interactomes. These include Hsp70s and their cochaperone Hsp40s in the ER (HspA5 and DNAJB11) and in the cytosol (HspA6, HspA8, HspA1A, DNAJA1, DNAJA2, and DNAJA3), Hsp90s and their cochaperones in the ER (Hsp90B1) and in the cytosol (Hsp90AB1, AHSA1, and UNC45A), Hsp60 subunits (CCT2, CCT5, and CCT7), and a protein disulfide isomerase (P4HB). Moreover, since GABAA receptors have several N-linked glycosylation sites, proteins that are involved in the N-linked glycoprotein maturation in the ER were identified, including glycosylation enzymes (DDOST, RPN1, RPN2, DPM1, GANAB, and UDP-glucose glycoprotein glucosyltransferase 1 [UGGT1]) and a lectin chaperone (CANX). Interactors that are involved in protein transport include translocon-related proteins (SSR1 and signal recognition particle 68 [SRP68]), a COPII subunit (SEC16A), COPI subunits (COPA and COPG2), and an endocytosis-related protein (CLINT1). In addition, we identified numerous proteolysis-related interactors. These include ERAD factors, including a number of ubiquitin E3 ligase complexes (HUWE1, TRIM21 [tripartite motif containing-21], UBR5, LTN1, UBE3C, CAND1, KLHL22, and DDB1), an ubiquitin E1-activating enzyme (UBA1), a deubiquitination enzyme (USP9X), retrotranslocation proteins (SEL1L and VCP), and other factors (ERLIN2).

      Comparison between interactomes of WT and misfolding-prone α1(A322D) variant–containing GABAA receptors

      Our proteomic analysis identified 125 interactors for WT GABAA receptors and 105 interactors for misfolding-prone α1(A322D)-containing receptors, and 54 proteins overlap within these two interactomes (Fig. 2B and Table S1). On one hand, WT and the variant share 54 interactors (Fig. 3B, top right area) to regulate the folding, assembly, degradation, and trafficking of GABAA receptors during their biogenesis in cells. Such common interactors include major chaperones and their cochaperones, such as HspA5 and DNAJB11, and CANX. On the other hand, WT GABAA receptors have 71 unique interactors (Fig. 3B, bottom right area), whereas the α1(A322D)-containing receptors have 51 unique interactors (Fig. 3B, top left area), indicating that WT and the variant also utilize distinct proteostasis networks for their biogenesis. Therefore, it is feasible to target the distinct interactors to adapt the proteostasis network to achieve selectivity for the misfolding-prone variant. For example, several ERAD factors, such as VCP and TRIM21, were recognized in the top left area in the 2D plot (Fig. 3B) since the α1(A322D) protein undergoes accelerated ERAD (also see later for the selective effect of TRIM21 on the α1(A322D) degradation).
      Furthermore, we defined (1) if an interactor has a SILAC ratio of α1(A322D)/WT α1 of at least 1.30, this interactor preferentially binds α1(A322D) variant–containing receptors and (2) if an interactor has a SILAC ratio of WT α1/α1(A322D) of at least 1.30, this interactor preferentially binds WT α1-containing receptors. Accordingly, 13 interactors were identified to preferentially bind α1(A322D)-containing receptors over WT (Fig. 3C, top left section above the dashed line), including three degradation factors (TRIM21, Hsp90B1, and XPNPEP3) and ten proteins with less known function in proteostasis maintenance. It is worth noting that the most enriched α1(A322D) variant–containing receptor interactor is SRRM1, encoding serine/arginine repetitive matrix protein 1. Moreover, 64 interactors were identified to preferentially bind WT α1 over α1(A322D)-containing receptors (Fig. 3C, bottom right section below the dashed line), including seven folding factors, 11 degradation factors, eight trafficking factors, and 38 proteins with less defined function in proteostasis maintenance. Top enriched WT α1-containing receptor interactors include CSE1L, GNAI1, EMD, PRKDC, GCN1L1, XPO1, SLC39A7, and HAX1, which merit future investigation.

      Verification of the role of selected GABAA receptor proteostasis network components

      The interactions between selected GABAA receptor proteostasis network components and WT α1 and α1(A322D)-containing receptors were verified using coimmunoprecipitation and Western blot analysis. We focus on molecular chaperones and ERAD factors because of their critical role in the protein QC of GABAA receptors in the ER. Previously, we showed that BiP (HspA5), an Hsp70 family chaperone in the ER lumen, interacts with WT α1 and α1(A322D)-containing GABAA receptors and promotes their maturation in the ER (
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ,
      • Di X.J.
      • Wang Y.J.
      • Cotter E.
      • Wang M.
      • Whittsette A.L.
      • Han D.Y.
      • et al.
      Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
      ,
      • Han D.Y.
      • Guan B.J.
      • Wang Y.J.
      • Hatzoglou M.
      • Mu T.W.
      L-Type calcium channel blockers enhance trafficking and function of epilepsy-associated α1(d219N) subunits of GABA(A) receptors.
      ). Here, we verified that HspA8, a Hsp70 family chaperone in the cytosol, and its cochaperones, DNAJA1 and DNAJA2, interact with WT α1 and α1(A322D)-containing GABAA receptors (Fig. 4A), suggesting that Hsp70s and their Hsp40 cochaperones coordinate the folding of GABAA receptors both in the ER and in the cytosol. Previously, we showed that CANX interacts with WT α1 and α1(A322D)-containing GABAA receptors in a glycosylation-dependent manner (
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ,
      • Di X.J.
      • Wang Y.J.
      • Cotter E.
      • Wang M.
      • Whittsette A.L.
      • Han D.Y.
      • et al.
      Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
      ,
      • Han D.Y.
      • Guan B.J.
      • Wang Y.J.
      • Hatzoglou M.
      • Mu T.W.
      L-Type calcium channel blockers enhance trafficking and function of epilepsy-associated α1(d219N) subunits of GABA(A) receptors.
      ). Here, we verified that UGGT1, a reglucosylation enzyme that rescues glycoproteins with minor folding defects with a client preference for large plasma membrane proteins (
      • Adams B.M.
      • Canniff N.P.
      • Guay K.P.
      • Larsen I.S.B.
      • Hebert D.N.
      Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.
      ,
      • Sousa M.
      • Parodi A.J.
      The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.
      ), interacts with α1 subunit–containing receptors (Fig. 4A), suggesting its potential role in folding glycosylated GABAA receptors. In addition, the interaction between α1 subunit–containing GABAA receptors and UNC45A, an Hsp90 cochaperone, was demonstrated (Fig. 4A).
      Figure thumbnail gr4
      Figure 4Verification of the role of selected GABAA receptor interactors. HEK293T cells stably expressing WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors (A) or mouse brain homogenates (B) were immunoprecipitated using an antibody against α1 subunits and then subjected to SDS-PAGE and Western blot analysis. Three replicates were carried out. Immunoglobulin G serves as a negative control during the coimmunoprecipitation. C, HEK293T cells stably expressing WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were transfected with nontargeting (NT) siRNA or siRNA against TRIM21. Forty-eight hours post-transfection, cells were lysed and subjected to Western blot analysis. β-actin serves as a loading control. Quantification of the normalized protein band intensity was shown on the right (n = 3). Student’s t test was used to evaluate the statistical significance. D, HEK293T cells expressing α1(A322D)β2γ2 receptors were transfected as in (C). Cycloheximide (CHX), a potent protein synthesis inhibitor, was added to the cell culture medium for the indicated time before cell lysis and Western blot analysis. Quantification of the normalized remaining α1 band intensity was shown on the right (n = 3). Two-way ANOVA followed by post hoc Tukey's test was used to evaluate the statistical significance. Data are presented as mean ± SD. ∗p < 0.05; ∗∗p < 0.01. AD, α1(A322D) variant; GABAA, gamma-aminobutyric acid type A; HEK293T, human embryonic kidney 293T cell line; IB, immunoblotting; IP, immunoprecipitation; TRIM21, tripartite motif containing-21.
      Furthermore, we verified the cellular interactions between GABAA receptors and three ubiquitin E3 ligases that we identified (LTN1, UBR5, and TRIM21) (Fig. 4A). Mammalian ubiquitin E3 ligases play a central role in the ubiquitination and targeting of their client proteins to the cellular clearance pathway (
      • Christianson J.C.
      • Carvalho P.
      Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis.
      ,
      • Christianson J.C.
      • Ye Y.
      Cleaning up in the endoplasmic reticulum: ubiquitin in charge.
      ,
      • Kumari D.
      • Brodsky J.L.
      The targeting of native proteins to the endoplasmic reticulum-associated degradation (ERAD) pathway: an expanding repertoire of regulated substrates.
      ). Therefore, these E3 ligases are promising candidates to direct GABAA receptors to the proteasome for degradation. In addition, the interaction between α1 subunit–containing GABAA receptors and ERLIN2, a potential ERAD factor, which was known to promote the degradation of inositol 1,4,5-trisphosphate receptors on the ER membrane (
      • Pearce M.M.
      • Wang Y.
      • Kelley G.G.
      • Wojcikiewicz R.J.
      SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells.
      ), was confirmed (Fig. 4A).
      To evaluate whether the interactions between GABAA receptors and the proteostasis network components that we identified are utilized in the mammalian central nervous system, we used mouse brain homogenates to carry out coimmunoprecipitation assay. Indeed, we demonstrated the endogenous interactions between α1 subunit–containing GABAA receptors and their selected interactors (Fig. 4B), consistent with the result that most interactors are well conserved between HEK293 cells and the nervous system (Fig. 2C).
      We selected TRIM21 (aka RO52/SSA1), a RING-type E3 ubiquitin ligase (
      • Wada K.
      • Kamitani T.
      Autoantigen Ro52 is an E3 ubiquitin ligase.
      ), for further characterization. TRIM21 is known to play a critical role in innate immunity and cell proliferation by ubiquitinating various proteins (
      • Alomari M.
      TRIM21 - a potential novel therapeutic target in cancer.
      ,
      • Kiss L.
      • James L.C.
      The molecular mechanisms that drive intracellular neutralization by the antibody-receptor and RING E3 ligase TRIM21.
      ). TRIM21 contains an N-terminal RING-finger domain, which is responsible for its ubiquitin E3 ligase activity, a B-box domain, a coiled coil domain, and a C-terminal SPRY domain, which is responsible to bind its client proteins (
      • Higgs R.
      • J N.G.
      • Ben Larbi N.
      • Breen E.P.
      • Fitzgerald K.A.
      • Jefferies C.A.
      The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.
      ). Knocking down TRIM21 using siRNA in HEK293T cells significantly increased the total α1(A322D) protein level (Fig. 4C, cf. lane 4 to lane 3) without an apparent influence on WT α1 subunits (Fig. 4C, cf. lane 2 to lane 1), indicating that TRIM21 targets misfolded α1(A322D) subunits for degradation more favorably. Moreover, cycloheximide (CHX), a potent protein synthesis inhibitor, was applied to the cells for the indicated time to determine the degradation kinetics of α1(A322D) proteins. Compared with nontargeting siRNA control, CHX-chase experiments showed that knocking down TRIM21 increased the remaining α1(A322D) protein levels from 39% to 63% at 0.5 h post CHX application (Fig. 4D, cf. lane 2 to lane 5), and from 12% to 30% at 1 h post CHX application (Fig. 4D, cf. lane 3 to lane 6), indicating that depleting TRIM21 attenuated the degradation of α1(A322D) subunits substantially. These results supported that TRIM21 positively regulates the ERAD of the misfolding-prone α1(A322D) subunits.

      Discussion

      The comprehensive profiling of the interactomes for GABAA receptors enables us to assemble a cellular model about how the proteostasis network regulates their biogenesis (Fig. 5). GABAA receptor subunits are cotranslationally targeted to the ER membrane (Fig. 5, state 1), which is mediated by the SRP complex (
      • Zhang X.
      • Shan S.O.
      Fidelity of cotranslational protein targeting by the signal recognition particle.
      ). The insertion of the TM domains of GABAA receptors into the lipid bilayer could be facilitated by the SEC61 translocon as well as the ER membrane complex (
      • Chitwood P.J.
      • Juszkiewicz S.
      • Guna A.
      • Shao S.
      • Hegde R.S.
      EMC is required to initiate accurate membrane protein topogenesis.
      ,
      • Guna A.
      • Volkmar N.
      • Christianson J.C.
      • Hegde R.S.
      The ER membrane protein complex is a transmembrane domain insertase.
      ,
      • Whittsette A.L.
      • Wang Y.-J.
      • Mu T.-W.
      The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors.
      ). Our SILAC analysis identified SRP68 and SSR1, which is the α subunit of a translocon-associated protein. GABAA receptor subunits have several Asn N-linked glycosylation sites in the ER lumen, which are in the Asn-X-Ser/Thr sequence motif (X can be any residue except Pro): the α1 subunit has two sites at N38 and N138; the β2 subunit has three sites at N32, N104, and N173; and the γ2 subunit has three sites at N52, N129, and N247. The oligosaccharyltransferase complex is responsible for the transfer of 14-monosaccharide residues Glc3Man9GlcNAc2 (Glc: glucose, Man: mannose, GlcNAc: N-acetylglucosamine) to the aforementioned Asn residues in GABAA receptors (Fig. 5, state 2) (
      • Mohorko E.
      • Glockshuber R.
      • Aebi M.
      Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation.
      ). Our SILAC analysis identified three oligosaccharyltransferase subunits, including DDOST, RPN1, and RPN2. N-linked glycans serve as protein maturation and QC tag (
      • Tannous A.
      • Pisoni G.B.
      • Hebert D.N.
      • Molinari M.
      N-linked sugar-regulated protein folding and quality control in the ER.
      ). The N-linked glycan is then trimmed by α-glucosidase I and α-glucosidase II sequentially to create monoglucosylated glycan (Fig. 5, state 3). Our SILAC analysis identified GANAB, the α subunit of ER α-glucosidase II. The monoglucosylated GABAA receptor subunit is the client of the membrane-bound lectin chaperone CANX for the folding process (Fig. 5, state 3 to state 4). In addition, heat shock proteins facilitate the folding of GABAA receptor subunits in a glycan-independent manner both in the ER lumen and in the cytosol (Fig. 5, state 2 to state 4). Our SILAC analysis identified major Hsp60, Hsp70, and Hsp90 family chaperones as well as their cochaperones, such as HspA5 (BiP) and its cochaperone DNAJB11 (ERdj3), and HspA8 (Hsc70) and its cochaperones DNAJA1/2/3. Since each GABAA subunit has a signature Cys loop in the ER lumen, protein disulfide isomerases play a critical role in assisting the formation of proper disulfide bonds. Our SILAC analysis identified P4HB, an ER protein disulfide isomerase.
      Figure thumbnail gr5
      Figure 5Cellular model of the proteostasis network for GABAA receptors. Interactors that were identified from our SILAC analysis are included in each step. Interactors that are expected to play a role in the folding and assembly of GABAA receptors are colored in green; those in their degradation, in orange; and those in their transport, in purple. The GABAA receptor subunit is cotranslationally translocated to the ER membrane (state 1). The OST complex, including DDOST, RPN1, and RPN2, installs N-linked glycans to the subunit (state 1 to state 2). The glycoprotein is trimmed by ER α-glucosidases, such as GANAB (state 2 to state 3), and subjected to calnexin (CANX) folding cycles in a glycan-dependent manner (state 3 to state 4). In parallel, heat shock proteins and their cochaperones facilitate the folding of the subunit both in the ER lumen (HspA5, DNAJB11, and P4HB) and in the cytosol (HspA1A, HspA6, HspA8, DNAJA1, DNAJA2, and DNAJA3) (state 2 to state 4). After coordinated folding efforts (state 4), the subunit faces three possible routes. First, if folded into the native state, the subunit assembles with other subunits to form a heteropentamer with the assistance from assembly factors (state 4 to state 5). The assembled receptors engage the COPII machinery, including SEC16A, for anterograde trafficking to the Golgi and to the plasma membrane (state 5 to state 6 and state 7). The COPI machinery, including COPA, COPG2, aids the recycle of the subunits from the Golgi back to the ER (state 6 to state 5). Second, non-native subunits are reglucosylated by UGGT1 for additional folding attempts (state 4 to state 3). Third, terminally misfolded subunits undergo ERAD pathway, being recognized, ubiquitinated, dislocated, and targeted to the proteasome for degradation (state 4 to state 8 and state 9). Ubiquitin E3 ligases (UBR5, UBE3C, TRIM21, LTN1, and HUWE1) and other ERAD factors (Hsp90B1, Hsp90AB1, AHSA1, SEL1L, VCP, and ERLIN2) potentially play a critical role in this process. Aggregated subunits could utilize the lysosome-related degradation pathway (state 4 to state 10). ER, endoplasmic reticulum; ERAD, ER-associated degradation; GABAA, gamma-aminobutyric acid type A; OST, oligosaccharyltransferase; SILAC, stable isotope labeling by amino acids in cell culture; UGGT1, UDP-glucose glycoprotein glucosyltransferase 1.
      After the collaborative folding efforts (Fig. 5, state 4), GABAA receptor subunits have three possible pathways. First, if they achieve the natively folded state, they can assemble with other subunits to form a pentameric receptor for further anterograde trafficking (Fig. 5, state 4 to state 5). Properly assembled pentameric GABAA receptors engage the trafficking factors to exit the ER, traffic through the Golgi, and travel en route to the plasma membrane (Fig. 5, state 5–6 and 7). Our SILAC analysis identified SEC16A in the COPII machinery that regulates the anterograde cargo protein vesicle transport from the ER to the Golgi (
      • Hutt D.M.
      • Balch W.E.
      Expanding proteostasis by membrane trafficking networks.
      ) as well as COPA and COPG2 in the COPI machinery that regulates the retrograde retrieval of cargo proteins from the Golgi to the ER for recycling (
      • Szul T.
      • Sztul E.
      COPII and COPI traffic at the ER-Golgi interface.
      ). Second, non-natively folded subunits are recognized by UGGT1, which was identified from our SILAC analysis. UGGT1 acts as a folding sensor and reglucosylates its substrates for re-entering the CANX folding cycles for additional folding attempts (Fig. 5, state 4 to state 3) (
      • Adams B.M.
      • Oster M.E.
      • Hebert D.N.
      Protein quality control in the endoplasmic reticulum.
      ). Third, terminally misfolded subunits are subjected to either the ERAD clearance pathway or the lysosome-related degradation pathway (
      • Oikonomou C.
      • Hendershot L.M.
      Disposing of misfolded ER proteins: a troubled substrate's way out of the ER.
      ). During the ERAD, misfolded subunits are recognized, ubiquitinated, dislocated from the ER into the cytosol, and targeted to the proteasome for degradation (Fig. 5, state 4–8 and 9). Our SILAC analysis identified ubiquitin E3 ligases (HUWE1, UBR5, UBE3C, TRIM21, and LTN1) and other important ERAD factors, such as SEL1L and VCP. If misfolded subunits tend to form large aggregates in the ER, they are likely to be targeted to the lysosome for degradation (Fig. 5, state 4 to state 10). Several lysosomal degradation pathways have been described, including macroautophagy, selective ER-phagy, and direct ER-to-lysosome-associated degradation (
      • Reggiori F.
      • Molinari M.
      ER-Phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.
      ). Our SILAC analysis identified SQSTM1 (p62), a marker protein during autophagy by acting as an autophagosome cargo protein.
      Previously, we reported that a number of proteostasis network components regulate the folding, degradation, and trafficking of GABAA receptors (
      • Di X.J.
      • Wang Y.J.
      • Han D.Y.
      • Fu Y.L.
      • Duerfeldt A.S.
      • Blagg B.S.
      • et al.
      Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.
      ,
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ,
      • Di X.J.
      • Wang Y.J.
      • Cotter E.
      • Wang M.
      • Whittsette A.L.
      • Han D.Y.
      • et al.
      Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
      ,
      • Fu Y.L.
      • Zhang B.
      • Mu T.W.
      LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors.
      ,
      • Han D.Y.
      • Di X.J.
      • Fu Y.L.
      • Mu T.W.
      Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.
      ), and most of them, such as BiP (HspA5), CANX, Grp94 (Hsp90B1), VCP, and SEL1L, were identified from this quantitative SILAC-based IP–MS/MS analysis, indicating the effectiveness of this proteomics approach. For example, we showed that BiP and CANX interacted with both WT α1 and α1(A322D)-containing GABAA receptors; furthermore, overexpression of BiP or CANX was sufficient to enhance the ER-to-Golgi trafficking efficiency of WT α1 and α1(A322D) subunits (
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ). Also the interaction between CANX and α1 subunits is dependent on the N-linked glycosylation since mutating the glycosylation sites in α1 subunits substantially decreases such interaction (
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ,
      • Han D.Y.
      • Guan B.J.
      • Wang Y.J.
      • Hatzoglou M.
      • Mu T.W.
      L-Type calcium channel blockers enhance trafficking and function of epilepsy-associated α1(d219N) subunits of GABA(A) receptors.
      ). These results indicated that BiP and CANX act as profolding chaperones to facilitate the productive folding of GABAA receptors. Regarding the ERAD factors, previously we showed that Grp94 interacted with α1 subunits and that depleting Grp94 decreased ubiquitinated α1 subunits and their degradation rate (
      • Di X.J.
      • Wang Y.J.
      • Han D.Y.
      • Fu Y.L.
      • Duerfeldt A.S.
      • Blagg B.S.
      • et al.
      Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.
      ). In addition, we demonstrated that knocking down SEL1L (a critical cofactor of the ubiquitin E3 ligase Hrd1 complex) or VCP (an ATPase that extracts misfolded proteins from the ER membrane to the cytosol) increased the total protein levels of α1(A322D) subunits (
      • Di X.J.
      • Wang Y.J.
      • Cotter E.
      • Wang M.
      • Whittsette A.L.
      • Han D.Y.
      • et al.
      Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
      • Han D.Y.
      • Di X.J.
      • Fu Y.L.
      • Mu T.W.
      Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.
      ). These results supported their critical role in targeting misfolded GABAA receptors to the ERAD pathway. Such knowledge assisted the integration of the interactors into the proteostasis network for GABAA receptors (Fig. 5).
      Here, we identified 176 interactors for α1 subunit–containing GABAA receptors in HEK293T cells, including 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping interactors. In addition to three GABAA subunits (GABRA1, GABRB2, and GABRG2), our α1 subunit–containing GABAA receptor interactomes showed 13 overlapping interactors with one other GABAA receptor proteomic study, including factors that regulate protein folding and degradation (DNAJA1, RPN2, SQSTM1, USP9X, DDB1, and PSMC2), transporters (SLC25A3, SLC25A4, and SLC25A5), an enzyme (PTPLAD1), and miscellaneous proteins (PHB2, IMMT, and EMD) (
      • Ge Y.
      • Kang Y.
      • Cassidy R.M.
      • Moon K.M.
      • Lewis R.
      • Wong R.O.L.
      • et al.
      Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.
      ,
      • Nakamura Y.
      • Morrow D.H.
      • Modgil A.
      • Huyghe D.
      • Deeb T.Z.
      • Lumb M.J.
      • et al.
      Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
      ). Among the 160 interactors that were not reported from previous proteomic analyses, previously two chaperones (HspA5 and CANX) were demonstrated to facilitate the folding of GABAA receptors (
      • Di X.J.
      • Han D.Y.
      • Wang Y.J.
      • Chance M.R.
      • Mu T.W.
      SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
      ,
      • Han D.Y.
      • Guan B.J.
      • Wang Y.J.
      • Hatzoglou M.
      • Mu T.W.
      L-Type calcium channel blockers enhance trafficking and function of epilepsy-associated α1(d219N) subunits of GABA(A) receptors.
      ), and three ERAD factors (Hsp90B1, VCP, and SEL1L) were shown to promote the ERAD of GABAA receptors (
      • Di X.J.
      • Wang Y.J.
      • Han D.Y.
      • Fu Y.L.
      • Duerfeldt A.S.
      • Blagg B.S.
      • et al.
      Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.
      ). Therefore, 155 novel interactors were identified from this study for α1-containing GABAA receptors. Literature mining enabled us to assemble a cellular model about how the proteostasis network could regulate the folding, degradation, and trafficking of GABAA receptors (Fig. 5), paving the foundation for future efforts to elucidate the biogenesis of GABAA receptors in mechanistic details, which is not well understood.
      Furthermore, our proteomic study identified 78 interactors with previously unknown function in protein folding, proteolysis, protein transport, and protein synthesis (Fig. 2G and Table S4). It would be of great interest to determine whether and how they could regulate GABAA receptor proteostasis in the future. For instance, RCN2, a Ca2+-binding protein in the ER lumen, is abundant in the central nervous system, and its gene expression is upregulated in patients with idiopathic absence epilepsies (
      • Helbig I.
      • Matigian N.A.
      • Vadlamudi L.
      • Lawrence K.M.
      • Bayly M.A.
      • Bain S.M.
      • et al.
      Gene expression analysis in absence epilepsy using a monozygotic twin design.
      ). Dedicator of cytokinesis protein 7 (DOCK7), which is localized to the developing axons, activates Rac1 and Rac3 small GTPases and regulates neuronal polarity; moreover, its variants cause developmental and epileptic encephalopathy 23 (DEE23) (
      • Perrault I.
      • Hamdan F.F.
      • Rio M.
      • Capo-Chichi J.M.
      • Boddaert N.
      • Décarie J.C.
      • et al.
      Mutations in DOCK7 in individuals with epileptic encephalopathy and cortical blindness.
      ,
      • Watabe-Uchida M.
      • John K.A.
      • Janas J.A.
      • Newey S.E.
      • Van Aelst L.
      The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18.
      ). SLC39A7 (ZIP-7) is a Zn2+ efflux transporter in the ER membrane; ZIP7 depletion causes neurodevelopmental impairments, and ZIP7 signaling pathway provides neuroprotective effects in recurrent seizures (
      • Ni H.
      • Zhang L.L.
      • Ren S.Y.
      • Sun B.L.
      Long-term expression of zinc transporters in hippocampus following penicillin-induced developmental seizures and its regulation by E-64d.
      ,
      • Yan G.
      • Zhang Y.
      • Yu J.
      • Yu Y.
      • Zhang F.
      • Zhang Z.
      • et al.
      Slc39a7/zip7 plays a critical role in development and zinc homeostasis in zebrafish.
      ).
      An apparent limitation of our current proteomic study is the expression of exogenous GABAA receptors in HEK293T cells, which is not within their native neuronal environment in the central nervous system. As such, some synaptic proteins were not recognized as interactors for GABAA receptors. Nonetheless, since the proteostasis network is well conserved between species and tissues (
      • Powers E.T.
      • Balch W.E.
      Diversity in the origins of proteostasis networks–a driver for protein function in evolution.
      ) and the majority of GABAA receptor interactors have comparable protein abundance levels between HEK293T cells and human brain (Fig. 2C), our proteomic study should be able to identify the main components of GABAA receptor interactomes. Indeed, endogenous interactions were demonstrated for selected interactors and GABAA receptors in mouse brain homogenates (Fig. 4B). For future efforts, it would be desirable to mimic the native neuronal environment of GABAA receptors by generating human-induced pluripotent stem cells carrying WT or pathogenic variant–containing receptors and differentiating such induced pluripotent stem cells into GABAergic neurons (
      • Yang N.
      • Chanda S.
      • Marro S.
      • Ng Y.H.
      • Janas J.A.
      • Haag D.
      • et al.
      Generation of pure GABAergic neurons by transcription factor programming.
      ).
      Loss of function of GABAA receptors is a prominent cause of genetic epilepsies, and recent advances in genetics have identified an increasing number of GABAA variants that are associated with epilepsy (
      • Fu X.
      • Wang Y.J.
      • Kang J.Q.
      • Mu T.W.
      GABAA Receptor Variants in Epilepsy.
      ,
      • Noebels J.L.
      The biology of epilepsy genes.
      ). Current treatments for epilepsy focus on relieving symptoms instead of targeting the underlying causes, leaving behind much unmet medical needs. Recently, we demonstrated that adapting the ER proteostasis network is a promising strategy to restore the functional surface expression of pathogenic GABAA variants (
      • Di X.J.
      • Wang Y.J.
      • Cotter E.
      • Wang M.
      • Whittsette A.L.
      • Han D.Y.
      • et al.
      Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
      ,
      • Fu Y.L.
      • Han D.Y.
      • Wang Y.J.
      • Di X.J.
      • Yu H.B.
      • Mu T.W.
      Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.
      ). Therefore, identification of the proteostasis network for GABAA receptors paves the foundation to adjust the cellular folding, assembly, degradation, and trafficking pathways to fine-tune the functional surface expression of GABAA receptors as a novel therapeutic strategy to ameliorate epilepsy. Furthermore, since WT receptors and the clinical variant-containing GABAA receptors have substantially differentiating cellular interacting networks, it is feasible to adapt the proteostasis network to selectively target pathogenic variants to restore their function with minimal disruption of WT proteins. For example, here we demonstrated that depleting TRIM21, an E3 ubiquitin ligase, attenuated the degradation of α1(A322D) variant without an apparent effect on WT receptors, indicating that inhibiting ERAD factors that interact preferentially with pathogenic variants can specifically prevent the excessive degradation of variant-containing receptors. Moreover, previously we showed that small-molecule proteostasis regulators, such as stress-independent activators of the ATF6 arm of the unfolded protein response, selectively rescued the functional surface expression of misfolding-prone GABAA variants by enhancing cellular folding capacity (
      • Wang M.
      • Cotter E.
      • Wang Y.J.
      • Fu X.
      • Whittsette A.L.
      • Lynch J.W.
      • et al.
      Pharmacological activation of ATF6 remodels the proteostasis network to rescue pathogenic GABA(A) receptors.
      ). Therefore, adapting the proteostasis network to specifically correct the function of pathogenic GABAA receptors is a promising strategy to be further developed for the potential treatment of genetic epilepsy.

      Experimental procedures

      Plasmids

      The pCMV6 plasmids containing human GABAA receptor α1 subunit (UniProt no.: P14867-1) (catalog no.: RC205390), β2 subunit (isoform 2, UniProt no.: P47870-1) (catalog no.: RC216424), γ2 subunit (isoform 2, UniProt no.: P18507-2) (catalog no.: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog no.: PS100001) were purchased from Origene. The missense mutation A322D in the GABAA receptor α1 subunit was constructed using a QuikChange II site-directed mutagenesis Kit (Agilent Genomics; catalog no.: 200523), and the complementary DNA sequences were confirmed by DNA sequencing.

      Antibodies

      The mouse monoclonal anti-GABRA1 antibody (catalog no.: MAB339) was obtained from Millipore. The rabbit polyclonal anti-DNAJB11 (catalog no.: GTX105619) antibody was obtained from GeneTex. The rabbit polyclonal anti-ERLIN2 antibody (catalog no.: PA5-21736) was obtained from Thermo Fisher Scientific. The rabbit polyclonal anti-LTN1 antibody (catalog no.: AP53685PU-N) was obtained from Novus Biologicals. The rabbit polyclonal anti-DNAJA1 antibody (catalog no.: AP5849C) and anti-HspA8 (catalog no.: AP2872A) antibody were obtained from Abgent. The rabbit polyclonal anti-DNAJA2 antibody (catalog no.: 12236-1-AP), anti-TRIM21 antibody (catalog no.: 12108-1-AP), and anti-UNC45A (catalog no.: 15479-1-AP) antibody were obtained from Proteintech. The rabbit monoclonal anti-UGGT1 antibody (catalog no.: 3543-1) was obtained from Epitomics. The mouse monoclonal anti-β-actin antibody (catalog no.: A1978) was obtained from Sigma. The rabbit polyclonal anti-UBR5 antibody (catalog no.: A300-573A) was obtained from Bethyl Laboratories. The light chain–specific goat antimouse secondary antibody (catalog no.: 115-035-174) was purchased from Jackson ImmunoResearch.

      Cell culture and transfection

      HEK293T cells (American Type Culture Collection; catalog no.: CRL-3216) were maintained in Dulbecco’s modified Eagle's medium (DMEM) (Fisher; catalog no.: SH3024301) with 10% heat-inactivated fetal bovine serum (FBS; Fisher; catalog no.: SH3039603HI), and 1% penicillin–streptomycin (Fisher; catalog no.: SV30010) at 37 °C in 5% CO2. Monolayers were passaged upon reaching confluency with 0.05% trypsin protease (Fisher; catalog no.: SH30236.01). HEK293T cells were grown in 6-well plates or 10-cm dishes and allowed to reach ∼70% confluency before transient transfection using TransIT-2020 (Mirus; catalog no.: MIR 5400) or siRNA treatment (50 nM) using the HiPerfect Transfection Reagent (Qiagen; catalog no.: 301707), according to the manufacturer’s instruction. The siRNA duplexes were obtained from Dharmacon: TRIM21 (catalog no.: J-006563-10-0005) and nontargeting siRNA (catalog no.: D-001810-01-20) as negative control. Forty-eight hours post-transfection, cells were harvested for further analysis.
      HEK293T cells stably expressing either WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were generated using the G-418 selection method. Briefly, cells were transfected with α1:β2:γ2 (0.3 μg:0.3 μg:0.3 μg) or α1(A322D):β2:γ2 (0.3 μg:0.3 μg:0.3 μg) plasmids in 6-well plates. Forty-eight hours post-transfection, cells were selected in DMEM with 10% FBS and 1% penicillin–streptomycin supplemented with 0.8 mg/ml G418 for 15 days. Afterward, cells were maintained in DMEM with 10% FBS and 1% penicillin–streptomycin supplemented with 0.4 mg/ml G418. The G418-resistant polyclonal cells stably expressing GABAA receptors were used for experiments.

      SDS-PAGE and Western blot

      Cells were harvested with 0.05% trypsin protease (Fisher; catalog no.: SH30236.01) and then lysed with the lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton X-100) supplemented with cOmplete Protease Inhibitor Cocktail (Roche; catalog no.: 4693159001). Cell lysates were cleared by centrifugation (15,000g, 10 min, 4 °C), and the supernatants were collected as total proteins. Protein concentration was determined by MicroBCA assay (Pierce; catalog no.: 23235). Equal amounts of total proteins were separated in 8% reducing SDS-PAGE gels, and Western blot analysis was performed using appropriate antibodies.

      Mouse brain homogenization

      C57BL/6J mice (Jackson Laboratory) at 6 to 10 weeks were sacrificed, and the cortex was isolated and homogenized in the homogenization buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, and 2% Triton X-100) supplemented with the Roche cOmplete Protease Inhibitor Cocktail. The sample was centrifuged at 800g for 10 min at 4 °C. The pellet was rehomogenized in the same homogenization buffer and centrifuged at 800g for 10 min at 4 °C. The combined supernatants were placed on a rotating device for 2 h at 4 °C and then centrifuged at 15,000g for 30 min at 4 °C. The resulting supernatant was collected as mouse brain homogenate, and its protein concentration was determined by a MicroBCA assay. These animal studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and carried out in agreement with the recommendation of the American Veterinary Medical Association Panel on Euthanasia.

      Immunoprecipitation

      For immunoprecipitation using cell lysates (500 μg) and the mouse brain homogenates (1 mg), they were precleared with 30 μl of protein A/G plus-agarose beads (Santa Cruz; catalog no.: sc-2003) and 1.0 μg of normal mouse immunoglobulin G (Santa Cruz; catalog no.: sc-2025) for 1 h at 4 °C to remove nonspecific binding proteins. The precleared cell lysates were incubated with 2.0 μg of mouse anti-α1 antibody for 1 h at 4 °C and then with 30 μl of protein A/G plus agarose beads overnight at 4 °C. Afterward, the beads were collected by centrifugation at 8000g for 30 s and washed three times with lysis buffer. The complex was eluted by incubation with 40 μl of 2× Laemmli sample buffer (Bio-Rad; catalog no.: 1610737) in the presence of DTT. The immunopurified eluents were separated in 8% SDS-PAGE gel, and Western blot analysis was performed using appropriate antibodies.

      SILAC-based quantitative proteomics analysis

      SILAC labeling

      SILAC is an in vivo labeling strategy for MS-based quantitative proteomics (
      • Mann M.
      Functional and quantitative proteomics using SILAC.
      ,
      • Harsha H.C.
      • Molina H.
      • Pandey A.
      Quantitative proteomics using stable isotope labeling with amino acids in cell culture.
      ). HEK293T cells stably expressing either WT α1β2γ2 or α1(A322D)β2γ2 GABAA receptors were labeled with heavy media (SILAC DMEM media [Pierce; catalog no.: 88420] plus 10% dialyzed FBS [Sigma; catalog no.: F0392], 1% penicillin–streptomycin, 0.01% 13C6 l-lysine–2 HCl [Pierce; catalog no.: 89988], 0.01% 13C6 l-arginine–HCl [Pierce; catalog no.: 88210], and 0.002% l-proline [Pierce; catalog no.: 88430]), whereas HEK293T cells that were transfected with EV plasmids were cultured in normal light media (SILAC DMEM media plus 10% dialyzed FBS, 1% penicillin–streptomycin, 0.01% l-lysine–2 HCl (Pierce; catalog no.: 88429), 0.01% l-arginine–HCl (Pierce; catalog no.: 88427), and 0.002% l-proline) for 14 days to ensure complete labeling.

      Coimmunoprecipitation and in-gel digestion

      Cells were then harvested with 0.05% trypsin and lysed in the lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, and 1% Triton X-100) supplemented with Roche cOmplete Protease Inhibitor Cocktail. Lysates were cleared by centrifugation (15,000g, 10 min, 4 °C). Protein concentration was determined by MicroBCA assay. The same amount of light and heavy cell lysates was mixed by 1:1. Six milligrams of total proteins were immunoprecipitated using a mouse monoclonal anti-GABAA receptor α1 subunit antibody. The immunoisolated complexes were separated by reducing SDS-PAGE and stained with Coomassie blue to visualize protein gel bands (Fig. 1C). The gel was washed in distilled water to remove excess background stain. The gel was then divided to six parts evenly, excised, and destained with 500 μl of 1:1 acetonitrile (ACN) and 100 mM ammonium bicarbonate (ABC) solution for 2 to 8 h. Afterward, 10 mM reductive Tris(2-carboxyethyl)phosphine was added for 30 min, and then free cysteines were alkylated with 55 mM iodoacetamide for 20 min in the dark. ACN and 100 mM ABC were used to dehydrate and rehydrate the gel pieces alternatively for three times. Gel pieces were swelled in 50 mM ABC containing freshly prepared 10 ng/μl trypsin (Promega, sequencing grade) and digested overnight. Peptides were extracted with 60% ACN/5% formic acid (FA). Digested peptides were cleaned through C18 spin columns (Thermo Pierce), dried in SpeedVac, and saved at −80 °C if not immediately analyzed.

      Tandem MS analysis

      Three biological replicates were analyzed by LC–MS/MS. Digested peptides were reconstituted with 0.1% FA and analyzed by LC–MS/MS. Separation of peptides via capillary liquid chromatography was performed using Waters nanoAquity system (Waters Corp). Mobile phase A (aqueous) contained 0.1% FA in 5% ACN, and mobile phase B (organic) contained 0.1% FA in 85% ACN. Separation was achieved using a C18 column (75 μm × 20 cm; Waters Corp; Ethylene Bridged Hybrid column; #BEH300) through a 300-min gradient of 6% to 45% mobile phase B at a flow rate of 300 nl/min. MS analysis was performed using a hybrid linear ion trap Orbitrap Velos mass spectrometer (LTQ-Orbitrap Velos; Thermo). Survey scans were operated at 60,000 resolution, followed by 20 collision-induced dissociation fragmentations.

      Database search

      Acquired tandem mass spectra were searched against UniProt human protein database with 20,244 protein entries, downloaded on May 5, 2011. A decoy database containing the reversed sequences of all proteins was appended to estimate the FDR (
      • Peng J.M.
      • Schwartz D.
      • Elias J.E.
      • Thoreen C.C.
      • Cheng D.M.
      • Marsischky G.
      • et al.
      A proteomics approach to understanding protein ubiquitination.
      ). Protein identification using SEQUEST (
      • Eng J.K.
      • McCormack A.L.
      • Yates J.R.
      An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database.
      ) or ProLuCID (
      • Xu T.
      • Venable J.D.
      • Park S.K.
      • Cociorva D.
      • Lu B.
      • Liao L.
      • et al.
      ProLuCID, a fast and sensitive tandem mass spectra-based protein identification program.
      ) and DTASelect (
      • Cociorva D.
      • Tabb D.L.
      • Yates J.R.
      Validation of tandem mass spectrometry database search results using DTASelect.
      ,
      • Tabb D.L.
      • McDonald W.H.
      • Yates J.R.
      DTASelect and contrast: tools for assembling and comparing protein identifications from shotgun proteomics.
      ) and quantification using Census (
      • Park S.K.
      • Venable J.D.
      • Xu T.
      • Yates III., J.R.
      A quantitative analysis software tool for mass spectrometry-based proteomics.
      ) were performed using the Integrated Proteomics Pipeline (IP2; Integrated Proteomics Applications). Mass accuracy was limited to 10 ppm for precursor ions and 0.6 Da for product ions, with tryptic enzyme specificity and up to two missed cleavages. Static modifications included carbamidomethylation on cysteines (57 Da), and variable modifications included oxidation on methionines (16 Da) in addition to the SILAC modifications, that is, heavy 13C6 on lysines and arginines (6 Da). DTASelect (
      • Cociorva D.
      • Tabb D.L.
      • Yates J.R.
      Validation of tandem mass spectrometry database search results using DTASelect.
      ,
      • Tabb D.L.
      • McDonald W.H.
      • Yates J.R.
      DTASelect and contrast: tools for assembling and comparing protein identifications from shotgun proteomics.
      ) was applied to generate search results for peptide-to-spectra matches with a maximum FDR of 5%, yielding a protein FDR of less than 1%. Protein quantification based on SILAC peptide pairs was performed with Census (
      • Park S.K.
      • Venable J.D.
      • Xu T.
      • Yates III., J.R.
      A quantitative analysis software tool for mass spectrometry-based proteomics.
      ). Census allows users to filter peptide ratio measurements based on a correlation threshold because the correlation coefficient (values between zero and one) represents the quality of the correlation between the unlabeled and labeled chromatograms and can be used to filter out poor quality measurements. In this study, only peptide ratios with correlation values greater than 0.5 were retained. High-confidence protein quantifications were based on two or more SILAC pairs for a given protein.
      The interactomes of WT α1 and α1(A322D)-containing GABAA receptors were identified using the SILAC ratio with arbitrary yet strict criteria to remove potential false positives. Only proteins with p < 0.05 were further considered. To be included as an interactor, it must (1) have a SILAC ratio of WT α1/EV or α1(A322D)/EV to be at least 1.30 and (2) have a Benjamini and Hochberg correction (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the false discovery rate - a practical and powerful approach to multiple testing.
      ) of FDR of no more than 0.10. For display purpose in Figure 3, B and C, if the SILAC ratio is greater than 4.0, it is displayed as 4.0; if a protein is only detected in one sample, the SILAC ratio of this protein in the other sample is artificially set to 1.0. The final list of the interactomes of α1 subunit–containing GABAA receptors and α1(A322D)-containing GABAA receptors is presented in Table S1.

      GO analysis

      Cellular component and biological process of GABAA receptor interactomes were analyzed using DAVID (https://david.ncifcrf.gov/) (
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
      ,
      • Huang da W.
      • Sherman B.T.
      • Lempicki R.A.
      Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.
      ).
      Protein abundance analysis was performed using the ProteomicsDB database (https://www.proteomicsdb.org/) (
      • Wilhelm M.
      • Schlegl J.
      • Hahne H.
      • Gholami A.M.
      • Lieberenz M.
      • Savitski M.M.
      • et al.
      Mass-spectrometry-based draft of the human proteome.
      ). RNA abundance analysis was carried out using the Human Protein Atlas database (https://www.proteinatlas.org/) (
      • Sjöstedt E.
      • Zhong W.
      • Fagerberg L.
      • Karlsson M.
      • Mitsios N.
      • Adori C.
      • et al.
      An atlas of the protein-coding genes in the human, pig, and mouse brain.
      ,
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ).

      CHX-chase assay

      HEK293T cells stably expressing α1(A322D)β2γ2 GABAA receptors were seeded at 2.5 × 105 cells per well in 6-well plates and incubated at 37 °C overnight. Cells were then transfected with indicated siRNA. Forty-eight hours post transfection, cells were incubated with CHX (100 μg/ml) (Enzo; catalog no.: ALX-380-269-G001), a potent protein synthesis inhibitor, to stop protein translation, and chased for the indicated time. Cells were then harvested and lysed for SDS-PAGE and Western blot analysis.

      Statistical analysis

      All data are presented as mean ± SD. Statistical significance was calculated using Student’s t test for two-group comparison. If more than two groups were compared, ANOVA followed by post hoc Tukey's test was used. A p value of less than 0.05 was considered statistically significant. ∗p < 0.05 and ∗∗p < 0.01.

      Data availability

      All data are contained within the article.

      Supporting information

      This article contains supporting information (six supporting tables).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This work was supported by the Clinical Translational Science Collaborative of Cleveland ( Clinical and Translational Science Award) (grant no.: UL1RR024989 ) from the National Center for Research Resources and the National Center for Advancing Translational Sciences of the National Institutes of Health .

      Author contributions

      Y.-J. W. and T.-W. M. conceptualization; Y.-J. W. and T.-W. M. formal analysis; Y.-J. W. and X.-J. D. data curation; Y.-J. W. and T.-W. M. writing–original draft; Y.-J. W., X.-J. D., and T.-W. M. writing–review & editing; Y.-J. W. and T.-W. M. supervision; T.-W. M. funding acquisition.

      Funding and additional information

      This work was supported by the National Institutes of Health (grant nos.: R01NS105789 and R01NS117176; to T.-W. M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

      Supporting information

      • Table S1

        List of proteins and their SILAC ratios from the quantitative proteomics experiments to identify GABAA receptor interactomes. Labeled with a unique entry number for each protein, first 54 proteins overlap within WT α1-containing GABAA receptor interactome and the α1(A322D)-containing GABAA receptor interactome. Number 55 to 125 are WT α1-containing GABAA receptor only interactors, whereas number 126 to 176 are α1(A322D)-containing GABAA receptor only interactors

      • Table S2

        List of protein abundance and RNA abundance of GABAA receptor interactors. Scores of protein abundance were retrieved from the ProteomicsDB database (https://www.proteomicsdb.org/). Protein comparison included data from HEK293 cells and human brain tissues. Scores of RNA abundance were retrieved from the Human Protein Atlas database (https://www.proteinatlas.org/). RNA comparison included data from HEK cells, SH-SY5Y cells and human brain tissues. Fpkm, fragments per kilobase of exon per million mapped fragments

      • Table S3

        List of cellular location and membrane proteins of GABAA receptor interactors. Entry number for each protein was the same as in Table S1 for overlapping, WT α1-containing GABAA receptor only, or α1(A322D)-containing GABAA receptor only interactors. In the column of Membrane protein, 1 means that the corresponding protein is indeed an integral membrane protein, whereas 0 means otherwise. Moreover, cellular location statistics and membrane protein statistics were presented as well, summarizing number of interactors in each GO term

      • Table S4

        List of biological process for GABAA receptor interactors. Entry number for each protein was the same as in Table S1 for overlapping, WT α1-containing GABAA receptor only, or α1(A322D)-containing GABAA receptor only interactors. In the column of Function 2, further available information was provided. Moreover, biological process statistics were presented as well, summarizing number of interactors in each category

      • Table S5

        List of enriched biological process for GABAA receptor interactors. Data were analyzed and retrieved from Database for Annotation, Visualization and Integrated Discovery (DAVID). Five functional clusters were presented from both WT α1-containing GABAA receptor and α1(A322D)-containing GABAA receptor interactors. GO terms, enriched genes, and FDR were included

      • Table S6

        List of the proteostasis network components among GABAA receptor interactors. Based on GO analysis and literature mining, three categories were included as the following: protein folding, proteolysis, and protein transport, for overlapping, WT α1-containing GABAA receptor only, or α1(A322D)-containing GABAA receptor only interactors

      References

        • Hartl F.U.
        • Bracher A.
        • Hayer-Hartl M.
        Molecular chaperones in protein folding and proteostasis.
        Nature. 2011; 475: 324-332
        • Balch W.E.
        • Morimoto R.I.
        • Dillin A.
        • Kelly J.W.
        Adapting proteostasis for disease intervention.
        Science. 2008; 319: 916-919
        • Hetz C.
        Adapting the proteostasis capacity to sustain brain healthspan.
        Cell. 2021; 184: 1545-1560
        • Powers E.T.
        • Gierasch L.M.
        The proteome folding problem and cellular proteostasis.
        J. Mol. Biol. 2021; 433167197
        • Bukau B.
        • Weissman J.
        • Horwich A.
        Molecular chaperones and protein quality control.
        Cell. 2006; 125: 443-451
        • Hetz C.
        • Zhang K.
        • Kaufman R.J.
        Mechanisms, regulation and functions of the unfolded protein response.
        Nat. Rev. Mol. Cell Biol. 2020; 21: 421-438
        • Needham P.G.
        • Guerriero C.J.
        • Brodsky J.L.
        Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases.
        Cold Spring Harbor Perspect. Biol. 2019; 11: a033928
        • Peters K.W.
        • Okiyoneda T.
        • Balch W.E.
        • Braakman I.
        • Brodsky J.L.
        • Guggino W.B.
        • et al.
        CFTR folding consortium: methods available for studies of CFTR folding and correction.
        Methods Mol. Biol. 2011; 742: 335-353
        • Feige M.J.
        • Behnke J.
        • Mittag T.
        • Hendershot L.M.
        Dimerization-dependent folding underlies assembly control of the clonotypic αβT cell receptor chains.
        J. Biol. Chem. 2015; 290: 26821-26831
        • Buck T.M.
        • Jordahl A.S.
        • Yates M.E.
        • Preston G.M.
        • Cook E.
        • Kleyman T.R.
        • et al.
        Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.
        Biochem. J. 2017; 474: 357-376
        • Delaney E.
        • Khanna P.
        • Tu L.
        • Robinson J.M.
        • Deutsch C.
        Determinants of pore folding in potassium channel biogenesis.
        Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 4620-4625
        • Kweon H.J.
        • Gu S.
        • Witham E.
        • Dhara M.
        • Yu H.
        • Mandon E.D.
        • et al.
        NACHO engages N-glycosylation ER chaperone pathways for α7 nicotinic receptor assembly.
        Cell Rep. 2020; 32108025
        • Fu Y.L.
        • Wang Y.J.
        • Mu T.W.
        Proteostasis maintenance of cys-loop receptors.
        Adv. Protein Chem. Struct. Biol. 2016; 103: 1-23
        • Macdonald R.L.
        • Olsen R.W.
        GABA(A) receptor channels.
        Annu. Rev. Neurosci. 1994; 17: 569-602
        • Akerman C.J.
        • Cline H.T.
        Refining the roles of GABAergic signaling during neural circuit formation.
        Trends Neurosci. 2007; 30: 382-389
        • Fu X.
        • Wang Y.J.
        • Kang J.Q.
        • Mu T.W.
        GABAA Receptor Variants in Epilepsy.
        Exon Publications, Brisbane (AU)2022
        • Hernandez C.C.
        • Macdonald R.L.
        A structural look at GABA(A) receptor mutations linked to epilepsy syndromes.
        Brain Res. 2019; 1714: 234-247
        • Hirose S.
        Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.
        Prog. Brain Res. 2014; 213: 55-85
        • Noebels J.L.
        The biology of epilepsy genes.
        Annu. Rev. Neurosci. 2003; 26: 599-625
        • Steinlein O.K.
        ion channel mutations in neuronal diseases: a genetics perspective.
        Chem. Rev. 2012; 112: 6334-6352
        • Dougherty D.A.
        Cys-loop neuroreceptors: structure to the rescue?.
        Chem. Rev. 2008; 108: 1642-1653
        • Laverty D.
        • Desai R.
        • Uchański T.
        • Masiulis S.
        • Stec W.J.
        • Malinauskas T.
        • et al.
        Cryo-EM structure of the human α1β3γ2 GABA(A) receptor in a lipid bilayer.
        Nature. 2019; 565: 516-520
        • Zhu S.
        • Noviello C.M.
        • Teng J.
        • Walsh Jr., R.M.
        • Kim J.J.
        • Hibbs R.E.
        Structure of a human synaptic GABAA receptor.
        Nature. 2018; 559: 67-72
        • Olzmann J.A.
        • Kopito R.R.
        • Christianson J.C.
        The mammalian endoplasmic reticulum-associated degradation system.
        Cold Spring Harbor Perspect. Biol. 2013; 5: a013185
        • Di X.J.
        • Wang Y.J.
        • Han D.Y.
        • Fu Y.L.
        • Duerfeldt A.S.
        • Blagg B.S.
        • et al.
        Grp94 protein delivers γ-aminobutyric acid type A (GABAA) receptors to Hrd1 protein-mediated endoplasmic reticulum-associated degradation.
        J. Biol. Chem. 2016; 291: 9526-9539
        • Gallagher M.J.
        • Shen W.Z.
        • Song L.Y.
        • Macdonald R.L.
        Endoplasmic reticulum retention and associated degradation of a GABA(A) receptor epilepsy mutation that inserts an aspartate in the M3 transmembrane segment of the alpha 1 subunit.
        J. Biol. Chem. 2005; 280: 37995-38004
        • Poliquin S.
        • Kang J.Q.
        Disruption of the ubiquitin-proteasome system and elevated endoplasmic reticulum stress in epilepsy.
        Biomedicines. 2022; 10: 647
        • Fregno I.
        • Molinari M.
        Proteasomal and lysosomal clearance of faulty secretory proteins: ER-associated degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD) pathways.
        Crit. Rev. Biochem. Mol. Biol. 2019; 54: 153-163
        • Wilkinson S.
        ER-Phagy: shaping up and destressing the endoplasmic reticulum.
        FEBS J. 2019; 286: 2645-2663
        • Arancibia-Cárcamo I.L.
        • Yuen E.Y.
        • Muir J.
        • Lumb M.J.
        • Michels G.
        • Saliba R.S.
        • et al.
        Ubiquitin-dependent lysosomal targeting of GABA(A) receptors regulates neuronal inhibition.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17552-17557
        • Davis K.M.
        • Sturt B.L.
        • Friedmann A.J.
        • Richmond J.E.
        • Bessereau J.L.
        • Grant B.D.
        • et al.
        Regulated lysosomal trafficking as a mechanism for regulating GABAA receptor abundance at synapses in Caenorhabditis elegans.
        Mol. Cell. Neurosci. 2010; 44: 307-317
        • Ge Y.
        • Kang Y.
        • Cassidy R.M.
        • Moon K.M.
        • Lewis R.
        • Wong R.O.L.
        • et al.
        Clptm1 limits forward trafficking of GABA(A) receptors to scale inhibitory synaptic strength.
        Neuron. 2018; 97: 596-610.e8
        • Han W.
        • Li J.
        • Pelkey K.A.
        • Pandey S.
        • Chen X.
        • Wang Y.X.
        • et al.
        Shisa7 is a GABA(A) receptor auxiliary subunit controlling benzodiazepine actions.
        Science. 2019; 366: 246-250
        • Han W.
        • Shepard R.D.
        • Lu W.
        Regulation of GABA(A)Rs by transmembrane accessory proteins.
        Trends Neurosciences. 2021; 44: 152-165
        • Lorenz-Guertin J.M.
        • Jacob T.C.
        GABA type a receptor trafficking and the architecture of synaptic inhibition.
        Developmental Neurobiol. 2018; 78: 238-270
        • Nakamura Y.
        • Morrow D.H.
        • Modgil A.
        • Huyghe D.
        • Deeb T.Z.
        • Lumb M.J.
        • et al.
        Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged γ-aminobutyric acid receptor, type A (GABAA), α2 subunit knock-in mouse.
        J. Biol. Chem. 2016; 291: 12394-12407
        • Heller E.A.
        • Zhang W.
        • Selimi F.
        • Earnheart J.C.
        • Ślimak M.A.
        • Santos-Torres J.
        • et al.
        The biochemical anatomy of cortical inhibitory synapses.
        PLoS One. 2012; 7e39572
        • Mann M.
        Functional and quantitative proteomics using SILAC.
        Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958
        • Thomas P.
        • Smart T.G.
        HEK293 cell line: a vehicle for the expression of recombinant proteins.
        J. Pharmacol. Toxicol. Methods. 2005; 51: 187-200
        • Gallagher M.J.
        • Ding L.
        • Maheshwari A.
        • Macdonald R.L.
        The GABA(A) receptor alpha 1 subunit epilepsy mutation A322D inhibits transmembrane helix formation and causes proteasomal degradation.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12999-13004
        • Di X.J.
        • Han D.Y.
        • Wang Y.J.
        • Chance M.R.
        • Mu T.W.
        SAHA enhances Proteostasis of epilepsy-associated alpha1(A322D)beta2gamma2 GABA(A) receptors.
        Chem. Biol. 2013; 20: 1456-1468
        • Benjamini Y.
        • Hochberg Y.
        Controlling the false discovery rate - a practical and powerful approach to multiple testing.
        J. R. Stat. Soc. Ser. B-Methodological. 1995; 57: 289-300
        • Wilhelm M.
        • Schlegl J.
        • Hahne H.
        • Gholami A.M.
        • Lieberenz M.
        • Savitski M.M.
        • et al.
        Mass-spectrometry-based draft of the human proteome.
        Nature. 2014; 509: 582-587
        • Sjöstedt E.
        • Zhong W.
        • Fagerberg L.
        • Karlsson M.
        • Mitsios N.
        • Adori C.
        • et al.
        An atlas of the protein-coding genes in the human, pig, and mouse brain.
        Science. 2020; 367eaay5947
        • Uhlén M.
        • Fagerberg L.
        • Hallström B.M.
        • Lindskog C.
        • Oksvold P.
        • Mardinoglu A.
        • et al.
        Proteomics. Tissue-based map of the human proteome.
        Science. 2015; 3471260419
        • Huang da W.
        • Sherman B.T.
        • Lempicki R.A.
        Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
        Nat. Protoc. 2009; 4: 44-57
        • Huang da W.
        • Sherman B.T.
        • Lempicki R.A.
        Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.
        Nucleic Acids Res. 2009; 37: 1-13
        • Weis K.
        • Griffiths G.
        • Lamond A.I.
        The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif.
        J. Biol. Chem. 1994; 269: 19142-19150
        • Helbig I.
        • Matigian N.A.
        • Vadlamudi L.
        • Lawrence K.M.
        • Bayly M.A.
        • Bain S.M.
        • et al.
        Gene expression analysis in absence epilepsy using a monozygotic twin design.
        Epilepsia. 2008; 49: 1546-1554
        • Schlingmann K.P.
        • Bandulik S.
        • Mammen C.
        • Tarailo-Graovac M.
        • Holm R.
        • Baumann M.
        • et al.
        Germline de novo mutations in ATP1A1 cause renal hypomagnesemia, refractory seizures, and intellectual disability.
        Am. J. Hum. Genet. 2018; 103: 808-816
        • Wu H.
        • Carvalho P.
        • Voeltz G.K.
        Here, there, and everywhere: the importance of ER membrane contact sites.
        Science. 2018; 361eaan5835
        • Di X.J.
        • Wang Y.J.
        • Cotter E.
        • Wang M.
        • Whittsette A.L.
        • Han D.Y.
        • et al.
        Proteostasis regulators restore function of epilepsy-associated GABA(A) receptors.
        Cell Chem. Biol. 2021; 28: 46-59.e7
        • Han D.Y.
        • Guan B.J.
        • Wang Y.J.
        • Hatzoglou M.
        • Mu T.W.
        L-Type calcium channel blockers enhance trafficking and function of epilepsy-associated α1(d219N) subunits of GABA(A) receptors.
        ACS Chem. Biol. 2015; 10: 2135-2148
        • Adams B.M.
        • Canniff N.P.
        • Guay K.P.
        • Larsen I.S.B.
        • Hebert D.N.
        Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2.
        eLife. 2020; 9e63997
        • Sousa M.
        • Parodi A.J.
        The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.
        EMBO J. 1995; 14: 4196-4203
        • Christianson J.C.
        • Carvalho P.
        Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis.
        EMBO J. 2022; 41e109845
        • Christianson J.C.
        • Ye Y.
        Cleaning up in the endoplasmic reticulum: ubiquitin in charge.
        Nat. Struct. Mol. Biol. 2014; 21: 325-335
        • Kumari D.
        • Brodsky J.L.
        The targeting of native proteins to the endoplasmic reticulum-associated degradation (ERAD) pathway: an expanding repertoire of regulated substrates.
        Biomolecules. 2021; 11: 1185
        • Pearce M.M.
        • Wang Y.
        • Kelley G.G.
        • Wojcikiewicz R.J.
        SPFH2 mediates the endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors and other substrates in mammalian cells.
        J. Biol. Chem. 2007; 282: 20104-20115
        • Wada K.
        • Kamitani T.
        Autoantigen Ro52 is an E3 ubiquitin ligase.
        Biochem. Biophys. Res. Commun. 2006; 339: 415-421
        • Alomari M.
        TRIM21 - a potential novel therapeutic target in cancer.
        Pharmacol. Res. 2021; 165105443
        • Kiss L.
        • James L.C.
        The molecular mechanisms that drive intracellular neutralization by the antibody-receptor and RING E3 ligase TRIM21.
        Semin. Cel. Dev. Biol. 2022; 126: 99-107
        • Higgs R.
        • J N.G.
        • Ben Larbi N.
        • Breen E.P.
        • Fitzgerald K.A.
        • Jefferies C.A.
        The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.
        J. Immunol. 2008; 181: 1780-1786
        • Zhang X.
        • Shan S.O.
        Fidelity of cotranslational protein targeting by the signal recognition particle.
        Annu. Rev. Biophys. 2014; 43: 381-408
        • Chitwood P.J.
        • Juszkiewicz S.
        • Guna A.
        • Shao S.
        • Hegde R.S.
        EMC is required to initiate accurate membrane protein topogenesis.
        Cell. 2018; 175: 1507-1519
        • Guna A.
        • Volkmar N.
        • Christianson J.C.
        • Hegde R.S.
        The ER membrane protein complex is a transmembrane domain insertase.
        Science. 2018; 359: 470-473
        • Whittsette A.L.
        • Wang Y.-J.
        • Mu T.-W.
        The endoplasmic reticulum membrane complex promotes proteostasis of GABAA receptors.
        iScience. 2022; 25: 104754
        • Mohorko E.
        • Glockshuber R.
        • Aebi M.
        Oligosaccharyltransferase: the central enzyme of N-linked protein glycosylation.
        J. Inherit. Metab. Dis. 2011; 34: 869-878
        • Tannous A.
        • Pisoni G.B.
        • Hebert D.N.
        • Molinari M.
        N-linked sugar-regulated protein folding and quality control in the ER.
        Semin. Cel. Dev. Biol. 2015; 41: 79-89
        • Hutt D.M.
        • Balch W.E.
        Expanding proteostasis by membrane trafficking networks.
        Cold Spring Harbor Perspect. Biol. 2013; 5: a013383
        • Szul T.
        • Sztul E.
        COPII and COPI traffic at the ER-Golgi interface.
        Physiology. 2011; 26: 348-364
        • Adams B.M.
        • Oster M.E.
        • Hebert D.N.
        Protein quality control in the endoplasmic reticulum.
        Protein J. 2019; 38: 317-329
        • Oikonomou C.
        • Hendershot L.M.
        Disposing of misfolded ER proteins: a troubled substrate's way out of the ER.
        Mol. Cell. Endocrinol. 2020; 500110630
        • Reggiori F.
        • Molinari M.
        ER-Phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.
        Physiol. Rev. 2022; 102: 1393-1448
        • Fu Y.L.
        • Zhang B.
        • Mu T.W.
        LMAN1 (ERGIC-53) promotes trafficking of neuroreceptors.
        Biomedicine. 2019; 511: 356-362
        • Han D.Y.
        • Di X.J.
        • Fu Y.L.
        • Mu T.W.
        Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.
        J. Biol. Chem. 2015; 290: 325-337
        • Perrault I.
        • Hamdan F.F.
        • Rio M.
        • Capo-Chichi J.M.
        • Boddaert N.
        • Décarie J.C.
        • et al.
        Mutations in DOCK7 in individuals with epileptic encephalopathy and cortical blindness.
        Am. J. Hum. Genet. 2014; 94: 891-897
        • Watabe-Uchida M.
        • John K.A.
        • Janas J.A.
        • Newey S.E.
        • Van Aelst L.
        The Rac activator DOCK7 regulates neuronal polarity through local phosphorylation of stathmin/Op18.
        Neuron. 2006; 51: 727-739
        • Ni H.
        • Zhang L.L.
        • Ren S.Y.
        • Sun B.L.
        Long-term expression of zinc transporters in hippocampus following penicillin-induced developmental seizures and its regulation by E-64d.
        Exp. Ther. Med. 2016; 12: 208-214
        • Yan G.
        • Zhang Y.
        • Yu J.
        • Yu Y.
        • Zhang F.
        • Zhang Z.
        • et al.
        Slc39a7/zip7 plays a critical role in development and zinc homeostasis in zebrafish.
        PLoS One. 2012; 7e42939
        • Powers E.T.
        • Balch W.E.
        Diversity in the origins of proteostasis networks–a driver for protein function in evolution.
        Nat. Rev. Mol. Cell Biol. 2013; 14: 237-248
        • Yang N.
        • Chanda S.
        • Marro S.
        • Ng Y.H.
        • Janas J.A.
        • Haag D.
        • et al.
        Generation of pure GABAergic neurons by transcription factor programming.
        Nat. Methods. 2017; 14: 621-628
        • Fu Y.L.
        • Han D.Y.
        • Wang Y.J.
        • Di X.J.
        • Yu H.B.
        • Mu T.W.
        Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.
        PLoS One. 2018; 13e0207948
        • Wang M.
        • Cotter E.
        • Wang Y.J.
        • Fu X.
        • Whittsette A.L.
        • Lynch J.W.
        • et al.
        Pharmacological activation of ATF6 remodels the proteostasis network to rescue pathogenic GABA(A) receptors.
        Cell Biosci. 2022; 12: 48
        • Harsha H.C.
        • Molina H.
        • Pandey A.
        Quantitative proteomics using stable isotope labeling with amino acids in cell culture.
        Nat. Protoc. 2008; 3: 505-516
        • Peng J.M.
        • Schwartz D.
        • Elias J.E.
        • Thoreen C.C.
        • Cheng D.M.
        • Marsischky G.
        • et al.
        A proteomics approach to understanding protein ubiquitination.
        Nat. Biotechnol. 2003; 21: 921-926
        • Eng J.K.
        • McCormack A.L.
        • Yates J.R.
        An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database.
        J. Am. Soc. Mass Spectrom. 1994; 5: 976-989
        • Xu T.
        • Venable J.D.
        • Park S.K.
        • Cociorva D.
        • Lu B.
        • Liao L.
        • et al.
        ProLuCID, a fast and sensitive tandem mass spectra-based protein identification program.
        Mol. Cell. Proteomics. 2006; 5S174-S174
        • Cociorva D.
        • Tabb D.L.
        • Yates J.R.
        Validation of tandem mass spectrometry database search results using DTASelect.
        in: Current Protocols in Bioinformatics. Wiley, NJ2006 (Unit 13.4)
        • Tabb D.L.
        • McDonald W.H.
        • Yates J.R.
        DTASelect and contrast: tools for assembling and comparing protein identifications from shotgun proteomics.
        J. Proteome Res. 2002; 1: 21-26
        • Park S.K.
        • Venable J.D.
        • Xu T.
        • Yates III., J.R.
        A quantitative analysis software tool for mass spectrometry-based proteomics.
        Nat. Methods. 2008; 5: 319-322