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Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse*

  • Yasuko Nakamura
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Danielle H. Morrow
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Amit Modgil
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Deborah Huyghe
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Tarek Z. Deeb
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Michael J. Lumb
    Affiliations
    Department of Neuroscience, Physiology and Pharmacology, University College, London WC1E 6BT, United Kingdom
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  • Paul A. Davies
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111
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  • Stephen J. Moss
    Correspondence
    To whom correspondence should be addressed. Tel.: 617-636-3976; Fax: 617-636-2413.
    Affiliations
    From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111

    Department of Neuroscience, Physiology and Pharmacology, University College, London WC1E 6BT, United Kingdom
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants NS051195, NS056359, NS081735, R21NS080064, and NS087662 from NINDS (to S. J. M.), National Institute of Mental Health Grant MH097446 (to P. A. D. and S. J. M.), and Department of Defense Grant AR140209 (to P. A. D. and S. J. M.). S. J. M. serves as a consultant for AstraZeneca and SAGE Therapeutics relationships that are regulated by Tufts University and do not impact this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental Tables S1 and S2.
    2 The abbreviations used are: GABAARγ-aminobutyric acid receptorGphngephyrinDGGCdentate gyrus granule cellpFpicofaradsIPSCspontaneous IPSCGEFGDP-GTP exchange factorIPimmunoprecipitationcoIPcoimmunoprecipitation.
Open AccessPublished:April 04, 2016DOI:https://doi.org/10.1074/jbc.M116.724443
      The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1–3, β, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

      Introduction

      GABAARs
      The abbreviations used are: GABAAR
      γ-aminobutyric acid receptor
      Gphn
      gephyrin
      DGGC
      dentate gyrus granule cell
      pF
      picofarad
      sIPSC
      spontaneous IPSC
      GEF
      GDP-GTP exchange factor
      IP
      immunoprecipitation
      coIP
      coimmunoprecipitation.
      are Cl-permeable ligand-gated ion channels that mediate the majority of fast synaptic inhibition in the central nervous system (CNS) (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ). They are also of therapeutic significance as they are the sites of action for barbiturates, benzodiazepines, general anesthetics, and neuroactive steroids (
      • Sieghart W.
      Allosteric modulation of GABAA receptors via multiple drug-binding sites.
      ). Consistent with their critical roles in regulating neuronal excitability, deficits in the activity of GABAARs contribute to a plethora of neurological disorders ranging from anxiety to schizophrenia (
      • Rudolph U.
      • Möhler H.
      GABAA receptor subtypes: therapeutic potential in Down syndrome, affective disorders, schizophrenia, and autism.
      ).
      Structurally, GABAARs can be assembled from 19 different subunits (α1–6, β1–3, γ1–3, δ, ϵ, θ, π, and ρ1–3). The majority of GABAARs are believed to be heteropentamers composed of two copies of a single α subunit, two copies of a single β subunit, and one copy of either γ or δ subunits (
      • Olsen R.W.
      • Sieghart W.
      International Union of Pharmacology. LXX. Subtypes of γ-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.
      ,
      • Patel B.
      • Mortensen M.
      • Smart T.G.
      Stoichiometry of δ subunit containing GABA(A) receptors.
      ). GABAARs containing α1–3 and γ are enriched at inhibitory synapses and mediate phasic inhibition, whereas those containing α4–6 and δ are found at extrasynaptic locales and mediate tonic inhibition (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ). Notably, subunit composition impacts the pharmacological and physiological properties of these varying receptor subtypes (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Verdoorn T.A.
      • Draguhn A.
      • Ymer S.
      • Seeburg P.H.
      • Sakmann B.
      Functional properties of recombinant rat GABAA receptors depend upon subunit composition.
      ,
      • Rudolph U.
      • Knoflach F.
      Beyond classical benzodiazepines: novel therapeutic potential of GABAA receptor subtypes.
      ). Moreover, GABAARs containing unique subunit combinations are selectively targeted to distinct types of inhibitory synapses. However, our understanding of the cellular mechanisms that neurons utilize to regulate GABAAR accumulation at inhibitory synapses is rudimentary. Importantly, the processes that regulate inhibitory synaptogenesis are distinct to those used to build excitatory synapses, which are largely dependent upon PDZ domain-mediated protein-protein interactions (
      • Feng W.
      • Zhang M.
      Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density.
      ).
      To identify proteins that are relevant for inhibitory synaptogenesis and maintenance, we created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced between amino acids 4 and 5 of the mature GABAAR α2 subunit (pHα2). Following purification on Myc and/or GFP matrices, GABAAR complexes were analyzed by mass spectrometry, and a stable complex of 174 interacting proteins was identified. Importantly, these included the GABAAR α1–5, β1–3, γ1–3, and δ subunits in addition to the previously identified GABAAR-associated proteins gephyrin (Gphn) and collybistin (Arhgef9). However, 149 of these proteins were novel GABAAR binding partners G-protein-coupled receptors (GPCRs); ion channel subunits; regulators of membrane trafficking and protein stability; modulators of protein phosphorylation; GTPases; and related exchange factors. Significantly, these interactions were confirmed using in vitro binding coupled with immunoprecipitation. Collectively, these results provide new insights into the components of the GABAAR proteome.

      Experimental Procedures

      Animals

      All animal protocols were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Institutional Animal Care and Use Committee of Tufts University.

      Antibodies and Expression Constructs

      The following antibodies were used for immunocytochemistry: C-terminal anti-α2 antibody was provided by Drs. V. Tretter and W. Sieghart (Medical University of Vienna); anti-gephyrin (1:1000, Synaptic Systems, catalog no. 147021); Alexa Fluor 568 and 647 secondaries (1:1000, Invitrogen). The following antibodies were used for Western blotting: anti-GABAAR α2 (1:500, PhosphoSolutions, catalog no. 822-GA2C); anti-GABAAR α4 (1:5000) antisera was raised against the intracellular domain of this subunit (379–421), as described previously (
      • Hörtnagl H.
      • Tasan R.O.
      • Wieselthaler A.
      • Kirchmair E.
      • Sieghart W.
      • Sperk G.
      Patterns of mRNA and protein expression for 12 GABAA receptor subunits in the mouse brain.
      ); anti-GABAAR β3 (1:1000, PhosphoSolutions, catalog no. 863-GB3C and 1:1000, NeuroMab, catalog no. 75-149); anti-collybistin (1:500, Synaptic Systems, catalog no. 261-003); anti-cul1 (1:2500, Abcam, catalog no. AB75817); anti-ephexin (1:1000, provided by Dr. M. E. Greenberg, Harvard University); anti-GAPDH (1:5000, Santa Cruz Biotechnology, catalog no. SC25778); anti-gephyrin (1:1000, C13B11, Synaptic Systems, catalog no. 147111); anti-GFP (1:1000, Synaptic Systems, catalog no. 132002); anti-Mfn2 (0.5 μg/ml, Abcam, catalog no. 56889); anti-mGluR5 (1:4000, Millipore, AB5675); anti-NR1 (1:1000, BD Biosciences); anti-PAK5 (1:1000, R&D Systems, catalog no. MAB4696); anti-Rab5 (1:1000, Abcam, catalog no. AB18211); anti-tubulin (1:10,000, Millipore, catalog no. 05661); and anti-HRP-conjugated secondary (1:10,000, Jackson ImmunoResearch, catalog nos. 715035150 and 715035152). The following constructs were used: GST fusion protein constructs encoding the large intracellular loop of GABAAR subunits α1, α2, β3, and γ2 as described previously (
      • Brandon N.J.
      • Jovanovic J.N.
      • Colledge M.
      • Kittler J.T.
      • Brandon J.M.
      • Scott J.D.
      • Moss S.J.
      A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor β subunits.
      ,
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.
      ). FLAG-ephexin was provided by M. E. Greenberg (Harvard University), as described previously (
      • Shamah S.M.
      • Lin M.Z.
      • Goldberg J.L.
      • Estrach S.
      • Sahin M.
      • Hu L.
      • Bazalakova M.
      • Neve R.L.
      • Corfas G.
      • Debant A.
      • Greenberg M.E.
      EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin.
      ). pHα2 and β3 constructs have been described previously (
      • Jacob T.C.
      • Michels G.
      • Silayeva L.
      • Haydon J.
      • Succol F.
      • Moss S.J.
      Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition.
      ,
      • Abramian A.M.
      • Comenencia-Ortiz E.
      • Vithlani M.
      • Tretter E.V.
      • Sieghart W.
      • Davies P.A.
      • Moss S.J.
      Protein kinase C phosphorylation regulates membrane insertion of GABAA receptor subtypes that mediate tonic inhibition.
      ), respectively.

      Creation of Myc-pHluorin GABAAR α2 Knock-in Mice

      pHα2 mice were generated by homologous recombination in embryonic stem (ES) cells (129Sv/Pas ES cells). A targeting vector was constructed to insert the pHluorin and Myc tag into exon 3 between amino acids 4 and 5 of the mature protein. The targeting vector consisted of a neomycin-positive selection cassette in intron 2 found ∼250 bp upstream of exon 3. An HSV-thymidine kinase-negative selection cassette was positioned at the 5′ end of the construct. The targeting vector was electroporated into 129Sv ES cells, and clones were screened by PCR and Southern blot analysis. ES cell clones were then expanded and selected for C57BL/6J blastocyst injections. The resulting chimeras were bred with wild type C57BL/6J mice. The neomycin cassette was subsequently excised by breeding with Cre mice.

      Cresyl Violet Stain

      pHα2 and WT mice (8–10 weeks old) were transcardially perfused with PBS followed by 2% paraformaldehyde in PBS. Dissected brains were post-fixed overnight and transferred to 30% sucrose solution. Brains were subsequently sliced into 40-μm sections and stored in cryoprotectant (30% sucrose, 30% ethylene glycol, 1% polyvinylpyrrolidone in PBS) at −20 °C until use. Sections were washed with PBS before processing. Slide-mounted sections were sequentially washed in 100% ethanol, 95% ethanol, distilled H2O and stained with cresyl violet (0.3% glacial acetic acid, 0.5% cresyl violet acetate). This was followed by further rinses in 95% ethanol, 100% ethanol and xylene. Images were acquired with Nikon E800 microscope at 1600 × 1200 resolution using a ×4 objective. Twelve sections and three animals per genotype were imaged.

      Western Blot Analysis

      Proteins separated by SDS-PAGE (8–10% gel) were transferred to PVDF membranes and blocked in 6% milk in PBST for 1 h. Membranes were further incubated with the appropriate primary antibody (5% milk in PBST), and after extensive washes, they were probed with HRP-conjugated secondary antibodies for 1 h. Western blots were developed using an enhanced chemiluminescence system as per the manufacturer's instructions (Amresco). Membranes were imaged (ChemiDoc MP, Bio-Rad) and analyzed using ImageJ (National Institutes of Health). Two-tailed unpaired t test or analyses of variance with Games-Howell post hoc test (for multiple comparisons with unequal variances) were performed to analyze data (GraphPad, SPSS). Graphs presented show means ± S.E. of the mean (S.E.).

      Immunocytochemistry

      Hippocampal neurons were prepared from E18 to E19 pHα2 mice and were used for experiments at 18 days in vitro. For immunocytochemistry experiments, cultures were fixed in 4% paraformaldehyde, 5% sucrose, permeabilized, and probed for the GABAAR α2 subunit and gephyrin and were subsequently stained with Alexa Fluor secondary antibodies. 3–5 neurons were imaged from three independent cultures.
      Fixed-cell images were acquired using a Nikon Eclipse Ti confocal microscope. Images were taken at 1024 × 1024 resolution with a ×60 objective. Calculation of the Pearson's coefficient was performed with the JaCOP (
      • Bolte S.
      • Cordelières F.P.
      A guided tour into subcellular colocalization analysis in light microscopy.
      ) plugin for ImageJ software (
      • Schneider C.A.
      • Rasband W.S.
      • Eliceiri K.W.
      NIH Image to ImageJ: 25 years of image analysis.
      ).

      Coimmunoprecipitation (coIP)

      To detect bound gephyrin and collybistin, brains were removed from isoflurane-anesthetized mice (8–10 weeks). Hippocampi from WT and pHα2 mice were lysed in lysis buffer containing 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2% Triton X-100, 5 mm EDTA, 10 mm NaF, 2 mm Na3VO4, 10 mm Na4P2O7, plus protease inhibitors. These samples were spun at 16,100 × g for 15 min at 4 °C, and the supernatant (or lysate) was incubated with 3 μg of Myc antibody overnight in lysis buffer (modified to 1% Triton X-100). The addition of protein G-Sepharose beads (GE Healthcare) for 4 h was followed by four quick washes (400 × g, 2 min, 4 °C) in 1.5 ml of lysis buffer. For GFP IPs, GFP-Trap beads (Chromotek, catalog no. Gta-200) were incubated with hippocampal lysate overnight. Bound proteins were detected by Western blotting. To detect bound mGluR5, KCTD12, and ephexin, hippocampal/cortical lysates prepared as above were pre-cleared overnight with agarose beads conjugated to IgG. These samples were incubated with GFP-Trap for 2 h and subsequently washed three times for 10 min in 1.5 ml of lysis buffer (modified to 0.2% Triton X-100 and centrifuged at 2500 × g, 2 min, 4 °C). Bound proteins were detected by Western blotting. For experiments using HEK293 cells, pre-cleared lysates were incubated with anti-FLAG conjugated beads (Sigma, catalog no. F3165) or GFP-Trap for 2 h and subsequently washed four times in lysis buffer. Bound proteins were detected by Western blotting. A minimum of three independent experiments were performed for all coIP experiments.

      Hippocampal Slice Preparation for Electrophysiology Recordings

      Coronal slices were prepared from male WT and pHα2 animals (8–10 weeks old). Isoflurane-anesthetized mice were decapitated, and brains were rapidly removed and put in an ice-cold cutting solution (126 mm NaCl, 2.5 mm KCl, 0.5 mm CaCl2, 2 mm MgCl2, 26 mm NaHCO3, 1.25 mm NaH2PO4, 10 mm glucose, 1.5 mm sodium pyruvate, and 3 mm kynurenic acid). 310-μm slices cut with a vibratome VT1000S (Leica Microsystems, St Louis, MO) were transferred to an incubation chamber filled with warmed (31 °C) oxygenated artificial cerebrospinal fluid (ACSF: 126 mm NaCl, 2.5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 26 mm NaHCO3, 1.25 mm NaH2PO4, 10 mm glucose, 1.5 mm sodium pyruvate, 1 mm glutamine, 3 mm kynurenic acid, and 5 μm GABA) and bubbled with 95% O2 to 5% CO2. Slices were allowed to recover for 1 h before recording.

      Electrophysiology Recordings

      After recovery, slices were transferred to a submerged recording chamber on the stage of an upright microscope (Nikon FN-1) with a ×40 water immersion objective equipped with DIC/IR optics. Slices were gravity-superfused with ACSF solution throughout experimentation and perfused at a rate of 2 ml/min with oxygenated (O2/CO2 95:5%) ACSF at 32 °C. Adequate O2 tension and pH 7.3–7.4 values were maintained by continuously bubbling the media with 95% O2, 5% CO2. Currents were recorded from the dentate gyrus granule cells (DGGCs) in coronal hippocampal slices. Patch pipettes (5–7 megohms) were pulled from borosilicate glass (World Precision Instruments) and filled with intracellular solution (140 mm CsCl, 1 mm MgCl2, 0.1 mm EGTA, 10 mm HEPES, 2 mm Mg-ATP, 4 mm NaCl, and 0.3 mm Na-GTP, pH 7.25). A 5-min period for stabilization after obtaining the whole-cell recording configuration was allowed before currents were recorded using an Axopatch 200B amplifier (Molecular Devices), low pass-filtered at 2 kHz, digitized at 20 kHz (Digidata 1440A; Molecular Devices), and stored for off-line analysis. The holding potential was −60 mV for all recordings.

      Electrophysiology Analysis

      Tonic current measurements were measured from an all-points histogram that was plotted for a 10-s period before and during picrotoxin application. A Gaussian fit to these points gave the mean current amplitude, and the difference between these two values was considered to be the tonic current and normalized to cell capacitance (pA/pF). Throughout the course of the experiment, series resistance and whole-cell capacitance were continually monitored and compensated. If series resistance increased by >20%, recordings were eliminated from the data analysis. Statistical significance was determined using Student's t test. Spontaneous IPSCs (sIPSCs) were analyzed using the mini-analysis software (version 5.6.4; Synaptosoft, Decatur, GA). sIPSCs were recorded for a minimum of 5 min. To detect sIPSCs, the minimum threshold detection was set to three times the value of baseline noise signal. The recording trace was visually inspected, and only sIPSC events with a stable baseline, sharp rising phase, and single peak were used to negate artifacts due to event summation. Only recordings with a minimum of 100 events fitting these criteria were analyzed. 8–10 cells were recorded from three animals of each genotype. Amplitude, decay, and frequency distributions of sIPSCs were examined by constructing all-point cumulative probability distributions and compared using the Mann-Whitney test and Kolmogorov-Smirnov test. Values of p < 0.05 were considered significant.

      Large Scale Immunoprecipitation for Mass Spectrometry Analysis

      Hippocampus and cortex of age-matched (8–10 weeks) and sex-matched WT and pHα2 mice (seven animals each) were prepared as above. Lysates were filtered and pre-cleared with agarose beads conjugated to IgG overnight. For tandem IPs, pre-cleared lysates were incubated with Myc antibody overnight. Sepharose beads were added and incubated at 4 °C for 4 h. These beads were washed (three times at 400 × g, 2 min, 4 °C), and the proteins were eluted off beads with 200 μg/ml c-Myc peptide (Alpha Diagnostics) in lysis buffer. The eluate was incubated with GFP-Trap for 1 h, followed by four washes (2500 × g, 2 min, 4 °C) in lysis buffer. Gels were run and stained with silver stain (Sigma), and gel bands of interest from pHα2 and the corresponding regions from WT mice were excised. For single IPs, pre-cleared lysates were incubated with GFP-Trap for 2 h, followed by four washes in lysis buffer (2500 × g, 2 min, 4 °C). Gels were run and stained with colloidal Coomassie (
      • Candiano G.
      • Bruschi M.
      • Musante L.
      • Santucci L.
      • Ghiggeri G.M.
      • Carnemolla B.
      • Orecchia P.
      • Zardi L.
      • Righetti P.G.
      Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis.
      ). Each gel lane (for pHα2 or WT IP) was cut into five pieces and sent to Taplin Mass Spectrometry Facility (Harvard University) for proteomic analysis.

      Mass Spectrometry Analysis

      Trypsin digestion, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and MS/MS spectra search in a mouse database (Uniprot) using the Sequest 28 analysis program was performed by Taplin Mass Spectrometry Facility (Harvard University). Peptide matches were considered true matches for ΔCN scores (Δ correlation) >0.2 and XCorr values (cross-correlation) of greater than 2, 2, 3, 4 for +1, +2, +3, +4 charged peptides, respectively (supplemental Tables 1 and 2). A particular protein would only be considered present if at least five such high quality peptides were detected. Three independent mass spectrometry experiments were performed. Proteins identified in pHα2 mice were compared with those found in WT animals to control for nonspecific binding of proteins. Proteins found at similar levels to a list of nonspecific binding proteins often found in mass spectrometry experiments were removed (
      • Mellacheruvu D.
      • Wright Z.
      • Couzens A.L.
      • Lambert J.P.
      • St-Denis N.A.
      • Li T.
      • Miteva Y.V.
      • Hauri S.
      • Sardiu M.E.
      • Low T.Y.
      • Halim V.A.
      • Bagshaw R.D.
      • Hubner N.C.
      • Al-Hakim A.
      • Bouchard A.
      • et al.
      The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.
      ). For tandem IP experiments, proteins were identified by a minimum of seven peptides. Peptides found in WT control IPs were removed from the final list of proteins displayed in Table 1. For GFP-Trap IPs, proteins listed in TABLE 2, TABLE 3, TABLE 4, TABLE 5, TABLE 6, TABLE 7 have been identified by a minimum of five peptide, or were at least 3-fold enriched in the pHα2 compared with WT IPs. Furthermore, these peptides were present in all three experiments. Proteins in TABLE 2, TABLE 3, TABLE 4, TABLE 5, TABLE 6, TABLE 7 were manually organized into broad functions through information from GeneCards, HUGO gene nomenclature committee, and the literature.
      TABLE 1Proteins identified with pHα2 identified using tandem myc/GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      GFP-AequoreaGreen fluorescent protein034
      Atp1a1ATA1_MOUSENa+/K+-transporting ATPase subunit α109
      Gabra1GBRA1_MOUSEGABAAR, subunit α1070
      Gabra2GBRA2_MOUSEGABAAR, subunit α2015
      Gabra3GBRA3_MOUSEGABAAR, subunit α308
      Gabra4GBRA4_MOUSEGABAAR, subunit α4014
      Gabra5GBRA5_MOUSEGABAAR, subunit α5011
      Gabrb1GBRB1_MOUSEGABAAR, subunit β1023
      Gabrb2GBRB2_MOUSEGABAAR, subunit β2017
      Gabrb3GBRB3_MOUSEGABAAR, subunit β3040
      Gabrg2GBRG2_MOUSEGABAAR, subunit γ2010
      TABLE 7Miscellaneous proteins associated with pHα2 identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      Appl1DP13A_MOUSEAdaptor protein, phosphotyrosine interaction, PH domain, and leucine zipper containing 129
      Armc10ARM10_MOUSEArmadillo repeat containing 1006
      Avl9AVL9_MOUSEAVL9 homolog (Saccharomyces cerevisiae)415
      Bcl2l13B2L13_MOUSEBCL2-like 13 (apoptosis facilitator)19
      Chchd3CHCH3_MOUSECoiled-coil-helix-coiled-coil-helix domain containing 3223
      Chchd6CHCH6_MOUSECoiled-coil-helix-coiled-coil-helix domain containing 6113
      CluCLUS_MOUSEClusterin114
      Cyc1CY1_MOUSECytochrome c-1625
      Eif2b5EI2BE_MOUSEEukaryotic translation initiation factor 2B subunit ϵ019
      Fam49aFA49A_MOUSEFamily with sequence similarity 49 member A623
      Fam49bFA49B_MOUSEFamily with sequence similarity 49 member B522
      Fam73bFA73B_MOUSEFamily with sequence similarity 73 member B17
      Hbs1lHBS1L_MOUSEHBS1-like translational GTPase19
      ImmtIMMT_MOUSEInner membrane protein, mitochondrial40210
      Kctd12KCD12_MOUSEPotassium channel tetramerization domain containing 1209
      Lhfpl4LHPL4_MOUSELipoma HMGIC fusion partner-like 406
      Lin7aLIN7A_MOUSELin-7 homolog A (Caenorhabditis elegans)29
      MogMOG_MOUSEMyelin oligodendrocyte glycoprotein110
      NbeaNBEA_MOUSENeurobeachin537
      Pgrmc1PGRC1_MOUSEProgesterone receptor membrane component 1210
      PhbPHB_MOUSEProhibitin211
      Phb2PHB2_MOUSEProhibitin 2311
      Plxdc1PLDX1_MOUSEPlexin domain containing 107
      Prrt2PRRT2_MOUSEProline-rich transmembrane protein 217
      Samm50SAM50_MOUSESAMM50 sorting and assembly machinery component117
      Shisa7SHSA7_MOUSEShisa family member 718
      Tmem132bF7BAB2_MOUSETransmembrane protein 132B06
      Ywhab1433B_MOUSETyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, β828
      Ywhag1433G_MOUSETyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, γ1042
      Ywhah1433F_MOUSETyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, η930
      Ywhaz1433Z_MOUSETyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, ζ1167
      Zer1ZER1_MOUSEZyg-11 related, cell cycle regulator210
      TABLE 6Miscellaneous enzyme activities associated with pHα2 identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      Acsbg1ACBG1_MOUSEAcyl-CoA synthetase bubblegum family member 11239
      Acsl3ACSL3_MOUSEAcyl-CoA synthetase long-chain family member 317
      Acsl4ACSL4_MOUSEAcyl-CoA synthetase long-chain family member 429
      Acss2ACSA_MOUSEAcyl-CoA synthetase short-chain family member 2213
      AdprhADPRH_MOUSEADP-ribosylarginine hydrolase06
      Aldh18a1P5CS_MOUSEAldehyde dehydrogenase 18 family member A1627
      Ca2CAH2_MOUSECarbonic anhydrase II210
      Capn2CAN2_MOUSECalpain 2, (m/II) large subunit212
      Cds2CDS2_MOUSECDP-diacylglycerol synthase 2115
      Cpt1aCPT1A_MOUSECarnitine palmitoyltransferase 1A (liver)113
      Dpp3DPP3_MOUSEDipeptidyl-peptidase 318
      Echs1ECHM_MOUSEEnoyl-CoA hydratase, short chain, 1, mitochondrial09
      Eci1ECI1_MOUSEEnoyl-CoA δ isomerase 1311
      Gfpt1GFPT1_MOUSEGlutamine-fructose-6-phosphate transaminase 1113
      Gstz1MAAI_MOUSEGlutathione S-transferase ζ106
      Gucy1a2F8VQK3_MOUSEGuanylate cyclase 1, soluble, α2415
      Hsd17b8DHB8_MOUSEHydroxysteroid (17-β) dehydrogenase 8518
      MpstTHTM_MOUSEMercaptopyruvate sulfurtransferase29
      MutMUTA_MOUSEMethylmalonyl-CoA mutase311
      Ndufs1NDUS1_MOUSENADH:ubiquinone oxidoreductase core subunit S135117
      Ndufs3NDUS3_MOUSENADH:ubiquinone oxidoreductase core subunit S3519
      Pank4PANK4_MOUSEPantothenate kinase 4110
      PfklK6PL_MOUSEPhosphofructokinase, liver2268
      Plcd1PLCD1_MOUSEPhospholipase C δ 1119
      Rpn2RPN2_MOUSERibophorin II413
      SrrSRR_MOUSESerine racemase06
      TarsSYTC_MOUSEThreonyl-tRNA synthetase928
      TABLE 5Regulators of GTP exchange and protein phosphorylation associated with pHα2 identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      Adrbk1ARBK1_MOUSEAdrenergic, β, receptor kinase 1427
      Arfgef3BIG3_MOUSEARFGEF family member 3111
      Atl1ATLA1_MOUSEAtlastin GTPase 118
      Dnm1lDNM1L_MOUSEDynamin 1-like1761
      Elmo1ELMO1_MOUSEEngulfment and cell motility 106
      Gnl1GNL1_MOUSEGuanine nucleotide-binding protein-like 1415
      Gpsm1GPSM1_MOUSEG-protein signaling modulator 1114
      Iqsec3IQEC3_MOUSEIQ motif and Sec7 domain 3013
      Lppr4LPPR4_MOUSEPhospholipid phosphatase-related 4867
      Mfn2MFN2_MOUSEMitofusin 2832
      Nedd4lNED4L_MOUSENeural precursor cell expressed, developmentally down-regulated 4-like, E3 ubiquitin protein ligase17
      NgefNGEF_MOUSENeuronal guanine nucleotide exchange factor537
      Opa1OPA1_MOUSEOptic atrophy 1 (autosomal dominant)1032
      Pak7PAK7_MOUSEp21 protein (Cdc42/Rac)-activated kinase 706
      Ppm1ePPM1E_MOUSEProtein phosphatase, Mg2+/Mn2+-dependent 1E17
      PtprdPTPRD_MOUSEProtein-tyrosine phosphatase, receptor type D632
      PtprsPTPRS_MOUSEProtein-tyrosine phosphatase, receptor type S629
      Rab14RAB14_MOUSERAB14, member RAS oncogene family831
      Rab1bRAB1B_MOUSERAB1B, member RAS oncogene family18
      Rab33bRB33B_MOUSERAB33B, member RAS oncogene family211
      Rab5aRAB5A_MOUSERAB5A, member RAS oncogene family111
      Rab5bRAB5B_MOUSERAB5B, member RAS oncogene family07
      Rhot1MIRO1_MOUSERas homolog family member T1, Miro1425
      Ric8aRIC8A_MOUSERIC8 guanine nucleotide exchange factor A210
      Tbc1d15TBC15_MOUSETBC1 domain family member 1518
      Tbc1d17TBC17_MOUSETBC1 domain family member 1729
      TABLE 4Regulators of protein trafficking, stability, and cytoskeletal targeting associated with pHα2 identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      Adam22ADA22_MOUSEADAM metallopeptidase domain 2209
      Add3ADDG_MOUSEAdducin 3417
      Afg3l2AFG32_MOUSEAFG3-like AAA ATPase 2842
      Cul1CUL1_MOUSECullin 1222
      Cul2CUL2_MOUSECullin 2317
      Cul3CUL3_MOUSECullin 3521
      Dcaf8DCAF8_MOUSEDDB1- and CUL4-associated factor 807
      Ddb1DDB1_MOUSEDamage-specific DNA-binding protein 1110
      Dnaja1DNJA1_MOUSEDnaJ heat shock protein family (Hsp40) member A1211
      Dync1i2DC1I2_MOUSEDynein, cytoplasmic 1, intermediate chain 2313
      Epn1EPN1_MOUSEEpsin 108
      Erlin1ERLN1_MOUSEEndoplasmic reticulum lipid raft-associated 1313
      Exoc3EXOC3_MOUSEExocyst complex component 3118
      Exoc7EXOC7_MOUSEExocyst complex component 7128
      Exoc8EXOC8_MOUSEExocyst complex component 8213
      Hook3HOOK3_MOUSEHook microtubule-tethering protein 318
      Ipo9IPO9_MOUSEImportin 9210
      Kbtbd7G5E8C2_MOUSEKelch repeat and BTB (POZ) domain containing 7013
      Kif3aKIF3A_MOUSEKinesin family member 3A726
      Lrrc7LRRC7_MOUSELeucine-rich repeat containing 717
      Magi3MAGI3_MOUSEMembrane-associated guanylate kinase, WW, and PDZ domain containing 3014
      Mapre2MARE2_MOUSEMicrotubule-associated protein RP/EB family member 2010
      NapaSNAA_MOUSENSF attachment protein α522
      NapbSNAB_MOUSENSF attachment protein β215
      NeflNFL_MOUSENeurofilament, light polypeptide311
      Ngly1NGLY1_MOUSEN-Glycanase 1010
      Os9OS9_MOUSEOsteosarcoma-amplified 9, endoplasmic reticulum lectin08
      Psmd9PSMD9_MOUSEProteasome 26S subunit, non-ATPase 929
      Scamp3SCAM3_MOUSESecretory carrier membrane protein 308
      Sec23bSC23B_MOUSESec23 homolog B, COPII coat complex component09
      Sqstm1SQSTM_MOUSESequestosome 118
      Sv2aSV2A_MOUSESynaptic vesicle glycoprotein 2A1564
      Sv2bSV2B_MOUSESynaptic vesicle glycoprotein 2B635
      Trim32TRI32_MOUSETripartite motif containing 32724
      Uchl1UCHL1_MOUSEUbiquitin C-terminal hydrolase L1722
      Usp9xUSP9X_MOUSEUbiquitin-specific peptidase 9, X-linked212
      Vps35VPS35_MOUSEVPS35 retromer complex component2169
      Vps52VPS52_MOUSEVPS52 GARP complex subunit221
      TABLE 3G-protein-coupled receptors, ion channels, and transporters associated with pHα2 identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      Abcf2ABCF2_MOUSEATP binding cassette subfamily F member 217
      Abcf3ABCF3_MOUSEATP binding cassette subfamily F member 3214
      Bai1BAI1_MOUSEAdhesion of G protein-coupled receptor B106
      Bai2BAI2_MOUSEAdhesion of G protein-coupled receptor B207
      Cacna1eCAC1E_MOUSECalcium channel, voltage-dependent, R type, α1E subunit115
      Cacnb1CACB1_MOUSECalcium channel, voltage-dependent, β1 subunit017
      Cacnb3CACB3_MOUSECalcium channel, voltage-dependent, β3 subunit114
      Cacnb4CACB4_MOUSECalcium channel, voltage-dependent, β4 subunit413
      Grm5GRM5_MOUSEGlutamate receptor, metabotropic 506
      Kcna1KCNA1_MOUSEPotassium channel, voltage-gated shaker-related subfamily A, member 1224
      Kcna2KCNA2_MOUSEPotassium channel, voltage-gated shaker-related subfamily A, member 229
      Kcna3KCNA3_MOUSEPotassium channel, voltage-gated shaker-related subfamily A, member 308
      Kcnb1KCNB1_MOUSEPotassium channel, voltage-gated Shab-related subfamily B, member 117
      Lphn3LPHN3_MOUSEAdhesion G protein-coupled receptor L3010
      Slc1a1EAA3_MOUSESolute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 107
      Slc1a3EAA1_MOUSESolute carrier family 1 (glial high affinity glutamate transporter), member 31855
      Slc24a2Q14BI1_MOUSESolute carrier family 24 (sodium/potassium/calcium exchanger), member 2013
      Slc25a11M2OM_MOUSESolute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11941
      Slc25a23SCMC3_MOUSESolute carrier family 25 (mitochondrial carrier; phosphate carrier), member 2307
      Slc25a3MPCP_MOUSESolute carrier family 25 (mitochondrial carrier; phosphate carrier), member 31649
      Slc25a4ADT1_MOUSESolute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 41460
      Slc25a5ADT2_MOUSESolute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 51350
      Slc27a1S27A1_MOUSESolute carrier family 27 (fatty acid transporter), member 1010
      Slc2a3GTR3_MOUSESolute carrier family 2 (facilitated glucose transporter), member 3211
      Slc4a10S4A10_MOUSESolute carrier family 4, sodium bicarbonate transporter, member 10013
      Ttyh3TTYH3_MOUSETweety family member 3213
      TABLE 2Known binding partners of GABAAR subunits and their closely associated proteins identified using GFP-Trap purification
      Gene symbolReferenceNameTotal peptide
      WTpHα2
      GFP_AequoreaGreen fluorescent protein2855
      Arhgef9ARHG9_MOUSECdc42 guanine nucleotide exchange factor 9, collybistin162
      Gabbr2GABR2_MOUSEγ-Aminobutyric acid (GABA) B receptor, 2216
      Gabra1GBRA1_MOUSEγ-Aminobutyric acid (GABA) A receptor, α110501
      Gabra2GBRA2_MOUSEγ-Aminobutyric acid (GABA) A receptor, α25341
      Gabra3GBRA3_MOUSEγ-Aminobutyric acid (GABA) A receptor, α33266
      Gabra4GBRA4_MOUSEγ-Aminobutyric acid (GABA) A receptor, α41369
      Gabra5GBRA5_MOUSEγ-Aminobutyric acid (GABA) A receptor, α53146
      Gabrb1GBRB1_MOUSEγ-Aminobutyric acid (GABA) A receptor, β17481
      Gabrb2GBRB2_MOUSEγ-Aminobutyric acid (GABA) A receptor, β26293
      Gabrb3GBRB3_MOUSEγ-Aminobutyric acid (GABA) A receptor, β37422
      GabrdGBRD_MOUSEγ-Aminobutyric acid (GABA) A receptor, δ080
      Gabrg1GBRG1_MOUSEγ-Aminobutyric acid (GABA) A receptor, γ10112
      Gabrg2Q3UVW2_MOUSEγ-Aminobutyric acid (GABA) A receptor, γ209
      Gabrg2GBRG2_MOUSEγ-Aminobutyric acid (GABA) A receptor, γ21198
      Gabrg3GBRG3_MOUSEγ-Aminobutyric acid (GABA) A receptor, γ3056
      GlrbGLRB_MOUSEGlycine receptor β06
      GphnGEPH_MOUSEGephyrin5140
      Nlgn1NLGN1_MOUSENeuroligin 1259
      Nlgn2NLGN2_MOUSENeuroligin 20117
      Nlgn3NLGN3_MOUSENeuroligin 3433
      Nlgn4lNLGN4_MOUSENeuroligin 406
      PrkacbKAPCB_MOUSEProtein kinase, cAMP-dependent, β catalytic subunit17
      PrkcaKPCA_MOUSEProtein kinase C, α1555
      PrkcgKPCG_MOUSEProtein kinase C, γ2795

      Glutathione S-transferase (GST) Production and Pulldown Assay

      GST fusion proteins expressed in Escherichia coli BL21 were induced (0.2 mm isopropyl 1-thio-β-d-galactopyranoside, 2 h), pelleted, and resuspended in buffer A (10 mm Tris-Cl, pH 7.4, 1 mm EDTA, pH 8.0, 1% Triton X-100). After sonication, 2.5× buffer B was added (20 mm HEPES, 100 mm KCl, 0.2 mm EDTA, 20% glycerol), and the lysate was spun down. Supernatants containing GST fusion proteins were immobilized on pre-swollen glutathione-agarose beads (Sigma). Beads were washed five times with buffer B and kept frozen until use.
      Hippocampal and cortical lysates (prepared as above) from male WT mice were pre-cleared with GST alone. These samples were then incubated with GST tagged to various GABAAR subunits immobilized on glutathione-agarose beads overnight. Beads were washed three times (400 × g, 2 min, 4 °C), and bound proteins were detected by immunoblotting. A minimum of three independent GST experiments was performed for each protein studied.

      Human Embryonic Kidney 293 (HEK293) Cell Transfection

      HEK293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37 °C and 5% CO2. HEK293 cells were cotransfected by electroporation (Bio-Rad) with 3 μg of plasmid DNA per construct 40–48 h before experimentation.

      Results

      Creation of a pHluorin/Myc-tagged GABAAR α2 Subunit Knock-in Mouse

      To date, our understanding of the mechanisms responsible for the formation and maintenance of inhibitory synapses has been limited. These issues are confounded by the structural diversity of GABAARs and technical limitations such as the paucity of high affinity subunit-selective antibodies. To overcome these limitations, mice were created in which pHluorin, a pH-sensitive GFP, and the Myc epitope (EQKLISEEDL, Fig. 1, A and E) were introduced into the GABAAR α2 subunit. These reporters were introduced into exon 3 of the GABAAR α2 subunit gene between the codons encoding amino acids 4 and 5 of the mature protein (pHα2). This location was chosen because studies in expression systems suggest that the respective modifications are functionally silent (
      • Jacob T.C.
      • Michels G.
      • Silayeva L.
      • Haydon J.
      • Succol F.
      • Moss S.J.
      Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition.
      ,
      • Jacob T.C.
      • Bogdanov Y.D.
      • Magnus C.
      • Saliba R.S.
      • Kittler J.T.
      • Haydon P.G.
      • Moss S.J.
      Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.
      ). pHα2 mice were created using homologous recombination in ES cells, blastocyst injection, and Cre-mediated excision of the neomycin selection marker (Fig. 1B). Mice were genotyped by PCR using primers that detect the presence of pHluorin insertion (Fig. 1C), and the respective mice were backcrossed on the C57BL/6J background in excess of 10 generations. The presence of the pHluorin and Myc reporters was confirmed by DNA sequencing (Fig. 1E).
      Figure thumbnail gr1
      FIGURE 1Construction of pHluorin-Myc-tagged GABAAR α2 mouse. A, schematic representation of pHluorin-Myc tagged at the N terminus of the GABAAR α2 subunit. B, illustrations of the targeting vector and the targeted α2 subunit gene with addition of pHluorin-myc into exon 3. C, genotyping for wild type (−/−), heterozygotes (+/−), and pHα2 (+/+) mice using primers flanking pHluorin. D, cresyl violet staining of hippocampus shows there are no gross morphological changes in the hippocampal anatomy of pHα2 mice. Scale bar, 500 μm. E, DNA and protein sequence of N-terminal segment of pHα2 knock-in mouse. pHluorin (green, italics) and Myc (red, underline) reporters are depicted.

      pHα2 Subunit Is Associated with Endogenous GABAAR Subunits and Known Receptor-associated Proteins

      pHα2 homozygotes were viable, bred normally, and did not exhibit any overt phenotypes. In addition, Nissl staining did not reveal any gross abnormalities in the structure of the hippocampus between WT and pHα2 mice (Fig. 1D). To confirm the expression of the pHα2 subunit, immunoblotting was utilized with α2 subunit antibodies. In accordance with the addition of pHluorin, the molecular mass of the α2 subunit was increased by ∼30 kDa in extracts prepared from pHα2 homozygotes compared with WT (Fig. 2A). However, there were no significant differences in the total expression levels of the GABAARs α4 and β3 subunit, GAPDH, gephyrin, NMDA receptor NR1 subunit, and tubulin in pHα2 mice compared with wild type animals (Fig. 2B; p > 0.05).
      Figure thumbnail gr2
      FIGURE 2Characterization of pHα2 mice. A, representative Western blots of hippocampal lysates from WT and pHα2 mice. The pHluorin-Myc tag increases the molecular weight of the GABAAR α2 subunit. B, pooled quantification of protein expression shows there are no significant differences in the total expression levels of GABAAR α4 (p = 0.80, t test, n = 5), β3 (p = 0.78, t test, n = 5), GAPDH (p = 0.99, t test, n = 4), gephyrin (p = 0.46, t test, n = 5), NMDAR NR1 (p = 0.09, t test, n = 5), and tubulin (Tub) (p = 0.99, t test, n = 4) between the two genotypes. Data represent means ± S.E.
      Plasma membrane accumulation of the α2 subunit is dependent upon oligomerization with receptor β subunits (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ,
      • Connolly C.N.
      • Wooltorton J.R.
      • Smart T.G.
      • Moss S.J.
      Subcellular localization of γ-aminobutyric acid type A receptors is determined by receptor β subunits.
      ). To test whether pHα2 subunits are associated with endogenous receptor β subunits, detergent-solubilized brain extracts were subjected to immunoprecipitation with Myc or GFP antibodies. As measured by immunoblotting, the α2 and β3 subunits were detected to immunoprecipitate with Myc or GFP antibodies from pHα2 but not WT brains (Fig. 3, A and B). Molecular, genetic, and biochemical approaches suggest that the multifunctional protein gephyrin and the GDP-GTP exchange factor collybistin play important roles in determining the synaptic accumulation of GABAARs (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ,
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.
      ,
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      Complex role of collybistin and gephyrin in GABAA receptor clustering.
      ). Consistent with this, both of these proteins were detected to immunoprecipitate with Myc/GFP antibodies from pHα2 but not WT brain extracts. Thus, in mouse brain pHα2 assembles with endogenous GABAAR subunits and is associated with gephyrin and collybistin.
      Figure thumbnail gr3
      FIGURE 3Localization of pHα2 at inhibitory synaptic sites. Gephyrin, collybistin, and the GABAAR β3 subunit coIP with pHα2. Hippocampal lysates from WT and pHα2 mice were incubated with Myc (A) or GFP (B) antibody, and bound proteins were detected by Western blotting. Immunoprecipitated pHα2 (GFP and α2 bands at ∼75 kDa) coimmunoprecipitated with GABAAR β3, gephyrin (Gphn), and collybistin (Cb). C, hippocampal neurons from pHα2 mice were stained for GABAAR α2 (red) and the inhibitory synaptic marker gephyrin (blue). Endogenous pHluorin fluorescence (green) colocalized with GABAAR α2 (Pearson's coefficient α2 0.89 ± 0.02, p < 0.001) and gephyrin (Pearson's coefficient gephyrin 0.76 ± 0.02, p < 0.005) staining at inhibitory synapses. n = 12 cells taken from three separate cultures. Scale bar, 30 μm.

      pHα2 Subunits Are Targeted to Functional Inhibitory Synapses

      In the brain, GABAARs containing α2 subunits are highly concentrated at inhibitory synapses (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ,
      • Essrich C.
      • Lorez M.
      • Benson J.A.
      • Fritschy J.M.
      • Lüscher B.
      Postsynaptic clustering of major GABAA receptor subtypes requires the γ2 subunit and gephyrin.
      ,
      • Kneussel M.
      • Brandstätter J.H.
      • Laube B.
      • Stahl S.
      • Müller U.
      • Betz H.
      Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice.
      ). To assess whether this synaptic targeting also occurs in pHα2 mice, 18 days in vitro hippocampal cultures produced from these mice were stained with α2 and gephyrin antibodies and imaged by confocal microscopy. Endogenous green fluorescence colocalized with GABAAR α2 subunit immunoreactivity (Fig. 3C; p < 0.001) at gephyrin-positive postsynaptic inhibitory specializations (Fig. 3C; p < 0.005).
      Next, we compared the properties of phasic and tonic inhibition in the dentate gyrus granule cells (DGGCs) of WT and pHα2 mice (Fig. 4). Examination of sIPSCs revealed that there was no significant difference in the amplitude (Fig. 4B; WT 68.7 ± 1.6 pA, n = 8; pHα2 67.3 ± 2.0 pA, n = 8, p = 0.06), frequency (Fig. 4C; WT 2.7 ± 0.4 Hz, n = 8; pHα2 2.8 ± 0.2 Hz, n = 8, p > 0.99), and decay time (Fig. 4D; WT 4.6 ± 0.1 ms, n = 8; pHα2 4.9 ± 0.1 ms, n = 8, p = 0.82) between genotypes. Similarly, the tonic current amplitude (Fig. 4F; WT 30.1 ± 7.0 pA, n = 9; pHα2 21.0 ± 6.0 pA, n = 10, p = 0.34) and current density (Fig. 4G; WT 1.3 ± 0.3 pA/pF n = 10; pHα2 0.8 ± 0.2 pA/pF n = 9, p = 0.18) were comparable between WT and pHα2 mice.
      Figure thumbnail gr4
      FIGURE 4Phasic and tonic inhibition are unperturbed in pHα2 mice. sIPSCs recorded from DGGCs of WT (black) and pHα2 (gray) mice (A) show no significant differences in their amplitude (p = 0.06, Kolmogorov-Smirnov test, n = 8 cells) (B), frequency (p > 0.99, Mann Whitney test, n = 8) (C), and decay time (p = 0.82, Kolmogorov-Smirnov test, n = 8) (D). Tonic current in DGGCs display no differences in amplitude (p = 0.34, t test, n = 9–10) (E) and current density (p = 0.18, t test, n = 9–10) (F) between genotypes (G).
      Collectively, these data suggest that GABAARs containing pHα2 subunits are targeted to inhibitory synapses, and their incorporation at these subcellular specializations does not have an impact on GABAergic inhibition.

      Isolation of GABAARs from the Brains of pHα2 Mice Using Two-step Tandem Affinity Purification

      To assess which proteins associate with GABAAR subunits in the brain, a two-step immunoaffinity purification protocol was performed. First, hippocampi and cortices from age/sex-matched WT and pHα2 mice were solubilized and exposed to Myc antibody followed by binding to G-Sepharose beads. After extensive washes, bound material was eluted with Myc peptide and exposed to immobilized GFP-Trap beads. Bound material was subsequently eluted using 2% SDS and subjected to SDS-PAGE followed by silver staining. Bands that were present in the pHα2 lane and the adjacent lane from WT mice were excised and subjected to LC-MS/MS (Fig. 5). Three independent purifications were performed for both WT controls and pHα2 animals. Table 1 shows a list of the proteins identified by MS analysis that associate with pHα2. Proteins listed were identified by a minimum of seven peptides. Furthermore, proteins that bound non-specifically (in WT controls) were removed. Using these criteria, the GABAAR α1, α3, α4, α5, β1, β2, β3, and γ2 subunits in addition to the α1 subunit of the Na+/K+-ATPase subunit copurified with the pHα2 (Table 1 and supplemental Table 1). Although there was some contamination between bands, the majority of GFP and α2 subunit peptides were identified in the major silver-stained product at ∼80 kDa. Atp1a1 was found at the 100-kDa region, α4 subunit at the 65-kDa region, and the rest were found in the 50–55-kDa region of the gel. Collectively, these results suggest that pHα2 is capable of assembling with the γ2 and multiple α and β subunit isoforms in the brain.
      Figure thumbnail gr5
      FIGURE 5Two-step purification to isolate pHα2 complexes. Detergent-solubilized hippocampal and cortical lysates of age- and sex-matched WT and pHα2 mice were immunoprecipitated with Myc followed by GFP-Trap and subjected to SDS-PAGE and silver staining (A). Representative silver-stained gel depicts bands of interest (arrow) that were excised from pHα2 and the corresponding WT lane for mass spectrometry analysis. Protein coverage of GABAAR α2 subunit (blue, underline) identified by MS analysis (B). Example of MS/MS spectrum for tryptic peptide identified as GABAAR α2 is shown (C). The sequence of the identified peptide is indicated.

      GFP-Trap Purification of GABAARs Reveals Their Association with Known Binding Partners

      To increase the probability of identifying proteins that are associated with the α2-containing GABAARs, a single purification with GFP-Trap was used. Lysates from hippocampi and cortices of age- and sex-matched WT and pHα2 mice were incubated with GFP-Trap beads. These samples were then subjected to SDS-PAGE followed by Coomassie staining (Fig. 6). The single step purification method led to an increased yield of protein compared with the tandem purification as indicated by the increased number of peptides identified and greater protein coverage for GABAAR α2 (Figs. 5B and 6B; GFP/myc IP 8.4%, GFP IP 43%). Three independent purifications were performed, and proteins identified by LC-MS/MS in all three experiments and found to be at least 3-fold enriched in the pHα2 samples are listed in TABLE 2, TABLE 3, TABLE 4, TABLE 5, TABLE 6, TABLE 7 and supplemental Table 2.
      Figure thumbnail gr6
      FIGURE 6Single-step purification to isolate pHα2 complexes. Detergent-solubilized hippocampal and cortical lysates from WT and pHα2 mice were immunoprecipitated with GFP antibodies and subjected to SDS-PAGE and colloidal Coomassie staining (A). Each gel lane was cut into five pieces and pooled for mass spectrometry analysis. Protein coverage of GABAAR α2 subunit (blue, underline) identified by MS analysis (B). Example of MS/MS spectrum for tryptic peptide identified as GABAAR α2 is shown (C). The sequence of the identified peptide is indicated.
      In common with tandem affinity purification, the single-step GFP purification resulted in the isolation of the GABAAR α1–5, β1–3, and γ2 subunits. However, in addition, the single step purification resulted in the isolation of γ1, γ3, and δ subunits (Table 2). Furthermore, a number of other previously verified interactions were confirmed, including binding of GABAARs or their closely associated proteins to gephyrin (Gphn), collybistin (Arhgef9), neuroligins 1–4 (Nlgn), PKC isoforms (Prkc), PKA (Prkacb), GABABR2 (Gabbr2), and glycine receptor β (Glrb) as described previously (
      • Tretter V.
      • Jacob T.C.
      • Mukherjee J.
      • Fritschy J.M.
      • Pangalos M.N.
      • Moss S.J.
      The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor α2 subunits to gephyrin.
      ,
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      Complex role of collybistin and gephyrin in GABAA receptor clustering.
      ,
      • Connolly C.N.
      • Krishek B.J.
      • McDonald B.J.
      • Smart T.G.
      • Moss S.J.
      Assembly and cell surface expression of heteromeric and homomeric γ-aminobutyric acid type A receptors.
      ,
      • Mukherjee J.
      • Kretschmannova K.
      • Gouzer G.
      • Maric H.M.
      • Ramsden S.
      • Tretter V.
      • Harvey K.
      • Davies P.A.
      • Triller A.
      • Schindelin H.
      • Moss S.J.
      The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin.
      ,
      • Hoon M.
      • Soykan T.
      • Falkenburger B.
      • Hammer M.
      • Patrizi A.
      • Schmidt K.F.
      • Sassoè-Pognetto M.
      • Löwel S.
      • Moser T.
      • Taschenberger H.
      • Brose N.
      • Varoqueaux F.
      Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.
      ,
      • Poulopoulos A.
      • Aramuni G.
      • Meyer G.
      • Soykan T.
      • Hoon M.
      • Papadopoulos T.
      • Zhang M.
      • Paarmann I.
      • Fuchs C.
      • Harvey K.
      • Jedlicka P.
      • Schwarzacher S.W.
      • Betz H.
      • Harvey R.J.
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      ,
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      Identification of a gephyrin binding motif on the glycine receptor β subunit.
      ,
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      Conserved phosphorylation of the intracellular domains of GABA(A) receptor β2 and β3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase.
      ,
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      ). Crucially, a key component of excitatory synapses, the highly abundant PSD95 family of proteins (
      • Sheng M.
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      The postsynaptic organization of synapses.
      ), was absent from these purifications.

      Identification of Novel Components of the GABAAR Proteome Using GFP-Trap Purification

      In addition to known interacting proteins as detailed in Table 2, 149 novel binding partners for GABAARs were identified in material purified from pHα2 animals. For brevity, these proteins were divided into five groups based on literature searches of their presumed functions: 1) G-protein coupled receptors (GPCRs), ion channels, and transporters (Table 3); 2) regulators of protein trafficking, stability, and cytoskeletal anchoring (Table 4); 3) regulators of GTP exchange and protein phosphorylation (Table 5); 4) miscellaneous enzymes (Table 6); and 5) miscellaneous proteins (Table 7). These various binding partners presumably act sequentially to control receptor assembly, forward trafficking in the secretory pathway, trafficking to and stabilization at inhibitory synapses, receptor endocytosis, and endocytic sorting followed by lysosomal or proteosomal degradation.

      Cullin1, Ephexin, KCTD12, Mitofusin2, mGluR5, PAK5/7, and Rab5 Bind to the Intracellular Loop of Specific GABAAR Subunits

      To confirm our MS findings, we examined the binding of selected hits to the intracellular domains of GABAAR subunits. Our initial studies focused on the GPCR mGluR5 (Grm5), the kinase PAK5/7 (Pak7), the GTPases mitofusin2 (Mfn2), and Rab5, the Rho guanine nucleotide exchange factor ephexin (Ngef) and regulator of ubiquitination cullin1 (Cul1) (TABLE 3, TABLE 4, TABLE 5). These proteins were chosen for their range in the total number of peptides identified by MS analysis as follows: from a lower number of peptides (e.g. mGluR5; 0 peptides WT and 6 peptides pHα2) to protein identified by a larger number of peptides (e.g. ephexin; 5 peptides WT and 37 peptides pHα2). In addition, GPCRs and the respective activities have all been previously implicated in regulating GABAAR membrane trafficking (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ). Furthermore, we also assessed the interaction of KCTD12 (Table 7), an auxiliary subunit of GABABRs previously implicated in regulating GABABR signaling and G-protein activation (
      • Schwenk J.
      • Metz M.
      • Zolles G.
      • Turecek R.
      • Fritzius T.
      • Bildl W.
      • Tarusawa E.
      • Kulik A.
      • Unger A.
      • Ivankova K.
      • Seddik R.
      • Tiao J.Y.
      • Rajalu M.
      • Trojanova J.
      • Rohde V.
      • et al.
      Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits.
      ). For these experiments, purified GST fusion proteins encoding the intracellular domains of the receptor α1, α2, β3, and γ2 subunits were exposed to detergent-solubilized brain extracts from WT mice, and bound material was subjected to immunoblotting. Cullin1, a component of an E3 ubiquitin ligase complex (
      • Petroski M.D.
      • Deshaies R.J.
      Function and regulation of cullin-RING ubiquitin ligases.
      ), bound to GST-β3 and γ2 compared with GST alone (Fig. 7A; β3 p < 0.05, γ2 p < 0.05) as did KCTD12 (Fig. 7C; β3 p < 0.05, γ2 p < 0.05). Likewise, mitofusin2, a GTPase localized at the outer mitochondrial membrane (
      • Stuppia G.
      • Rizzo F.
      • Riboldi G.
      • Del Bo R.
      • Nizzardo M.
      • Simone C.
      • Comi G.P.
      • Bresolin N.
      • Corti S.
      MFN2-related neuropathies: Clinical features, molecular pathogenesis and therapeutic perspectives.
      ), bound β3 and γ2 (Fig. 7D; β3 p < 0.001, γ2 p < 0.0001). The GTPase Rab5 is found at endosomes, phagosomes, caveosome, and the plasma membrane (
      • Stenmark H.
      Rab GTPases as coordinators of vesicle traffic.
      ) and has been shown to colocalize with the GABAAR β3 subunit (
      • Smith K.R.
      • Muir J.
      • Rao Y.
      • Browarski M.
      • Gruenig M.C.
      • Sheehan D.F.
      • Haucke V.
      • Kittler J.T.
      Stabilization of GABA(A) receptors at endocytic zones is mediated by an AP2 binding motif within the GABA(A) receptor β3 subunit.
      ). Consistent with these results, Rab5 bound GST-β3 and γ2 (Fig. 7G; β3 p < 0.0001, γ2 p < 0.05). In contrast to this, PAK5/7, a poorly described serine/threonine kinase and downstream effector protein for the Rho GTPase Cdc42 (
      • Wells C.M.
      • Jones G.E.
      The emerging importance of group II PAKs.
      ), bound solely to GST-γ2 (Fig. 7F; γ2 p < 0.05). Furthermore, the RhoGEF ephexin (
      • Shi L.
      • Fu A.K.
      • Ip N.Y.
      Multiple roles of the Rho GEF ephexin1 in synapse remodeling.
      ) bound α2 and β3 (Fig. 7B; α2 p < 0.05, β3 p < 0.0001). Finally, the metabotropic glutamate receptor (mGluR5) previously shown to colocalize with GABAAR subunit α1 (
      • Besheer J.
      • Hodge C.W.
      Pharmacological and anatomical evidence for an interaction between mGluR5- and GABA(A) α1-containing receptors in the discriminative stimulus effects of ethanol.
      ) bound α1, α2, β3, and γ2 (Fig. 7E; α1 p < 0.05, α2 p < 0.05, β3 p < 0.0001; γ2 p < 0.001). Collectively, these data suggest that proteins that copurify with pHα2 from brain extracts bind to the major intracellular domain of specific GABAAR subunits.
      Figure thumbnail gr7
      FIGURE 7Cullin1, ephexin, KCTD12, mitofusin2, mGluR5, PAK5/7 and Rab5 bind the intracellular loop of specific GABAARs. Detergent-solubilized hippocampal and cortical lysates from WT mice were incubated with GST or GST tagged to the large intracellular loop of various GABAARs. Bound proteins including Cul1 (A), ephexin (B), KCTD12 (C), Mfn2 (D), mGluR5 (E), PAK5/7 (F) and Rab5 (G) were detected by immunoblotting. The upper panels show representative immunoblots; the lower panels show Ponceau staining depicting the relative amounts of GST utilized. Graphs show pooled quantification of immunoblots. *, p < 0.05; **, p < 0.001; ***, p < 0.0001 compared with GST alone and #, p < 0.05; ##, p < 0.001; ###, p < 0.0001 compared with other subunits, analysis of variance with Games-Howell post hoc test (due to differences in variance), n = 3–8. Data are means ± S.E.

      mGluR5, Ephexin, and KCTD12 Coimmunoprecipitate with GABAARs

      To extend our studies using fusion proteins, detergent-solubilized brains from WT and pHα2 mice were subjected to immunoprecipitation with GFP antibody. Immunoblotting revealed that β3, mGluR5, KCTD12, ephexin, and GFP immunoprecipitated from pHα2 but not WT mice (Fig. 8A).
      Figure thumbnail gr8
      FIGURE 8Ephexin, KCTD12, and mGluR5 bind pHα2. A, hippocampal and cortical lysates from WT and pHα2 mice immunoprecipitated with GFP-Trap beads. Bound proteins were immunoblotted with mGluR5, GFP, ephexin, β3, and KCTD12 antibodies. B and C, transfection of HEK293 cells with a combination of plasmids encoding pHα2, β3, FLAG-ephexin, and empty vector. Cell lysates were immunoprecipitated with FLAG (B) or GFP (C) and bound proteins were detected by Western blotting.
      The potential interaction of ephexin with GABAARs was of particular interest because ephexin belongs to the same family of GDP-GTP exchange factors (GEFs) as collybistin, a molecule that plays a key role in determining the formation of hippocampal inhibitory synapses (
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      Complex role of collybistin and gephyrin in GABAA receptor clustering.
      ,
      • Papadopoulos T.
      • Korte M.
      • Eulenburg V.
      • Kubota H.
      • Retiounskaia M.
      • Harvey R.J.
      • Harvey K.
      • O'Sullivan G.A.
      • Laube B.
      • Hülsmann S.
      • Geiger J.R.
      • Betz H.
      Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice.
      ). To further corroborate our findings in pHα2 mice, we expressed FLAG-ephexin, pHα2, and β3 in HEK293 cells. Reciprocal immunoprecipitation with FLAG and GFP antibodies revealed the robust association of ephexin with GABAARs in HEK293 cells (Fig. 8B).
      Together, these studies demonstrate that proteins identified by mass spectroscopy can be validated in the brain and in expression systems.

      Discussion

      Inhibitory fast synaptic transmission is critically dependent upon the accumulation and stabilization of selected GABAAR subtypes at inhibitory postsynaptic specializations. To further elucidate the processes neurons utilize to regulate the synaptic accumulation of these critical ligand-gated ion channels, we have created mice in which the α2 subunit is modified with pHluorin and Myc reporters by targeting the respective gene using homologous recombination. These reporters were introduced between residues 4 and 5 of the mature subunit. pHα2 homozygotes were viable and did not exhibit any overt phenotypes but exhibited endogenous fluorescence at inhibitory synapses. Moreover, the properties of sIPSCs and tonic currents, the unitary events that underlie phasic and tonic inhibitory synaptic transmission, were similar between genotypes. Importantly, gephyrin and collybistin, which were previously reported to associate with GABAAR α2 in HEK293 cells (
      • Saiepour L.
      • Fuchs C.
      • Patrizi A.
      • Sassoè-Pognetto M.
      • Harvey R.J.
      • Harvey K.
      Complex role of collybistin and gephyrin in GABAA receptor clustering.
      ), could be shown to coimmunoprecipitate in brain lysates, highlighting the necessity for the tagged protein to enable high affinity purifications.
      Consensus opinion suggests that the α1–3 subunits are components of synaptic GABAARs and that the anxiolytic and sedative properties of benzodiazepines are mediated by specific receptor subtypes containing individual α subunit isoforms. Therefore, we assessed which receptor subunits associate with pHα2 using tandem purification on Myc and GFP antibodies followed by LC-MS/MS. This approach revealed that the pHα2 subunit copurified with α1, α3, α4, α5, β1–3, and γ2 subunits. Using GFP-Trap alone, we further detected association with the γ1, γ3, and δ subunits. Although these results are not quantitative and do not discriminate between surface and intracellular populations, our results do suggest the existence of multiple receptor subtypes with mixed α and/or β subunits, supporting previous observations of the coexistence of different α subunits in a single receptor complex (
      • del Río J.C.
      • Araujo F.
      • Ramos B.
      • Ruano D.
      • Vitorica J.
      Prevalence between different α subunits performing the benzodiazepine binding sites in native heterologous GABA(A) receptors containing the α2 subunit.
      ,
      • Benke D.
      • Fakitsas P.
      • Roggenmoser C.
      • Michel C.
      • Rudolph U.
      • Mohler H.
      Analysis of the presence and abundance of GABAA receptors containing two different types of α subunits in murine brain using point-mutated α subunits.
      ,
      • Benke D.
      • Michel C.
      • Mohler H.
      GABA(A) receptors containing the α4-subunit: prevalence, distribution, pharmacology, and subunit architecture in situ.
      ,
      • Duggan M.J.
      • Pollard S.
      • Stephenson F.A.
      Immunoaffinity purification of GABAA receptor α-subunit iso-oligomers. Demonstration of receptor populations containing α1 α2, α1 α3, and α2 α3 subunit pairs.
      ,
      • Pollard S.
      • Thompson C.L.
      • Stephenson F.A.
      Quantitative characterization of α6 and α1 α6 subunit-containing native γ-aminobutyric acid A receptors of adult rat cerebellum demonstrates two α subunits per receptor oligomer.
      ). Consistent with our results, previous studies to identify proteins associated with the GABAAR α5 subunit through MS analysis exclusively identified other GABAAR subunits, including α1–3, α5, β1–3, and γ2 (
      • Ju Y.H.
      • Guzzo A.
      • Chiu M.W.
      • Taylor P.
      • Moran M.F.
      • Gurd J.W.
      • MacDonald J.F.
      • Orser B.A.
      Distinct properties of murine α5 γ-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy.
      ). A more recent investigation into the proteins associated with the GABAAR α1 subunit isolated 18 associated proteins via MS analysis, more than half of which were other GABAAR subunits (
      • Heller E.A.
      • Zhang W.
      • Selimi F.
      • Earnheart J.C.
      • Ślimak M.A.
      • Santos-Torres J.
      • Ibañez-Tallon I.
      • Aoki C.
      • Chait B.T.
      • Heintz N.
      The biochemical anatomy of cortical inhibitory synapses.
      ), further supporting the possibility of a more heterogeneous population of receptors than originally predicted (
      • Olsen R.W.
      • Sieghart W.
      International Union of Pharmacology. LXX. Subtypes of γ-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update.
      ,
      • Rudolph U.
      • Möhler H.
      Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics.
      ). It is important to note that some of these subunit interactions may represent “non-productive” or non-functional receptor assembly intermediates that are not present on the plasma membrane (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ,
      • Connolly C.N.
      • Krishek B.J.
      • McDonald B.J.
      • Smart T.G.
      • Moss S.J.
      Assembly and cell surface expression of heteromeric and homomeric γ-aminobutyric acid type A receptors.
      ,
      • Gorrie G.H.
      • Vallis Y.
      • Stephenson A.
      • Whitfield J.
      • Browning B.
      • Smart T.G.
      • Moss S.J.
      Assembly of GABAA receptors composed of α1 and β2 subunits in both cultured neurons and fibroblasts.
      ). Because GABAARs are a major target for pharmacological agents such as benzodiazepines, barbiturate, neurosteroids, and general anesthetics (
      • Sieghart W.
      Allosteric modulation of GABAA receptors via multiple drug-binding sites.
      ), the heterogeneity of these receptors may have major implications in the design of subunit-selective drugs for therapeutic use.
      In addition to receptor subunits, we also isolated the known GABAAR binding partners gephyrin, collybistin, PKC, PKA, and GABABR2. To the best of our knowledge, this is the first time that these respective protein-protein interactions have been simultaneously demonstrated for GABAARs in their native environment. The use of a single GFP-Trap protein purification yielded a 174-multiprotein complex comprising 149 novel protein components that copurified with pHα2 compared with material isolated from WT mice. Novel components of the GABAAR complex include other receptors, proteins required for trafficking, ubiquitination/degradation, GTPases and their regulators, cytoskeletal components, and a host of enzymes. Significantly, the PSD95 family of proteins, which is enriched in excitatory synapses (
      • Sheng M.
      • Kim E.
      The postsynaptic organization of synapses.
      ), was absent from these purifications.
      As an initial means of assessing the significance of our MS experiments, we tested the interaction of selected proteins from brain extracts with GST fusion proteins encoding the intracellular domains of GABAAR subunits. Our studies focused on mGluR5, PAK5/7, mitofusin2, Rab5, ephexin. and cullin1 due to the availability of suitable antibodies. All of the proteins bound to the intracellular domains of the receptor α1, α2, β3, or γ2 subunits, confirming the veracity of our GFP-Trap purifications.
      We further validated some of the MS results by demonstrating that mGluR5, KCTD12, and ephexin coIP with pHα2 from brain lysates. We are particularly interested in ephexin due to some similarities with collybistin. Collybistin is a member of the Dbl family of GEFs necessary for the proper clustering of gephyrin and gephyrin-dependent GABAARs (
      • Papadopoulos T.
      • Korte M.
      • Eulenburg V.
      • Kubota H.
      • Retiounskaia M.
      • Harvey R.J.
      • Harvey K.
      • O'Sullivan G.A.
      • Laube B.
      • Hülsmann S.
      • Geiger J.R.
      • Betz H.
      Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice.
      ). Like collybistin, ephexin also belongs to the Dbl family of GEFs and therefore has a similar domain structure to collybistin. Studies on ephexin have described its role in axon guidance in retina ganglion cells (
      • Shamah S.M.
      • Lin M.Z.
      • Goldberg J.L.
      • Estrach S.
      • Sahin M.
      • Hu L.
      • Bazalakova M.
      • Neve R.L.
      • Corfas G.
      • Debant A.
      • Greenberg M.E.
      EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin.
      ) and dispersal of synaptic acetylcholine receptor clusters in the neuromuscular junction through its capacity to activate Rho family GTPases (
      • Shi L.
      • Butt B.
      • Ip F.C.
      • Dai Y.
      • Jiang L.
      • Yung W.H.
      • Greenberg M.E.
      • Fu A.K.
      • Ip N.Y.
      Ephexin1 is required for structural maturation and neurotransmission at the neuromuscular junction.
      ). Numerous regulators of the actin cytoskeleton such as the Rho family GTPases have been demonstrated to be critical for synapse remodeling at excitatory synapses (
      • Tada T.
      • Sheng M.
      Molecular mechanisms of dendritic spine morphogenesis.
      ). In addition, similar roles for the regulation of the actin cytoskeleton at inhibitory GABAergic synapses have only more recently begun to emerge (
      • Smith K.R.
      • Davenport E.C.
      • Wei J.
      • Li X.
      • Pathania M.
      • Vaccaro V.
      • Yan Z.
      • Kittler J.T.
      GIT1 and βPIX are essential for GABA(A) receptor synaptic stability and inhibitory neurotransmission.
      ). Although how ephexin, other GTPases, and GTPase regulators identified here may affect GABAARs remains to be seen, it is tantalizing to speculate that they may have similarly important roles at inhibitory synapses.
      Typical contaminants such as highly abundant proteins (e.g. actin, tubulin, and ribosomal proteins) and proteins that bind unfolded proteins (e.g. heat shock proteins) are commonly found in affinity-purified protein preparations (
      • Gingras A.C.
      • Gstaiger M.
      • Raught B.
      • Aebersold R.
      Analysis of protein complexes using mass spectrometry.
      ). Our use of proper WT controls removed many of these contaminants. Furthermore, the requirement for the detection of proteins from three different experiments unveiled protein binding partners that may weakly but stably form a complex with pHα2. Thus, potential pHα2-associated proteins cannot readily be discarded due to a low number of total peptides discovered. Indeed, although only six peptides were identified for mGluR5, we demonstrated that it was robustly coimmunoprecipitated with pHα2 (Fig. 8A).
      Previously described GABAAR-associated proteins have been demonstrated to be essential for regulatory processes crucial for GABAAR function (
      • Jacob T.C.
      • Moss S.J.
      • Jurd R.
      GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.
      ,
      • Luscher B.
      • Fuchs T.
      • Kilpatrick C.L.
      GABAA receptor trafficking-mediated plasticity of inhibitory synapses.
      ,
      • Charych E.I.
      • Liu F.
      • Moss S.J.
      • Brandon N.J.
      GABA(A) receptors and their associated proteins: implications in the etiology and treatment of schizophrenia and related disorders.
      ). The characterization of the protein components that form the inhibitory synaptic complex described here have wide-ranging ramifications for the understanding of GABAAR activity and trafficking and therefore its role in synaptic transmission and plasticity. The vast majority of proteins purified here are novel putatively GABAAR-associated proteins, indicating that the inhibitory synapse is likely to be far more complex than initially appreciated. Thus, the challenge still remains to elucidate the effects of these associations on GABAARs. Considering the crucial role of GABAAR in brain function, it is of fundamental importance to ascertain the underpinning mechanisms that govern these receptors thereby clarifying its role in CNS health and disease.

      Author Contributions

      Y. N. conducted most of the experiments, analyzed the results, and co-wrote paper. D. H. M. performed PCRs to sequence the mouse and collybistin coIPs, produced GSTs, and provided technical assistance. A. M. performed electrophysiology experiments. D. H. produced GSTs and performed some GST experiments. T. Z. D. performed some electrophysiological experiments. P. A. D. and S. J. M. conceived and coordinated the study and wrote the paper with Y. N. M. J. L. created the pHα2 mouse. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      The FLAG-ephexin construct and ephexin antibody were the generous gifts from Prof. Michael Greenberg (Harvard University). The C-terminal anti-α2 antibody was provided by Dr. Verena Tretter and Prof. Werner Sieghart (Medical University of Vienna).

      Supplementary Material

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