title: Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.
abstract: 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.
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Danielle H
authors.initials: DH
authors.lastname: Morrow
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Amit
authors.initials: A
authors.lastname: Modgil
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Deborah
authors.initials: D
authors.lastname: Huyghe
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
authors.affiliation: the Department of Neuroscience, Physiology and Pharmacology, University College, London WC1E 6BT, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lumb
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Davies
authors.affiliation: From the Department of Neuroscience, Tufts University School of Medicine, Boston Massachusetts 02111 and the Department of Neuroscience, Physiology and Pharmacology, University College, London WC1E 6BT, United Kingdom Stephen.Moss@Tufts.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Moss
mesh_terms: D000818:Animals; D015153:Blotting, Western; D055724:Electrophysiological Phenomena; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053444:Inhibitory Postsynaptic Potentials; D013058:Mass Spectrometry; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism* / Q000502:physiology
chemical_list: C485187:PHluorin; D020543:Proteome; D011963:Receptors, GABA-A; D049452:Green Fluorescent Proteins
keywords: GABA receptor; ion channel; mass spectrometry (MS); neurobiology; synapse