title: PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density.
abstract: The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158;
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Levy
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Austin
authors.initials: A
authors.lastname: Hou
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Christine
authors.initials: C
authors.lastname: Winters
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Rita
authors.initials: R
authors.lastname: Azzam
authors.affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institutes of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Alioscka A
authors.initials: AA
authors.lastname: Sousa
authors.affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institutes of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Leapman
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158;
authors.forename: Roger A
authors.initials: RA
authors.lastname: Nicoll
authors.affiliation: Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; treese@mbl.edu.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Reese
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000076122:Disks Large Homolog 4 Protein; D055785:Gene Knockdown Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D057907:Post-Synaptic Density / Q000378:metabolism*; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate
keywords: AMPAR; EM tomography; MAGUKs; NMDAR; knockdown