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Sample records for ampa receptor structure

  1. Structural and pharmacological characterization of phenylalanine-based AMPA receptor antagonists at kainate receptors

    DEFF Research Database (Denmark)

    Venskutonyte, Raminta; Frydenvang, Karla; Valadés, Elena Antón;

    2012-01-01

    . A new series of phenylalanine derivatives that target iGluRs was reported to bind AMPA receptors. Herein we report our studies of these compounds at the kainate receptors GluK1-3. Several compounds bind with micromolar affinity at GluK1 and GluK3, but do not bind GluK2. The crystal structure of the most...

  2. AMPA receptor ligands

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Mellor, Ian

    2004-01-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player in the f...

  3. Uncompetitive antagonism of AMPA receptors

    DEFF Research Database (Denmark)

    Andersen, Trine F; Tikhonov, Denis B; Bølcho, Ulrik;

    2006-01-01

    Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. ...... polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors....

  4. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives.

    Science.gov (United States)

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn

    2013-01-01

    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders.

  5. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism.

    Science.gov (United States)

    Chen, Lei; Dürr, Katharina L; Gouaux, Eric

    2014-08-29

    AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating.

  6. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes.

    Science.gov (United States)

    Dawe, G Brent; Musgaard, Maria; Aurousseau, Mark R P; Nayeem, Naushaba; Green, Tim; Biggin, Philip C; Bowie, Derek

    2016-03-16

    Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.

  7. Forster Resonance Energy Transfer (FRET) Analysis of Dual CFP/YFP Labeled AMPA Receptors Reveals Structural Rearrangement within the C-Terminal Domain during Receptor Activation

    DEFF Research Database (Denmark)

    Zachariassen, Linda Grønborg; Katchan, Mila; Plested, Andrew;

    2014-01-01

    AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. AMPARs are formed by homo- or heterotetramers of GluA1 to GluA4 sub- units. A recent X-ray crystal structure of a full-length homomeric GluA2 A...

  8. Novel bivalent positive allosteric modulators of AMPA receptor.

    Science.gov (United States)

    Lavrov, M I; Grigor'ev, V V; Bachurin, S O; Palyulin, V A; Zefirov, N S

    2015-01-01

    A positive allosteric modulator of AMPA receptors has been designed using computer-aided molecular modeling techniques. It possessed a record high experimentally confirmed potency in the picomolar concentration range and belongs to a new type of bivalent AMPA receptor ligands containing bicyclo[3.3.1]nonane scaffold. The suggested structure could serve as a basis for further optimization and development of drugs for the treatment of neurodegenerative diseases, cognition enhancement, and improvement of memory.

  9. ABP: a novel AMPA receptor binding protein.

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    Srivastava, S; Ziff, E B

    1999-04-30

    We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor.

  10. Agonist discrimination between AMPA receptor subtypes

    DEFF Research Database (Denmark)

    Coquelle, T; Christensen, J K; Banke, T G

    2000-01-01

    The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H...

  11. A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures

    DEFF Research Database (Denmark)

    Bundgaard Christiansen, Gitte; Harbak, Barbara; E. Hede, Susanne;

    2015-01-01

    -mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two...... information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing...

  12. 3-Substituted phenylalanines as selective AMPA- and kainate receptor ligands

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Pickering, Darryl S; Nielsen, Birgitte;

    2009-01-01

    On the basis of X-ray structures of ionotropic glutamate receptor constructs in complex with amino acid-based AMPA and kainate receptor antagonists, a series of rigid as well as flexible biaromatic alanine derivatives carrying selected hydrogen bond acceptors and donors have been synthesized in o...

  13. AMPA receptor inhibition by synaptically released zinc.

    Science.gov (United States)

    Kalappa, Bopanna I; Anderson, Charles T; Goldberg, Jacob M; Lippard, Stephen J; Tzounopoulos, Thanos

    2015-12-22

    The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.

  14. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea;

    2003-01-01

    -methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational...... design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects....

  15. Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Chao-Yin Chen

    Full Text Available Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM, concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M-1 s-1 and 0.58 s-1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM was similar at all kainate concentrations (3-100 µM, demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.

  16. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    DEFF Research Database (Denmark)

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;

    2007-01-01

    Dimeric positive allosteric modulators of ionotropic glutamate receptors were designed, synthesized, and characterized pharmacologically in electrophysiological experiments. The designed compounds are dimers of arylpropylsulfonamides and have been constructed without a linker. The monomeric...

  17. Sucrose ingestion induces rapid AMPA receptor trafficking.

    Science.gov (United States)

    Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B

    2013-04-03

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

  18. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    DEFF Research Database (Denmark)

    Madsen, U; Sløk, F A; Stensbøl, T B;

    2000-01-01

    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2...

  19. Rational Design of a Novel AMPA Receptor Modulator through a Hybridization Approach

    Science.gov (United States)

    2015-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a family of glutamate ion channels of considerable interest in excitatory neurotransmission and associated disease processes. Here, we demonstrate how exploitation of the available X-ray crystal structure of the receptor ligand binding domain enabled the development of a new class of AMPA receptor positive allosteric modulators (7) through hybridization of known ligands (5 and 6), leading to a novel chemotype with promising pharmacological properties. PMID:25893038

  20. Differences in AMPA and Kainate Receptor Interactomes Facilitate Identification of AMPA Receptor Auxiliary Subunit GSG1L

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    Natalie F. Shanks

    2012-06-01

    Full Text Available AMPA receptor (AMPA-R complexes consist of channel-forming subunits, GluA1-4, and auxiliary proteins, including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. The combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors, we compared the proteins that copurify with each receptor type in the rat brain. This interactome study identified the majority of known interacting proteins and, more importantly, provides candidates for additional studies. We validate the claudin homolog GSG1L as a newly identified binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes, and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function.

  1. Ganglioside Regulation of AMPA Receptor Trafficking

    Science.gov (United States)

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  2. Ganglioside regulation of AMPA receptor trafficking.

    Science.gov (United States)

    Prendergast, Jillian; Umanah, George K E; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G; Cole, Robert N; Huganir, Richard L; Dawson, Ted M; Dawson, Valina L; Schnaar, Ronald L

    2014-09-24

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

  3. Computational study of the evolutionary relationships of the ionotropic receptors NMDA, AMPA and kainate in four species of primates.

    OpenAIRE

    Moreno-Pedraza, Francy Johanna; Grupo de Bioquímica Molecular Computacional y Bioinformática, Departamento de Bioquímica, Facultad de Ciencias. Pontificia Universidad Javeriana, Carrera 7 No. 43-82 Ed. 52, Bogotá,; Lareo, Leonardo René; Grupo de Bioquímica Molecular Computacional y Bioinformática, Departamento de Bioquímica, Facultad de Ciencias. Pontificia Universidad Javeriana, Carrera 7 No. 43-82 Ed. 52, Bogotá,; Reyes-Montaño, Edgar Antonio; Grupo de Investigación en Proteínas (GRIP) Departamento de Química, Universidad Nacional de Colombia, Ciudad Universitaria, Edificio 451, Bogotá

    2010-01-01

    Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA and kainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91 sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites, multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that s...

  4. Dual-specific Phosphatase-6 (Dusp6) and ERK Mediate AMPA Receptor-induced Oligodendrocyte Death*

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    Domercq, Maria; Alberdi, Elena; Sánchez-Gómez, Maria Victoria; Ariz, Usue; Pérez-Samartín, Alberto; Matute, Carlos

    2011-01-01

    Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter. PMID:21300799

  5. Effects of cyclothiazide on GluR1/AMPA receptors

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    Fucile, Sergio; Miledi, Ricardo; Eusebi, Fabrizio

    2006-01-01

    Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, is used frequently to block the desensitization of both native and heterologously expressed AMPA receptors. Specifically, CTZ is known to produce a fast inhibition of AMPA receptor desensitization and a much slower potentiation of the AMPA current. By using patch-clamp techniques, the effects of CTZ were studied in HEK 293 cells stably transfected with the rat flip GluR1 subunit. Upon CTZ treatment, we found an increased apparent affinity for the agonist, a slow whole-cell current potentiation, a fast inhibition of desensitization, and a lengthening of single-channel openings. Furthermore, we show that CTZ alters the channel gating events modifying the relative contribution of different single-channel classes of conductance (γ), increasing and decreasing, respectively, the contributions of γM (medium) and γL (low) without altering that of the γH (high) conductance channels. We also present a kinetic model that predicts well all of the experimental findings of CTZ action. Finally, we suggest a protocol for standard cell treatment with CTZ to attain maximal efficacy of CTZ on GluR1 receptors. PMID:16473938

  6. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    Directory of Open Access Journals (Sweden)

    Lucas R. Watterson

    2013-12-01

    Full Text Available Positive allosteric modulators (PAMs of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications.

  7. Channel-lining residues of the AMPA receptor M2 segment: structural environment of the Q/R site and identification of the selectivity filter.

    Science.gov (United States)

    Kuner, T; Beck, C; Sakmann, B; Seeburg, P H

    2001-06-15

    In AMPA receptor channels, a single amino acid residue (Q/R site) of the M2 segment controls permeation of calcium ions, single-channel conductance, blockade by intracellular polyamines, and permeation of anions. The structural environment of the Q/R site and its positioning with regard to a narrow constriction were probed with the accessibility of substituted cysteines to positively and negatively charged methanethiosulfonate reagents, applied from the extracellular and cytoplasmic sides of the channel. The accessibility patterns confirm that the M2 segment forms a pore loop with the Q/R site positioned at the tip of the loop (position 0) facing the extracellular vestibule. Cytoplasmically accessible residues on the N- and C-terminal sides of position 0 form the ascending alpha-helical (-8 to -1) and descending random coil (+1 to +6) components of the loop, respectively. Substitution of a glycine residue at position +2 with alanine strongly decreased the permeability of organic cations, indicating that position +2 contributes to the narrow constriction. The anionic 2-sulfonatoethyl-methanethiosufonate reacted with a cysteine at position 0 only from the external side and with cysteines at positions +1 to +4 only from the cytoplasmic side. These results suggest that charge selectivity occurs external to the constriction (+2) and possibly involves interactions of ions with the negative electrostatic potential created by the dipole of the alpha-helix formed by the ascending limb of the loop.

  8. AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S;

    1998-01-01

    was blocked was seen as early as 5 DIV since 10 microM MK-801 did not completely block the response whereas 10 microM NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity...

  9. AMPA receptor potentiation can prevent ethanol-induced intoxication.

    Science.gov (United States)

    Jones, Nicholas; Messenger, Marcus J; O'Neill, Michael J; Oldershaw, Anna; Gilmour, Gary; Simmons, Rosa M A; Iyengar, Smriti; Libri, Vincenzo; Tricklebank, Mark; Williams, Steve C R

    2008-06-01

    We present a substantial series of behavioral and imaging experiments, which demonstrate, for the first time, that increasing AMPA receptor-mediated neurotransmission via administration of potent and selective biarylsulfonamide AMPA potentiators LY404187 and LY451395 reverses the central effects of an acutely intoxicating dose of ethanol in the rat. Using pharmacological magnetic resonance imaging (phMRI), we observed that LY404187 attenuated ethanol-induced reductions in blood oxygenation level dependent (BOLD) in the anesthetized rat brain. A similar attenuation was apparent when measuring local cerebral glucose utilization (LCGU) via C14-2-deoxyglucose autoradiography in freely moving conscious rats. Both LY404187 and LY451395 significantly and dose-dependently reversed ethanol-induced deficits in both motor coordination and disruptions in an operant task where animals were trained to press a lever for food reward. Both prophylactic and acute intervention treatment with LY404187 reversed ethanol-induced deficits in motor coordination. Given that LY451395 and related AMPA receptor potentiators/ampakines are tolerated in both healthy volunteers and elderly patients, these data suggest that such compounds may form a potential management strategy for acute alcohol intoxication.

  10. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses.

    Science.gov (United States)

    Wang, Xulong; Lippi, Giordano; Carlson, David M; Berg, Darwin K

    2013-12-01

    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

  11. Auxiliary Subunits: Shepherding AMPA Receptors to the Plasma Membrane

    Directory of Open Access Journals (Sweden)

    Simon C. Haering

    2014-08-01

    Full Text Available Ionotropic glutamate receptors (iGluRs are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors (AMPARs mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP and long-term depression (LTD. Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs, suppressor of lurcher (SOL, cornichon homologues (CNIHs, synapse differentiation-induced gene I (SynDIG I, cysteine-knot AMPAR modulating proteins 44 (CKAMP44, and germ cell-specific gene 1-like (GSG1L protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.

  12. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

    DEFF Research Database (Denmark)

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L;

    2013-01-01

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling...

  13. mTOR is essential for corticosteroid effects on hippocampal AMPA receptor function and fear memory

    NARCIS (Netherlands)

    Xiong, H.; Cassé, F.; Zhou, Y.; Zhou, M.; Xiong, Z.Q.; Joëls, M.; Martin, S.; Krugers, H.J.

    2015-01-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging flu

  14. Hormonal regulation of AMPA receptor trafficking and memory formation

    Directory of Open Access Journals (Sweden)

    Harmen J Krugers

    2009-10-01

    Full Text Available Humans and rodents retain memories for stressful events very well. The facilitated retention of these memories is normally very useful. However, in susceptible individuals a variety of pathological conditions may develop in which memories related to stressful events remain inappropriately present, such as in post-traumatic stress disorder. The memory enhancing effects of stress are mediated by hormones, such as norepinephrine and glucocorticoids which are released during stressful experiences. Here we review recently identified molecular mechanisms that underlie the effects of stress hormones on synaptic efficacy and learning and memory. We discuss AMPA receptors as major target for stress hormones and describe a model in which norepinephrine and glucocorticoids are able to strengthen and prolong different phases of stressful memories.

  15. Synthesis and in vitro pharmacology at AMPA and kainate preferring glutamate receptors of 4-heteroarylmethylidene glutamate analogues

    DEFF Research Database (Denmark)

    Valgeirsson, Jon; Christensen, Jeppe K; Kristensen, Anders S;

    2003-01-01

    2-Amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (1) is a potent AMPA receptor agonist with moderate affinity for native kainic acid (KA) receptors, whereas (S)-E-4-(2,2-dimethylpropylidene)glutamic acid (3) show high affinity for the GluR5 subtype of KA receptors and much lower...... affinity for the GluR2 subtype of AMPA receptors. As an attempt to develop new pharmacological tools for studies of GluR5 receptors, (S)-E-4-(2-thiazolylmethylene)glutamic acid (4a) was designed as a structural hybrid between 1 and 3. 4a was shown to be a potent GluR5 agonist and a high affinity ligand...

  16. Effects of Food Restriction and Sucrose Intake on Synaptic Delivery of AMPA Receptors in Nucleus Accumbens

    OpenAIRE

    Peng, Xing-Xiang; Ziff, Edward B.; Carr, Kenneth D.

    2011-01-01

    Insertion and removal of AMPA receptors from the synaptic membrane underlie dynamic tuning of synaptic transmission and enduring changes in synaptic strength. Preclinical addiction research suggests that AMPA receptor trafficking plays an important role in nucleus accumbens (NAc) neuroplasticity underlying the compulsive and persistent quality of drug-seeking. Considering the parallels between drug addiction and compulsive eating, plus the supranormal reward properties of sucrose, and the rol...

  17. NMDA and AMPA receptors contribute to the maintenance of substance P-induced thermal hyperalgesia.

    Science.gov (United States)

    Nakayama, Tomohiro; Naono, Rumi; Ikeda, Tetsuya; Nishimori, Toshikazu

    2010-05-01

    It is well known that intrathecal administration of substance P (SP) induces thermal hyperalgesia, but the mechanisms underlying the maintenance of SP-induced thermal hyperalgesia remain to be clarified. Thus, to clarify the receptors involved in the maintenance of SP-induced thermal hyperalgesia, the effect of administering SP or glutamate receptor agonists, NMDA or AMPA, under SP-induced thermal hyperalgesia was investigated. Also, the effect of pretreatment with protein kinase inhibitors on scratching behavior by NMDA or AMPA under SP-induced thermal hyperalgesia was examined. Under SP-induced thermal hyperalgesia, the number of scratchings following SP administration was time-dependently suppressed, whereas the number of scratchings after NMDA or AMPA administration was markedly enhanced and SP-induced thermal hyperalgesia was attenuated by pretreatment with NMDA or AMPA receptor antagonist. Furthermore, pretreatment with kinase inhibitors significantly attenuated the enhancement of scratching behavior by NMDA or AMPA under SP-induced thermal hyperalgesia. These findings indicate that SP-induced thermal hyperalgesia may be maintained through the enhanced responsiveness of NMDA or AMPA receptors, but not the receptor of SP, mediated by kinases.

  18. Mechanisms for Antagonistic Regulation of AMPA and NMDA-D1 Receptor Complexes at Postsynaptic Sites

    Science.gov (United States)

    Schumann, Johann; Scheler, Gabriele

    2004-01-01

    From the analysis of these pathways we conclude that postsynaptic processes that regulate synaptic transmission undergo significant cross-talk with respect to glutamatergic and neuromodulatory (dopamine) signals. The main hypothesis is that of a compensatory regulation, a competitive switch between the induction of increased AMPA conductance by CaMKII-dependent phosphorylation and reduced expression of PP2A, and increased D1 receptor sensitivity and expression by increased PKA, PP2A and decreased PP-1/calcineurin expression. Both types of plasticity are induced by NMDA receptor activation and increased internal calcium, they require different internal conditions to become expressed. Specifically we propose that AMPA regulation and D1 regulation are inversely coupled;The net result may be a bifurcation of synaptic state into predominantly AMPA or NMDA-D1 synapses. This could have functional consequences: stable connections for AMPA and conditional gating for NMDA-D1 synapses.

  19. Actin-dependent mechanisms in AMPA receptor trafficking

    Directory of Open Access Journals (Sweden)

    Jonathan G Hanley

    2014-11-01

    Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.

  20. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus.

    Science.gov (United States)

    Mitsushima, Dai; Ishihara, Kouji; Sano, Akane; Kessels, Helmut W; Takahashi, Takuya

    2011-07-26

    The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

  1. Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex

    NARCIS (Netherlands)

    Kooijmans, Roxana N; Self, Matthew W; Wouterlood, Floris G; Beliën, Jeroen A M; Roelfsema, Pieter R

    2014-01-01

    Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density a

  2. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  3. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus.

    Science.gov (United States)

    Schmidt, Mathias V; Trümbach, Dietrich; Weber, Peter; Wagner, Klaus; Scharf, Sebastian H; Liebl, Claudia; Datson, Nicole; Namendorf, Christian; Gerlach, Tamara; Kühne, Claudia; Uhr, Manfred; Deussing, Jan M; Wurst, Wolfgang; Binder, Elisabeth B; Holsboer, Florian; Müller, Marianne B

    2010-12-15

    Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.

  4. Induction of Increased Intraceilular Calcium in Astrocytes by Glutamate through Activating NMDA and AMPA Receptors

    Institute of Scientific and Technical Information of China (English)

    张蕲; 胡波; 孙圣刚; 童萼塘

    2003-01-01

    To study the effect of glutamate on the intracellular calcium signal of pure cultured ratastrocytes and the role of NMDA and AMPA receptors in the procedure, the change of calcium sig-nal was investigated by monitoring the fluctuation of intracellular Ca2+ concentration ([Ca2+]i) onthe basis of Fura-2 single cell fluorescent ratio (F345/F380). The changes in the effect of glutamateon the intracellular calcium signal were observed after blockage of NMDA and(or) AMPA recep-tors. It was found that L-glutamate could induce an increased [Ca2+]i in most of the cells in concen-tration- and time-dependent manner. D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5, a selec-tive antagonist of the NMDA receptor) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selec-tive antagonist of the AMPA receptor) could abolish the effects of NMDA and AMPA respectively.Th.e treatment of D-AP-5 and CNQX simultaneously or respectively could attenuate the effect of L-glutamate at varying degrees. All these indicated that glutamate could modulate intracellular Ca2+of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA re-ceptors took part in the complex mechanisms.

  5. NMDA and AMPA receptors mediate intracellular calcium increase in rat cortical astrocytes

    Institute of Scientific and Technical Information of China (English)

    Bo HU; Sheng-gang SUN; E-tang TONG

    2004-01-01

    AIM: To study the effect of glutamate on the intracellular calcium signal of pure cultured rat astrocytes and the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors in the procedure. METHODS: The fluorescence of calcium was measured by Fura-2/AM (F345/F380).RESULTS: L-Glutamate induced [Ca2+]i increase in most of the cells in concentration- and time-dependent manner.NMDA 50 mmol/L induced the fluorescence increase by almost three to four times, while the effect of AMPA 50mmol/L was just half of that of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP-5; a selective antagonist of the NMDA receptor). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, a selective antagonist of the AMPA receptor)abolished the effects of NMDA and AMPA, respectively. D-AP-5 and CNQX simultaneously or respectively attenuated the effect of L-glutamate at different degrees, but could not abolish it entirely. CONCLUSION: Glutamate modulated intracellular Ca2+ of pure cultured rat astrocytes through different pathways. The activation of NMDA and AMPA receptors took part in the complex mechanisms.

  6. mTOR Is Essential for Corticosteroid Effects on Hippocampal AMPA Receptor Function and Fear Memory

    Science.gov (United States)

    Xiong, Hui; Casse, Frédéric; Zhou, Yang; Zhou, Ming; Xiong, Zhi-Qi; Joëls, Marian; Martin, Stéphane; Krugers, Harm J.

    2015-01-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging fluorescent recovery after photobleaching approach…

  7. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-containing NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Li-Jun Li

    2016-10-01

    Full Text Available NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs, but not GluN2B-containing NMDA receptors (GluN2BRs, to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  8. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    Science.gov (United States)

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation.

  9. Synthesis and enantiopharmacology of new AMPA-kainate receptor agonists

    DEFF Research Database (Denmark)

    Conti, P; De Amici, M; De Sarro, G

    1999-01-01

    . The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice...

  10. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    Science.gov (United States)

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  11. Studies on Aryl-Substituted Phenylalanines: Synthesis, Activity, and Different Binding Modes at AMPA Receptors

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Frydenvang, Karla Andrea; Pickering, Darryl S;

    2016-01-01

    A series of racemic aryl-substituted phenylalanines was synthesized and evaluated in vitro at recombinant rat GluA1−3, at GluK1−3, and at native AMPA receptors. The individual enantiomers of two target compounds, (RS)-2-amino-3-(3,4-dichloro-5-(5-hydroxypyridin-3-yl)phenyl)- propanoic acid (37...

  12. C-terminal interactors of the AMPA receptor auxiliary subunit Shisa9.

    Directory of Open Access Journals (Sweden)

    Anna R Karataeva

    Full Text Available Shisa9 (initially named CKAMP44 has been identified as auxiliary subunit of the AMPA-type glutamate receptors and was shown to modulate its physiological properties. Shisa9 is a type-I transmembrane protein and contains a C-terminal PDZ domain that potentially interacts with cytosolic proteins. In this study, we performed a yeast two-hybrid screening that yielded eight PDZ domain-containing interactors of Shisa9, which were independently validated. The identified interactors are known scaffolding proteins residing in the neuronal postsynaptic density. To test whether C-terminal scaffolding interactions of Shisa9 affect synaptic AMPA receptor function in the hippocampus, we disrupted these interactions using a Shisa9 C-terminal mimetic peptide. In the absence of scaffolding interactions of Shisa9, glutamatergic AMPA receptor-mediated synaptic currents in the lateral perforant path of the mouse hippocampus had a faster decay time, and paired-pulse facilitation was reduced. Furthermore, disruption of the PDZ interactions between Shisa9 and its binding partners affected hippocampal network activity. Taken together, our data identifies novel interaction partners of Shisa9, and shows that the C-terminal interactions of Shisa9 through its PDZ domain interaction motif are important for AMPA receptor synaptic and network functions.

  13. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

    Science.gov (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B

    2002-05-01

    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  14. Opposite modulation of brain stimulation reward by NMDA and AMPA receptors in the ventral tegmental area.

    Directory of Open Access Journals (Sweden)

    Charles eDucrot

    2013-10-01

    Full Text Available Previous studies have shown that blockade of ventral midbrain (VM glutamate N-Methyl-D-Aspartate (NMDA receptors induces reward, stimulates forward locomotion and enhances brain stimulation reward. Glutamate induces two types of excitatory response on VM neurons, a fast and short lasting depolarisation mediated by a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA receptors and a longer lasting depolarization mediated by NMDA receptors. A role for the two glutamate receptors in modulation of VM neuronal activity is evidenced by the functional change in AMPA and NMDA synaptic responses that result from repeated exposure to reward. Since both receptors contribute to the action of glutamate on VM neuronal activity, we studied the effects of VM AMPA and NMDA receptor blockade on reward induced by electrical brain stimulation. Experiments were performed on rats trained to self-administer electrical pulses in the medial posterior mesencephalon. Reward thresholds were measured with the curve-shift paradigm before and for two hours after bilateral VM microinjections of the AMPA antagonist, NBQX (2,3,-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(fquinoxaline-7-sulfonamide, 0, 80, and 800 pmol/0.5ul/side and of a single dose (0.825 nmol/0.5ul/side of the NMDA antagonist, PPPA (2R,4S-4-(3-Phosphonopropyl-2-piperidinecarboxylic acid. NBQX produced a dose-dependent increase in reward threshold with no significant change in maximum rate of responding. Whereas PPPA injected at the same VM sites produced a significant time dependent decrease in reward threshold and increase in maximum rate of responding. We found a negative correlation between the magnitude of the attenuation effect of NBQX and the enhancement effect of PPPA; moreover, NBQX and PPPA were most effective when injected respectively into the anterior and posterior VM. These results suggest that glutamate acts on different receptor sub-types, most likely located on different VM neurons, to modulate

  15. Opposite modulation of brain stimulation reward by NMDA and AMPA receptors in the ventral tegmental area.

    Science.gov (United States)

    Ducrot, Charles; Fortier, Emmanuel; Bouchard, Claude; Rompré, Pierre-Paul

    2013-01-01

    Previous studies have shown that blockade of ventral tegmental area (VTA) glutamate N-Methyl-D-Aspartate (NMDA) receptors induces reward, stimulates forward locomotion and enhances brain stimulation reward. Glutamate induces two types of excitatory response on VTA neurons, a fast and short lasting depolarization mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors and a longer lasting depolarization mediated by NMDA receptors. A role for the two glutamate receptors in modulation of VTA neuronal activity is evidenced by the functional change in AMPA and NMDA synaptic responses that result from repeated exposure to reward. Since both receptors contribute to the action of glutamate on VTA neuronal activity, we studied the effects of VTA AMPA and NMDA receptor blockade on reward induced by electrical brain stimulation. Experiments were performed on rats trained to self-administer electrical pulses in the medial posterior mesencephalon. Reward thresholds were measured with the curve-shift paradigm before and for 2 h after bilateral VTA microinjections of the AMPA antagonist, NBQX (2,3,-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide, 0, 80, and 800 pmol/0.5 μl/side) and of a single dose (0.825 nmol/0.5 μl/side) of the NMDA antagonist, PPPA (2R,4S)-4-(3-Phosphonopropyl)-2-piperidinecarboxylic acid). NBQX produced a dose-dependent increase in reward threshold with no significant change in maximum rate of responding. Whereas PPPA injected at the same VTA sites produced a significant time dependent decrease in reward threshold and increase in maximum rate of responding. We found a negative correlation between the magnitude of the attenuation effect of NBQX and the enhancement effect of PPPA; moreover, NBQX and PPPA were most effective when injected, respectively, into the anterior and posterior VTA. These results suggest that glutamate acts on different receptor sub-types, most likely located on different VTA neurons, to

  16. Differential effects of two chronic diazepam treatment regimes on withdrawal anxiety and AMPA receptor characteristics.

    Science.gov (United States)

    Allison, Claire; Pratt, Judith A

    2006-03-01

    Withdrawal from chronic benzodiazepines is associated with increased anxiety and seizure susceptibility. Neuroadaptive changes in neural activity occur in limbo-cortical structures although changes at the level of the GABA(A) receptor do not provide an adequate explanation for these functional changes. We have employed two diazepam treatment regimes known to produce differing effects on withdrawal aversion in the rat and examined whether withdrawal-induced anxiety was accompanied by changes in AMPA receptor characteristics. Rats were given 28 days treatment with diazepam by the intraperitoneal (i.p.) route (5 mg/kg) and the subcutaneous (s.c.) route (15 mg/kg). Withdrawal anxiety in the elevated plus maze was evident in the group withdrawn from chronic s.c. diazepam (relatively more stable plasma levels) but not from the chronic i.p. group (fluctuating daily plasma levels). In the brains of these rats, withdrawal anxiety was accompanied by increased [3H]Ro48 8587 binding in the hippocampus and thalamus, and decreased GluR1 and GluR2 subunit mRNA expression in the amygdala (GluR1 and GluR2) and cortex (GluR1). The pattern of changes was different in the chronic i.p. group where in contrast to the chronic s.c. group, there was reduced [3H]Ro48 8587 binding in the hippocampus and no alterations in GluR1 and GluR2 subunit expression in the amygdala. While both groups showed reduced GluR1 mRNA subunit expression in the cortex overall, only the agranular insular cortex exhibited marked reductions following chronic i.p. diazepam. Striatal GluR2 mRNA expression was increased in the i.p. group but not the s.c. group. Taken together, these data are consistent with differential neuroadaptive processes in AMPA receptor plasticity being important in withdrawal from chronic benzodiazepines. Moreover, these processes may differ both at a regional and receptor function level according to the behavioral manifestations of withdrawal.

  17. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

    Directory of Open Access Journals (Sweden)

    Sayyed Alireza Talaei

    2015-06-01

    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  18. Competitive antagonism of AMPA receptors by ligands of different classes

    DEFF Research Database (Denmark)

    Hogner, Anders; Greenwood, Jeremy R; Liljefors, Tommy;

    2003-01-01

    that ATPO and DNQX stabilize an open form of the ligand-binding core by different sets of interactions. Computational techniques are used to quantify the differences between these two ligands and to map the binding site. The isoxazole moiety of ATPO acts primarily as a spacer, and other scaffolds could......-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) in complex with the ligand-binding core of the receptor. Comparison with the only previous structure of the ligand-binding core in complex with an antagonist, 6,7-dinitro-2,3-quinoxalinedione (DNQX) (Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165-181), reveals...

  19. Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus.

    Science.gov (United States)

    Castilho, A F; Liberal, J T; Baptista, F I; Gaspar, J M; Carvalho, A L; Ambrósio, A F

    2012-09-06

    Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

  20. Bi-directional modulation of AMPA receptor unitary conductance by synaptic activity

    Directory of Open Access Journals (Sweden)

    Matthews Paul

    2004-11-01

    Full Text Available Abstract Background Knowledge of how synapses alter their efficiency of communication is central to the understanding of learning and memory. The most extensively studied forms of synaptic plasticity are long-term potentiation (LTP and its counterpart long-term depression (LTD of AMPA receptor-mediated synaptic transmission. In the CA1 region of the hippocampus, it has been shown that LTP often involves a rapid increase in the unitary conductance of AMPA receptor channels. However, LTP can also occur in the absence of any alteration in AMPA receptor unitary conductance. In the present study we have used whole-cell dendritic recording, failures analysis and non-stationary fluctuation analysis to investigate the mechanism of depotentiation of LTP. Results We find that when LTP involves an increase in unitary conductance, subsequent depotentiation invariably involves the return of unitary conductance to pre-LTP values. In contrast, when LTP does not involve a change in unitary conductance then depotentiation also occurs in the absence of any change in unitary conductance, indicating a reduction in the number of activated receptors as the most likely mechanism. Conclusions These data show that unitary conductance can be bi-directionally modified by synaptic activity. Furthermore, there are at least two distinct mechanisms to restore synaptic strength from a potentiated state, which depend upon the mechanism of the previous potentiation.

  1. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    Science.gov (United States)

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  2. Modification of the philanthotoxin-343 polyamine moiety results in different structure-activity profiles at muscle nicotinic ACh, NMDA and AMPA receptors

    DEFF Research Database (Denmark)

    Mellor, I R; Brier, T J; Pluteanu, F;

    2003-01-01

    -isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups...... of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most...... analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor...

  3. AMPA Receptor-Induced Local Brain-Derived Neurotrophic Factor Signaling Mediates Motor Recovery after Stroke

    OpenAIRE

    Clarkson, Andrew N.; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S. Thomas

    2011-01-01

    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb contr...

  4. Ontogeny of AMPA and NMDA receptor gene expression in the developing sheep white matter and cerebral cortex.

    Science.gov (United States)

    Dean, Justin M; Fraser, Mhoyra; Shelling, Andrew N; Bennet, Laura; George, Sherly; Shaikh, Shamim; Scheepens, Arjan; Gunn, Alistair J

    2005-10-03

    This study examined the hypothesis that the high prevalence of white matter injury in premature infants is associated with increased expression of calcium-permeable forms of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors in pre-myelinating white matter. We characterized expression of subunits of the AMPA, and for reference, the N-methyl-d-aspartate (NMDA), glutamate receptors at 0.5, 0.65, 0.85, and term gestation in the ovine fetal white matter and cerebral cortex. There was a low expression of the critical calcium-impermeable AMPA receptor GluR2 subunit in subcortical white matter both absolutely and relative to other AMPA subunits throughout gestation. In contrast, GluR2 subunit mRNA expression fell in the cerebral cortex with increasing gestation whereas protein expression increased. These findings suggest a vulnerability of subcortical white matter to AMPA receptor-mediated calcium toxicity throughout the second half of gestation. Thus, the hypothesis that AMPA receptor-mediated glutamate toxicity contributes to brain damage in premature infants needs to be revised.

  5. Precision therapy for a new disorder of AMPA receptor recycling due to mutations in ATAD1

    Science.gov (United States)

    Ahrens-Nicklas, Rebecca C.; Umanah, George K.E.; Sondheimer, Neal; Deardorff, Matthew A.; Wilkens, Alisha B.; Conlin, Laura K.; Santani, Avni B.; Nesbitt, Addie; Juulsola, Jane; Ma, Erica; Dawson, Ted M.; Dawson, Valina L.

    2017-01-01

    Objective: ATAD1 encodes Thorase, a mediator of α-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptor recycling; in this work, we characterized the phenotype resulting from ATAD1 mutations and developed a targeted therapy in both mice and humans. Methods: Using exome sequencing, we identified a novel ATAD1 mutation (p.E276X) as the etiology of a devastating neurologic disorder characterized by hypertonia, seizures, and death in a consanguineous family. We postulated that pathogenesis was a result of excessive AMPA receptor activity and designed a targeted therapeutic approach using perampanel, an AMPA-receptor antagonist. Results: Perampanel therapy in ATAD1 knockout mice reversed behavioral defects, normalized brain MRI abnormalities, prevented seizures, and prolonged survival. The ATAD1 patients treated with perampanel showed improvement in hypertonicity and resolution of seizures. Conclusions: This work demonstrates that identification of novel monogenic neurologic disorders and observation of response to targeted therapeutics can provide important insights into human nervous system functioning. PMID:28180185

  6. Estudio computacional de las relaciones evolutivas de los receptores ionotrópicos NMDA, AMPA y kainato en cuatro especies de primates

    Directory of Open Access Journals (Sweden)

    Francy Johanna Moreno-Pedraza

    2010-12-01

    Full Text Available Computational study of the evolutionary relationships of the ionotropic receptors NMDA, AMPA and kainate in four species ofprimates. Objective. To identify the influence of changes on the secondary structure and evolutionary relationship of NMDA, AMPA andkainate receptors in Homo sapiens, Pan troglodytes, Pongo pygmaeus and Macaca mulatta. Materials and methods. We identified 91sequences for NMDA, AMPA and kainate receptors and analyzed with software for predicting secondary structure, phosphorylation sites,multiple alignments, selection of protein evolution models and phylogenetic prediction. Results. We found that subunits GLUR5, NR2A,NR2C and NR3A showed structural changes in the C-terminal region and formation or loss of phosphorylation sites in this zone.Additionally the phylogenetic prediction suggests that the NMDA NR2 subunits are the closest to the ancestral node that gives rise to theother subunits. Conclusions. Changes in structure and phosphorylation sites in GLUR5, NR2A, NR2C and NR3A subunits suggestvariations in the interaction of the C-terminal region with kinase proteins and with proteins with PDZ domains, which could affect thetrafficking and anchoring of the subunits. On the other hand, the phylogenetic prediction suggests that the changes that occurred in the NR2subunits gave rise to the other subunits of glutamate ionotropic receptors, primarily because the NMDA and particularly the NR2D subunitsare the most closely related to the ancestral node that possibly gave rise to the iGluRs.

  7. Role of AMPA and NMDA receptors and back-propagating action potentials in spike timing-dependent plasticity.

    Science.gov (United States)

    Fuenzalida, Marco; Fernández de Sevilla, David; Couve, Alejandro; Buño, Washington

    2010-01-01

    The cellular mechanisms that mediate spike timing-dependent plasticity (STDP) are largely unknown. We studied in vitro in CA1 pyramidal neurons the contribution of AMPA and N-methyl-d-aspartate (NMDA) components of Schaffer collateral (SC) excitatory postsynaptic potentials (EPSPs; EPSP(AMPA) and EPSP(NMDA)) and of the back-propagating action potential (BAP) to the long-term potentiation (LTP) induced by a STDP protocol that consisted in pairing an EPSP and a BAP. Transient blockade of EPSP(AMPA) with 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) during the STDP protocol prevented LTP. Contrastingly LTP was induced under transient inhibition of EPSP(AMPA) by combining SC stimulation, an imposed EPSP(AMPA)-like depolarization, and BAP or by coupling the EPSP(NMDA) evoked under sustained depolarization (approximately -40 mV) and BAP. In Mg(2+)-free solution EPSP(NMDA) and BAP also produced LTP. Suppression of EPSP(NMDA) or BAP always prevented LTP. Thus activation of NMDA receptors and BAPs are needed but not sufficient because AMPA receptor activation is also obligatory for STDP. However, a transient depolarization of another origin that unblocks NMDA receptors and a BAP may also trigger LTP.

  8. Synthesis and Pharmacology of Mono-, Di-, and Trialkyl-Substituted 7-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides Combined with X-ray Structure Analysis to Understand the Unexpected Structure-Activity Relationship at AMPA Receptors

    DEFF Research Database (Denmark)

    Larsen, Anja Probst; Francotte, Pierre; Frydenvang, Karla;

    2016-01-01

    Positive allosteric modulators of 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA)-type ionotropic glutamate receptors are promising compounds for treatment of neurological disorders, for example, Alzheimer's disease. Here, we report synthesis and pharmacological evaluation...

  9. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

    Science.gov (United States)

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

    2002-01-01

    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  10. An AMPA receptor potentiator modulates hippocampal expression of BDNF: an in vivo study.

    Science.gov (United States)

    Mackowiak, Marzena; O'Neill, Michael J; Hicks, Caroline A; Bleakman, David; Skolnick, Phil

    2002-07-01

    AMPA receptor activation has been demonstrated to increase the neuronal expression of brain derived neurotrophic factor (BDNF). In the present study, we investigated the effect of a novel AMPA receptor potentiator (LY404187) and its active isomer (LY451646) on the expression of BDNF protein and mRNA, as well as TrkB mRNA in rat hippocampus. LY404187 administered for 7 days (1 mg/kg) significantly increased the number of BDNF immunopositive cells in the dentate gyrus, but not other hippocampal subfields. Chronic treatment (7 days) with LY451646 (0.5 mg/kg, comparable to 1 mg/kg of LY404187) increased the level of both BDNF and TrkB mRNA expression in the dentate gyrus, CA3 and CA4 of the hippocampus. However, chronic treatment with lower doses of LY451646 (0.125 and 0.25 mg/kg) decreased the level of BDNF and TrkB mRNA in hippocampus, whilst the highest used dose of LY451646 (1 mg/kg) had no effect on BDNF and TrkB mRNA in hippocampus. In contrast, acute treatment with LY451646 produced an increase in BDNF mRNA levels at doses of 0.125 and 0.25 mg/kg in the hippocampus (CA4, CA3 and dentate gyrus, but not in CA1). LY451646 at 0.5 mg/kg had no effect, but at 1.0 mg/kg decreased the level of BDNF mRNA in hippocampus. Acute treatment with LY451646 did not affect the TrkB receptor mRNA levels in hippocampus. Our results demonstrate that biarylpropylsulfonamide AMPA receptor potentiators are capable of modulating the expression of BDNF and TrkB mRNA in a dose- and time-dependent manner. The increase in both BDNF protein and mRNA expression in the dentate gyrus but not in CA1 indicates a specific role of AMPA receptors in the regulation of BDNF expression in this hippocampal subfield. The regulation of BDNF expression by biarylpropylsulfonamids such as LY451646 may have important therapeutical implications for this class of molecule in the treatment of depression and other CNS disorders.

  11. Shape-induced asymmetric diffusion in dendritic spines allows efficient synaptic AMPA receptor trapping.

    Science.gov (United States)

    Kusters, Remy; Kapitein, Lukas C; Hoogenraad, Casper C; Storm, Cornelis

    2013-12-17

    Dendritic spines are the primary postsynaptic sites of excitatory neurotransmission in the brain. They exhibit a remarkable morphological variety, ranging from thin protrusions, to stubby shapes, to bulbous mushroom shapes. The remodeling of spines is thought to regulate the strength of the synaptic connection, which depends vitally on the number and the spatial distribution of AMPA-type glutamate receptors (AMPARs). We present numerical and analytical analyses demonstrating that this shape strongly affects AMPAR diffusion. We report a pronounced suppression of the receptor exit rate out of spines with decreasing neck radius. Thus, mushroomlike spines become highly effective at retaining receptors in the spine head. Moreover, we show that the postsynaptic density further enhances receptor trapping, particularly in mushroomlike spines local exocytosis in the spine head, in contrast to release at the base, provides rapid and specific regulatory control of AMPAR concentration at synapses.

  12. (S)-homo-AMPA, a specific agonist at the mGlu6 subtype of metabotropic glutamic acid receptors

    DEFF Research Database (Denmark)

    Ahmadian, H; Nielsen, B; Bräuner-Osborne, Hans;

    1997-01-01

    of the spectroscopic configurational assignments. The activities of 6 and 7 at ionotropic EAA (iGlu) receptors and at mGlu1-7 were studied. (S)-Homo-AMPA (6) was shown to be a specific agonist at mGlu6 (EC50 = 58 +/- 11 microM) comparable in potency with the endogenous mGlu agonist (S)-glutamic acid (EC50 = 20 +/- 3......Our previous publication (J. Med. Chem. 1996, 39, 3188-3194) described (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (Homo-AMPA) as a highly selective agonist at the mGlu6 subtype of metabotropic excitatory amino acid (EAA) receptors. Homo-AMPA has already become a standard agonist...... microM). Although Homo-AMPA did not show significant effects at iGlu receptors, (R)-Homo-AMPA (7), which was inactive at mGlu1-7, turned out to be a weak N-methyl-D-aspartic acid (NMDA) receptor antagonist (IC50 = 131 +/- 18 microM)....

  13. AMPA receptor modulators have different impact on hippocampal pyramidal cells and interneurons.

    Science.gov (United States)

    Xia, Y-F; Arai, A C

    2005-01-01

    Positive modulators of AMPA receptors enhance synaptic plasticity and memory encoding. Facilitation of AMPA receptor currents not only results in enhanced activation of excitatory neurons but also increases the activity of inhibitory interneurons by up-modulating their excitatory input. However, little is known about the effects of these modulators on cells other than pyramidal neurons and about their impact on local microcircuits. This study examined the effects of members from three subfamilies of modulators (mainly CX516, CX546 and cyclothiazide) on excitatory synaptic responses in four classes of hippocampal CA1 neurons and on excitatory and disynaptically induced inhibitory field potentials in hippocampal slices. Effects on excitatory postsynaptic currents (EPSCs) were examined in pyramidal cells, in two types of inhibitory interneurons located in stratum radiatum and oriens, and in stratum radiatum giant cells, a novel type of excitatory neuron. With CX516, increases in EPSC amplitude in pyramidal cells were two to three times larger than in interneurons and six times larger than in radiatum giant cells. The effects of CX546 on response duration similarly were largest in pyramidal cells. However, this drug also strongly differentiated between stratum oriens and radiatum interneurons with increases being four times larger in the latter. In contrast, cyclothiazide had similar effects on response duration in all cell types. In field recordings, CX516 was several times more potent in enhancing excitatory postsynaptic potentials (EPSPs) than feedback or feedforward circuits, as expected from its larger influence on pyramidal cells. In contrast, BDP-20, a CX546 analog, was more potent in enhancing feedforward inhibition than either EPSPs or feedback inhibition. This preference for feedforward over feedback circuits is probably related to its higher potency in stratum radiatum versus oriens interneurons. Taken together, AMPA receptor modulators differ substantially

  14. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  15. AMPA and GABA receptor antagonists and their interaction in rats with a genetic form of absence epilepsy

    NARCIS (Netherlands)

    Kaminski, R.M.; Rijn, C.M. van; Turski, W.A.; Czuczwar, S.J.; Luijtelaar, E.L.J.M. van

    2001-01-01

    The effects of combined and single administration of the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 7,8-methylenedioxy-1-(4-aminophenyl)-4-methyl-3-acetyl-4,5-dihydro-2,3 -benzodiazepine (LY 300164), and of the GABAB receptor antagonist -aminopropyl-n-butyl-phosp

  16. Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist.

    Science.gov (United States)

    Laprell, L; Hüll, K; Stawski, P; Schön, C; Michalakis, S; Biel, M; Sumser, M P; Trauner, D

    2016-01-20

    Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

  17. Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse.

    Science.gov (United States)

    Panicker, Sandip; Brown, Keith; Nicoll, Roger A

    2008-01-22

    Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

  18. The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

    Science.gov (United States)

    Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

    2012-09-06

    The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents.

  19. Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function

    Science.gov (United States)

    McGee, Thomas P.; Bats, Cécile

    2015-01-01

    AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability of recombinant CP-AMPARs, while increasing polyamine-dependent rectification. To examine the effects of GSG1L on native AMPARs, we manipulated its expression in cerebellar and hippocampal neurons. Transfection of GSG1L into mouse cultured cerebellar stellate cells that lack this protein increased the inward rectification of mEPSCs. Conversely, shRNA-mediated knockdown of endogenous GSG1L in rat cultured hippocampal pyramidal neurons led to an increase in mEPSC amplitude and in the underlying weighted mean single-channel conductance, revealing that GSG1L acts to suppress current flow through native CP-AMPARs. Thus, our data suggest that GSG1L extends the functional repertoire of AMPAR auxiliary subunits, which can act not only to enhance but also diminish current flow through their associated AMPARs. SIGNIFICANCE STATEMENT Calcium-permeable AMPA receptors (CP-AMPARs) are an important group of receptors for the neurotransmitter glutamate. These receptors contribute to various forms of

  20. Evidence for loss of synaptic AMPA receptors in anterior piriform cortex of aged mice.

    Science.gov (United States)

    Gocel, James; Larson, John

    2013-01-01

    It has been suggested that age-related impairments in learning and memory may be due to age-related deficits in long-term potentiation of glutamatergic synaptic transmission. For example, olfactory discrimination learning is significantly affected by aging in mice and this may be due, in part, to diminished synaptic plasticity in piriform cortex. In the present study, we tested for alterations in electrophysiological properties and synaptic transmission in this simple cortical network. Whole-cell recordings were made from principal neurons in slices of anterior piriform cortex from young (3-6 months old) and old (24-28 months) C57Bl/6 mice. Miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were collected from cells in presence of tetrodotoxin (TTX) and held at -80 mV in voltage-clamp. Amplitudes of mEPSCs were significantly reduced in aged mice, suggesting that synaptic AMPA receptor expression is decreased during aging. In a second set of experiments, spontaneous excitatory postsynaptic currents (s/mEPSCs) were recorded in slices from different cohorts of young and old mice, in the absence of TTX. These currents resembled mEPSCs and were similarly reduced in amplitude in old mice. The results represent the first electrophysiological evidence for age-related declines in glutamatergic synaptic function in the mammalian olfactory system.

  1. Phenotype of mice with inducible ablation of GluA1 AMPA receptors during late adolescence: relevance for mental disorders.

    Science.gov (United States)

    Inta, Dragos; Vogt, Miriam A; Elkin, Hasan; Weber, Tillmann; Lima-Ojeda, Juan M; Schneider, Miriam; Luoni, Alessia; Riva, Marco A; Gertz, Karen; Hellmann-Regen, Julian; Kronenberg, Golo; Meyer-Lindenberg, Andreas; Sprengel, Rolf; Gass, Peter

    2014-04-01

    Adolescence is characterized by important molecular and anatomical changes with relevance for the maturation of brain circuitry and cognitive function. This time period is of critical importance in the emergence of several neuropsychiatric disorders accompanied by cognitive impairment, such as affective disorders and schizophrenia. The molecular mechanisms underlying these changes at neuronal level during this specific developmental stage remains however poorly understood. GluA1-containing AMPA receptors, which are located predominantly on hippocampal neurons, are the primary molecular determinants of synaptic plasticity. We investigated here the consequences of the inducible deletion of GluA1 AMPA receptors in glutamatergic neurons during late adolescence. We generated mutant mice with a tamoxifen-inducible deletion of GluA1 under the control of the CamKII promoter for temporally and spatially restricted gene manipulation. GluA1 ablation during late adolescence induced cognitive impairments, but also marked hyperlocomotion and sensorimotor gating deficits. Unlike the global genetic deletion of GluA1, inducible GluA1 ablation during late adolescence resulted in normal sociability. Deletion of GluA1 induced redistribution of GluA2 subunits, suggesting AMPA receptor trafficking deficits. Mutant animals showed increased hippocampal NMDA receptor expression and no change in striatal dopamine concentration. Our data provide new insight into the role of deficient AMPA receptors specifically during late adolescence in inducing several cognitive and behavioral alterations with possible relevance for neuropsychiatric disorders.

  2. Effects of 2,3-benzodiazepine AMPA receptor antagonists on dopamine turnover in the striatum of rats with experimental parkinsonism.

    Science.gov (United States)

    Megyeri, Katalin; Marko, Bernadett; Sziray, Nora; Gacsalyi, Istvan; Juranyi, Zsolt; Levay, Gyorgy; Harsing, Laszlo G

    2007-03-15

    Although levodopa is the current "gold standard" for treatment of Parkinson's disease, there has been disputation on whether AMPA receptor antagonists can be used as adjuvant therapy to improve the effects of levodopa. Systemic administration of levodopa, the precursor of dopamine, increases brain dopamine turnover rate and this elevated turnover is believed to be essential for successful treatment of Parkinson's disease. However, long-term treatment of patients with levodopa often leads to development of dyskinesia. Therefore, drugs that feature potentiation of dopamine turnover rate and are able to reduce daily levodopa dosages might be used as adjuvant in the treatment of patients suffering from Parkinson's disease. To investigate such combined treatment, we have examined the effects of two non-competitive AMPA receptor antagonists, GYKI-52466 and GYKI-53405, alone or in combination with levodopa on dopamine turnover rate in 6-hydroxydopamine-lesioned striatum of the rat. We found here that repeated administration of levodopa, added with the peripheral DOPA decarboxylase inhibitor carbidopa, increased dopamine turnover rate after lesioning the striatum with 6-hydroxydopamine. Moreover, combination of levodopa with GYKI-52466 or GYKI-53405 further increased dopamine turnover enhanced by levodopa administration while the AMPA receptor antagonists by themselves failed to influence striatal dopamine turnover. We concluded from the present data that potentiation observed between levodopa and AMPA receptor antagonists may reflect levodopa-sparing effects in clinical treatment indicating the therapeutic potential of such combination in the management of Parkinson's disease.

  3. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  4. Identification of an ionotropic glutamate receptor AMPA1/GRIA1 polymorphism in crossbred beef cows differing in fertility

    Science.gov (United States)

    A proposed functional polymorphism in the ionotropic glutamate receptor AMPA1 (GRIA1) has been reported to influence antral follicle numbers and fertility in cows. Repeat Breeder cows that fail to produce a calf in multiple seasons have been reported to have reduced numbers of small (1-3 mm) antral ...

  5. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

    Directory of Open Access Journals (Sweden)

    Amanda Lorraine Wright

    2012-04-01

    Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

  6. Differential roles for NSF and GRIP/ABP in AMPA receptor cycling.

    Science.gov (United States)

    Braithwaite, Steven P; Xia, Houhui; Malenka, Robert C

    2002-05-14

    alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) stability and movement at synapses are important factors controlling synaptic strength. Here, we study the roles of proteins [N-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPARs. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or N-methyl-d-aspartate (NMDA). Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPARs that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPAR trafficking.

  7. The AAA+ ATPase, Thorase Regulates AMPA Receptor-Dependent Synaptic Plasticity and Behavior

    Science.gov (United States)

    Zhang, Jianmin; Wang, Yue; Chi, Zhikai; Keuss, Matthew J.; Pai, Ying-Min Emily; Kang, Ho Chul; Shin, Jooho; Bugayenko, Artem; Wang, Hong; Xiong, Yulan; Pletnikov, Mikhail V.; Mattson, Mark P.; Dawson, Ted M.; Dawson, Valina L.

    2011-01-01

    SUMMARY The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase, Thorase, that regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory. PMID:21496646

  8. The effect of AMPA receptor blockade on spatial information acquisition, consolidation and expression in juvenile rats.

    Science.gov (United States)

    Tzakis, Nikolaos; Bosnic, Tim; Ritchie, Thomas; Dixon, Kaylyn; Holahan, Matthew R

    2016-09-01

    Improvement on spatial tasks in rats is observed during a late, postnatal developmental period (post-natal day (PND) 18 - PND 20). The developmental emergence of this spatial function occurs in conjunction with hippocampal connectivity changes and enhanced hippocampal-AMPA receptor-mediated synaptic responses. The current work investigated the effect of AMPAr blockade on the emergence and long-term storage of spatial information in juvenile rats and associated neural activity patterns in the dorsal hippocampus CA1 region. Male, Long Evans rats between the ages of PND 18 and PND 20 were systemically (i.p.) administered the AMPAr antagonist, NBQX, (0, 5 or 10mg/kg) every day prior to hidden platform water maze training (PND 18, 19 and 20), every day immediately post-training or immediately before the probe test (PND 41). NBQX administration prior to training prolonged latencies, pathlength and increased thigmotaxis during the acquisition phase. Administration of NBQX immediately posttraining had no effect on the day-to-day performance. When given a probe test 3weeks later, the saline group across all conditions spent more time in the target quadrant. Rats treated with pretraining 5mg NBQX dose showed a preference for the target quadrant while the posttraining and pretesting 5mg NBQX doses impaired the target quadrant preference. Groups injected with 10mg of NBQX pretraining, posttraining or pretesting did not show a preference for the target quadrant. c-Fos labeling in the CA1 reflected these differences in probe performance in that groups showing greater than chance dwell time in the target quadrant showed more c-Fos labeling in the CA1 region than groups that did not show a target quadrant preference. These findings provide support for the critical role of AMPA receptor-mediated function in the organization and long-term storage of spatial memories acquired during the juvenile period.

  9. [EFfect of quinazolone-alkyl-carboxylic acid derivatives on the transmembrane Ca2+ ion flux mediated by AMPA receptors].

    Science.gov (United States)

    Szárics, Eva; LaszTóczi, Bálint; Nyikos, Lajos; Barabás, Péter; Kovács, Ilona; Skuban, Nina; Nagy, Péter I; Kökösi, József; Takácsné, Novák Krisztina; Kardos, Julianna

    2002-01-01

    The excitatory neurotransmitter, Glu, plays a crucial role in many sensory and motor functions as well as in brain development, learning and memory and it is also involved in the pathogenesis of a number of neurological disorders, including epilepsy, Alzheimer's and Parkinson's diseases. Therefore, the study of Glu receptors (GluRs) is of therapeutical importance. We showed here by fluorescence monitoring of transmembrane Ca2+ ion fluxes in response to (S)-alpha-amino-3-hidroxi-5-metil-4-izoxazol propionic acid ((S)-AMPA) on the time scale of 0.00004-10 s that Ca2+ ion influx proceeds through faster and slower desensitizing receptors. Pharmacological isolation of the slower and faster desensitizing AMPA receptor was possible by fluorescence monitoring of Ca2+ ion translocation in response to (S)-AMPA in the presence and absence of various 2-methyl-4-oxo-3H-quinazoline-3-alkyl-carboxilic acid derivatives (Qxs): the acetic acid Q1 inhibits the slower desensitizing receptor response specifically, while the acetyl-piperidine Q5 is a more potent inhibitor of the faster desensitizing receptor response. In addition, spontaneous interictal activity, as induced by high [K+] conditions in hippocampal slices, was reduced significantly by Q5, suggesting a possible anticonvulsant property of Q5. Substitutions of Qxs into the GluR2 S1S2 binding core were consistent with their effect by causing variable degree of S1S2 bridging interaction as one of the main determinants of AMPA receptor agonist activity. The exploitation of differences between similar receptors will be important in the development and use of drugs with high pharmacological specificity.

  10. Reinforcement-related regulation of AMPA glutamate receptor subunits in the ventral tegmental area enhances motivation for cocaine.

    Science.gov (United States)

    Choi, Kwang Ho; Edwards, Scott; Graham, Danielle L; Larson, Erin B; Whisler, Kimberly N; Simmons, Diana; Friedman, Allyson K; Walsh, Jessica J; Rahman, Zia; Monteggia, Lisa M; Eisch, Amelia J; Neve, Rachael L; Nestler, Eric J; Han, Ming-Hu; Self, David W

    2011-05-25

    Chronic cocaine use produces numerous biological changes in brain, but relatively few are functionally associated with cocaine reinforcement. Here we show that daily intravenous cocaine self-administration, but not passive cocaine administration, induces dynamic upregulation of the AMPA glutamate receptor subunits GluR1 and GluR2 in the ventral tegmental area (VTA) of rats. Increases in GluR1 protein and GluR1(S845) phosphorylation are associated with increased GluR1 mRNA in self-administering animals, whereas increased GluR2 protein levels occurred despite substantial decreases in GluR2 mRNA. We investigated the functional significance of GluR1 upregulation in the VTA on cocaine self-administration using localized viral-mediated gene transfer. Overexpression of GluR1(WT) in rat VTA primarily infected dopamine neurons (75%) and increased AMPA receptor-mediated membrane rectification in these neurons with AMPA application. Similar GluR1(WT) overexpression potentiated locomotor responses to intra-VTA AMPA, but not NMDA, infusions. In cocaine self-administering animals, overexpression of GluR1(WT) in the VTA markedly increased the motivation for cocaine injections on a progressive ratio schedule of cocaine reinforcement. In contrast, overexpression of protein kinase A-resistant GluR1(S845A) in the VTA reduced peak rates of cocaine self-administration on a fixed ratio reinforcement schedule. Neither viral vector altered sucrose self-administration, and overexpression of GluR1(WT) or GluR1(S845A) in the adjacent substantia nigra had no effect on cocaine self-administration. Together, these results suggest that dynamic regulation of AMPA receptors in the VTA during cocaine self-administration contributes to cocaine addiction by acting to facilitate subsequent cocaine use.

  11. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

    Science.gov (United States)

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L

    2012-04-01

    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  12. Functional characterization of Tet-AMPA [tetrazolyl-2-amino-3-(3-hydroxy-5-methyl- 4-isoxazolyl)propionic acid] analogues at ionotropic glutamate receptors GluR1-GluR4. The molecular basis for the functional selectivity profile of 2-Bn-Tet-AMPA

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Christesen, Thomas; Bølcho, Ulrik;

    2007-01-01

    Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-i......Pr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible...

  13. Blockade of the AMPA receptor prevents CA1 hippocampal injury following severe but transient forebrain ischemia in adult rats.

    Science.gov (United States)

    Buchan, A M; Li, H; Cho, S; Pulsinelli, W A

    1991-11-11

    The cytoprotective effect of NBQX, a selective AMPA receptor antagonist, was tested following 10 min of severe forebrain ischemia using the 4-vessel occlusion model. Immediately, and at 15 and 30 min following reperfusion, adult Wistar rats received intraperitoneal injections of either saline (n = 5), 1 mg lithium chloride (n = 17) or 30 mg/kg of the lithium salt of NBQX (n = 18). In saline-treated animals 82 +/- 12% of CA1 hippocampal neurons were lost. Of those treated with lithium 70 +/- 23% were injured, while those given NBQX sustained only 40 +/- 34% CA1 necrosis (P less than 0.01). Twelve of 18 NBQX-treated animals had less than 30% CA1 injury as compared with 1 of 17 lithium-treated animals. The AMPA receptor may play a more important role than the NMDA receptor in selective ischemic necrosis of hippocampal neurons.

  14. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  15. DCP-LA stimulates AMPA receptor exocytosis through CaMKII activation due to PP-1 inhibition.

    Science.gov (United States)

    Kanno, Takeshi; Yaguchi, Takahiro; Nagata, Tetsu; Tanaka, Akito; Nishizaki, Tomoyuki

    2009-10-01

    The linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) by inhibiting protein phosphatase-1 (PP-1). DCP-LA induced a transient huge facilitation of synaptic transmission monitored from the CA1 region of rat hippocampal slices, which was largely inhibited by the CaMKII inhibitor KN-93. DCP-LA potentiated kainate-evoked whole-cell membrane currents for Xenopus oocytes expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors composed of the GluR1, GluR3, GluR1/GluR2, GluR1/GluR3, and GluR1/GluR2/GluR3 subunits, and the potentiation was significantly inhibited by KN-93. A similar potentiation was still found with mutant GluR1 (S831A) receptor lacking CaMKII phosphorylation site. The GluR1 and GluR2 subunits formed AMPA receptors in the rat hippocampus, and DCP-LA increased expression of both the subunits on the plasma membrane. The DCP-LA action was blocked by KN-93 and the exocytosis inhibitor botulinum toxin type A, but not by the endocytosis inhibitor phenylarsine oxide. DCP-LA, thus, appears to activate CaMKII through PP-1 inhibition, that stimulates AMPA receptor exocytosis to increase expression of the receptors on the plasma membrane, responsible for potentiate AMPA receptor responses and facilitation of hippocampal synaptic transmission.

  16. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

    Science.gov (United States)

    Lu, Wei; Ziff, Edward B

    2005-08-04

    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

  17. Synaptic transmission and plasticity require AMPA receptor anchoring via its N-terminal domain

    Science.gov (United States)

    Watson, Jake F; Ho, Hinze; Greger, Ingo H

    2017-01-01

    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and are selectively recruited during activity-dependent plasticity to increase synaptic strength. A prerequisite for faithful signal transmission is the positioning and clustering of AMPARs at postsynaptic sites. The mechanisms underlying this positioning have largely been ascribed to the receptor cytoplasmic C-termini and to AMPAR-associated auxiliary subunits, both interacting with the postsynaptic scaffold. Here, using mouse organotypic hippocampal slices, we show that the extracellular AMPAR N-terminal domain (NTD), which projects midway into the synaptic cleft, plays a fundamental role in this process. This highly sequence-diverse domain mediates synaptic anchoring in a subunit-selective manner. Receptors lacking the NTD exhibit increased mobility in synapses, depress synaptic transmission and are unable to sustain long-term potentiation (LTP). Thus, synaptic transmission and the expression of LTP are dependent upon an AMPAR anchoring mechanism that is driven by the NTD. DOI: http://dx.doi.org/10.7554/eLife.23024.001 PMID:28290985

  18. Impaired associative fear learning in mice with complete loss or haploinsufficiency of AMPA GluR1 receptors

    Directory of Open Access Journals (Sweden)

    Michael Feyder

    2007-12-01

    Full Text Available There is compelling evidence that L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA glutamate receptors containing the GluR1 subunit contribute to the molecular mechanisms associated with learning. AMPA GluR1 glutamate receptor knockout mice (KO exhibit abnormal hippocampal and amygdala plasticity, and deficits on various assays for cognition including Pavlovian fear conditioning. Here we examined associative fear learning in mice with complete absence (KO or partial loss (heterozygous mutant, HET of GluR1 on multiple fear conditioning paradigms. After multi-trial delay or trace conditioning, KO displayed impaired tone and context fear recall relative to WT, whereas HET were normal. After one-trial delay conditioning, both KO and HET showed impaired tone and context recall. HET and KO showed normal nociceptive sensitivity in the hot plate and tail flick tests. These data demonstrate that the complete absence of GluR1 subunit-containing receptors prevents the formation of associative fear memories, while GluR1 haploinsufficiency is sufficient to impair one-trial fear learning. These findings support growing evidence of a major role for GluR1-containing AMPA receptors in amygdalamediated forms of learning and memory.

  19. Alteration of AMPA Receptor-Mediated Synaptic Transmission by Alexa Fluor 488 and 594 in Cerebellar Stellate Cells.

    Science.gov (United States)

    Maroteaux, Matthieu; Liu, Siqiong June

    2016-01-01

    The fluorescent dyes, Alexa Fluor 488 and 594 are commonly used to visualize dendritic structures and the localization of synapses, both of which are critical for the spatial and temporal integration of synaptic inputs. However, the effect of the dyes on synaptic transmission is not known. Here we investigated whether Alexa Fluor dyes alter the properties of synaptic currents mediated by two subtypes of AMPA receptors (AMPARs) at cerebellar stellate cell synapses. In naive mice, GluA2-lacking AMPAR-mediated synaptic currents displayed an inwardly rectifying current-voltage (I-V) relationship due to blockade by cytoplasmic spermine at depolarized potentials. We found that the inclusion of 100 µm Alexa Fluor dye, but not 10 µm, in the pipette solution led to a gradual increase in the amplitude of EPSCs at +40 mV and a change in the I-V relationship from inwardly rectifying to more linear. In mice exposed to an acute stress, AMPARs switched to GluA2-containing receptors, and 100 µm Alexa Fluor 594 did not alter the I-V relationship of synaptic currents. Therefore, a high concentration of Alexa Fluor dye changed the I-V relationship of EPSCs at GluA2-lacking AMPAR synapses.

  20. Natural reward experience alters AMPA and NMDA receptor distribution and function in the nucleus accumbens.

    Directory of Open Access Journals (Sweden)

    Kyle K Pitchers

    Full Text Available Natural reward and drugs of abuse converge upon the mesolimbic system which mediates motivation and reward behaviors. Drugs induce neural adaptations in this system, including transcriptional, morphological, and synaptic changes, which contribute to the development and expression of drug-related memories and addiction. Previously, it has been reported that sexual experience in male rats, a natural reward behavior, induces similar neuroplasticity in the mesolimbic system and affects natural reward and drug-related behavior. The current study determined whether sexual experience causes long-lasting changes in mating, or ionotropic glutamate receptor trafficking or function in the nucleus accumbens (NAc, following 3 different reward abstinence periods: 1 day, 1 week, or 1 month after final mating session. Male Sprague Dawley rats mated during 5 consecutive days (sexual experience or remained sexually naïve to serve as controls. Sexually experienced males displayed facilitation of initiation and performance of mating at each time point. Next, intracellular and membrane surface expression of N-methyl-D-aspartate (NMDA: NR1 subunit and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA: GluA1, GluA2 subunits receptors in the NAc was determined using a bis(sulfosuccinimidylsuberate (BS(3 protein cross-linking assay followed by Western Blot analysis. NR1 expression was increased at 1 day abstinence both at surface and intracellular, but decreased at surface at 1 week of abstinence. GluA2 was increased intracellularly at 1 week and increased at the surface after 1 month of abstinence. Finally, whole-cell patch clamp electrophysiological recordings determined reduced AMPA/NMDA ratio of synaptic currents in NAc shell neurons following stimulation of cortical afferents in sexually experienced males after all reward abstinence periods. Together, these data show that sexual experience causes long-term alterations in glutamate receptor expression and

  1. Parvalbumin-containing interneurons in rat hippocampus have an AMPA receptor profile suggestive of vulnerability to excitotoxicity.

    Science.gov (United States)

    Moga, Diana; Hof, Patrick R; Vissavajjhala, Prabhakar; Moran, Thomas M; Morrison, John H

    2002-05-01

    alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate excitatory neurotransmission in the central nervous system, and contain combinations of four subunits (GluR1-4). We developed a GluR3-specific monoclonal antibody and quantified the cellular distribution of GluR3 in rat hippocampus. GluR3 immunoreactivity was detected in all pyramidal neurons and most interneurons. In addition, we found a subset of parvalbumin (PV)-containing interneurons in the hippocampus and neocortex that was notable for its intense GluR3 immunoreactivity and lack of GluR2 immunoreactivity. Such an expression pattern of AMPA receptor subunits is likely to make these interneurons selectively vulnerable to excitotoxicity.

  2. Calcium-permeable AMPA receptors in the nucleus accumbens regulate depression-like behaviors in the chronic neuropathic pain state.

    Science.gov (United States)

    Goffer, Yossef; Xu, Duo; Eberle, Sarah E; D'amour, James; Lee, Michelle; Tukey, David; Froemke, Robert C; Ziff, Edward B; Wang, Jing

    2013-11-27

    Depression is a salient emotional feature of chronic pain. Depression alters the pain threshold and impairs functional recovery. To date, however, there has been limited understanding of synaptic or circuit mechanisms that regulate depression in the pain state. Here, we demonstrate that depression-like behaviors are induced in a rat model of chronic neuropathic pain. Using this model, we show that chronic pain selectively increases the level of GluA1 subunits of AMPA-type glutamate receptors at the synapses of the nucleus accumbens (NAc), a key component of the brain reward system. We find, in addition, that this increase in GluA1 levels leads to the formation of calcium-permeable AMPA receptors (CPARs). Surprisingly, pharmacologic blockade of these CPARs in the NAc increases depression-like behaviors associated with pain. Consistent with these findings, an AMPA receptor potentiator delivered into the NAc decreases pain-induced depression. These results show that transmission through CPARs in the NAc represents a novel molecular mechanism modulating the depressive symptoms of pain, and thus CPARs may be a promising therapeutic target for the treatment of pain-induced depression. More generally, these findings highlight the role of central glutamate signaling in pain states and define the brain reward system as an important region for the regulation of depressive symptoms of pain.

  3. Activation of AMPA receptors in the suprachiasmatic nucleus phase-shifts the mouse circadian clock in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Yasutaka Mizoro

    Full Text Available The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor is well elucidated, much less is known about a role of AMPAR (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN.

  4. Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells.

    Science.gov (United States)

    Li, Qiang; Yan, Haidun; Wilson, Wilkie A; Swartzwelder, H Scott

    2010-06-25

    Although the endogenous cannabinoid system modulates a variety of physiological and pharmacological processes, the specific role of cannabinoid CB1 receptors in the modulation of glutamatergic neurotransmission and neural plasticity is not well understood. Using whole-cell patch clamp recording techniques, evoked or spontaneous excitatory postsynaptic currents (eEPSCs or sEPSCs) were recorded from visualized, layer II/III pyramidal cells in frontal cortical slices from rat brain. Bath application of the CB1 receptor agonist, WIN 55212-2 (WIN), reduced the amplitude of NMDA receptor-mediated EPSCs in a concentration-dependent manner. When co-applied with the specific CB1 antagonists, AM251 or AM281, WIN did not suppress NMDA receptor-mediated EPSCs. WIN also reduced the amplitude of evoked AMPA receptor-mediated EPSCs, an effect that was also reversed by AM251. Both the frequency and amplitude of spontaneous AMPA receptor-mediated EPSCs were significantly reduced by WIN. In contrast, WIN reduced the frequency, but not the amplitude of miniature EPSCs, suggesting that the suppression of glutamatergic activity by CB1 receptors in the frontal neocortex is mediated by a presynaptic mechanism. Taken together, these data indicate a critical role for endocannabinoid signaling in the regulation of excitatory synaptic transmission in frontal neocortex, and suggest a possible neuronal mechanism whereby THC regulates cortical function.

  5. Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity.

    Science.gov (United States)

    Klaassen, Remco V; Stroeder, Jasper; Coussen, Françoise; Hafner, Anne-Sophie; Petersen, Jennifer D; Renancio, Cedric; Schmitz, Leanne J M; Normand, Elisabeth; Lodder, Johannes C; Rotaru, Diana C; Rao-Ruiz, Priyanka; Spijker, Sabine; Mansvelder, Huibert D; Choquet, Daniel; Smit, August B

    2016-03-02

    Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission.

  6. AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke.

    Science.gov (United States)

    Clarkson, Andrew N; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S Thomas

    2011-03-09

    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb control when administered after a delay from the stroke. Conversely, AMPAR antagonists impair motor recovery. The contributions of AMPARs to recovery are mediated by release of brain-derived neurotrophic factor (BDNF) in periinfarct cortex, as blocking local BDNF function in periinfarct cortex blocks AMPAR-mediated recovery and prevents the normal pattern of motor recovery. In contrast to a delayed AMPAR role in motor recovery, early administration of AMPAR agonists after stroke increases stroke damage. These findings indicate that the role of glutamate signaling through the AMPAR changes over time in stroke: early potentiation of AMPAR signaling worsens stroke damage, whereas later potentiation of the same signaling system improves functional recovery.

  7. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Anubhuti Goel

    Full Text Available Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+-permeable AMPA receptors (CP-AMPARs. However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1 subunit at the serine 845 (S845 site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants, which is a substrate of cAMP-dependent kinase (PKA, show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  8. Phosphorylation of AMPA receptors is required for sensory deprivation-induced homeostatic synaptic plasticity.

    Science.gov (United States)

    Goel, Anubhuti; Xu, Linda W; Snyder, Kevin P; Song, Lihua; Goenaga-Vazquez, Yamila; Megill, Andrea; Takamiya, Kogo; Huganir, Richard L; Lee, Hey-Kyoung

    2011-03-31

    Sensory experience, and the lack thereof, can alter the function of excitatory synapses in the primary sensory cortices. Recent evidence suggests that changes in sensory experience can regulate the synaptic level of Ca(2+)-permeable AMPA receptors (CP-AMPARs). However, the molecular mechanisms underlying such a process have not been determined. We found that binocular visual deprivation, which is a well-established in vivo model to produce multiplicative synaptic scaling in visual cortex of juvenile rodents, is accompanied by an increase in the phosphorylation of AMPAR GluR1 (or GluA1) subunit at the serine 845 (S845) site and the appearance of CP-AMPARs at synapses. To address the role of GluR1-S845 in visual deprivation-induced homeostatic synaptic plasticity, we used mice lacking key phosphorylation sites on the GluR1 subunit. We found that mice specifically lacking the GluR1-S845 site (GluR1-S845A mutants), which is a substrate of cAMP-dependent kinase (PKA), show abnormal basal excitatory synaptic transmission and lack visual deprivation-induced homeostatic synaptic plasticity. We also found evidence that increasing GluR1-S845 phosphorylation alone is not sufficient to produce normal multiplicative synaptic scaling. Our study provides concrete evidence that a GluR1 dependent mechanism, especially S845 phosphorylation, is a necessary pre-requisite step for in vivo homeostatic synaptic plasticity.

  9. Activity-Dependent Ubiquitination of GluA1 and GluA2 Regulates AMPA Receptor Intracellular Sorting and Degradation

    OpenAIRE

    Jocelyn Widagdo; Ye Jin Chai; Margreet C. Ridder; Yu Qian Chau; Richard C. Johnson; Pankaj Sah; Richard L. Huganir; Victor Anggono

    2015-01-01

    AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1-4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ...

  10. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death.

    Science.gov (United States)

    Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B

    2007-03-28

    Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.

  11. Increase of AMPA receptor glutamate receptor 1 subunit and B-cell receptor-associated protein 31 gene expression in hippocampus of fatigued mice.

    Science.gov (United States)

    Kamakura, Masaki; Tamaki, Keisuke; Sakaki, Toshiyuki; Yoneda, Yukio

    2005-10-14

    Central fatigue is an indispensable biosignal for maintaining life, but the neuronal and molecular mechanisms involved remain unclear. In this study, we searched for genes differentially expressed in the hippocampus of fatigued mice to elucidate the mechanisms underlying fatigue. Mice were forced to swim in an adjustable-current water pool, and the maximum swimming time (endurance) until fatigue was measured thrice. Fatigued and nonfatigued mice with equal swimming capacity and body weight were compared. We found that the genes of GluR1 and B-cell receptor-associated protein 31 (Bap31), which acts as a transport molecule in the secretory pathway or as a mediator of apoptosis, were upregulated in the hippocampus of fatigued mice, and increases of GluR1 and Bap31 were confirmed by Northern blotting and real-time PCR. No change of gene expression of AMPA receptor subunits other than GluR1 was observed. These results suggest that a compositional change of AMPA receptor (increase of GluR1) and upregulation of the Bap31 gene may be implicated in fatigue in mice.

  12. Hippocampal GluA1-containing AMPA receptors mediate context-dependent sensitization to morphine.

    Science.gov (United States)

    Xia, Yan; Portugal, George S; Fakira, Amanda K; Melyan, Zara; Neve, Rachael; Lee, H Thomas; Russo, Scott J; Liu, Jie; Morón, Jose A

    2011-11-09

    Glutamatergic systems, including AMPA receptors (AMPARs), are involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are not fully understood. In the present study, we investigated the effects of repeated morphine administration on AMPAR expression, synaptic plasticity, and context-dependent behavioral sensitization to morphine. We found that morphine treatment produced changes of synaptic AMPAR expression in the hippocampus, a brain area that is critically involved in learning and memory. These changes could be observed 1 week after the treatment, but only when mice developed context-dependent behavioral sensitization to morphine in which morphine treatment was associated with drug administration environment. Context-dependent behavioral sensitization to morphine was also associated with increased basal synaptic transmission and disrupted hippocampal long-term potentiation (LTP), whereas these effects were less robust when morphine administration was not paired with the drug administration environment. Interestingly, some effects may be related to the prior history of morphine exposure in the drug-associated environment, since alterations of AMPAR expression, basal synaptic transmission, and LTP were observed in mice that received a saline challenge 1 week after discontinuation of morphine treatment. Furthermore, we demonstrated that phosphorylation of GluA1 AMPAR subunit plays a critical role in the acquisition and expression of context-dependent behavioral sensitization, as this behavior is blocked by a viral vector that disrupts GluA1 phosphorylation. These data provide evidence that glutamatergic signaling in the hippocampus plays an important role in context-dependent sensitization to morphine and supports further investigation of glutamate-based strategies for treating opiate addiction.

  13. Drug-driven AMPA receptor redistribution mimicked by selective dopamine neuron stimulation.

    Directory of Open Access Journals (Sweden)

    Matthew T C Brown

    Full Text Available BACKGROUND: Addictive drugs have in common that they cause surges in dopamine (DA concentration in the mesolimbic reward system and elicit synaptic plasticity in DA neurons of the ventral tegmental area (VTA. Cocaine for example drives insertion of GluA2-lacking AMPA receptors (AMPARs at glutamatergic synapes in DA neurons. However it remains elusive which molecular target of cocaine drives such AMPAR redistribution and whether other addictive drugs (morphine and nicotine cause similar changes through their effects on the mesolimbic DA system. METHODOLOGY/PRINCIPAL FINDINGS: We used in vitro electrophysiological techniques in wild-type and transgenic mice to observe the modulation of excitatory inputs onto DA neurons by addictive drugs. To observe AMPAR redistribution, post-embedding immunohistochemistry for GluA2 AMPAR subunit was combined with electron microscopy. We also used a double-floxed AAV virus expressing channelrhodopsin together with a DAT Cre mouse line to selectively express ChR2 in VTA DA neurons. We find that in mice where the effect of cocaine on the dopamine transporter (DAT is specifically blocked, AMPAR redistribution was absent following administration of the drug. Furthermore, addictive drugs known to increase dopamine levels cause a similar AMPAR redistribution. Finally, activating DA VTA neurons optogenetically is sufficient to drive insertion of GluA2-lacking AMPARs, mimicking the changes observed after a single injection of morphine, nicotine or cocaine. CONCLUSIONS/SIGNIFICANCE: We propose the mesolimbic dopamine system as a point of convergence at which addictive drugs can alter neural circuits. We also show that direct activation of DA neurons is sufficient to drive AMPAR redistribution, which may be a mechanism associated with early steps of non-substance related addictions.

  14. Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.

    OpenAIRE

    Dennis, Siobhan; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.

    2011-01-01

    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensi...

  15. Oxygen/glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and A3 Receptors

    OpenAIRE

    Dennis, Siobhan H.; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.

    2011-01-01

    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighbouring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+ resulting in delayed cell death. However, it is unclear if the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivit...

  16. Insights into the mechanisms of ifosfamide encephalopathy: drug metabolites have agonistic effects on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors and induce cellular acidification in mouse cortical neurons.

    Science.gov (United States)

    Chatton, J Y; Idle, J R; Vågbø, C B; Magistretti, P J

    2001-12-01

    Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

  17. Lessons from crystal structures of kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Frydenvang, Karla Andrea; Pickering, Darryl S;

    2017-01-01

    synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...... structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full...

  18. Phenobarbital but not diazepam reduces AMPA/Kainate receptor mediated currents and exerts opposite actions on initial seizures in the neonatal rat hippocampus

    Directory of Open Access Journals (Sweden)

    Romain eNardou

    2011-07-01

    Full Text Available Diazepam (DZP and phenobarbital (PB are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. Yet, their efficacy is variable with occasional failure or even aggravation of recurrent seizures questioning whether other mechanisms are not involved in their actions. We have now compared the effects of DZP and PB on ictal-like events (ILEs in an in vitro model of mirror focus (MF. Using the three-compartment chamber with the two immature hippocampi and their commissural fibers placed in 3 different compartments, kainate was applied to one hippocampus and PB or DZP to the contralateral one, either after one ILE or after many recurrent ILEs that produce an epileptogenic MF. We report that in contrast to PB, DZP aggravated propagating ILEs from the start and did not prevent the formation of MF. PB reduced and DZP increased the network driven Giant Depolarising Potentials suggesting that PB may exert additional actions that are not mediated by GABA signalling. In keeping with this, PB but not DZP reduced field potentials recorded in the presence of GABA and NMDA receptor antagonists. These effects are mediated by a direct action on AMPA/Kainate receptors since PB: i reduced AMPA/Kainate receptor mediated currents induced by focal applications of glutamate ; ii reduced the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature EPSCs; iii augmented the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in MF. Therefore, PB exerts its anticonvulsive actions partly by reducing AMPA/Kainate receptors mediated EPSCs in addition to the pro-GABA effects. We suggest that PB may have advantage over DZP in the treatment of initial neonatal seizures since the additional reduction of glutamate receptors mediated signals may reduce the severity of neonatal seizures.

  19. Role of AMPA and GluR5 kainate receptors in the development and expression of amygdala kindling in the mouse.

    Science.gov (United States)

    Rogawski, M A; Kurzman, P S; Yamaguchi, S I; Li, H

    2001-01-01

    The role of AMPA and GluR5-containing kainate receptors in the development and expression of amygdala kindling was examined using the selective 2,3-benzodiazepine AMPA receptor antagonist GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine] and the decahydroisoquinoline mixed AMPA receptor and GluR5 kainate receptor antagonist LY293558 {(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline- 3-carboxy lic acid)}. Administration of GYKI 52466 (5-40 mg/kg, intraperitoneally) and LY293558 (10-40 mg/kg, intraperitoneally) prior to daily kindling stimulation in mice produced a dose-dependent suppression of the rate of development of behavioral kindled seizure activity and reduced the duration of the stimulation-induced electrographic afterdischarge. In drug-free stimulation sessions after the initial drug-treatment sessions, there was an acceleration in the rate of kindling development compared with the rate during the preceding drug-administration period; the "rebound" rate was also greater than the kindling rate in saline-treated control animals. In fully kindled animals, both GYKI 52466 and LY293558 produced a dose-dependent suppression of evoked seizures (ED(50), 19.3 and 16.7 mg/kg, respectively). Although AMPA receptors appear to be critical to the expression of kindled seizures, since kindling development progressed despite the suppression of behavioral seizure activity, AMPA receptors are less important to the kindling process. LY293558 was modestly less effective at suppressing behavioral seizures during kindling and was not superior to GYKI 52466 in retarding the overall extent of kindling development, indicating that GluR5 kainate receptors do not contribute to epileptogenesis in this model.

  20. Requirement of AMPA receptor stimulation for the sustained antidepressant activity of ketamine and LY341495 during the forced swim test in rats.

    Science.gov (United States)

    Koike, Hiroyuki; Chaki, Shigeyuki

    2014-09-01

    Ketamine, a non-competitive N-methyl-d-aspartate receptor antagonist, and group II metabotropic glutamate (mGlu2/3) receptor antagonists produce antidepressant effects in animal models of depression, which last for at least 24h, through the transient increase in glutamate release, leading to activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor. Both ketamine and an mGlu2/3 receptor antagonist reportedly increase the expression of GluR1, an AMPA receptor subunit, within 24h, which may account for the sustained enhancement of excitatory synaptic transmission following ketamine administration. However, whether the sustained increase in AMPA receptor-mediated synaptic transmission is associated with the antidepressant effects of ketamine and mGlu2/3 receptor antagonists has not yet been investigated. In the present study, to address this question, we tested whether AMPA receptor stimulation at 24h after a single injection of ketamine or an mGlu2/3 receptor antagonist, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY341495) was necessary for the antidepressant effect of these compounds using a forced swim test in rats. A single injection of ketamine or LY341495 at 24h before the test significantly decreased the immobility time. An AMPA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), administered 30min prior to the test significantly and dose-dependently reversed the antidepressant effects of ketamine and LY341495, while NBQX itself had no effect on the immobility time. Our findings suggest that AMPA receptor stimulation at 24h after a single injection of ketamine or LY341495 is required to produce the anti-immobility effects of these compounds. Moreover, the present results provide additional evidence that an mGlu2/3 receptor antagonist may share some of neural mechanisms with ketamine to exert antidepressant effects.

  1. A new phenylalanine derivative acts as an antagonist at the AMPA receptor GluA2 and introduces partial domain closure

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Frydenvang, Karla; Contreras-Sanz, Alberto;

    2011-01-01

    In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and G...

  2. Studies on an (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor antagonist IKM-159

    DEFF Research Database (Denmark)

    Juknaite, Lina; Sugamata, Yutaro; Tokiwa, Kazuya;

    2013-01-01

    IKM-159 was developed and identified as a member of a new class of heterotricyclic glutamate analogs that act as AMPA receptor-selective antagonists. However, it was not known which enantiomer of IKM-159 was responsible for its pharmacological activities. Here, we report in vivo and in vitro neur...

  3. Administration of a PTEN inhibitor BPV(pic) attenuates early brain injury via modulating AMPA receptor subunits after subarachnoid hemorrhage in rats.

    Science.gov (United States)

    Chen, Yujie; Luo, Chunxia; Zhao, Mingyue; Li, Qiang; Hu, Rong; Zhang, John H; Liu, Zhi; Feng, Hua

    2015-02-19

    The aim of this study was to investigate whether the phosphatase and tensin homolog deleted on chromosome ten (PTEN) inhibitor dipotassium bisperoxo(pyridine-2-carboxyl) oxovanadate (BPV(pic)) attenuates early brain injury by modulating α-amino-3-hydroxy-5-methyl-4-isoxa-zolep-propionate (AMPA) receptor subunits after subarachnoid hemorrhage (SAH). A standard intravascular perforation model was used to produce the experimental SAH in Sprague-Dawley rats. BPV(pic) treatment (0.2mg/kg) was evaluated for effects on neurological score, brain water content, Evans blue extravasation, hippocampal neuronal death and AMPA receptor subunits alterations after SAH. We found that BPV(pic) is effective in attenuating BBB disruption, lowering edema, reducing hippocampal neural death and improving neurological outcomes. In addition, the AMPA receptor subunit GluR1 protein expression at cytomembrane was downregulated, whereas the expression of GluR2 and GluR3 was upregulated after BPV(pic) treatment. Our results suggest that PTEN inhibited by BPV(pic) plays a neuroprotective role in SAH pathophysiology, possibly by alterations in glutamate AMPA receptor subunits.

  4. AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2/3 subunits in most interneurons.

    Science.gov (United States)

    Leranth, C; Szeidemann, Z; Hsu, M; Buzsáki, G

    1996-02-01

    Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are assembled from the four subunits GluR1, 2, 3, 4 (or GluRA, B, C, D). AMPA channels that do not contain the GluR2 subunit are permeable to calcium. Recent studies indicate that excitotoxic as well as epileptic and ischemic cell damage may be mediated not only by N-methyl-Daspartate receptors, but also by AMPA receptors. The majority of interneurons in the hippocampus are resistant, but subsets of interneurons are consistently damaged in different disease states. Single immunolabeling using antibodies against AMPA receptor subunits, together with double immunolabeling for calcium-binding proteins (parvalbumin, calbindin and calretinin) and the neuropeptide somatostatin, were performed to study GluR1-4 immunoreactivity in interneuronal populations and principal cells. The ultrastructure of GluR1-4 labeled neurons was also examined using electron microscopy. With the exception of calbindin-positive interneurons, GluR2/3 was absent from hippocampal interneurons in both rat and monkey. In the rat, interneurons were more strongly immunoreactive against GluR1 than principal cells. In the monkey, immunoreactivity for GluR4 in interneurons was stronger than for GluR1. All GluR subunits were confined to spines, dendritic membrane and cytoplasm surrounding the nucleus but absent from axons and presynaptic terminals. Our findings suggest that hippocampal principal cells and interneurons express different complements of AMPA receptor subunits. Furthermore, the absence of GluR2 and/or GluR3 in both vulnerable and resistant interneurons subtypes indicates that knowledge of receptor subunit composition is not sufficient to predict neuronal vulnerability.

  5. Anti-AMPA-Receptor Encephalitis Presenting as a Rapid-Cycling Bipolar Disorder in a Young Woman with Turner Syndrome

    Directory of Open Access Journals (Sweden)

    Giuseppe Quaranta

    2015-01-01

    Full Text Available Background. Autoimmune encephalitis is a disorder characterised by the subacute onset of seizures, short-term memory loss, and psychiatric and behavioural symptoms. Initially, it was recognised as a paraneoplastic disorder, but recently a subgroup of patients without systemic cancer was identified. Case Description. We describe a 20-year-old woman with Turner syndrome presenting with a treatment-resistant rapid cycling bipolar disorder with cognitive impairment. She was diagnosed with anti-AMPA-receptor encephalitis. She showed marked improvement after starting memantine and valproic acid. Conclusion. This case description emphasises the importance of timely recognition of autoimmune limbic encephalitis in patients with psychiatric manifestations and a possible predisposition to autoimmune conditions, in order to rule out malignancy and to quickly initiate treatment.

  6. Domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation

    Directory of Open Access Journals (Sweden)

    Charles R. Midgett

    2012-01-01

    Full Text Available Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs, the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD, and of multiple conformations of the ligand-binding domain (LBD. Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPARs, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

  7. Differential localization of delta glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses.

    Science.gov (United States)

    Landsend, A S; Amiry-Moghaddam, M; Matsubara, A; Bergersen, L; Usami, S; Wenthold, R J; Ottersen, O P

    1997-01-15

    The delta 2 glutamate receptors are prominently expressed in Purkinje cells and are thought to play a key role in the induction of cerebellar long-term depression. The synaptic and subsynaptic localization of delta receptors in rat cerebellar cortex was investigated with sensitive and high-resolution immunogold procedures. After postembedding incubation with an antibody raised to a C-terminal peptide of delta 2, high gold particle densities occurred in all parallel fiber synapses with Purkinje cell dendritic spines, whereas other synapses were consistently devoid of labeling. Among the types of immunonegative synapse were climbing fiber synapses with spines and parallel fiber synapses with dendritic stems of interneurons. At the parallel fiber-spine synapse, gold particles signaling delta receptors were restricted to the postsynaptic specialization. By the use of double labeling with two different gold particle sizes, it was shown that delta and AMPA GluR2/3 receptors were colocalized along the entire extent of the postsynaptic specialization without forming separate domains. The distribution of gold particles representing delta receptors was consistent with a cytoplasmic localization of the C terminus and an absence of a significant presynaptic pool of receptor molecules. The present data suggest that the delta 2 receptors are targeted selectively to a subset of Purkinje cell spines and that they are coexpressed with ionotropic receptors in the postsynaptic specialization. This arrangement could allow for a direct interaction between the two classes of receptor.

  8. Inhibition of AMPA Receptors by Polyamine Toxins is Regulated by Agonist Efficacy and Stargazin

    DEFF Research Database (Denmark)

    Poulsen, Mette H; Lucas, Simon; Strømgaard, Kristian;

    2014-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels mediating the majority of fast excitatory synaptic transmission in the central nervous system (CNS). Polyamine toxins derived from spiders and wasps are use- and voltage-dependent...... explored the effect of the TARP γ-2 (also known as stargazin) on the inhibitory potency of three structurally different polyamine toxins at Ca(2+)-permeable homomeric GluA1 AMPARs expressed in oocytes. We find that polyamine toxin IC50 is differentially affected by presence of stargazin depending...... on the efficacy of the agonists used to activate GluA1. Co-assembly of GluA1 receptors with stargazin increases the potency of the polyamine toxins when activated by the weak partial agonist kainate, but has no effect in presence of full-agonist L-glutamate (Glu) and partial agonist (RS)-willardiine....

  9. The Prefrontal Dectin-1/AMPA Receptor Signaling Pathway Mediates The Robust and Prolonged Antidepressant Effect of Proteo-β-Glucan from Maitake

    Science.gov (United States)

    Bao, Hongkun; Ran, Pengzhan; Zhu, Ming; Sun, Lijuan; Li, Bai; Hou, Yangyang; Nie, Jun; Shan, Liping; Li, Hongliang; Zheng, Shangyong; Xu, Xiufeng; Xiao, Chunjie; Du, Jing

    2016-01-01

    Proteo-β-glucan from Maitake (PGM) is a strong immune regulator, and its receptor is called Dectin-1. Cumulative evidence suggests that AMPA receptors are important for the treatment of depression. Here, we report that PGM treatment leads to a significant antidepressant effect in the tail suspension test and forced swim test after sixty minutes of treatment in mice. After five consecutive days of PGM treatment, this antidepressant effect remained. PGM treatment did not show a hyperactive effect in the open field test. PGM significantly enhanced the expression of its receptor Dectin-1, as well as p-GluA1(S845) and GluA1, but not GluA2 or GluA3 in the prefrontal cortex (PFC) after five days of treatment. The Dectin-1 inhibitor Laminarin was able to block the antidepressant effect of PGM. At the synapses of PFC, PGM treatment significantly up-regulated the p-GluA1(S845), GluA1, GluA2, and GluA3 levels. Moreover, PGM’s antidepressant effects and the increase of p-GluA1(S845)/GluA1 lasted for 3 days after stopping treatment. The AMPA-specific antagonist GYKI 52466 was able to block the antidepressant effect of PGM. This study identified PGM as a novel antidepressant with clinical potential and a new antidepressant mechanism for regulating prefrontal Dectin-1/AMPA receptor signalling. PMID:27329257

  10. Consolidation of remote fear memories involves Corticotropin-Releasing Hormone (CRH) receptor type 1-mediated enhancement of AMPA receptor GluR1 signaling in the dentate gyrus.

    Science.gov (United States)

    Thoeringer, Christoph K; Henes, Kathrin; Eder, Matthias; Dahlhoff, Maik; Wurst, Wolfgang; Holsboer, Florian; Deussing, Jan M; Moosmang, Sven; Wotjak, Carsten T

    2012-02-01

    Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca(2+)-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.

  11. Regulated RalBP1 binding to RalA and PSD-95 controls AMPA receptor endocytosis and LTD.

    Directory of Open Access Journals (Sweden)

    Kihoon Han

    2009-09-01

    Full Text Available Long-term depression (LTD is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.

  12. Extinction of morphine-dependent conditioned behavior is associated with increased phosphorylation of the GluR1 subunit of AMPA receptors at hippocampal synapses

    OpenAIRE

    Billa, Sophie K.; Sinha, Namita; Rudrabhatla, Sri Rajyalakshmi; Morón, Jose A.

    2008-01-01

    In abstinent opiate addicts, relapse can be triggered by exposure to environmental cues associated with drug use; thus, the disruption of these learned associations may be an effective approach for reducing relapse. Interestingly, glutamatergic systems are thought to be involved in opiate-induced behavioral plasticity. In this study, changes in expression and phosphorylation levels of AMPA glutamate receptor subunits (GluR1, GluR2) in the hippocampus were investigated in rats showing a condit...

  13. Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiu-Li [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Ding, Fan [Office of Scientific R& D, Tsinghua University, Beijing (China); Li, Hui; Tan, Xiao-Qiu [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Liu, Xiao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Cao, Ji-Min, E-mail: caojimin@126.com [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Gao, Xue, E-mail: longlongnose@163.com [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China)

    2015-05-29

    The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation → ROS generation → c-Src phosphorylation → NF-κB activation → TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation. - Highlights: • AMPAR is expressed in RAW264.7 macrophages and is upregulated by AMPA stimulation. • Activation of AMPAR stimulates TNF-α release in macrophages through the ROS-cSrc-NFκB signaling cascade. • Macrophage AMPAR signaling may play an important role in inflammation.

  14. Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

    Science.gov (United States)

    McNair, L F; Kohlmeier, K A

    2015-06-01

    Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the

  15. Lessons from crystal structures of kainate receptors.

    Science.gov (United States)

    Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2017-01-01

    Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor structure and how they bind agonists, antagonists and ions. The first structure of the ligand-binding domain of the GluK1 subunit was reported in 2005, seven years after publication of the crystal structure of a soluble construct of the ligand-binding domain of the AMPA-type subunit GluA2. Today, a full-length structure has been determined of GluK2 by cryo electron microscopy to 7.6 Å resolution as well as 84 high-resolution crystal structures of N-terminal domains and ligand-binding domains, including agonist and antagonist bound structures, modulatory ions and mutations. However, there are still many unanswered questions and challenges in front of us. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

  16. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans.

    Science.gov (United States)

    Wu, Ye; Arai, Amy C; Rumbaugh, Gavin; Srivastava, Anand K; Turner, Gillian; Hayashi, Takashi; Suzuki, Erika; Jiang, Yuwu; Zhang, Lilei; Rodriguez, Jayson; Boyle, Jackie; Tarpey, Patrick; Raymond, F Lucy; Nevelsteen, Joke; Froyen, Guy; Stratton, Mike; Futreal, Andy; Gecz, Jozef; Stevenson, Roger; Schwartz, Charles E; Valle, David; Huganir, Richard L; Wang, Tao

    2007-11-13

    Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs) mediate the majority of excitatory synaptic transmission in the CNS and are essential for the induction and maintenance of long-term potentiation and long-term depression, two cellular models of learning and memory. We identified a genomic deletion (0.4 Mb) involving the entire GRIA3 (encoding iGluR3) by using an X-array comparative genomic hybridization (CGH) and four missense variants (G833R, M706T, R631S, and R450Q) in functional domains of iGluR3 by sequencing 400 males with X-linked mental retardation (XLMR). Three variants were found in males with moderate MR and were absent in 500 control males. Expression studies in HEK293 cells showed that G833R resulted in a 78% reduction of iGluR3 due to protein misfolding. Whole-cell recording studies of iGluR3 homomers in HEK293 cells revealed that neither iGluR3-M706T (S2 domain) nor iGluR3-R631S (near channel core) had substantial channel function, whereas R450Q (S1 domain) was associated with accelerated receptor desensitization. When forming heteromeric receptors with iGluR2 in HEK293 cells, all four iGluR3 variants had altered desensitization kinetics. Our study provides the genetic and functional evidence that mutant iGluR3 with altered kinetic properties is associated with moderate cognitive impairment in humans.

  17. A single high dose of dexamethasone affects the phosphorylation state of glutamate AMPA receptors in the human limbic system

    Science.gov (United States)

    Lopes, M W; Leal, R B; Guarnieri, R; Schwarzbold, M L; Hoeller, A; Diaz, A P; Boos, G L; Lin, K; Linhares, M N; Nunes, J C; Quevedo, J; Bortolotto, Z A; Markowitsch, H J; Lightman, S L; Walz, R

    2016-01-01

    Glucocorticoids (GC) released during stress response exert feedforward effects in the whole brain, but particularly in the limbic circuits that modulates cognition, emotion and behavior. GC are the most commonly prescribed anti-inflammatory and immunosuppressant medication worldwide and pharmacological GC treatment has been paralleled by the high incidence of acute and chronic neuropsychiatric side effects, which reinforces the brain sensitivity for GC. Synapses can be bi-directionally modifiable via potentiation (long-term potentiation, LTP) or depotentiation (long-term depression, LTD) of synaptic transmission efficacy, and the phosphorylation state of Ser831 and Ser845 sites, in the GluA1 subunit of the glutamate AMPA receptors, are a critical event for these synaptic neuroplasticity events. Through a quasi-randomized controlled study, we show that a single high dexamethasone dose significantly reduces in a dose-dependent manner the levels of GluA1-Ser831 phosphorylation in the amygdala resected during surgery for temporal lobe epilepsy. This is the first report demonstrating GC effects on key markers of synaptic neuroplasticity in the human limbic system. The results contribute to understanding how GC affects the human brain under physiologic and pharmacologic conditions. PMID:27959333

  18. Embryonic expression of zebrafish AMPA receptor genes: zygotic gria2alpha expression initiates at the midblastula transition.

    Science.gov (United States)

    Lin, Wei-Hsiang; Wu, Chan-Hwa; Chen, Yu-Chia; Chow, Wei-Yuan

    2006-09-19

    The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the central nervous system of vertebrates. Expression profiles of 8 AMPAR genes were studied by RT-PCR analyses to elucidate the properties of AMPARs during early zebrafish development. Transcripts of all AMPAR genes are detected at the time of fertilization, suggesting maternal transcriptions of zebrafish AMPAR genes. The amounts of gria1 and gria2 transcripts are several-fold higher than that of gria3 and gria4 between 10 and 72 hpf (hour postfertilization). The edited gria2alpha transcript decreases during gastrulation period, suggesting that zygotic expression of gria2alpha begins around the time of midblastula transition. Relative to the amount of beta-actin, the amounts of AMPAR transcripts increase significantly after the completion of neurulation. The amounts of gria2 transcripts exceed the total amounts of the remaining AMPAR transcripts after 36 hpf, suggesting increases in the representation of low Ca2+ permeable AMPARs during neuronal maturation. Many but not all of the known mammalian protein-protein interaction motifs are preserved in the C-terminal domains (CTD) of zebrafish AMPARs. Before 16 hpf, the embryos express predominantly the alternative splice forms encoding longer CTD. Representations of the short CTD splice forms of gria2 and gria4alpha increase after 24 hpf, when neurulation is nearly completed.

  19. Activation of AMPA receptor in the infralimbic cortex facilitates extinction and attenuates the heroin-seeking behavior in rats.

    Science.gov (United States)

    Chen, Weisheng; Wang, Yiqi; Sun, Anna; Zhou, Linyi; Xu, Wenjin; Zhu, Huaqiang; Zhuang, Dingding; Lai, Miaojun; Zhang, Fuqiang; Zhou, Wenhua; Liu, Huifen

    2016-01-26

    Infralimbic cortex (IL) is proposed to suppress cocaine seeking after extinction, but whether the IL regulates the extinction and reinstatement of heroin-seeking behavior is unknown. To address this issue, the male SD rats were trained to self-administer heroin under a FR1 schedule for consecutive 14 days, then the rats underwent 7 daily 2h extinction session in the operant chamber. The activation of IL by microinjection PEPA, an allosteric AMPA receptor potentiator into IL before each of extinction session facilitated the extinction responding after heroin self-administration, but did not alter the locomotor activity in an open field testing environment. Other rats were first trained under a FR1 schedule for heroin self-administration for 14 days, followed by 14 days of extinction training, and reinstatement of heroin-seeking induced by cues was measured for 2h. Intra-IL microinjecting of PEPA at 15min prior to test inhibited the reinstatement of heroin-seeking induced by cues. Moreover, the expression of GluR1 in the IL and NAc remarkably increased after treatment with PEPA during the reinstatement. These finding suggested that activation of glutamatergic projection from IL to NAc shell may be involved in the extinction and reinstatement of heroin-seeking.

  20. Synthetic and endogenous cannabinoids protect retinal neurons from AMPA excitotoxicity in vivo, via activation of CB1 receptors: Involvement of PI3K/Akt and MEK/ERK signaling pathways.

    Science.gov (United States)

    Kokona, Despina; Thermos, Kyriaki

    2015-07-01

    Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways.

  1. NMDA and AMPA/kainate glutamatergic receptors in the prelimbic medial prefrontal cortex modulate the elaborated defensive behavior and innate fear-induced antinociception elicited by GABAA receptor blockade in the medial hypothalamus.

    Science.gov (United States)

    de Freitas, Renato Leonardo; Salgado-Rohner, Carlos José; Biagioni, Audrey Francisco; Medeiros, Priscila; Hallak, Jaime Eduardo Cecílio; Crippa, José Alexandre S; Coimbra, Norberto Cysne

    2014-06-01

    The aim of the present study was to investigate the involvement of N-methyl-d-aspartate (NMDA) and amino-3-hydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptors of the prelimbic (PL) division of the medial prefrontal cortex (MPFC) on the panic attack-like reactions evoked by γ-aminobutyric acid-A receptor blockade in the medial hypothalamus (MH). Rats were pretreated with NaCl 0.9%, LY235959 (NMDA receptor antagonist), and NBQX (AMPA/kainate receptor antagonist) in the PL at 3 different concentrations. Ten minutes later, the MH was treated with bicuculline, and the defensive responses were recorded for 10 min. The antagonism of NMDA receptors in the PL decreased the frequency and duration of all defensive behaviors evoked by the stimulation of the MH and reduced the innate fear-induced antinociception. However, the pretreatment of the PL cortex with NBQX was able to decrease only part of defensive responses and innate fear-induced antinociception. The present findings suggest that the NMDA-glutamatergic system of the PL is critically involved in panic-like responses and innate fear-induced antinociception and those AMPA/kainate receptors are also recruited during the elaboration of fear-induced antinociception and in panic attack-related response. The activation of the glutamatergic neurotransmission of PL division of the MPFC during the elaboration of oriented behavioral reactions elicited by the chemical stimulation of the MH recruits mainly NMDA receptors in comparison with AMPA/kainate receptors.

  2. Regulation of AMPA receptor function by the human memory-associated gene KIBRA.

    Science.gov (United States)

    Makuch, Lauren; Volk, Lenora; Anggono, Victor; Johnson, Richard C; Yu, Yilin; Duning, Kerstin; Kremerskothen, Joachim; Xia, Jun; Takamiya, Kogo; Huganir, Richard L

    2011-09-22

    KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.

  3. AMPA Receptor Phosphorylation and Synaptic Colocalization on Motor Neurons Drive Maladaptive Plasticity below Complete Spinal Cord Injury.

    Science.gov (United States)

    Huie, J Russell; Stuck, Ellen D; Lee, Kuan H; Irvine, Karen-Amanda; Beattie, Michael S; Bresnahan, Jacqueline C; Grau, James W; Ferguson, Adam R

    2015-01-01

    Clinical spinal cord injury (SCI) is accompanied by comorbid peripheral injury in 47% of patients. Human and animal modeling data have shown that painful peripheral injuries undermine long-term recovery of locomotion through unknown mechanisms. Peripheral nociceptive stimuli induce maladaptive synaptic plasticity in dorsal horn sensory systems through AMPA receptor (AMPAR) phosphorylation and trafficking to synapses. Here we test whether ventral horn motor neurons in rats demonstrate similar experience-dependent maladaptive plasticity below a complete SCI in vivo. Quantitative biochemistry demonstrated that intermittent nociceptive stimulation (INS) rapidly and selectively increases AMPAR subunit GluA1 serine 831 phosphorylation and localization to synapses in the injured spinal cord, while reducing synaptic GluA2. These changes predict motor dysfunction in the absence of cell death signaling, suggesting an opportunity for therapeutic reversal. Automated confocal time-course analysis of lumbar ventral horn motor neurons confirmed a time-dependent increase in synaptic GluA1 with concurrent decrease in synaptic GluA2. Optical fractionation of neuronal plasma membranes revealed GluA2 removal from extrasynaptic sites on motor neurons early after INS followed by removal from synapses 2 h later. As GluA2-lacking AMPARs are canonical calcium-permeable AMPARs (CP-AMPARs), their stimulus- and time-dependent insertion provides a therapeutic target for limiting calcium-dependent dynamic maladaptive plasticity after SCI. Confirming this, a selective CP-AMPAR antagonist protected against INS-induced maladaptive spinal plasticity, restoring adaptive motor responses on a sensorimotor spinal training task. These findings highlight the critical involvement of AMPARs in experience-dependent spinal cord plasticity after injury and provide a pharmacologically targetable synaptic mechanism by which early postinjury experience shapes motor plasticity.

  4. AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus.

    Science.gov (United States)

    Catania, M V; Bellomo, M; Giuffrida, R; Giuffrida, R; Stella, A M; Albanese, V

    1998-11-01

    Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

  5. Differential dendritic targeting of AMPA receptor subunit mRNAs in adult rat hippocampal principal neurons and interneurons.

    Science.gov (United States)

    Cox, David J; Racca, Claudia

    2013-06-15

    In hippocampal neurons, AMPA receptors (AMPARs) mediate fast excitatory postsynaptic responses at glutamatergic synapses, and are involved in various forms of synaptic plasticity. Dendritic local protein synthesis of selected AMPAR subunit mRNAs is considered an additional mechanism to independently and rapidly control the strength of individual synapses. We have used fluorescent in situ hybridization and immunocytochemistry to analyze the localization of AMPAR subunit (GluA1-4) mRNAs and their relationship with the translation machinery in principal cells and interneurons of the adult rat hippocampus. The mRNAs encoding all four AMPAR subunits were detected in the somata and dendrites of CA3 and CA1 pyramidal cells and those of six classes of CA1 γ-aminobutyric acid (GABA)ergic interneurons. GluA1-4 subunit mRNAs were highly localized to the apical dendrites of pyramidal cells, whereas in interneurons they were present in multiple dendrites. In contrast, in the dentate gyrus, GluA1-4 subunit mRNAs were virtually restricted to the somata and were absent from the dendrites of granule cells. These different regional and cell type-specific labeling patterns also correlated with the localization of markers for components of the protein synthesis machinery. Our results support the local translation of GluA1-4 mRNAs in dendrites of hippocampal pyramidal cells and CA1 interneurons but not in granule cells of the dentate gyrus. Furthermore, the regional and cell type-specific differences we observed suggest that each cell type uses distinct ways of regulating the local translation of AMPAR subunits.

  6. Involvement of AMPA/kainate and GABAA receptors in topiramate neuroprotective effects against methylphenidate abuse sequels involving oxidative stress and inflammation in rat isolated hippocampus.

    Science.gov (United States)

    Motaghinejad, Majid; Motevalian, Manijeh

    2016-08-01

    Abuses of methylphenidate (MPH) as psychostimulant cause neural damage of brain cells. Neuroprotective properties of topiramate (TPM) have been indicated in several studies but its exact mechanism of action remains unclear. The current study evaluates protective role of various doses of TPM and its mechanism of action in MPH induced oxidative stress and inflammation. The neuroprotective effects of various doses of TPM against MPH induced oxidative stress and inflammation were evaluated and then the action of TPM was studied in presence of domoic acid (DOM), as AMPA/kainate receptor agonist and bicuculline (BIC) as GABAA receptor antagonist, in isolated rat hippocampus. Open Field Test (OFT) was used to investigate motor activity changes. Oxidative, antioxidant and inflammatory factors were measured in isolated hippocampus. TPM (70 and 100mg/kg) decreased MPH induced motor activity disturbances and inhibit MPH induced oxidative stress and inflammation. On the other hand pretreatment of animals with DOM or BIC, inhibit this effect of TPM and potentiate MPH induced motor activity disturbances and increased lipid peroxidation, mitochondrial oxidized form of glutathione (GSSG) level, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in isolated hippocampal cells and decreased reduced form of glutathione (GSH) level, superoxide dismutase, glutathione peroxidase and glutathione reductase activity. It seems that TPM can protect cells of hippocampus from oxidative stress and neuroinflammation and it could be partly by activation of GABAA receptor and inhibition of AMPA/kainite receptor.

  7. Central nitric oxide modulates hindquarter vasodilation elicited by AMPA receptor stimulation in the NTS of conscious rats.

    Science.gov (United States)

    Dias, Ana Carolina Rodrigues; Colombari, Eduardo

    2006-05-01

    Microinjection of S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in the nucleus of the solitary tract (NTS) of conscious rats causes hypertension, bradycardia, and vasoconstriction in the renal, mesenteric, and hindquarter vascular beds. In the hindquarter, the initial vasoconstriction is followed by vasodilation with AMPA doses >5 pmol/100 nl. To test the hypothesis that this vasodilation is caused by activation of a nitroxidergic pathway in the NTS, we examined the effect of pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 nmol/100 nl, microinjected into the NTS) on changes in mean arterial pressure, heart rate, and regional vascular conductance (VC) induced by microinjection of AMPA (10 pmol/100 nl in the NTS) in conscious rats. AMPA increased hindquarter VC by 18 +/- 4%, but after pretreatment with L-NAME, AMPA reduced hindquarter VC by 16 +/- 7% and 17 +/- 9% (5 and 15 min after pretreatment, P NTS activates both vasodilatatory and vasoconstrictor mechanisms and that the vasodilatatory mechanism depends on production of nitric oxide in the NTS.

  8. High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.

    Science.gov (United States)

    Baude, A; Nusser, Z; Molnár, E; McIlhinney, R A; Somogyi, P

    1995-12-01

    The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold

  9. AMPA receptor trafficking in inflammation-induced dorsal horn central sensitization

    Institute of Scientific and Technical Information of China (English)

    Yuan-Xiang Tao

    2012-01-01

    Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal horn central sensitization (a specific form of synaptic plasticity) that is associated with persistent pain.Recent studies have shown that peripheral inflammation drives changes in α-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid receptor (AMPAR) subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain.Here,we review current evidence to illustrate how spinal cord AMPARs participate in the dorsal horn central sensitization associated with persistent pain.Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.

  10. Acute stress causes rapid synaptic insertion of Ca2+ -permeable AMPA receptors to facilitate long-term potentiation in the hippocampus.

    Science.gov (United States)

    Whitehead, Garry; Jo, Jihoon; Hogg, Ellen L; Piers, Thomas; Kim, Dong-Hyun; Seaton, Gillian; Seok, Heon; Bru-Mercier, Gilles; Son, Gi Hoon; Regan, Philip; Hildebrandt, Lars; Waite, Eleanor; Kim, Byeong-Chae; Kerrigan, Talitha L; Kim, Kyungjin; Whitcomb, Daniel J; Collingridge, Graham L; Lightman, Stafford L; Cho, Kwangwook

    2013-12-01

    The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+ -permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

  11. Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

    DEFF Research Database (Denmark)

    Vogensen, S B; Jensen, H S; Stensbøl, T B;

    2000-01-01

    tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50......) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed...

  12. The antidepressant-like effects of glutamatergic drugs ketamine and AMPA receptor potentiator LY 451646 are preserved in bdnf⁺/⁻ heterozygous null mice.

    Science.gov (United States)

    Lindholm, Jesse S O; Autio, Henri; Vesa, Liisa; Antila, Hanna; Lindemann, Lothar; Hoener, Marius C; Skolnick, Phil; Rantamäki, Tomi; Castrén, Eero

    2012-01-01

    Accumulating evidence suggests that biogenic amine-based antidepressants act, at least in part, via regulation of brain-derived neurotrophic factor (BDNF) signaling. Biogenic amine-based antidepressants increase BDNF synthesis and activate its signaling pathway through TrkB receptors. Moreover, the antidepressant-like effects of these molecules are abolished in BDNF deficient mice. Glutamate-based drugs, including the NMDA antagonist ketamine, and the AMPA receptor potentiator LY 451646, mimic the effects of antidepressants in preclinical tests with high predictive validity. In humans, a single intravenous dose of ketamine produces an antidepressant effect that is rapid, robust and persistent. In this study, we examined the role of BDNF in expression of the antidepressant-like effects of ketamine and an AMPA receptor potentiator (LY 451646) in the forced swim test (FST). Ketamine and LY 451646 produced antidepressant-like effects in the FST in mice at 45 min after a single injection, but no effects were observed one week after a single ketamine injection. As previously reported, the effects of imipramine in the forced swim test were blunted in heterozygous BDNF knockout (bdnf(+/-)) mice. However ketamine and LY 451646 produced similar antidepressant-like responses in wildtype and bdnf(+/-) mice. Neither ketamine nor LY 451646 significantly influenced the levels BDNF or TrkB phosphorylation in the hippocampus when assessed at 45 min or 7 days after the drug administration. These data demonstrate that under the conditions tested, neither ketamine nor the AMPA-potentiator LY 451656 activate BDNF signaling, but produce a characteristic antidepressant-like response in heterozygous bdnf(+/-) mice. These data indicate that unlike biogenic amine-based agents, BDNF signaling does not play a pivotal role in the antidepressant effects of glutamate-based compounds. This article is part of a Special Issue entitled 'Anxiety and Depression'.

  13. Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning.

    Science.gov (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce

    2010-11-01

    Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

  14. Efecto neuroprotector de los cannabinoides sobre la muerte neuronal inducida por Ampa en la médula espinal: Activación conjunta de los receptores CB1 y CB2

    Directory of Open Access Journals (Sweden)

    Carmen Guaza

    2005-03-01

    Full Text Available La sobreactivación de receptores de glutamato, como el receptor AMPA, induce la muerte neural por un proceso denominado excitotoxicidad, el cual ha sido claramente implicado en enfermedades agudas del sistema nerviso central (SNC, particularmente con daño axonal.

  15. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells

    OpenAIRE

    Yu LIU; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-01-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca2+ and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the durati...

  16. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.

    Directory of Open Access Journals (Sweden)

    Andrew R Gallimore

    2016-01-01

    Full Text Available The expression of long-term depression (LTD in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins-glutamate receptor-interacting protein (GRIP and protein interacting with C kinase 1 (PICK1-regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process.

  17. Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after cocaine exposure: When, how and why?

    Directory of Open Access Journals (Sweden)

    Marina E Wolf

    2012-06-01

    Full Text Available In animal models of drug addiction, cocaine exposure has been shown to increase levels of calcium-permeable AMPA receptors (CP-AMPARs in two brain regions that are critical for motivation and reward - the ventral tegmental area (VTA and the nucleus accumbens (NAc. This review compares CP-AMPAR plasticity in the two brain regions and addresses its functional significance. In VTA dopamine neurons, cocaine exposure results in synaptic insertion of high conductance CP-AMPARs in exchange for lower conductance calcium-impermeable AMPARs (CI-AMPARs. This plasticity is rapid (hours, GluA2-dependent, and can be observed with a single cocaine injection. In addition to strengthening synapses and altering Ca2+ signaling, CP-AMPAR insertion affects subsequent induction of plasticity at VTA synapses. However, CP-AMPAR insertion is unlikely to mediate the increased dopamine cell activity that occurs during early withdrawal from cocaine exposure. Within the VTA, the group I metabotropic glutamate receptor mGluR1 exerts a negative influence on CP-AMPAR accumulation. Acutely, mGluR1 stimulation elicits a form of LTD resulting from CP-AMPAR removal and CI-AMPAR insertion. In medium spiny neurons (MSNs of the NAc, extended access cocaine self-administration is required to increase CP-AMPAR levels. This is first detected after approximately a month of withdrawal and then persists. Once present in NAc synapses, CP-AMPARs mediate the expression of incubation of cue-induced cocaine craving. The mechanism of their accumulation may be GluA1-dependent, which differs from that observed in the VTA. However, similar to VTA, mGluR1 stimulation removes CP-AMPARs from MSN synapses. Loss of mGluR1 tone during cocaine withdrawal may contribute to CP-AMPAR accumulation in the NAc. Thus, results in both brain regions point to the possibility of using positive modulators of mGluR1 as a treatment for cocaine addiction.

  18. Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

    Science.gov (United States)

    Akbarian, S.; Smith, M. A.; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA

  19. Synthesis and biological evaluation of analogues of 7-chloro-4,5-dihydro-4- oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (TQX-173) as novel selective AMPA receptor antagonists.

    Science.gov (United States)

    Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Calabri, Francesca Romana; Filacchioni, Guido; Galli, Alessandro; Costagli, Chiara; Carlà, Vincenzo

    2004-01-01

    In recent papers (Catarzi, D.; et al. J. Med. Chem. 2000, 43, 3824-3826; 2001, 44, 3157-3165) we reported chemical and biological studies on 4,5-dihydro-4-oxo-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylates (TQXs) bearing different nitrogen-containing heterocycles at position-8. In particular, from these studies it emerged that both the 7-chloro-4,5-dihydro-4-oxo-8-(1,2,4-triazol-4-yl)-1,2,4-triazolo[1,5-a] quinoxaline-2-carboxylic acid TQX-173 (compound B) and its corresponding ethyl ester (compound A) were the most active and selective compounds of this series. In pursuing our investigation on the structure-activity relationships of these TQX derivatives, different electron-withdrawing groups (CF(3), NO(2)) were introduced at position 7 on the TQX ring system, replacing the 7-chloro substituent of B and of other selected 8-heteroaryltriazoloquinoxaline-2-carboxylates previously described. All the newly synthesized compounds were biologically evaluated for their binding at the Gly/NMDA, AMPA, and KA high-affinity receptors. Gly/NMDA binding assays were performed to assess the selectivity of the reported compounds toward the AMPA receptor. Compounds endowed with micromolar binding affinity for the KA high-affinity binding site were also evaluated for their binding at the KA low-affinity receptor. Some selected compounds were also tested for their functional antagonist activity at the AMPA and NMDA receptor-ion channel complex. The results obtained in this study have pointed out that 4,5-dihydro-7-nitro-4-oxo-8-(3-carboxypyrrol-1-yl)-1,2,4-triazolo[1,5-a]quinoxaline-2-carboxylic acid (9b) and its corresponding ethyl ester (9a) are the most potent and selective AMPA receptor antagonists reported to date among the TQX series.

  20. Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.

    Science.gov (United States)

    Dennis, Siobhan H; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G; Henley, Jeremy M; Mellor, Jack R

    2011-08-17

    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

  1. Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells.

    Science.gov (United States)

    Lajeunesse, Francis; Kröger, Helmut; Timofeev, Igor

    2013-01-01

    Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-D-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size.

  2. PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors.

    Science.gov (United States)

    Gerace, E; Masi, A; Resta, F; Felici, R; Landucci, E; Mello, T; Pellegrini-Giampietro, D E; Mannaioni, G; Moroni, F

    2014-10-01

    An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100μM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting

  3. Enhanced odor discrimination and impaired olfactory memory by spatially controlled switch of AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Derya R Shimshek

    2005-11-01

    Full Text Available Genetic perturbations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs are widely used to dissect molecular mechanisms of sensory coding, learning, and memory. In this study, we investigated the role of Ca2+-permeable AMPARs in olfactory behavior. AMPAR modification was obtained by depletion of the GluR-B subunit or expression of unedited GluR-B(Q, both leading to increased Ca2+ permeability of AMPARs. Mice with this functional AMPAR switch, specifically in forebrain, showed enhanced olfactory discrimination and more rapid learning in a go/no-go operant conditioning task. Olfactory memory, however, was dramatically impaired. GluR-B depletion in forebrain was ectopically variable ("mosaic" among individuals and strongly correlated with decreased olfactory memory in hippocampus and cortex. Accordingly, memory was rescued by transgenic GluR-B expression restricted to piriform cortex and hippocampus, while enhanced odor discrimination was independent of both GluR-B variability and transgenic GluR-B expression. Thus, correlated differences in behavior and levels of GluR-B expression allowed a mechanistic and spatial dissection of olfactory learning, discrimination, and memory capabilities.

  4. A role for calcium-permeable AMPA receptors in synaptic plasticity and learning.

    Directory of Open Access Journals (Sweden)

    Brian J Wiltgen

    Full Text Available A central concept in the field of learning and memory is that NMDARs are essential for synaptic plasticity and memory formation. Surprisingly then, multiple studies have found that behavioral experience can reduce or eliminate the contribution of these receptors to learning. The cellular mechanisms that mediate learning in the absence of NMDAR activation are currently unknown. To address this issue, we examined the contribution of Ca(2+-permeable AMPARs to learning and plasticity in the hippocampus. Mutant mice were engineered with a conditional genetic deletion of GluR2 in the CA1 region of the hippocampus (GluR2-cKO mice. Electrophysiology experiments in these animals revealed a novel form of long-term potentiation (LTP that was independent of NMDARs and mediated by GluR2-lacking Ca(2+-permeable AMPARs. Behavioral analyses found that GluR2-cKO mice were impaired on multiple hippocampus-dependent learning tasks that required NMDAR activation. This suggests that AMPAR-mediated LTP interferes with NMDAR-dependent plasticity. In contrast, NMDAR-independent learning was normal in knockout mice and required the activation of Ca(2+-permeable AMPARs. These results suggest that GluR2-lacking AMPARs play a functional and previously unidentified role in learning; they appear to mediate changes in synaptic strength that occur after plasticity has been established by NMDARs.

  5. Oxygen/glucose Deprivation Induces a Reduction in Synaptic AMPA Receptors on Hippocampal CA3 Neurons Mediated by mGluR1 and A3 Receptors

    Science.gov (United States)

    Dennis, Siobhan H.; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G.; Henley, Jeremy M.; Mellor, Jack R.

    2011-01-01

    Summary Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighbouring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+ resulting in delayed cell death. However, it is unclear if the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 minute OGD protocol a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by mGluR1 or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalisation of AMPARs after OGD. We also show a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists indicating that AMPARs are degraded following internalisation. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection. PMID:21849555

  6. Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

    Science.gov (United States)

    McCartney, Amber J; Zolov, Sergey N; Kauffman, Emily J; Zhang, Yanling; Strunk, Bethany S; Weisman, Lois S; Sutton, Michael A

    2014-11-11

    Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

  7. Blocking GABA(A) inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus.

    Science.gov (United States)

    Crépel, V; Khazipov, R; Ben-Ari, Y

    1997-04-01

    We have investigated the conditions required to evoke polysynaptic responses in the isolated CA1 region of hippocampal slices from Wistar adult rats. Experiments were performed with extracellular and whole cell recording techniques. In the presence of bicuculline (10 microM), 6-cyano-7-nitroquinoxaline-2-3-dione (10 microM), glycine (10 microM), and a low external concentration of Mg2+ (0.3 mM), electrical stimulation of the Schaffer collaterals/commissural pathway evoked graded N-methyl-D-aspartate (NMDA)-receptor-mediated late field potentials in the stratum radiatum of the CA1 region. These responses were generated via polysynaptic connections because their latency varied strongly and inversely with the stimulation intensity and they were abolished by a high concentration of divalent cations (7 mM Ca2+). These responses likely were driven by local collateral branches of CA1 pyramidal cell axons because focal application of tetrodotoxin (30 microM) in the stratum oriens strongly reduced the late synaptic component and antidromic stimulation of CA1 pyramidal cells could evoke the polysynaptic response. Current-source density analysis suggested that the polysynaptic response was generated along the proximal part of the apical dendrites of CA1 pyramidal cells (50-150 microm below the pyramidal cell layer in the stratum radiatum). In physiological concentration of Mg2+ (1.3 mM), the pharmacologically isolated NMDA-receptor-mediated polysynaptic response was abolished. In control artificial cerebrospinal fluid (with physiological concentration of Mg2+), bicuculline ( 10 microM) generated a graded polysynaptic response. Under these conditions, this response was mediated both by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/NMDA receptors. In the presence of D-2-amino-5-phosphonovalerate (50 microM), the polysynaptic response could be mediated by AMPA receptors, although less efficiently. In conclusion, suppression of gamma-aminobutyric acid

  8. Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior.

    Science.gov (United States)

    Choi, Jeong Il; Svensson, Camilla I; Koehrn, Fred J; Bhuskute, Aditi; Sorkin, Linda S

    2010-05-01

    In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca(2+) permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100microg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5microg) and LY294002; 50 and 100microg) and Akt (Akt inhibitor IV; 3microg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and alpha-motor neurons (peak 45min) and later (peak 2h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

  9. Differential effect of NMDA and AMPA receptor blockade on protein synthesis in the rat infarct borderzone

    DEFF Research Database (Denmark)

    Christensen, Thomas; Bruhn, T; Frank, L

    1996-01-01

    treated with either saline, MK-801 (5 mg/kg i.p.) or NBQX (30 mg/kg i.p. x 3) were subjected to permanent MCAO. Regional CPSR and volumes of gray matter structures displaying normal CPSR were measured in coronal cryosections of the brain by quantitative autoradiography following an i.v. bolus injection....... Treatment with MK-801 significantly increased the volume of tissue with normal CPSR in the ischemic hemisphere compared to controls, whereas this was not seen with NBQX treatment. The results suggest that MK-801 and NBQX have different effects on peri-infarct protein synthesis after MCAO. Since both...

  10. Methylphenidate amplifies long-term potentiation in rat hippocampus CA1 area involving the insertion of AMPA receptors by activation of β-adrenergic and D1/D5 receptors.

    Science.gov (United States)

    Rozas, C; Carvallo, C; Contreras, D; Carreño, M; Ugarte, G; Delgado, R; Zeise, M L; Morales, B

    2015-12-01

    Methylphenidate (MPH, Ritalin©) is widely used in the treatment of Attention Deficit Hyperactivity Disorder and recently as a drug of abuse. Although the effect of MPH has been studied in brain regions such as striatum and prefrontal cortex (PFC), the hippocampus has received relatively little attention. It is known that MPH increases the TBS-dependent Long Term Potentiation (LTP) in the CA1 area. However, the cellular and molecular mechanisms involved in this process are still unknown. Using field potential recordings and western blot analysis in rat hippocampal slices of young rats, we found that acute application of MPH enhances LTP in CA3-CA1 synapses in a dose-dependent manner with an EC50 of 73.44±6.32 nM. Using specific antagonists and paired-pulse facilitation protocols, we observed that the MPH-dependent increase of LTP involves not only β-adrenergic receptors activation but also post-synaptic D1/D5 dopamine receptors. The inhibition of PKA with PKI, suppressed the facilitation of LTP induced by MPH consistent with an involvement of the adenyl cyclase-cAMP-PKA dependent cascade downstream of the activation of D1/D5 receptors. In addition, samples of CA1 areas taken from slices potentiated with MPH presented an increase in the phosphorylation of the Ser845 residue of the GluA1 subunit of AMPA receptors compared to control slices. This effect was reverted by SCH23390, antagonist of D1/D5 receptors, and PKI. Moreover, we found an increase of surface-associated functional AMPA receptors. We propose that MPH increases TBS-dependent LTP in CA3-CA1 synapses through a polysynaptic mechanism involving activation of β-adrenergic and D1/D5 dopaminergic receptors and promoting the trafficking and insertion of functional AMPA receptors to the plasma membrane.

  11. Ca2+ imaging of mouse neocortical interneurone dendrites: Contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+dynamics

    Science.gov (United States)

    Goldberg, Jesse H; Yuste, Rafael; Tamas, Gabor

    2003-01-01

    In this second study, we have combined two-photon calcium imaging with whole-cell recording and anatomic reconstructions to directly characterize synaptically evoked calcium signals in three types of mouse V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). We observed that subthreshold synaptic activation evoked calcium signals locally restricted to individual dendritic compartments. These signals were mediated by NMDA receptors (NMDARs) in AD and IS cells, whereas in FS cells, calcium-permeable AMPA receptors (CP-AMPARs) provided an additional and kinetically distinct influx. Furthermore, even a single, subthreshold synaptic activation evoked a larger dendritic calcium influx than backpropagating action potentials. Our results demonstrate that NMDARs dominate subthreshold calcium dynamics in interneurones and reveal the functional contribution of CP-AMPARs to a specific subclass of cortical interneurone. These data highlight different strategies in dendritic signal processing by distinct classes of interneurones. PMID:12844507

  12. Effect of curcumin on AMPA and kainate receptor-mediated calcium influx in cultured rat hippocampal neurons%姜黄素对AMPA/KA受体介导大鼠海马神经元钙内流的影响

    Institute of Scientific and Technical Information of China (English)

    杜鹏; 彭伟锋; 刘剑英; 林豪杰; 马昱; 汪昕; 范薇

    2009-01-01

    目的 探讨姜黄素对α-氨基-3-羧基-5-甲基异恶唑-4-丙酸(AMPA)/海人酸(KA)受体介导大鼠海马神经元钙内流的影响.方法 选用胚胎17dSD鼠分离海马,离体培养海马神经元,借助活体钙荧光染色和激光共聚焦钙成像技术观察100μmol/LKA刺激海马神经元内钙的变化,不同浓度(5、10、15、30、50 μmol/L)姜黄素预孵育海马神经元30min对100μmol/L KA刺激下细胞内钙变化的影响,15 μmol/L姜黄素对不同浓度(10、30、50、100、200、300 μmol/L)KA刺激海马神经元内钙变化的影响.应用钴染色技术观察(30、100 μmol/L KA)刺激后海马神经元钴阳性染色细胞变化.姜黄素预孵育30min对KA刺激导致钴阳性染色细胞变化的影响.结果 不同浓度姜黄素预孵育30 min均可以明显缓解100 μmol/L或30 μmol/L KA导致的细胞内钙升高程度.差异均有统计学意义(P<0.05),其中15 μmol/L姜黄素作用最为明显.30μmol/L或100 μmol/LKA刺激均可以引起海马神经元钴染色阳性细胞增加,15 μmol/L姜黄素预处理30 min后明显减少钴染色阳性细胞,差异有统计学意义(P<0.05),而其他浓度(5 μmol/L或30 μmol/L)姜黄素未见明显影响.结论 一定浓度的姜黄素可以影响AMPA/KA受体介导大鼠海马神经元钙内流.这可能是姜黄素抗癫痫作用的一个机制.%Objective To investigate the effect of curcumin on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) receptor-mediated calcium influx in cultured rat hippocampal neurons. Methods The hippocampal neurons from SD rat embryos (17 days old) were cultured for 9 days, and fluorescent calcium chelator and confocal microscopy calcium imaging were used to observe the changes in intracellular free calcium in the neurons following stimulation with 100 μmol/L KA. The effect of curcumin pretreatment at different concentrations (10, 30, 50, 100, 200 and 300 μmol/L) for 30 min on 100 μmol/L KA

  13. Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties

    DEFF Research Database (Denmark)

    Campiani, G; Morelli, E; Nacci, V;

    2001-01-01

    (S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound...

  14. Enhanced Long-Term and Impaired Short-Term Spatial Memory in GluA1 AMPA Receptor Subunit Knockout Mice: Evidence for a Dual-Process Memory Model

    Science.gov (United States)

    Sanderson, David J.; Good, Mark A.; Skelton, Kathryn; Sprengel, Rolf; Seeburg, Peter H.; Rawlins, J. Nicholas P.; Bannerman, David M.

    2009-01-01

    The GluA1 AMPA receptor subunit is a key mediator of hippocampal synaptic plasticity and is especially important for a rapidly-induced, short-lasting form of potentiation. GluA1 gene deletion impairs hippocampus-dependent, spatial working memory, but spares hippocampus-dependent spatial reference memory. These findings may reflect the necessity of…

  15. Mechanism of partial agonism at the GluR2 AMPA receptor: Measurements of lobe orientation in solution.

    Science.gov (United States)

    Maltsev, Alexander S; Ahmed, Ahmed H; Fenwick, Michael K; Jane, David E; Oswald, Robert E

    2008-10-07

    The mechanism by which the binding of a neurotransmitter to a receptor leads to channel opening is a central issue in molecular neurobiology. The structure of the agonist binding domain of ionotropic glutamate receptors has led to an improved understanding of the changes in structure that accompany agonist binding and have provided important clues about the link between these structural changes and channel activation and desensitization. However, because the binding domain has exhibited different structures under different crystallization conditions, understanding the structure in the absence of crystal packing is of considerable importance. The orientation of the two lobes of the binding domain in the presence of a full agonist, an antagonist, and several partial agonists was measured using NMR spectroscopy by employing residual dipolar couplings. For some partial agonists, the solution conformation differs from that observed in the crystal. A model of channel activation based on the results is discussed.

  16. Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons

    DEFF Research Database (Denmark)

    Jensen, J B; Lund, Trine Meldgaard; Timmermann, D B;

    2001-01-01

    of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during...

  17. Episodic sucrose intake during food restriction increases synaptic abundance of AMPA receptors in nucleus accumbens and augments intake of sucrose following restoration of ad libitum feeding.

    Science.gov (United States)

    Peng, X-X; Lister, A; Rabinowitsch, A; Kolaric, R; Cabeza de Vaca, S; Ziff, E B; Carr, K D

    2015-06-01

    Weight-loss dieting often leads to loss of control, rebound weight gain, and is a risk factor for binge pathology. Based on findings that food restriction (FR) upregulates sucrose-induced trafficking of glutamatergic AMPA receptors to the nucleus accumbens (NAc) postsynaptic density (PSD), this study was an initial test of the hypothesis that episodic "breakthrough" intake of forbidden food during dieting interacts with upregulated mechanisms of synaptic plasticity to increase reward-driven feeding. Ad libitum (AL) fed and FR subjects consumed a limited amount of 10% sucrose, or had access to water, every other day for 10 occasions. Beginning three weeks after return of FR rats to AL feeding, when 24-h chow intake and rate of body weight gain had normalized, subjects with a history of sucrose intake during FR consumed more sucrose during a four week intermittent access protocol than the two AL groups and the group that had access to water during FR. In an experiment that substituted noncontingent administration of d-amphetamine for sucrose, FR subjects displayed an enhanced locomotor response during active FR but a blunted response, relative to AL subjects, during recovery from FR. This result suggests that the enduring increase in sucrose consumption is unlikely to be explained by residual enhancing effects of FR on dopamine signaling. In a biochemical experiment which paralleled the sucrose behavioral experiment, rats with a history of sucrose intake during FR displayed increased abundance of pSer845-GluA1, GluA2, and GluA3 in the NAc PSD relative to rats with a history of FR without sucrose access and rats that had been AL throughout, whether they had a history of episodic sucrose intake or not. A history of FR, with or without a history of sucrose intake, was associated with increased abundance of GluA1. A terminal 15-min bout of sucrose intake produced a further increase in pSer845-GluA1 and GluA2 in subjects with a history of sucrose intake during FR

  18. Association of the AMPA receptor-related postsynaptic density proteins GRIP and ABP with subsets of glutamate-sensitive neurons in the rat retina.

    Science.gov (United States)

    Gábriel, Robert; de Souza, Sunita; Ziff, Edward B; Witkovsky, Paul

    2002-07-22

    We used specific antibodies against two postsynaptic density proteins, GRIP (glutamate receptor interacting protein) and ABP (AMPA receptor-binding protein), to study their distribution in the rat retina. In the central nervous system, it has been shown that both proteins bind strongly to the AMPA glutamate receptor (GluR) 2/3 subunits, but not other GluRs, through a set of three PDZ domains. Western blots detected a single GRIP protein that was virtually identical in retina and brain, whereas retinal ABP corresponded to only one of three ABP peptides found in brain. The retinal distributions of GluR2/3, GRIP, and ABP immunoreactivity (IR) were similar but not identical. GluR2/3 immunoreactivity (IR) was abundant in both plexiform layers and in large perikarya. ABP IR was concentrated in large perikarya but was sparse in the plexiform layers, whereas GRIP IR was relatively more abundant in the plexiform layers than in perikarya. Immunolabel for these three antibodies consisted of puncta ABP IR was examined by double labeling subclasses of retinal neuron with characteristic marker proteins, e.g., calbindin. GRIP, ABP, and GluR2/3 IR were detected in horizontal cells, dopaminergic and glycinergic AII amacrine cells and large ganglion cells. Immunolabel was absent in rod bipolar and weak or absent in cholinergic amacrine cells. By using the tyramide method of signal amplification, a colocalization of GluR2/3 was found with either GRIP or ABP in horizontal cell terminals, and perikarya of amacrine and ganglion cells. Our results show that ABP and GRIP colocalize with GluR2/3 in particular subsets of retinal neuron, as was previously established for certain neurons in the brain.

  19. Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

    Directory of Open Access Journals (Sweden)

    Zhe eJin

    2014-01-01

    Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

  20. Orchestrated regulation of Nogo receptors, LOTUS, AMPA receptors and BDNF in an ECT model suggests opening and closure of a window of synaptic plasticity.

    Directory of Open Access Journals (Sweden)

    Max Nordgren

    Full Text Available Electroconvulsive therapy (ECT is an efficient and relatively fast acting treatment for depression. However, one severe side effect of the treatment is retrograde amnesia, which in certain cases can be long-term. The mechanisms behind the antidepressant effect and the amnesia are not well understood. We hypothesized that ECT causes transient downregulation of key molecules needed to stabilize synaptic structure and to prevent Ca2+ influx, and a simultaneous increase in neurotrophic factors, thus providing a short time window of increased structural synaptic plasticity. Here we followed regulation of NgR1, NgR3, LOTUS, BDNF, and AMPA subunits GluR1 and GluR2 flip and flop mRNA levels in hippocampus at 2, 4, 12, 24, and 72 hours after a single episode of induced electroconvulsive seizures (ECS in rats. NgR1 and LOTUS mRNA levels were transiently downregulated in the dentate gyrus 2, 4, 12 and 4, 12, 24 h after ECS treatment, respectively. GluR2 flip, flop and GluR1 flop were downregulated at 4 h. GluR2 flip remained downregulated at 12 h. In contrast, BDNF, NgR3 and GluR1 flip mRNA levels were upregulated. Thus, ECS treatment induces a transient regulation of factors important for neuronal plasticity. Our data provide correlations between ECS treatment and molecular events compatible with the hypothesis that both effects and side effects of ECT may be caused by structural synaptic rearrangements.

  1. Pharmacological and structural characterization of conformationally restricted (S)-glutamate analogues at ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Juknaite, Lina; Venskutonyte, Raminta; Assaf, Zeinab

    2012-01-01

    A2 and the kainate receptor GluK3. These structures show that CBG-IV interacts with the binding pocket in the same way as (S)-glutamate. The binding affinities reveal that CBG-IV has high affinity at the AMPA and kainate receptor subtypes. Appreciable binding affinity of CBG-IV was not observed......Conformationally restricted glutamate analogues have been pharmacologically characterized at AMPA and kainate receptors and the crystal structures have been solved of the ligand (2S,1'R,2'S)-2-(2'-carboxycyclobutyl)glycine (CBG-IV) in complex with the ligand binding domains of the AMPA receptor Glu...... at NMDA receptors, where the introduction of the carbocyclic ring is expected to lead to a steric clash with binding site residues. CBG-IV was demonstrated to be an agonist at both GluA2 and the kainate receptor GluK1. CBG-IV showed high affinity binding to GluK1 compared to GluA2, GluK2 and GluK3, which...

  2. Effects of ketamine-midazolam anesthesia on the expression of NMDA and AMPA receptor subunit in the peri-infarction of rat brain

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yue-lin; ZHANG Peng-bo; QIU Shu-dong; LIU Yong; TIAN Ying-fang; WANG Ying

    2006-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors play an important role in the neurons death induced by ischemia.The mitigating effect of intravenous anesthetics on ischemic neuron injury is related to their influence on NMDA receptors. This study was performed to investigate the effect of ketamine-midazolam anesthesia on the NMDA and AMPA receptor subunits expression in the peri-infarction of ischemic rat brain and explore its potential mechanism of neuroprotection.This study was supported by National Natural Science Foundation of China (NSFC) (No.30200291).Methods Thirty Sprague Dawley (SD) rats were subjected to permanent middle cerebral artery occlusion under ketamine/atropine (100/0.05 mg/kg) or ketamine-midazolam/atropine (60/50/0.05 mg/kg) intraperitoneal anesthesia (n=15 each). Twenty-four hours after ischemia, five rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally and infarct size was measured. Twenty-four and 72 hours after ischemia, four rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally. After staining the brain tissue slices with toluidine blue, the survived neurons in the peri-infarction were observed. Also, the expression level of NMDA receptors 1 (NR1), NMDA receptors 2A (NR2A), NMDA receptors 2B (NR2B) and AMPA (GluR1 subunit) were determined by grayscale analysis in immunohistochemical stained slices.Results Compared with ketamine anesthesia, ketamine-midazolam anesthesia produced not only smaller infarct size [(24.1±4.6)% vs (38.4±4.2)%, P<0.05], but also higher neuron density (24 hours: 846± 16 vs 756±24,P<0.05; 72 hours: 882±22 vs 785± 18, P<0.05) and lower NR2A (24 hours: 123.0±4.9 vs 95.0±2.5, P<0.05; 72 hours: 77.8±4.1 vs 54.2±3.9, P<0.05) and NR2B (24 hours: 98.5±2.7 vs 76.3±2.4, P<0.05; 72hours: 67.2

  3. Long-term changes in brain following continuous phencyclidine administration: An autoradiographic study using flunitrazepam, ketanserin, mazindol, quinuclidinyl benzilate, piperidyl-3,4-{sup 3}H(N)-TCP, and AMPA receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Ellison, Gaylord; Keys, Alan; Noguchi, Kevin [Univ. of California Los Angeles, Dept. of Psychology, Los Angeles, CA (United States)

    1999-05-01

    Phencyclidine induces a model psychosis which can persist for prolonged periods and presents a strong drug model of schizophrenia. When given continuously for several days to rats, phencyclidine and other N-methyl-D-aspartate (NMDA) antagonists induce neural degeneration in a variety of limbic structures, including retrosplenial cortex, hippocampus, septohippocampal projections, and piriform cortex. In an attempt to further clarify the mechanisms underlying these degeneration patterns, autoradiographic studies using a variety of receptor ligands were conducted in animals 21 days after an identical dosage of the continuous phencyclidine administration employed in the previous degeneration studies. The results indicated enduring alterations in a number of receptors: these included decreased piperidyl-3,4-{sup 3}H(N)-TCP (TCP), flunitrazepam, and mazindol binding in many of the limbic regions in which degeneration has been reported previously. Quinuclidinyl benzilate and (AMPA) binding were decreased in anterior cingulate and piriform cortex, and in accumbens and striatum. Piperidyl-3,4-{sup 3}H(N)-TCP binding was decreased in most hippocampal regions. Many of these long-term alterations would not have been predicted by prior studies of the neurotoxic effects of continuous phencyclidine, and these results do not suggest a unitary source for the neurotoxicity. Whereas retrosplenial cortex, the structure which degenerates earliest, showed minimal alterations, some of the most consistent, long term alterations were in structures which evidence no immediate signs of neural degeneration, such as anterior cingulate cortex and caudate nucleus. In these structures, some of the receptor changes appeared to develop gradually (they were not present immediately after cessation of drug administration), and thus were perhaps due to changed input from regions evidencing neurotoxicity. Some of these findings, particularly in anterior cingulate, may have implications for models of

  4. Excitatory synapses are stronger in the hippocampus of Rett syndrome mice due to altered synaptic trafficking of AMPA-type glutamate receptors.

    Science.gov (United States)

    Li, Wei; Xu, Xin; Pozzo-Miller, Lucas

    2016-03-15

    Deficits in long-term potentiation (LTP) at central excitatory synapses are thought to contribute to cognitive impairments in neurodevelopmental disorders associated with intellectual disability and autism. Using the methyl-CpG-binding protein 2 (Mecp2) knockout (KO) mouse model of Rett syndrome, we show that naïve excitatory synapses onto hippocampal pyramidal neurons of symptomatic mice have all of the hallmarks of potentiated synapses. Stronger Mecp2 KO synapses failed to undergo LTP after either theta-burst afferent stimulation or pairing afferent stimulation with postsynaptic depolarization. On the other hand, basal synaptic strength and LTP were not affected in slices from younger presymptomatic Mecp2 KO mice. Furthermore, spine synapses in pyramidal neurons from symptomatic Mecp2 KO are larger and do not grow in size or incorporate GluA1 subunits after electrical or chemical LTP. Our data suggest that LTP is occluded in Mecp2 KO mice by already potentiated synapses. The higher surface levels of GluA1-containing receptors are consistent with altered expression levels of proteins involved in AMPA receptor trafficking, suggesting previously unidentified targets for therapeutic intervention for Rett syndrome and other MECP2-related disorders.

  5. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells.

    Science.gov (United States)

    Liu, Yu; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-07-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca(2+) and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the duration of action potentials can suppress the expression of GluR2-containing AMPARs at stellate cell synapses. We find that large-conductance Ca(2+)-activated potassium (BK) channels mediate a large proportion of the depolarization-evoked noninactivating potassium current in stellate cells. Pharmacological blockade of BK channels prolonged the action potential duration in postsynaptic stellate cells and altered synaptic AMPAR subtype from GluR2-lacking to GluR2-containing Ca(2+)-impermeable AMPARs. An L-type channel blocker abolished an increase in Ca(2+) entry that was associated with spike broadening and also prevented the BK channel blocker-induced switch in AMPAR phenotype. Thus blocking BK potassium channels prolongs the action potential duration and increases the expression of GluR2-containing receptors at the synapse by enhancing Ca(2+) entry in cerebellar stellate cells.

  6. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    Science.gov (United States)

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  7. Perampanel: A Selective AMPA Antagonist for Treating Seizures

    OpenAIRE

    Krauss, Gregory L.

    2013-01-01

    Perampanel is a selective, noncompetitive AMPA receptor antagonist that has recently been approved for treating localization-related epilepsy. This article reviews the pharmacology, clinical development, efficacy, and safety/tolerability of perampanel.

  8. Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

    DEFF Research Database (Denmark)

    Andersen, Trine F; Vogensen, Stine B; Jensen, Lars S;

    2005-01-01

    Polyamines and polyamine toxins are biologically important molecules, having modulatory effects on nucleotides and proteins. The wasp toxin, philanthotoxin-433 (PhTX-433), is a non-selective and uncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic...

  9. Involvement of AMPA/Kainate Glutamate Receptor in the Extinction and Reinstatement of Morphine-Induced Conditioned Place Preference: A Behavioral and Molecular Study.

    Science.gov (United States)

    Siahposht-Khachaki, Ali; Fatahi, Zahra; Yans, Asal; Khodagholi, Fariba; Haghparast, Abbas

    2017-03-01

    Glutamate receptors in mesolimbic areas such as the nucleus accumbens, ventral tegmental area, prefrontal cortex (PFC), and hippocampus (HIP) are a component of the mechanisms of drug-induced reward and can modulate the firing pattern of dopaminergic neurons in the reward system. In addition, several lines of study have indicated that cAMP response element-binding protein (CREB) and c-fos have important role in morphine-induced conditioned place preference (CPP) induced by drugs of abuse, such as morphine, cocaine, nicotine, and alcohol. Therefore, in the present study, we investigated the changes in phosphorylated CREB (p-CREB) and c-fos induction within the nucleus accumbens (NAc), HIP, and PFC after intracerebroventricular (ICV) administration of different doses of CNQX or vehicle during extinction period or reinstatement of morphine-induced CPP. In all groups, the CPP procedure was done; afterward, the conditioning scores were recorded by Ethovision software. After behavioral test recording, we dissected out the NAc, HIP, and PFC regions and measured the p-CREB/CREB ratio and c-fos level by Western blot analysis. Our results showed that administration of CNQX significantly shortened the extinction of morphine CPP. Besides, ICV microinjection of CNQX following extinction period decreased the reinstatement of morphine CPP in extinguished rats. In molecular section, in treatment group, all mentioned factors were dose-dependently decreased in comparison with vehicle group (DMSO) after ICV microinjection of different doses of CNQX but not in pre-extinction microinjection. These findings suggested that antagonism of AMPA receptor decreased p-CREB/CREB ratio and c-fos level in the PFC, NAc, and HIP. Modulation of the drug memory reconsolidation may be useful for faster extinction of drug-induced reward and attenuation of drug-seeking behavior.

  10. DHEAS increases levels of GluR2/3 and GluR2, AMPA receptor subunits, in C57BL/6 mice hippocampus El DHEAS incrementa la expresión de GluR2/3 y GLUR2 del receptor AMPA en el hipocampo de ratones C57/BL6

    Directory of Open Access Journals (Sweden)

    Diego Sepúlveda Falla

    2010-05-01

    Full Text Available

    Dehydroepiandrosterone sulfate (DHEA-S is a neurosteroid that has effects such as neuromodulator of synaptic transmission and neuroprotection. The specific signaling pathways for these effects are not elucidated yet. Given that, some neurosteroids act through the activation of ionotropic glutamate receptors, therefore the effect of DHEA-S on the subunits GluR2  and GluR3 of the AMPA receptor was evaluated.  Either DHEA-S or a control substance was administered to C57/BL6 mice. Subunit expression of the AMPA receptor was analyzed by Western blotting.

     

     

    Results show that long-term DHEA-S administration to C57/BL6 mice, increases the protein levels of the subunits GluR2 and GluR2/3 of the AMPA receptors located in the hippocampus.

  11. Cortical kindling induces elevated levels of AMPA and GABA receptor subunit mRNA within the amygdala/piriform region and is associated with behavioral changes in the rat.

    Science.gov (United States)

    Henderson, Amy K; Galic, Michael A; Teskey, G Campbell

    2009-11-01

    Cortical kindling causes alterations within the motor cortex and results in long-standing motor deficits. Less attention has been directed to other regions that also participate in the epileptiform activity. We examined if cortical kindling could induce changes in excitatory and inhibitory receptor subunit mRNA in the amygdala/piriform regions and if such changes are associated with behavioral deficits. After cortical kindling, amygdala/piriform regions were dissected to analyze mRNA levels of NMDA, AMPA, and GABA receptor subunits using reverse transcription polymerase chain reaction, or rats were subjected to a series of behavioral tests. Kindled rats had significantly greater amounts of GluR1 and GluR2 AMPA receptor mRNA, and alpha1 and alpha2 GABA receptor subunit mRNA, compared with sham controls, which was associated with greater anxiety-like behaviors in the elevated plus maze and reduced freezing behaviors in the fear conditioning task. In summary, cortical kindling produces dynamic receptor subunit changes in regions in addition to the seizure focus.

  12. Molecular pharmacology of the AMPA agonist, (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA] and the AMPA antagonist, (R)-APPA

    DEFF Research Database (Denmark)

    Ebert, B; Madsen, U; Lund, Trine Meldgaard

    1994-01-01

    The heterocyclic analogue of (S)-glutamic acid, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [(S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, (R)-AMPA, is virtually inactive. We have previously characterized (RS)-2-amino-3-(3-hydroxy-...

  13. Therapeutic window of opportunity for the neuroprotective effect of valproate versus the competitive AMPA receptor antagonist NS1209 following status epilepticus in rats.

    Science.gov (United States)

    Langer, Melanie; Brandt, Claudia; Zellinger, Christina; Löscher, Wolfgang

    2011-01-01

    Epileptogenesis, i.e., the process leading to epilepsy, is a presumed consequence of brain insults including head trauma, stroke, infections, tumors, status epilepticus (SE), and complex febrile seizures. Typically, brain insults produce morphological and functional alterations in the hippocampal formation, including neurodegeneration in CA1, CA3, and, most consistently, the dentate hilus. Most of these alterations develop gradually, over several days, after the insult, providing a therapeutic window of opportunity for neuroprotective agents in the immediate post-injury period. We have previously reported that prolonged (four weeks) treatment with the antiepileptic drug valproate (VPA) after SE prevents hippocampal damage and most of the behavioral alterations that occur after brain insult, but not the development of spontaneously occurring seizures. These data indicated that VPA, although not preventing epilepsy, might be an effective disease-modifying treatment following brain insult. The present study was designed to (1) determine the therapeutic window for the neuroprotective effect of VPA after SE; (2) compare the efficacy of different intermittent i.p. versus continuous i.v. VPA treatment protocols; and (3) compare VPA with the glutamate (AMPA) receptor antagonist NS1209. As in our previous study with VPA, SE was induced by sustained electrical stimulation of the basolateral amygdala in rats and terminated after 4 h by diazepam. In vehicle controls, >90% of the animals developed significant neurodegeneration in the dentate hilus, whereas damage in CA1 and CA3 was more variable. Hilar parvalbumin-expressing interneurons were more sensitive to the effects of seizures than somatostatin-stained hilar interneurons or hilar mossy cells. Among the various VPA treatment protocols, continuous infusion of VPA for 24 immediately following the SE was the most effective neuroprotective treatment, preventing most of the neuronal damage. Infusion with NS1209 for 24 h

  14. Eating 'Junk-Food' Produces Rapid and Long-Lasting Increases in NAc CP-AMPA Receptors: Implications for Enhanced Cue-Induced Motivation and Food Addiction.

    Science.gov (United States)

    Oginsky, Max F; Goforth, Paulette B; Nobile, Cameron W; Lopez-Santiago, Luis F; Ferrario, Carrie R

    2016-12-01

    Urges to eat are influenced by stimuli in the environment that are associated with food (food cues). Obese people are more sensitive to food cues, reporting stronger craving and consuming larger portions after food cue exposure. The nucleus accumbens (NAc) mediates cue-triggered motivational responses, and activations in the NAc triggered by food cues are stronger in people who are susceptible to obesity. This has led to the idea that alterations in NAc function similar to those underlying drug addiction may contribute to obesity, particularly in obesity-susceptible individuals. Motivational responses are mediated in part by NAc AMPA receptor (AMPAR) transmission, and recent work shows that cue-triggered motivation is enhanced in obesity-susceptible rats after 'junk-food' diet consumption. Therefore, here we determined whether NAc AMPAR expression and function is increased by 'junk-food' diet consumption in obesity-susceptible vs -resistant populations using both outbred and selectively bred models of susceptibility. In addition, cocaine-induced locomotor activity was used as a general 'read out' of mesolimbic function after 'junk-food' consumption. We found a sensitized locomotor response to cocaine in rats that gained weight on a 'junk-food' diet, consistent with greater responsivity of mesolimbic circuits in obesity-susceptible groups. In addition, eating 'junk-food' increased NAc calcium-permeable-AMPAR (CP-AMPAR) function only in obesity-susceptible rats. This increase occurred rapidly, persisted for weeks after 'junk-food' consumption ceased, and preceded the development of obesity. These data are considered in light of enhanced cue-triggered motivation and striatal function in obesity-susceptible rats and the role of NAc CP-AMPARs in enhanced motivation and addiction.

  15. AMPA Receptor-mTOR Activation Is Required for the Antidepressant-like Effects of Sarcosine during the Forced Swim Test in rats: Insertion of AMPA Receptor may Play a Role

    Directory of Open Access Journals (Sweden)

    Kuang-Ti eChen

    2015-06-01

    Full Text Available Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-D-aspartate receptor (NMDAR coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons. A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(fquinoxaline (NBQX pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR–mTOR signaling pathway activity and facilitating AMPAR membrane insertion.Highlights:- A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the

  16. AMPA Receptor-mTOR Activation is Required for the Antidepressant-Like Effects of Sarcosine during the Forced Swim Test in Rats: Insertion of AMPA Receptor may Play a Role.

    Science.gov (United States)

    Chen, Kuang-Ti; Tsai, Mang-Hung; Wu, Ching-Hsiang; Jou, Ming-Jia; Wei, I-Hua; Huang, Chih-Chia

    2015-01-01

    Sarcosine, an endogenous amino acid, is a competitive inhibitor of the type I glycine transporter and an N-methyl-d-aspartate receptor (NMDAR) coagonist. Recently, we found that sarcosine, an NMDAR enhancer, can improve depression-related behaviors in rodents and humans. This result differs from previous studies, which have reported antidepressant effects of NMDAR antagonists. The mechanisms underlying the therapeutic response of sarcosine remain unknown. This study examines the role of mammalian target of rapamycin (mTOR) signaling and α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) activation, which are involved in the antidepressant-like effects of several glutamatergic system modulators. The effects of sarcosine in a forced swim test (FST) and the expression levels of phosphorylated mTOR signaling proteins were examined in the absence or presence of mTOR and AMPAR inhibitors. In addition, the influence of sarcosine on AMPAR trafficking was determined by analyzing the phosphorylation of AMPAR subunit GluR1 at the PKA site (often considered an indicator for GluR1 membrane insertion in neurons). A single injection of sarcosine exhibited antidepressant-like effects in rats in the FST and rapidly activated the mTOR signaling pathway, which were significantly blocked by mTOR inhibitor rapamycin or the AMPAR inhibitor 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) pretreatment. Moreover, NBQX pretreatment eliminated the ability of sarcosine to stimulate the phosphorylated mTOR signaling proteins. Furthermore, GluR1 phosphorylation at its PKA site was significantly increased after an acute in vivo sarcosine treatment. The results demonstrated that sarcosine exerts antidepressant-like effects by enhancing AMPAR-mTOR signaling pathway activity and facilitating AMPAR membrane insertion. Highlights-A single injection of sarcosine rapidly exerted antidepressant-like effects with a concomitant increase in the activation of the mammalian

  17. Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reverses subcronic PCP-induced deficits in the novel object recognition task in rats

    DEFF Research Database (Denmark)

    Nielsen, Trine Damgaard; Larsen, Dorrit Bjerg; Hansen, Suzanne Lisbet;

    2010-01-01

    deficit in female Lister hooded rats in teh novel object recognition (NOR) task. Here we show that positive modulation of AMPA receptor (AMPAR) mediated glutamate transmission alleviates cognitive deficits induced by sub-chronic PCP treatment. Female Lister hooded rats were treated sub......-cbronic PCP treatment induced a significant decrease in the discrimination index (DI) and both ampakines CX546 and CX516 were able to reverse this diruption of object memory in rats in the novel object recognition task. These data suggest that positive AMPAR modulation may represent a mechanism for treatment...

  18. Exendin-4 promotes the membrane trafficking of the AMPA receptor GluR1 subunit and ADAM10 in the mouse neocortex.

    Science.gov (United States)

    Ohtake, Nobuaki; Saito, Mieko; Eto, Masaaki; Seki, Kenjiro

    2014-05-01

    Glucagon-like peptide-1 (GLP-1) is a novel treatment modality for type 2 diabetes mellitus. However, GLP-1 has been suggested as a therapeutic target for Alzheimer's disease (AD). In rodent studies, GLP-1 reduces amyloid beta (Aβ) and facilitates synaptic plasticity. Therefore, in the present study, we investigated how GLP-1 facilitates synaptic plasticity and reduces the Aβ in vivo. Exendin-4, a GLP-1 receptor agonist that can cross the blood brain barrier, was subcutaneously administered to adult mice. We then extracted the total and the plasma membrane proteins from the mouse neocortex. Exendin-4 significantly increased the phosphorylation level of cAMP response element-binding protein (CREB). Consistently, the expression level of brain-derived neurotrophic factor (BDNF), a transcriptional target of CREB, was increased. Furthermore, exendin-4 increased the membrane protein level of the AMPA receptor GluR1 subunit and postsynaptic density protein-95 (PSD-95), whereas GluR2 was unaffected. These exendin-4-dependent increases in membrane GluR1, total PSD-95 and BDNF were abrogated by pretreatment with temozolomide (TMZ), a DNA-alkylating agent, indicating that these alterations were dependent on exendin-4-induced transcriptional activity. In addition, we found that exendin-4 increased the level of the α-C terminal fragment (α-CTF) of amyloid precursor protein (APP). Furthermore, protein levels of both mature and immature ADAM10, the α-secretase of APP in the plasma membrane, were increased, whereas the total mature and immature ADAM10 levels were unchanged. These exendin-4-dependent increases in α-CTF and ADAM10 were not affected by TMZ. These findings suggested that GLP-1 facilitates the GluR1 membrane insertion through CREB activation and increases α-secretase activity through ADAM10 membrane trafficking. Upregulation of GluR1 and ADAM10 at the plasma membrane were also observed in mice with intracerebroventricular administration of Aβ oligomer

  19. Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by the AMPA/KA Receptor: Comparison between Immature and Mature Primary Cultures of Neurons and Glial Cells from Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Helena T. Hogberg

    2011-01-01

    Full Text Available Domoic acid (DomA is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have shown increased sensitivity to DomA-induced toxicity, and prenatal exposure has been associated with e.g. decreased brain GABA levels, and increased glutamate levels. Here, we evaluated DomA-induced toxicity in immature and mature primary cultures of neurons and glial cells from rat cerebellum by measuring the mRNA levels of selected genes. Moreover, we assessed if the induced toxicity was mediated by the activation of the AMPA/KA and/or the NMDA receptor. The expression of all studied neuronal markers was affected after DomA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment, as the effects were observed at lower concentrations and at earlier time points than for the immature cultures. The DomA effects were completely prevented by the antagonist of the AMPA/KA receptor (NBQX, while the antagonist of the NMDA receptor (APV partly blocked the DomA-induced effects. Interestingly, the DomA-induced effect was also partly prevented by the neurotransmitter GABA. DomA exposure also affected the mRNA levels of the astrocytic markers in mature cultures. These DomA-induced effects were reduced by the addition of NBQX, APV, and GABA.

  20. Synthesis and structure-activity studies on acidic amino acids and related diacids as NMDA receptor ligands

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B;

    1994-01-01

    The 3-isoxazolol amino acids (S)-2-amino-3-(3-hydroxy-5-methyl-4- isoxazolyl)propionic acid [(S)-AMPA, 2] and (R,S)-2-amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid (AMAA, 5a) (Figure 1) are potent and specific agonists at the AMPA and N-methyl-D-aspartic acid (NMDA) subtypes, respectively......, of (S)-glutamic acid (1) receptors. A number of amino acids and diacids structurally related to AMAA were synthesized and tested electrophysiologically and in receptor-binding assays. The hydroxymethyl analogue 7c of AMAA was an NMDA agonist approximately equipotent with AMAA in the [3H...... by molecular mechanics calculations. Compound 7a possesses extra steric bulk and shows significant restriction of conformational flexibility compared to AMAA and 7c, which may be determining factors for the observed differences in biological activity. Although the nitrogen atom of quinolinic acid (6) has very...

  1. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    Science.gov (United States)

    Takeuchi, Yusuke; Morise, Jyoji; Morita, Ippei; Takematsu, Hiromu; Oka, Shogo

    2015-01-01

    The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  2. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    Directory of Open Access Journals (Sweden)

    Yusuke Takeuchi

    Full Text Available The AMPA-type glutamate receptor (AMPAR, which is a tetrameric complex composed of four subunits (GluA1-4 with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc, human natural killer-1 (HNK-1 carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413 within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

  3. Age-dependent modifications of AMPA receptor subunit expression levels and related cognitive effects in 3xTg-AD mice

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    Pamela eCantanelli

    2014-08-01

    Full Text Available GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of AMPA receptors (AMPARs, the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q to R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease. With qRT-PCR, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.] and old (12 m.o.a Tg-AD mice and made comparisons with levels found in age-matched wild type (WT mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for short- and long-term spatial memory with the Morris Water Maze (MWM navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in maintaining cognition in pre-symptomatic 3xTg-AD mice.

  4. KIS, a kinase associated with microtubule regulators, enhances translation of AMPA receptors and stimulates dendritic spine remodeling.

    Science.gov (United States)

    Pedraza, Neus; Ortiz, Raúl; Cornadó, Alba; Llobet, Artur; Aldea, Martí; Gallego, Carme

    2014-10-15

    Local regulation of protein synthesis allows a neuron to rapidly alter the proteome in response to synaptic signals, an essential mechanism in synaptic plasticity that is altered in many neurological diseases. Synthesis of many synaptic proteins is under local control and much of this regulation occurs through structures termed RNA granules. KIS is a protein kinase that associates with stathmin, a modulator of the tubulin cytoskeleton. Furthermore, KIS is found in RNA granules and stimulates translation driven by the β-actin 3'UTR in neurites. Here we explore the physiological and molecular mechanisms underlying the action of KIS on hippocampal synaptic plasticity in mice. KIS downregulation compromises spine development, alters actin dynamics, and reduces postsynaptic responsiveness. The absence of KIS results in a significant decrease of protein levels of PSD-95, a postsynaptic scaffolding protein, and the AMPAR subunits GluR1 and GluR2 in a CPEB3-dependent manner. Underlying its role in spine maturation, KIS is able to suppress the spine developmental defects caused by CPEB3 overexpression. Moreover, either by direct or indirect mechanisms, KIS counteracts the inhibitory activity of CPEB3 on the GluR2 3'UTR at both mRNA translation and polyadenylation levels. Our study provides insights into the mechanisms that mediate dendritic spine morphogenesis and functional synaptic maturation, and suggests KIS as a link regulating spine cytoskeleton and postsynaptic activity in memory formation.

  5. Structure-activity relationship study of spider polyamine toxins as inhibitors of ionotropic glutamate receptors.

    Science.gov (United States)

    Xiong, Xiao-Feng; Poulsen, Mette H; Hussein, Rama A; Nørager, Niels G; Strømgaard, Kristian

    2014-12-01

    The spider polyamine toxins Joro spider toxin-3 (JSTX-3) and Nephila polyamine toxins-1 and -8 (NPTX-1 and NPTX-8) are isolated from the venom of the orb-weaver spider Nephila clavata (Joro spider). They share a high degree of structural resemblance, their aromatic head groups being the only difference, and were recently found to be very potent open-channel blockers of ionotropic glutamate (iGlu) receptors. In this study we designed and synthesized a collection of 24 analogues of these toxins using a recently developed solid-phase synthetic methodology. Systematic variation in two regions of the toxins and subsequent evaluation of biological activity at AMPA and NMDA subtypes of iGlu receptors provided succinct information on structure-activity relationships. In particular, one set of analogues were found to display exquisite selectivity and potency for AMPA receptors relative to the natural products. Thus, this systematic SAR study has provided new pharmacological tools for studies of iGlu receptors.

  6. NMDA antagonist, but not nNOS inhibitor, requires AMPA receptors in the ventromedial prefrontal cortex (vmPFC) to induce antidepressant-like effects

    DEFF Research Database (Denmark)

    Pereira, V. S.; Wegener, Gregers; Joca, S. R.

    2013-01-01

    Depressed individuals and stressed animals show enhanced levels of glutamate and neuronal nitric oxide synthase (nNOS) activity in limbic structures, including the vmPFC. Systemic administration of glutamatergic NMDA receptor antagonists or inhibitors of nitric oxide (NO) synthesis induces antide...

  7. Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type hori-zontal cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or dihydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R- and G-cones to LHCs, respectively.

  8. Environmental Risk Limits for AMPA

    NARCIS (Netherlands)

    Traas TP; Smit CE; SEC

    2003-01-01

    In dit rapport worden milieurisicogrenzen (MTR, VR en SRC-eco) afgeleid voor de stof aminomethyl-fosfonzuur (AMPA, de primaire metaboliet van het herbicide glyfosaat en fosfonaten, o.a ingredienten van wasmiddelen. De MTR voor AMPA is 79,7 ug/L. MTR's voor bodem en sediment konden niet worden

  9. Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

    Science.gov (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce

    2012-07-01

    Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.

  10. Role of Hippocampal 5-HT1A Receptor and Its Modulation to NMDA Receptor and AMPA Receptor in Depression Induced by Chronic Unpredictable Mild Stress%应激性抑郁样行为发生中海马5-羟色胺1A受体的作用及其对NMDA受体和AMPA受体的调节

    Institute of Scientific and Technical Information of China (English)

    问黎敏; 安书成; 刘慧

    2012-01-01

    为探讨慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)诱发抑郁样行为发生中海马5-羟色胺1A受体(5-hydroxytryptamine receptor 1A,5-HT1AR)表达与作用,及其对谷氨酸N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体和α-氨基羟甲基异恶唑丙酸(α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid,AMPA)受体的影响.通过建立CUMS动物模型,给应激抑郁模型大鼠海马微量注射5-HT1A受体激动剂、给正常大鼠海马微量注射5-HT1A受体拮抗剂,测量大鼠体重变化率,并采用糖水偏爱测试、旷场实验和悬尾实验等方法对大鼠进行行为学检测,运用Western blot和ELISA方法检测大鼠海马组织中5-HT1AR和NMDAR和AMPAR的关键亚基的表达以及磷酸化水平.结果显示,与对照组相比,CUMS组大鼠表现出抑郁样行为,海马5-HT1AR、AMPA受体的GluR2/3亚基表达及磷酸化明显降低,NMDA受体的NR1和NR2B亚基表达及磷酸化显著增加;正常大鼠海马微量注射5-HT1A受体拮抗剂WAY100635,动物行为学表现及AMPA受体、NMDA受体表达及磷酸化水平均与CUMS组相同;注射5-HT1A受体激动剂8-OH-DPAT能逆转应激诱导的上述改变.以上结果表明,CUMS诱发抑郁榉行为与海马5-HT1AR表达下降,AMPAR表达量及磷酸化水平降低,NMDAR表达量及磷酸化水平升高有关.5-HT通过5-HT1AR产生抗抑郁作用.5-HT1AR激动剂抗抑郁作用与降低NMDAR表达量及磷酸化水平,提高AMPAR表达量及磷酸化水平密切相关.%Stressors markedly influence central neurochemical and hormonal processes and thus play a pivotal role in the occurrence of depressive illnesses. As the center for stress response and the potential target for stressfulprovocation, the hippocampus is becoming a focus in depression research. Although a large number of behavioral paradigms have been proposed as animal models of depression, only a few are considered potentially useful research tools with

  11. Expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR2/3 receptors in the developing rat pineal gland.

    Science.gov (United States)

    Kaur, C; Sivakumar, V; Ling, E A

    2005-10-01

    The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate (GluR2/3) receptors and N-methyl-D-aspartate receptor subtype 1 (NMDAR1) was carried out by immunohistochemistry, double immunofluorescence and real-time RT-PCR analysis in the pineal glands of 1-day to 6-wk-old rats in the present study. GluR2/3 immunopositive cells were distributed throughout the pineal gland and showed branching processes in all age groups. The NMDAR1 immunoreactivity, however, was observed in fewer branched cells. A constitutive mRNA expression of NMDAR1, GluR2 and GluR3 was detected in the pineal glands of various ages and showed no significant difference between the age groups studied. Immunohistochemical and double immunofluorescence results showed that the GluR2/3 were mainly expressed and co-localized with OX-42-positive microglia/macrophages and the glial fibrillary acidic protein (GFAP)-positive astrocytes. Co-localization of NMDAR1 with OX-42- and GFAP-positive cells was much less. The expression of these receptors on the glial cells suggests that they may be involved in the development and growth of the pineal gland in the early postnatal period (1 day to 3 wk) and subsequently in the regulation of melatonin synthesis.

  12. AMPA experimental communications systems

    Science.gov (United States)

    Beckerman, D.; Fass, S.; Keon, T.; Sielman, P.

    1982-01-01

    The program was conducted to demonstrate the satellite communication advantages of Adaptive Phased Array Technology. A laboratory based experiment was designed and implemented to demonstrate a low earth orbit satellite communications system. Using a 32 element, L-band phased array augmented with 4 sets of weights (2 for reception and 2 for transmission) a high speed digital processing system and operating against multiple user terminals and interferers, the AMPA system demonstrated: communications with austere user terminals, frequency reuse, communications in the face of interference, and geolocation. The program and experiment objectives are described, the system hardware and software/firmware are defined, and the test performed and the resultant test data are presented.

  13. Group I mGluR activation reverses cocaine-induced accumulation of calcium-permeable AMPA receptors in nucleus accumbens synapses via a protein kinase C-dependent mechanism.

    Science.gov (United States)

    McCutcheon, James E; Loweth, Jessica A; Ford, Kerstin A; Marinelli, Michela; Wolf, Marina E; Tseng, Kuei Y

    2011-10-12

    Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+ -ermeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of "incubated" cue-induced cocaine craving. Using patch-clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 d of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50 μm) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+ -impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

  14. Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Kristensen, B W; Noraberg, J; Zimmer, J

    2001-10-26

    The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration

  15. 内源性大麻素对海马神经元 AMPA 受体GluR2的作用%Effect of endocannabinoids pretreatment on the expression of AMPA receptor GluR2 subunit in the hippocampal pyramidal neurons of mouse

    Institute of Scientific and Technical Information of China (English)

    刘曌宇; 高杨; 孙思斯; 陈绍洋; 王强

    2015-01-01

    目的:探索内源性大麻素预处理对小鼠全脑缺血再灌注损伤后海马神经元α-氨基-3羟基-5-甲基-4-异恶唑丙酸(AMPA)受体2亚基(GluR2)表达的影响及机制。方法雄性C57BL/6小鼠随机分为假手术组、模型组、2-花生酰基甘油(2-AG)处理组、大麻素1型受体( CB1R)拮抗剂( AM251)+2-AG组和溶剂组。2-AG 处理组腹腔注射2-AG 5 mg/kg;AM251+2-AG 组腹腔注射AM2511 mg/kg,30 min后腹腔给予2-AG 5 mg/kg;溶剂组腹腔给予0.1 ml二甲基亚砜( DMSO)。各组小鼠在预处理后30 min采用夹闭双侧颈总动脉20 min行再灌注的方法制备全脑缺血再灌注损伤模型。再灌注2 h取材行Western blot及免疫荧光检测。结果 GluR2高表达于正常小鼠海马CA1区锥体神经元;C57小鼠全脑缺血20 min,再灌后2 h海马组织GluR2表达明显下调(P<0.05);与模型组比较,2-AG处理组海马组织GluR2明显增高(P<0.05);与2-AG处理组比较,AM251+2-AG组海马组织GluR2明显下降(P<0.05)。结论内源性大麻素2-AG作用于神经元CB1R,逆转全脑缺血损伤导致的海马神经元GluR2表达下降。%Objective Whether endocannabinoids pretreatment affects the expression of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor 2 subunit (GluR2) in mice after global cerebral ischemia-reperfusion (I/R) injury and its mechanism are investigated.Methods Adult male C57BL/6 mice were randomly divided into five groups:sham group, I/R group, 2-arachidonoyl glycerol (2-AG) group, cannabinoid 1 receptor (CB1R) antagonist(AM251)+2-AG group and vehicle group.The mice in 2-AG group were injected 2-AG with a dose of 5 mg/kg intraperitoneally.AM251 was injected intraperitoneally with a dose of 1 mg/kg 30 min before pretreatment.2-AG was administered in AM251+2-AG group mice with a dose of 5 mg/kg.A total of 0.1 ml Dimethylsulfoxide (DMSO

  16. Role of hippocampal AMPA receptors in antidepressant effect of ketamine in rats%海马AMPA受体在氯胺酮对大鼠抗抑郁效应中的作用

    Institute of Scientific and Technical Information of China (English)

    杨春; 高志勤; 杨春; 周志强; 杨建军; 徐建国

    2012-01-01

    Objective To evaluate the role of hippocampal AMPA receptors in the antidepressant effect of ketamine in rats.Methods Thirty male Wistar rats aged 2 months weighing 180-220 g were randomly divided into 3 groups (n =10 each):control group (group C); ketamine group (group K) and AMPA receptor antagonist NBQX group (group N).The animals were forced to swim for 15 min on the 1st day.On the 2nd day,NBQX 10 mg/kg was injected intrapefitoneally in group N; 30 min later,normal saline was injected intraperitoneally in group C,while ketamine 10 mg/kg was injected intraperitoneally in groups K and N.The forced swimming test was performed again for 5 min at 30 min after administration and the immobility time of the rats was recorded.Then the animals were sacrificed and the hippocampus was removed for determination of the expression of phosphorylated rapamycin (p-mTOR) and phosphorylated glutamate receptor 1 (p-GluR1).Results Compared with group C,the immobility time was significantly shortened and the expression of p-mTOR and p-GluR1 up-regulated in group K,and the immobility time was significantly shortened,the expression of p-mTOR up-regulated and the expression of p-GluR1 down-regulated in group N (P < 0.05).Compared with group K,the immobility time was significantly prolonged and the expression of p-mTOR and p-GluR1 down-regulated in group N (P < 0.05 ).Conclusion AMPA receptors in hippocampus are involved in the antidepressant effect of ketamine in rats and the inhibition of mTOR and GluR1 activities may be involved in the mechanism.%目的 评价海马α-氨基-3-羟基-5-甲基-4-异恶唑基丙酸(AMPA)受体在氯胺酮对大鼠抗抑郁效应中的作用.方法 雄性Wistar大鼠30只,2月龄,体重180~220 g,采用随机数字表法,将其随机均分为3组(n=10):对照组(C组)、氯胺酮组(K组)和AMPA受体拮抗剂NBQX组(N组).行强迫游泳实验15 min建立大鼠抑郁模型.于第2天N组腹腔注射NBQX 10 mg/kg;30 min

  17. Alterations in Hippocampal Oxidative Stress, Expression of AMPA Receptor GluR2 Subunit and Associated Spatial Memory Loss by Bacopa monnieri Extract (CDRI-08) in Streptozotocin-Induced Diabetes Mellitus Type 2 Mice.

    Science.gov (United States)

    Pandey, Surya P; Singh, Hemant K; Prasad, S

    2015-01-01

    Bacopa monnieri extract has been implicated in the recovery of memory impairments due to various neurological disorders in animal models and humans. However, the precise molecular mechanism of the role of CDRI-08, a well characterized fraction of Bacopa monnieri extract, in recovery of the diabetes mellitus-induced memory impairments is not known. Here, we demonstrate that DM2 mice treated orally with lower dose of CDRI-08 (50- or 100 mg/kg BW) is able to significantly enhance spatial memory in STZ-DM2 mice and this is correlated with a significant decline in oxidative stress and up regulation of the AMPA receptor GluR2 subunit gene expression in the hippocampus. Treatment of DM2 mice with its higher dose (150 mg/kg BW or above) shows anti-diabetic effect in addition to its ability to recover the spatial memory impairment by reversing the DM2-induced elevated oxidative stress and decreased GluR2 subunit expression near to their values in normal and CDRI-08 treated control mice. Our results provide evidences towards molecular basis of the memory enhancing and anti diabetic role of the Bacopa monnieri extract in STZ-induced DM2 mice, which may have therapeutic implications.

  18. Radiosynthesis and preliminary PET evaluation of (18)F-labeled 2-(1-(3-fluorophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile for imaging AMPA receptors.

    Science.gov (United States)

    Yuan, Gengyang; Jones, Graham B; Vasdev, Neil; Liang, Steven H

    2016-10-01

    To prompt the development of (18)F-labeled positron emission tomography (PET) tracers for the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, we have prepared (18)F-labeled 2-(1-(3-fluorophenyl)-2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile ([(18)F]8). The radiosynthesis was achieved by a one-pot two-step method that utilized a spirocyclic hypervalent iodine(III) mediated radiofluorination to prepare the (18)F-labeled 1-bromo-3-fluorobenzene ([(18)F]15) intermediate with K(18)F. A subsequent copper(I) iodide mediated coupling reaction was carried out with 2-(2-oxo-5-(pyrimidin-2-yl)-1,2-dihydropyridin-3-yl)benzonitrile (10) to [(18)F]8 in 10±2% uncorrected radiochemical yield relative to starting (18)F-fluoride with >99% radiochemical purity and 29.6±7.4Gbq/μmol specific activity at the time of injection. PET imaging studies with the title radiotracer in normal mice demonstrated good brain uptake (peak standardized uptake value (SUV)=2.3±0.1) and warrants further in vivo validation.

  19. Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology.

    Science.gov (United States)

    Frølund, Bente; Greenwood, Jeremy R; Holm, Mai M; Egebjerg, Jan; Madsen, Ulf; Nielsen, Birgitte; Bräuner-Osborne, Hans; Stensbøl, Tine B; Krogsgaard-Larsen, Povl

    2005-09-15

    Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.

  20. Positive allosteric modulation of AMPA receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action

    DEFF Research Database (Denmark)

    Fitzpatrick, Ciarán Martin; Larsen, Maria; Madsen, Louise

    2016-01-01

    Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR)....

  1. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    Science.gov (United States)

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  2. Three-dimensional structure of the ligand-binding core of GluR2 in complex with the agonist (S)-ATPA: implications for receptor subunit selectivity.

    Science.gov (United States)

    Lunn, Marie-Louise; Hogner, Anders; Stensbøl, Tine B; Gouaux, Eric; Egebjerg, Jan; Kastrup, Jette S

    2003-02-27

    Two X-ray structures of the GluR2 ligand-binding core in complex with (S)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid ((S)-ATPA) have been determined with and without Zn(2+) ions. (S)-ATPA induces a domain closure of ca. 21 degrees compared to the apo form. The tert-butyl moiety of (S)-ATPA is buried in a partially hydrophobic pocket and forces the ligand into the glutamate-like binding mode. The structures provide new insight into the molecular basis of agonist selectivity between AMPA and kainate receptors.

  3. Mechanism and site of inhibition of AMPA receptors: substitution of one and two methyl groups at the 4-aminophenyl ring of 2,3-benzodiazepine and implications in the "E" site.

    Science.gov (United States)

    Wang, Congzhou; Wu, Andrew; Shen, Yu-Chuan; Ettari, Roberta; Grasso, Silvana; Niu, Li

    2015-08-19

    2,3-Benzodiazepines are a well-known group of compounds for their potential antagonism against AMPA receptors. It has been previously reported that the inhibitory effect of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety can be enhanced by simply adding a chlorine atom at position 3 of the 4-aminophenyl ring. Here we report that adding a methyl group at position 3 on the 4-aminophenyl ring, termed as BDZ-11-7, can similarly enhance the inhibitory activity, as compared with the unsubstituted one or BDZ-11-2. Our kinetic studies have shown that BDZ-11-7 is a noncompetitive antagonist of GluA2Q homomeric receptors and prefers to inhibit the closed-channel state. However, adding another methyl group at position 5 on the 4-aminophenyl ring, termed as BDZ-11-6, fails to yield extra inhibition on GluA2Q receptors. Instead, BDZ-11-6 exhibits a diminished inhibition of GluA2Q. Site interaction test indicates the two compounds, BDZ-11-6 and BDZ-11-7, bind to the same site on GluA2Q, which is also the binding site for their prototype, BDZ-11-2. Based on the results from this and our earlier studies, we propose that the binding site that accommodates the 4-aminophenyl ring must contain two interactive points, with one preferring polar groups like chlorine and the other preferring nonpolar groups such as a methyl group. Either adding a chlorine or a methyl group may enhance the inhibitory activity of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety. Adding any two of the same group on positions 3 and 5 of the 4-aminophenyl ring, however, significantly reduces the interaction between these 2,3-benzodiazepines and their binding site, because one group is always repelled by one interactive point. We predict therefore that adding a chlorine atom at position 3 and a methyl group at position 5 of the 4-aminophenyl ring of 2,3-benzodiazepine derivatives with a 7,8-ethylenedioxy moiety may produce a new compound that is more potent.

  4. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  5. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits.

    Directory of Open Access Journals (Sweden)

    Suleman Hussain

    Full Text Available Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density. We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2 and glutamate receptor subunit 1 (GluA1. Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  6. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits.

    Science.gov (United States)

    Hussain, Suleman; Davanger, Svend

    2015-01-01

    Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  7. How Ca2+-permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD.

    Science.gov (United States)

    Hell, Johannes W

    2016-04-26

    Both synaptic long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical for memory formation. Dell'Acqua and co-workers now demonstrate that transient postsynaptic incorporation of Ca(2+)-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is required for LTD in the exemplary hippocampal CA1 region in 2-week-old mice. Mechanistically, LTD depends on AKAP150-anchored protein kinase A (PKA) to promote the initial functional recruitment of CP-AMPARs during LTD induction and on AKAP150-anchored protein phosphatase 2B (PP2B) to trigger their subsequent removal as part of the lasting depression of synaptic transmission.

  8. Object-in-place associative recognition memory depends on glutamate receptor neurotransmission within two defined hippocampal-cortical circuits: a critical role for AMPA and NMDA receptors in the hippocampus, perirhinal, and prefrontal cortices.

    Science.gov (United States)

    Barker, Gareth Robert Issac; Warburton, Elizabeth Clea

    2015-02-01

    Object-in-place associative recognition memory depends on an interaction between the hippocampus (HPC), perirhinal (PRH), and medial prefrontal (mPFC) cortices, yet the contribution of glutamate receptor neurotransmission to these interactions is unknown. NMDA receptors (NMDAR) in the HPC were critical for encoding of object-in-place memory but not for single-item object recognition. Next, a disconnection procedure was used to examine the importance of "concurrent" glutamate neurotransmission in the HPC-mPFC and HPC-PRH. Contralateral unilateral infusions of NBQX (AMPAR antagonist), into the HPC-mPFC, or HPC-PRH, either before acquisition or test, impaired object-in-place performance. Thus, both circuits are necessary for encoding and retrieval. Crossed unilateral AP5 (NMDAR antagonist) infusions into the HPC-mPFC or HPC-PRH impaired encoding, but not retrieval. Specifically crossed HPC-mPFC infusions impaired both short-term (5 min) and longer term (1 h) memory while HPC-PRH infusions impaired longer term memory only. This delay-dependent effect of AP5 in the HPC-PRH on object-in-place memory, accords with its effects in the PRH, on single item object recognition memory, thereby suggesting that a single PRH synaptic plasticity mechanism underpins different recognition memory processes. Further, blocking excitatory neurotransmission in any pair of structures within the networks impaired "both" encoding and retrieval, thus object-in-place memory clearly requires network interdependency across multiple structures.

  9. Attenuation of ketamine-induced impairment in verbal learning and memory in healthy volunteers by the AMPA receptor potentiator PF-04958242.

    Science.gov (United States)

    Ranganathan, M; DeMartinis, N; Huguenel, B; Gaudreault, F; Bednar, M M; Shaffer, C L; Gupta, S; Cahill, J; Sherif, M A; Mancuso, J; Zumpano, L; D'Souza, D C

    2017-02-28

    There is a need to develop treatments for cognitive impairment associated with schizophrenia (CIAS). The significant role played by N-methyl-d-aspartate receptors (NMDARs) in both the pathophysiology of schizophrenia and in neuronal plasticity suggests that facilitation of NMDAR function might ameliorate CIAS. One strategy to correct NMDAR hypofunction is to stimulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as AMPAR and NMDAR functioning are coupled and interdependent. In rats and nonhuman primates (NHP), AMPAR potentiators reduce spatial working memory deficits caused by the nonselective NMDAR antagonist ketamine. The current study assessed whether the AMPAR potentiator PF-04958242 would attenuate ketamine-induced deficits in verbal learning and memory in humans. Healthy male subjects (n=29) participated in two randomized treatment periods of daily placebo or PF-04958242 for 5 days separated by a washout period. On day 5 of each treatment period, subjects underwent a ketamine infusion for 75 min during which the effects of PF-04958242/placebo were assessed on ketamine-induced: (1) impairments in verbal learning and recall measured by the Hopkins Verbal Learning Test; (2) impairments in working memory on a CogState battery; and (3) psychotomimetic effects measured by the Positive and Negative Syndrome Scale and Clinician-Administered Dissociative Symptoms Scale. PF-04958242 significantly reduced ketamine-induced impairments in immediate recall and the 2-Back and spatial working memory tasks (CogState Battery), without significantly attenuating ketamine-induced psychotomimetic effects. There were no pharmacokinetic interactions between PF-04958242 and ketamine. Furthermore, PF-04958242 was well tolerated. 'High-impact' AMPAR potentiators like PF-04958242 may have a role in the treatment of the cognitive symptoms, but not the positive or negative symptoms, associated with schizophrenia. The excellent concordance between the

  10. Nuclear respiratory factor 1 co-regulates AMPA glutamate receptor subunit 2 and cytochrome c oxidase: tight coupling of glutamatergic transmission and energy metabolism in neurons.

    Science.gov (United States)

    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

    2009-03-01

    Neuronal activity, especially of the excitatory glutamatergic type, is highly dependent on energy from the oxidative pathway. We hypothesized that the coupling existed at the transcriptional level by having the same transcription factor to regulate a marker of energy metabolism, cytochrome c oxidase (COX) and an important subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors, GluR2 (Gria2). Nuclear respiratory factor 1 (NRF-1) was a viable candidate because it regulates all COX subunits and potentially activates Gria2. By means of in silico analysis, electrophoretic mobility shift and supershift, chromatin immunoprecipitation, and promoter mutational assays, we found that NRF-1 functionally bound to Gria2 promoter. Silencing of NRF-1 with small interference RNA prevented the depolarization-stimulated up-regulation of Gria2 and COX, and over-expression of NRF-1 rescued neurons from tetrodotoxin-induced down-regulation of Gria2 and COX transcripts. Thus, neuronal activity and energy metabolism are tightly coupled at the molecular level, and NRF-1 is a critical agent in this process.

  11. Ethanol exposure during the third trimester equivalent does not affect GABAA or AMPA receptor-mediated spontaneous synaptic transmission in rat CA3 pyramidal neurons

    OpenAIRE

    Baculis, Brian Charles; Valenzuela, Carlos Fernando

    2015-01-01

    Background Ethanol exposure during the rodent equivalent to the 3rd trimester of human pregnancy (i.e., first 1–2 weeks of neonatal life) has been shown to produce structural and functional alterations in the CA3 hippocampal sub-region, which is involved in associative memory. Synaptic plasticity mechanisms dependent on retrograde release of brain-derived neurotrophic factor (BDNF) driven by activation of L-type voltage-gated Ca2+ channels (L-VGCCs) are thought to play a role in stabilization...

  12. Structure of Leptin Receptor Related with Obesity

    DEFF Research Database (Denmark)

    Toleikis, Zigmantas

    The hormone leptin is central to obesity, but the molecular processes underlying the activation of the leptin receptor are unknown. To further the understanding of the system, an atomic resolution structure of this cytokine type I receptor in the unbound inactive form and in the activated bound...... of the receptor, while the D5 domain is the central leptin-binding domain, implicated in the first steps of activation. Both domains are characterized by a fibronectin type III fold and both contain a conserved WSXWS motif (X represents an unconserved amino acid residue), a distinct feature of the cytokine...... receptors. This motif is thought to play a major role in correct folding and activation of the receptor. The complex between leptin and the D5CA domain was analyzed using nuclear magnetic resonance spectroscopy and the amino acid residues implicated in the binding were determined. To investigate which parts...

  13. Ganglioside Regulation of AMPA Receptor Trafficking

    OpenAIRE

    Prendergast, Jillian; Umanah, George K. E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.; Schnaar, Ronald L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside G...

  14. Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Kristensen, Bjarne Winther; Noraberg, J; Zimmer, J

    2001-01-01

    ) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed...... by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity...

  15. Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Bernardino, Liliana; Xapelli, Sara; Silva, Ana P

    2005-01-01

    The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects...... of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol....... By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed...

  16. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    Directory of Open Access Journals (Sweden)

    Li Q

    2013-07-01

    Full Text Available Qingli Li,1,2 Mark J Lambrechts,1 Qiuyang Zhang,1 Sen Liu,1 Dongxia Ge,1 Rutie Yin,2 Mingrong Xi,2 Zongbing You1 1Departments of Structural and Cellular Biology and Orthopaedic Surgery, Tulane Cancer Center and Louisiana Cancer Research Consortium, Tulane Center for Stem Cell Research and Regenerative Medicine, and Tulane Center for Aging, Tulane University Health Sciences Center, New Orleans, LA, USA; 2Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, People’s Republic of China Abstract: Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA, are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. Keywords: serine hydroxymethyltransferase, prostate cancer, apoptosis

  17. 3D structure of muscle dihydropyridine receptor

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2015-01-01

    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  18. Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and dopamine D1 receptors regulate GluA1 trafficking in striatal neurons.

    Science.gov (United States)

    Tukey, David S; Ziff, Edward B

    2013-12-06

    Regulation of striatal medium spiny neuron synapses underlies forms of motivated behavior and pathological drug seeking. A primary mechanism for increasing synaptic strength is the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynapse, a process mediated by GluA1 AMPAR subunit phosphorylation. We have examined the role of converging glutamate and dopamine inputs in regulating biochemical cascades upstream of GluA1 phosphorylation. We focused on the role of Ca(2+)-permeable AMPARs (CPARs), which lack the GluA2 AMPAR subunit. Under conditions that prevented depolarization, stimulation of CPARs activated neuronal nitric oxide synthase and production of cGMP. CPAR-dependent cGMP production was sufficient to induce synaptic insertion of GluA1, detected by confocal microscopy, through a mechanism dependent on GluA1 Ser-845 phosphorylation. Dopamine D1 receptors, in contrast, stimulate GluA1 extra synaptic insertion. Simultaneous activation of dopamine D1 receptors and CPARs induced additive increases in GluA1 membrane insertion, but only CPAR stimulation augmented CPAR-dependent GluA1 synaptic insertion. This incorporation into the synapse proceeded through a sequential two-step mechanism; that is, cGMP-dependent protein kinase II facilitated membrane insertion and/or retention, and protein kinase C activity was necessary for synaptic insertion. These data suggest a feed-forward mechanism for synaptic priming whereby an initial stimulus acting independently of voltage-gated conductance increases striatal neuron excitability, facilitating greater neuronal excitation by a subsequent stimulus.

  19. Size and receptor density of glutamatergic synapses: a viewpoint from left-right asymmetry of CA3-CA1 connections

    Directory of Open Access Journals (Sweden)

    Yoshiaki Shinohara

    2009-07-01

    Full Text Available Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left-right asymmetry of hippocampal CA3-CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supra-linear (GluR1 AMPA or inverse (NR2B NMDAR relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.

  20. 钩藤碱对甲基苯丙胺条件性位置偏爱大鼠AMPA受体蛋白改变的影响%The effect of rhynchophylline on AMPA receptors expression in methamphetamine dependent rats

    Institute of Scientific and Technical Information of China (English)

    林晓亮; 汤伟; 陈文倩; 翁建霖; 莫志贤

    2010-01-01

    Objective To study changes of AMPA receptors expression in nucleus accumbens and hypothalamus of methamphetamine dependent rats,and the therapeutical effect of rhynchophylline.Methods SPF male rata were randomly divided into normal control group,model group of methamphetamine,low dose of rhynchophylline group and high dose of rhynchophylline group(n=8 in each group).Experiment of conditioned place preference(CPP)was used to build the model of methamphetamine dependent rata.Western blotting was used to examine the changes of GluR2/3 subunits expression.The time of staying in drug-paired compartment of rats was used independent-samples t test to gather statistics,and the photodensity of proteinum strap was used One-Way ANOVA to gather statistics.Results Compare with rats in normal control group(the time of staying in drug-paired compartment of rats was(383.00±38.20)s),the rats produced CPP after treated with methamphetamine(the time of staying in drug-paired compartment of rats was(536.20±57.49)s),and low(30mg/kg) and high (60 ms/kg)dose of rhynchophylline(the time of staying in drug-paired compartment of rats were(299.80±15.96)s and(189.40±59.02)s)both could eliminate CPP effect.Compare with rats in normal control group (the ratio of value of average gray scale were(0.54±0.04)INT·mm~2 and (0.70±0.04)INT·mm~2),GluR2/3 subunits expression in nucleus aecumbens increased significantly in model group(the ratio of value of average gray seale was(0.89±0.03)INT·mm~2)and low dose of rhynchophylline group(the ratio of value of average gray seale was (0.93±0.03)INT·mm~2,P0.05).Conclusion GluR2/3 subunits expression of methamphetamine-induced CPP rats increased in nucleus accumbens but decreased in hypothalamus.High dose of rhynchophylline can reverse such changes and rebound the expression to normal level.%目的 观察甲基苯丙胺成瘾大鼠伏隔核及下丘脑中AMPA受体表达的改变及钩藤碱对其的干预作用.方法 SPF级雄性SD大鼠分为空

  1. Assessment of structurally diverse philanthotoxin analogues for inhibitory activity on ionotropic glutamate receptor subtypes

    DEFF Research Database (Denmark)

    Frølund, Sidsel; Bella, Angelo; Kristensen, Anders Skov;

    2010-01-01

    -electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 n......M. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM....

  2. Modifikasi Mekanisme Koufopanos pada Kinetika Reaksi Pirolisis Ampas Tebu (Bagasse)

    OpenAIRE

    Erawati, Emi; Sediawan, Wahyudi Budi; Mulyono, Panut

    2014-01-01

    Ampas tebu merupakan produk samping dari ekstraksi gula. Ampas tebu yang dihasilkan di pabrik gula sekitar 13% dari tebu yang digiling. Tujuan penelitian ini adalah menentukan energi aktivasi dan pre-exponential factor pada persamaan kinetika reaksi pirolisis ampas tebu. Pirolisis dilakukan dalam reaktor yang terbuat dari pipa besi jenis 5737 dengan diameter 7,62 cm dan panjang 37 cm. Reaktor ini dimasukkan ke dalam furnace yang berdiameter 15,24 cm dan panjang 40 cm. Seratus lima puluh...

  3. Decreased AMPA GluR2, but not GluR3, mRNA expression in rat amygdala and dorsal hippocampus following morphine-induced behavioural sensitization.

    Science.gov (United States)

    Sepehrizadeh, Zargham; Bahrololoumi Shapourabadi, Mina; Ahmadi, Shamseddin; Hashemi Bozchlou, Saeed; Zarrindast, Mohammad-Reza; Sahebgharani, Mousa

    2008-11-01

    1. Repeated administration of psychostimulants and micro-opioid receptor agonists elicits a progressive enhancement of drug-induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor function. 2. In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state-dependent memory in a passive avoidance test were measured in the presence or absence of 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine. 3. The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state-dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed. 4. These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium-permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.

  4. Crystallization and Structure Determination of Superantigens and Immune Receptor Complexes.

    Science.gov (United States)

    Rödström, Karin E J; Lindkvist-Petersson, Karin

    2016-01-01

    Structure determination of superantigens and the complexes they form with immune receptors have over the years provided insight in their modes of action. This technique requires growing large and highly ordered crystals of the superantigen or receptor-superantigen complex, followed by exposure to X-ray radiation and data collection. Here, we describe methods for crystallizing superantigens and superantigen-receptor complexes using the vapor diffusion technique, how the crystals may be optimized, and lastly data collection and structure determination.

  5. Adrenergic Receptors From Molecular Structure to in vivo function.

    Science.gov (United States)

    Hein, L; Kobilka, B K

    1997-07-01

    Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145). © 1997, Elsevier Science Inc.

  6. Primary Structure of Nicotinic Acetylcholine Receptor

    Science.gov (United States)

    1986-08-01

    quantities of starting material (for reviews of receptor, see Popot and Changeux, 1984; Stroud and Finer-Moore, 1985). This work led to the...Cloning of the Acetylcholine Receptor. Cold Spring Harbor Symp. on Quant. Biol. XLVIH: 71-78. 15. Popot , J-L. and Changeux, J-P. (1984) The

  7. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S;

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs...

  8. Class I Cytokine Receptors: Structure and function in the Membrane

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard

    Class I cytokine receptors are involved in important biological functions of both physiological and pathological nature in mammals. However, the molecular details of the cross-membrane signal transduction through these receptors remain obscure. One of the major reasons for this is the lack...... of structural knowledge on their membrane-embedded transmembrane domains (TMDs), which connect the extracellular ligand binding domains to the intracellular signaling platforms. The overall aim of this thesis work was to improve our understanding of the class I cytokine receptor signaling across the membrane...... ample material of high quality for structural studies with NMR spectroscopy of several class I cytokine receptor TMDs. Furthermore, the structure of a class I cytokine receptor TMD in DHPC micelles was solved with solution-state NMR spectroscopy. Additionally, since structural studies of intact proteins...

  9. Leaching of glyphosate and AMPA under two soil management practices in Burgundy vineyards (Vosne-Romanee, 21-France)

    Energy Technology Data Exchange (ETDEWEB)

    Landry, David [UMR 1229 INRA/Universite de Bourgogne, Microbiologie et Geochimie des sols, Centre des Sciences de la Terre, Universite de Bourgogne, 6 bd Gabriel 21000 Dijon (France)]. E-mail: david.landry@u-bourgogne.fr; Dousset, Sylvie [UMR 1229 INRA/Universite de Bourgogne, Microbiologie et Geochimie des sols, Centre des Sciences de la Terre, Universite de Bourgogne, 6 bd Gabriel 21000 Dijon (France); Fournier, Jean-Claude [UMR 1229 INRA/Universite de Bourgogne, INRA, 17 rue Sully, 21000 Dijon (France); Andreux, Francis [UMR 1229 INRA/Universite de Bourgogne, Microbiologie et Geochimie des sols, Centre des Sciences de la Terre, Universite de Bourgogne, 6 bd Gabriel 21000 Dijon (France)

    2005-11-15

    Some drinking water reservoirs under the vineyards of Burgundy are contaminated with herbicides. Thus the effectiveness of alternative soil management practices, such as grass cover, for reducing the leaching of glyphosate and its metabolite, AMPA, through soils was studied. The leaching of both molecules was studied in structured soil columns under outdoor conditions for 1 year. The soil was managed under two vineyard soil practices: a chemically treated bare calcosol, and a vegetated calcosol. After 680 mm of rainfall, the vegetated calcosol leachates contained lower amounts of glyphosate and AMPA (0.02% and 0.03%, respectively) than the bare calcosol leachates (0.06% and 0.15%, respectively). No glyphosate and only low amounts of AMPA (<0.01%) were extracted from the soil. Glyphosate, and to a greater extent, AMPA, leach through the soils; thus, both molecules may be potential contaminants of groundwater. However, the alternative soil management practice of grass cover could reduce groundwater contamination by the pesticide. - Glyphosate and AMPA leached in greater amounts through a chemically treated bare calcosol than through a vegetated calcosol.

  10. Structure-Function Studies on the Prolactin Receptor

    DEFF Research Database (Denmark)

    Haxholm, Gitte Wolfsberg

    Class 1 Cytokine receptors are involved in important biological functions mediated through complex networks of intracellular signaling. However, the molecular details of how signaling is regulated are poorly understood. One of the primary reasons for this limited knowledge is the lack of structural...... information on the intracellular domains (ICDs) of these receptors. The overall aim of this study was to obtain an improved understanding of cytokine receptor signaling through structure-function studies on the prolactin receptor (PRLR). The primary focus of this thesis was to structurally characterize...... the PRLR-ICD and the ICD of the related growth hormone receptor (GHR). We showed that both ICDs were intrinsically disordered throughout their entire lengths and that they associated with lipids characteristic of the inner plasma membrane leaflet through conserved motifs, implicating the membrane...

  11. Structural basis for ligand recognition of incretin receptors

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Parthier, Christoph; Reedtz-Runge, Steffen

    2010-01-01

    been solved recently by X-ray crystallography. The crystal structures reveal a similar fold of the ECD and a similar mechanism of ligand binding, where the ligand adopts an α-helical conformation. Residues in the C-terminal part of the ligand interact directly with the ECD and hydrophobic interactions...... appear to be the main driving force for ligand binding to the ECD of incretin receptors. Obviously, the-still missing-structures of full-length incretin receptors are required to construct a complete picture of receptor function at the molecular level. However, the progress made recently in structural...

  12. Synaptic plasticity, AMPA-R trafficking, and Ras-MAPK signaling

    Institute of Scientific and Technical Information of China (English)

    Yun GU; Ruth L STORNETTA

    2007-01-01

    Synaptic modification of transmission is a general phenomenon expressed at al-most every excitatory synapse in the mammalian brain. Over the last three decades,much has been discovered about the cellular, synaptic, molecular, and signalingmechanisms responsible for controlling synaptic transmission and plasticity. Here,we present a brief review of these mechanisms with emphasis on the currentunderstanding of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid recep-tor (AMPA-R) trafficking and Ras-mitogen-activated protein kinase (MAPK)signaling events involved in controlling synaptic transmission.

  13. Effect of AMPA receptors on learning and momery of the hippocampal dentate gyrus in free-moving conscious rats%AMPA受体对清醒大鼠海马齿状回区学习记忆功能的影响

    Institute of Scientific and Technical Information of China (English)

    孙志刚; 姜岩; 包金锁; 金秀吉; 金元哲; 金清华

    2009-01-01

    the changes of PS were significantly attenuated, the concentrations of Glu were still significantly increased (P < 0.05) by local-microinjection of DNQX. The establishment of conditioned reflex was significantly prevented by DNQX. Conclusion AMPA receptor shows the trigger action in the hippocampal learning-dependent LTP.

  14. Structure of the [delta]-opioid receptor bound to naltrindole

    Energy Technology Data Exchange (ETDEWEB)

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K. (Stanford-MED)

    2012-07-11

    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  15. Study of bioengineered zebra fish olfactory receptor 131-2: receptor purification and secondary structure analysis.

    Science.gov (United States)

    Leck, Kwong-Joo; Zhang, Shuguang; Hauser, Charlotte A E

    2010-11-25

    How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs). Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.

  16. Study of bioengineered zebra fish olfactory receptor 131-2: receptor purification and secondary structure analysis.

    Directory of Open Access Journals (Sweden)

    Kwong-Joo Leck

    Full Text Available How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs. Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.

  17. LYRA, a webserver for lymphocyte receptor structural modeling

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Anderson, Mads Valdemar; Jespersen, Martin Closter

    2015-01-01

    a complete and automated method for building of B- and T-cell receptor structural models starting from their amino acid sequence alone. The webserver is freely available and easy to use for non-specialists. Upon submission, LYRA automatically generates alignments using ad hoc profiles, predicts...... the structural class of each hypervariable loop, selects the best templates in an automatic fashion, and provides within minutes a complete 3D model that can be downloaded or inspected online. Experienced users can manually select or exclude template structures according to case specific information. LYRA......The accurate structural modeling of B- and T-cell receptors is fundamental to gain a detailed insight in the mechanisms underlying immunity and in developing new drugs and therapies. The LYRA (LYmphocyte Receptor Automated modeling) web server (http://www.cbs.dtu.dk/services/LYRA/) implements...

  18. A structural biology perspective on NMDA receptor pharmacology and function.

    Science.gov (United States)

    Regan, Michael C; Romero-Hernandez, Annabel; Furukawa, Hiro

    2015-08-01

    N-methyld-aspartate receptors (NMDARs) belong to the large family of ionotropic glutamate receptors (iGluRs), which are critically involved in basic brain functions as well as multiple neurological diseases and disorders. The NMDARs are large heterotetrameric membrane protein complexes. The extensive extracellular domains recognize neurotransmitter ligands and allosteric compounds and translate the binding information to regulate activity of the transmembrane ion channel. Here, we review recent advances in the structural biology of NMDARs with a focus on pharmacology and function. Structural analysis of the isolated extracellular domains in combination with the intact heterotetrameric NMDAR structure provides important insights into how this sophisticated ligand-gated ion channel may function.

  19. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K. (Stanford); (NIH); (D.E. Shaw); (Hanyang); (UTSMC)

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  20. Structure of a human rhinovirus complexed with its receptor molecule.

    OpenAIRE

    1993-01-01

    Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. ...

  1. In silico discovery of novel Retinoic Acid Receptor agonist structures

    Directory of Open Access Journals (Sweden)

    Samuels Herbert H

    2001-06-01

    Full Text Available Abstract Background Several Retinoic Acid Receptors (RAR agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia. Results We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually to the structure of the receptor with the Internal Coordinates Mechanics (ICM program. Thirty ligand candidates were tested in vitro. Conclusions Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

  2. Structure and dynamics of a constitutively active neurotensin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Krumm, Brian E. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Lee, Sangbae [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Bhattacharya, Supriyo [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Botos, Istvan [National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Diabetes and; White, Courtney F. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Du, Haijuan [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Vaidehi, Nagarajan [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Grisshammer, Reinhard [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services

    2016-12-07

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

  3. Structure and function of the human megalin receptor

    DEFF Research Database (Denmark)

    Dagil, Robert

    Megalin is an endocytic lipoprotein receptor expressed widely throughout the body, ranging from the proximal tubule in the kidneys to the cochlea in the inner ear. Megalin is known to bind over 50 different ligands and is involved in protein clearance of the renal ultrafiltrate via endocytosis...... was studied using NMR spectroscopy. The structure of the tenth CR domain from the human megalin receptor was solved using NMR spectroscopy and a HADDOCK model of the complex between this domain and gentamicin was determined. The structural complex showed that a Trp residue and three Asp residues from megalin...

  4. Enhancing VTA Cav1.3 L-type Ca(2+) channel activity promotes cocaine and mood-related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens.

    Science.gov (United States)

    Martínez-Rivera, A; Hao, J; Tropea, T F; Giordano, T P; Kosovsky, M; Rice, R C; Lee, A; Huganir, R L; Striessnig, J; Addy, N A; Han, S; Rajadhyaksha, A M

    2017-02-14

    Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca(2+) channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms.Molecular Psychiatry advance online publication, 14

  5. An alternating GluN1-2-1-2 subunit arrangement in mature NMDA receptors.

    Directory of Open Access Journals (Sweden)

    Morgane Riou

    Full Text Available NMDA receptors (NMDARs form glutamate-gated ion channels that play a critical role in CNS physiology and pathology. Together with AMPA and kainate receptors, NMDARs are known to operate as tetrameric complexes with four membrane-embedded subunits associating to form a single central ion-conducting pore. While AMPA and some kainate receptors can function as homomers, NMDARs are obligatory heteromers composed of homologous but distinct subunits, most usually of the GluN1 and GluN2 types. A fundamental structural feature of NMDARs, that of the subunit arrangement around the ion pore, is still controversial. Thus, in a typical NMDAR associating two GluN1 and two GluN2 subunits, there is evidence for both alternating 1/2/1/2 and non-alternating 1/1/2/2 arrangements. Here, using a combination of electrophysiological and cross-linking experiments, we provide evidence that functional GluN1/GluN2A receptors adopt the 1/2/1/2 arrangement in which like subunits are diagonal to one another. Moreover, based on the recent crystal structure of an AMPA receptor, we show that in the agonist-binding and pore regions, the GluN1 subunits occupy a "proximal" position, closer to the central axis of the channel pore than that of GluN2 subunits. Finally, results obtained with reducing agents that differ in their membrane permeability indicate that immature (intracellular and functional (plasma-membrane inserted pools of NMDARs can adopt different subunit arrangements, thus stressing the importance of discriminating between the two receptor pools in assembly studies. Elucidating the quaternary arrangement of NMDARs helps to define the interface between the subunits and to understand the mechanism and pharmacology of these key signaling receptors.

  6. The perception of gibberellins: clues from receptor structure.

    Science.gov (United States)

    Ueguchi-Tanaka, Miyako; Matsuoka, Makoto

    2010-10-01

    The discovery of GID1, a soluble receptor for gibberellins (GAs), has revealed new insights into how GA is perceived. X-ray analysis has demonstrated similarities in the tertiary structure of GID1 to hormone sensitive lipase (HSL), and the GA-binding pocket of GID1 corresponds to the active site of HSL. X-ray analysis has also revealed the structural basis of the GA-GID1 interaction, and evolutionary aspects of GID1 have been discovered by comparison to GID1 from non-flowering plants. Recent studies have also demonstrated the complexity of GA signaling in Arabidopsis, which is mediated by three GID1 and five DELLA proteins. Finally, mechanistic and structural similarities for hormone signaling are compared for GA, auxin and abscisic acid, three hormones where the receptor protein structure was recently described.

  7. Nuclear hormone receptor co-repressors: Structure and function

    OpenAIRE

    2012-01-01

    Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered “hub proteins” that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined ...

  8. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  9. Study of structure function correlation of chemokine receptor CXCR4

    Institute of Scientific and Technical Information of China (English)

    ZHENG Hong; Stephen C PEIPER; ZHU Xi-hua

    2002-01-01

    Objective: To explore the correlation between structure domains and functions of chemokine receptor CXCR4. Methods: After the establishment of wild type chemokine receptor CXCR4 and CXCR2 expressing cell lines, 5 CXCR4/CXCR2 chimeras, 2 CXCR4 mutants were stably expressed on CHO cell line.Binding activities of all variants with the ligand, recombinant human SDF-1β, signal transduction ability after stimulation and their function as coreceptor for HIV-1 were studied with ligand-binding assay, Cytosensor/microphysiometry and cell-cell reporter gene fusion assay. Results: Among all 7 changed CXCR4 receptors, 3 chimeras (2444a, 4442, 4122), and 1 mutant (CXCR4-Tr) bond with SDF-1β in varying degrees, of which only 2444a totally and CXCR4-Tr partially maintain signaling. All changed receptors except for 4222 could act as coreceptors for HIV-1(LAI) in varying degrees. Conclusion: Several structure domains of CXCR4 are involved in the binding with SDF-1β, among which, N-terminal extracellular domain has high affinity of binding with SDF-1β, and the 3rd extracellular loop contributes to the binding, too. Although the C-terminal intracellular domain has no association with the maintenance of the overall structure of the receptor and ligand binding capability, the signaling is decreased when this domain is truncated. For CXCR4 signaling, not only is the conserved motif DRY box needed, but also the characterized conformation of the whole molecule must be formed when activation is required. There are some overlaps between SDF-1β binding domains and coreceptor function domains in molecular structure of CXCR4.

  10. Receptors for myelin inhibitors: Structures and therapeutic opportunities.

    Science.gov (United States)

    Cao, Zixuan; Gao, Ying; Deng, Kangwen; Williams, Gareth; Doherty, Patrick; Walsh, Frank S

    2010-01-01

    Many studies have indicated that the inability of adult mammalian central nervous system (CNS) to regenerate after injury is partly due to the existence of growth-inhibitory molecules associated with CNS myelin. Studies over the years have led to the identification of multiple myelin-associated inhibitors, among which Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (Omgp) represent potentially major contributors to CNS axon regeneration failure. Here we review in vitro and in vivo investigations into these inhibitory ligands and their functional mechanisms, focusing particularly on the neuronal receptors that mediate the inhibitory signals from these myelin molecules. A better understanding of the receptors for myelin-associated inhibitors could provide opportunities to decipher the mechanism of restriction in CNS regeneration, and lead to the development of potential therapeutic targets in neurodegenerative diseases and neurological injury. We will discuss the structures of the receptors and therapeutic opportunities that might arise based on this information.

  11. Structure-based rationale for interleukin 5 receptor antagonism.

    Science.gov (United States)

    Ishino, Tetsuya; Harrington, Adrian E; Gopi, Hosahudya; Chaiken, Irwin

    2008-01-01

    Human interleukin 5 (IL5) is the major hematopoietin that stimulates the proliferation, migration and activation of eosinophils and is implicated in the pathogenesis of inflammatory and other myeloproliferative diseases. IL5 functions through the signaling of a common receptor subunit beta (beta c), in a receptor activation process that requires initial recruitment of an IL5 specific receptor subunit alpha (IL5Ralpha), for cytokine presentation to beta c. Important advances have been made to understand molecular mechanisms of cytokine recognition and receptor antagonism. Mutational studies indicate that a pair of charge complementary regions play an essential role in specific interaction between IL5Ralpha and IL5. Moreover, peptide studies with the IL5 system have identified a cyclic peptide inhibitor, AF17121, which binds specifically to IL5Ralpha by mimicking the cytokine. A key receptor-recognition pharmacophore has been identified in this peptide inhibitor, and sites of inhibitor recognition can be proposed in the homology-deduced structural model of IL5Ralpha. These results provide an experimental platform to derive enhanced-potency peptidomimetic inhibitors. Such inhibitors have potential use as tools to evaluate the role of eosinophilia in disease and as potential leads to antagonists to treat hyper-eosinophilic diseases such as eosinophilic esophagitis, asthma and chronic myeloproliferative leukemias.

  12. Signaling by postsynaptic AMPA receptors in glutamatergic synapse maturation

    OpenAIRE

    2010-01-01

    Excitatory transmission in the brain is largely mediated by synapses containing the neurotransmitter glutamate. Neuronal circuitry is first established early in brain development requiring the formation of vast numbers of glutamatergic synapses at individual sites of contact made between presynaptic axons and postsynaptic dendrites. Despite mounting efforts in the last decade to identify the complex molecular mechanisms underlying initial synaptogenesis and the subsequent steps of synapse m...

  13. Fate and availability of glyphosate and AMPA in agricultural soil.

    Science.gov (United States)

    Simonsen, Louise; Fomsgaard, Inge S; Svensmark, Bo; Spliid, Niels Henrik

    2008-06-01

    The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT(50) values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, (14)C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 +/- 0.002% and 0.005 +/- 0.001% of the applied radioactivity was measured in rape and barley, respectively.

  14. μ Opioid receptor: novel antagonists and structural modeling

    Science.gov (United States)

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

    2016-02-01

    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

  15. Central nicotinic receptors: structure, function, ligands, and therapeutic potential.

    Science.gov (United States)

    Romanelli, M Novella; Gratteri, Paola; Guandalini, Luca; Martini, Elisabetta; Bonaccini, Claudia; Gualtieri, Fulvio

    2007-06-01

    The growing interest in nicotinic receptors, because of their wide expression in neuronal and non-neuronal tissues and their involvement in several important CNS pathologies, has stimulated the synthesis of a high number of ligands able to modulate their function. These membrane proteins appear to be highly heterogeneous, and still only incomplete information is available on their structure, subunit composition, and stoichiometry. This is due to the lack of selective ligands to study the role of nAChR under physiological or pathological conditions; so far, only compounds showing selectivity between alpha4beta2 and alpha7 receptors have been obtained. The nicotinic receptor ligands have been designed starting from lead compounds from natural sources such as nicotine, cytisine, or epibatidine, and, more recently, through the high-throughput screening of chemical libraries. This review focuses on the structure of the new agonists, antagonists, and allosteric ligands of nicotinic receptors, it highlights the current knowledge on the binding site models as a molecular modeling approach to design new compounds, and it discusses the nAChR modulators which have entered clinical trials.

  16. Alternative Splicing of AMPA subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys following Chronic Ethanol Self-Administration

    Directory of Open Access Journals (Sweden)

    Glen eAcosta

    2012-01-01

    Full Text Available Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA subunit variant and kainate (GRIK subunit mRNA expression were studied in the orbitofrontal cortex (OFC, dorsolateral prefrontal cortex (DLPFC and anterior cingulate cortex (ACC of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and blood ethanol concentrations averaged over the six months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted.

  17. Stereoselective effects of AMOA on non-NMDA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Wahl, P; Nielsen, B; Krogsgaard-Larsen, P

    1992-01-01

    a nearly parallel shift to the right of the dose-response curve for kainate-induced currents. AMOA was found to have two different effects on AMPA receptors: 1) currents elicited by low concentrations of AMPA (6 microM) were inhibited by AMOA with an IC50 value of 160 +/- 19 microM and 2) currents elicited...

  18. Crystal Structure of the Human Laminin Receptor Precursor

    Energy Technology Data Exchange (ETDEWEB)

    Jamieson,K.; Wu, J.; Hubbard, S.; Meruelo, D.

    2008-01-01

    The human laminin receptor (LamR) interacts with many ligands, including laminin, prions, Sindbis virus, and the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and has been implicated in a number of diseases. LamR is overexpressed on tumor cells, and targeting LamR elicits anti-cancer effects. Here, we report the crystal structure of human LamR, which provides insights into its function and should facilitate the design of novel therapeutics targeting LamR.

  19. Structural dynamics and thermostabilization of neurotensin receptor 1.

    Science.gov (United States)

    Lee, Sangbae; Bhattacharya, Supriyo; Tate, Christopher G; Grisshammer, Reinhard; Vaidehi, Nagarajan

    2015-04-16

    The neurotensin receptor NTSR1 binds the peptide agonist neurotensin (NTS) and signals preferentially via the Gq protein. Recently, Grisshammer and co-workers reported the crystal structure of a thermostable mutant NTSR1-GW5 with NTS bound. Understanding how the mutations thermostabilize the structure would allow efficient design of thermostable mutant GPCRs for protein purification, and subsequent biophysical studies. Using microsecond scale molecular dynamics simulations (4 μs) of the thermostable mutant NTSR1-GW5 and wild type NTSR1, we have elucidated the structural and energetic factors that affect the thermostability and dynamics of NTSR1. The thermostable mutant NTSR1-GW5 is found to be less flexible and less dynamic than the wild type NTSR1. The point mutations confer thermostability by improving the interhelical hydrogen bonds, hydrophobic packing, and receptor interactions with the lipid bilayer, especially in the intracellular regions. During MD, NTSR1-GW5 becomes more hydrated compared to wild type NTSR1, with tight hydrogen bonded water clusters within the transmembrane core of the receptor, thus providing evidence that water plays an important role in improving helical packing in the thermostable mutant. Our studies provide valuable insights into the stability and functioning of NTSR1 that will be useful in future design of thermostable mutants of other peptide GPCRs.

  20. Androgen receptor: structure, role in prostate cancer and drug discovery.

    Science.gov (United States)

    Tan, M H Eileen; Li, Jun; Xu, H Eric; Melcher, Karsten; Yong, Eu-leong

    2015-01-01

    Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2-3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.

  1. A combined computational and structural model of the full-length human prolactin receptor

    Science.gov (United States)

    Bugge, Katrine; Papaleo, Elena; Haxholm, Gitte W.; Hopper, Jonathan T. S.; Robinson, Carol V.; Olsen, Johan G.; Lindorff-Larsen, Kresten; Kragelund, Birthe B.

    2016-05-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than 40 different receptor chains, and reveals that the extracellular domain is merely the tip of a molecular iceberg.

  2. Exploring the GluR2 ligand-binding core in complex with the bicyclical AMPA analogue (S)-4-AHCP

    DEFF Research Database (Denmark)

    Nielsen, Bettina B; Pickering, Darryl S; Greenwood, Jeremy R;

    2005-01-01

    The X-ray structure of the ionotropic GluR2 ligand-binding core (GluR2-S1S2J) in complex with the bicyclical AMPA analogue (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid [(S)-4-AHCP] has been determined, as well as the binding pharmacology of this construct...

  3. Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

    Science.gov (United States)

    Tatulian, Suren A

    2015-09-15

    The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.

  4. Crystal Structure of an LSD-Bound Human Serotonin Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wacker, Daniel; Wang, Sheng; McCorvy, John D.; Betz, Robin M.; Venkatakrishnan, A.J.; Levit, Anat; Lansu, Katherine; Schools, Zachary L.; Che, Tao; Nichols, David E.; Shoichet, Brian K.; Dror, Ron O.; Roth, Bryan L. (UNCSM); (UNC); (Stanford); (Stanford-MED); (UCSF)

    2017-01-01

    The prototypical hallucinogen LSD acts via serotonin receptors, and here we describe the crystal structure of LSD in complex with the human serotonin receptor 5-HT2B. The complex reveals conformational rearrangements to accommodate LSD, providing a structural explanation for the conformational selectivity of LSD’s key diethylamide moiety. LSD dissociates exceptionally slow from both 5-HT2BR and 5-HT2AR—a major target for its psychoactivity. Molecular dynamics (MD) simulations suggest that LSD’s slow binding kinetics may be due to a “lid” formed by extracellular loop 2 (EL2) at the entrance to the binding pocket. A mutation predicted to increase the mobility of this lid greatly accelerates LSD’s binding kinetics and selectively dampens LSD-mediated β-arrestin2 recruitment. This study thus reveals an unexpected binding mode of LSD; illuminates key features of its kinetics, stereochemistry, and signaling; and provides a molecular explanation for LSD’s actions at human serotonin receptors.

  5. Crystal Structure of the Human Cannabinoid Receptor CB1.

    Science.gov (United States)

    Hua, Tian; Vemuri, Kiran; Pu, Mengchen; Qu, Lu; Han, Gye Won; Wu, Yiran; Zhao, Suwen; Shui, Wenqing; Li, Shanshan; Korde, Anisha; Laprairie, Robert B; Stahl, Edward L; Ho, Jo-Hao; Zvonok, Nikolai; Zhou, Han; Kufareva, Irina; Wu, Beili; Zhao, Qiang; Hanson, Michael A; Bohn, Laura M; Makriyannis, Alexandros; Stevens, Raymond C; Liu, Zhi-Jie

    2016-10-20

    Cannabinoid receptor 1 (CB1) is the principal target of Δ(9)-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

  6. A combined computational and structural model of the full-length human prolactin receptor

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Papaleo, Elena; Haxholm, Gitte Wolfsberg;

    2016-01-01

    The prolactin receptor is an archetype member of the class I cytokine receptor family, comprising receptors with fundamental functions in biology as well as key drug targets. Structurally, each of these receptors represent an intriguing diversity, providing an exceptionally challenging target...... for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small......-angle X-ray scattering, native mass spectrometry and NMR spectroscopy. Along with previously published data, these are integrated by molecular modelling to generate a full receptor structure. The result provides the first full view of a class I cytokine receptor, exemplifying the architecture of more than...

  7. Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains

    OpenAIRE

    2011-01-01

    Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is respons...

  8. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti

  9. Crystal structure of pentapeptide-independent chemotaxis receptor methyltransferase (CheR) reveals idiosyncratic structural determinants for receptor recognition.

    Science.gov (United States)

    Batra, Monu; Sharma, Rajesh; Malik, Anjali; Dhindwal, Sonali; Kumar, Pravindra; Tomar, Shailly

    2016-12-01

    Chemotactic methyltransferase, CheR catalyse methylation of specific glutamate residues in the cytoplasmic domain of methyl-accepting chemotactic protein receptors (MCPRs). The methylation of MCPRs is essential for the chemical sensing and chemotactic bacterial mobility towards favorable chemicals or away from unfavorable ones. In this study, crystal structure of B. subtilis CheR (BsCheR) in complex with S-adenosyl-l-homocysteine (SAH) has been determined to 1.8Å resolution. This is the first report of crystal structure belonging to the pentapeptide-independent CheR (PICheR) class. Till date, only one crystal structure of CheR from S. typhimurium (StCheR) belonging to pentapeptide-dependent CheR (PDCheR) class is available. Structural analysis of BsCheR reveals a helix-X-helix motif (HXH) with Asp53 as the linker residue in the N-terminal domain. The key structural features of the PDCheR β-subdomain involved in the formation of a tight complex with the pentapeptide binding motif in MCPRs were found to be absent in the structure of BsCheR. Additionally, isothermal titration calorimetry (ITC) experiments were performed to investigate S-adenosyl-(l)-methionine (SAM) binding affinity and KD was determined to be 0.32mM. The structure of BsCheR reveals that mostly residues of the large C-terminal domain contribute to SAH binding, with contributions of few residues from the linker region and the N-terminal domain. Structural investigations and sequence analysis carried out in this study provide critical insights into the distinct receptor recognition mechanism of the PDCheR and PICheR methyltransferase classes.

  10. The Histamine H3 Receptor: Structure, Pharmacology, and Function.

    Science.gov (United States)

    Nieto-Alamilla, Gustavo; Márquez-Gómez, Ricardo; García-Gálvez, Ana-Maricela; Morales-Figueroa, Guadalupe-Elide; Arias-Montaño, José-Antonio

    2016-11-01

    Among the four G protein-coupled receptors (H1-H4) identified as mediators of the biologic effects of histamine, the H3 receptor (H3R) is distinguished for its almost exclusive expression in the nervous system and the large variety of isoforms generated by alternative splicing of the corresponding mRNA. Additionally, it exhibits dual functionality as autoreceptor and heteroreceptor, and this enables H3Rs to modulate the histaminergic and other neurotransmitter systems. The cloning of the H3R cDNA in 1999 by Lovenberg et al. allowed for detailed studies of its molecular aspects. In this work, we review the characteristics of the H3R, namely, its structure, constitutive activity, isoforms, signal transduction pathways, regional differences in expression and localization, selective agonists, antagonists and inverse agonists, dimerization with other neurotransmitter receptors, and the main presynaptic and postsynaptic effects resulting from its activation. The H3R has attracted interest as a potential drug target for the treatment of several important neurologic and psychiatric disorders, such as Alzheimer and Parkinson diseases, Gilles de la Tourette syndrome, and addiction.

  11. Structure of the homodimeric androgen receptor ligand-binding domain

    Science.gov (United States)

    Nadal, Marta; Prekovic, Stefan; Gallastegui, Nerea; Helsen, Christine; Abella, Montserrat; Zielinska, Karolina; Gay, Marina; Vilaseca, Marta; Taulès, Marta; Houtsmuller, Adriaan B.; van Royen, Martin E.; Claessens, Frank; Fuentes-Prior, Pablo; Estébanez-Perpiñá, Eva

    2017-01-01

    The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor. PMID:28165461

  12. Solution structure of the coxsackievirus and adenovirus receptor domain 2

    OpenAIRE

    Jiang, Shaokai; Caffrey, Michael

    2007-01-01

    The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR–D1 and CAR–D2. In the present work, the solution structure of CAR–D2, consisting of residues 142–235 of human CAR, has been determined by NMR spectroscopy. CAR–D2 is shown to be a β-sandwich motif comprised of two β-sheets, which are stabilized by two disulfide bonds. The first β-sheet is comprised of β...

  13. Structure of the LDL receptor extracellular domain at endosomalpH

    Energy Technology Data Exchange (ETDEWEB)

    Rudenko, Gabby; Henry, Lisa; Henderson, Keith; Ichtchenko,Konstantin; Brown, Michael S.; Goldstein, Joseph L.; Deisenhofer, Johann

    2002-09-05

    The structure of the low-density lipoprotein receptor extracellular portion has been determined. The document proposes a mechanism for the release of lipoprotein in the endosome. Without this release, the mechanism of receptor recycling cannot function.

  14. Genomic organization of a receptor from sea anemones, structurally and evolutionary related to glycoprotein hormone receptors from mamals

    DEFF Research Database (Denmark)

    Vibede, N; Hauser, Frank; Williamson, M

    1998-01-01

    Abstract Cnidarians (e.g., sea anemones and corals) are the lowest animal group having a nervous system. Previously, we cloned a receptor from sea anemones that showed a strong structural similarity to the glycoprotein hormone (TSH, FSH, LH/CG) receptors from mammals. Here, we determine the genomic...... organization of this sea anemone receptor. The receptor gene contains eight introns that are all localized within a region coding for the large extracellular N terminus. These introns occur at the same positions and have the same intron phasing as eight introns in the genes coding for the mammalian...

  15. Behavioural and neuronal activation after microinjections of AMPA and NMDA into the perifornical lateral hypothalamus in rats.

    Science.gov (United States)

    Li, Frederick W; Deurveilher, Samuel; Semba, Kazue

    2011-10-31

    The perifornical lateral hypothalamic area (PeFLH), which houses orexin/hypocretin (OX) neurons, is thought to play an important role in arousal, feeding, and locomotor activity. The present study examined behavioural effects of activating PeFLH neurons with microinjections of ionotropic glutamate receptor agonists. Three separate unilateral microinjections of either (1) AMPA (1 and 2mM in 0.1 μL artificial cerebrospinal fluid, ACSF) and ACSF, or (2) NMDA (1 and 10mM in 0.1 μL ACSF), and ACSF were made into the PeFLH of adult male rats. Following each injection, the rats were placed into an open field for behavioural scoring for 45 min. Rats were perfused after the third injection for immunohistochemistry for c-Fos and OX to assess the level of activation of OX neurons. Behavioural analyses showed that, as compared to ACSF conditions, AMPA injections produced a dose-dependent increase in locomotion and rearing that persisted throughout the 45 min recording period, and an increase in drinking. Injection of NMDA at 10mM, but not 1mM, induced a transient increase in locomotion and an increase in feeding. Histological analyses showed that while both agonists increased the number of neurons immunoreactive for c-Fos in the PeFLH, only AMPA increased the number of neurons immunoreactive for both c-Fos and OX. There were positive correlations between the number of c-Fos/OX-immunoreactive neurons and the amounts of locomotion, rearing, and drinking. These results support the role of ionotropic glutamate receptors on OX and other neurons in the PeFLH in the regulation of locomotor and ingestive behaviours.

  16. Structural basis for simvastatin competitive antagonism of complement receptor 3

    DEFF Research Database (Denmark)

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei;

    2016-01-01

    The complement system is an important part of the innate immune response to infection, but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor (CR)3 have been widely sought, but a structural basis for their mode of action is not available. We...... report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg2+ ion. Simvastatin antagonizes I domain binding...... to the complement fragments iC3b and C3d, but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μM simvastatin...

  17. Crystal Structures of the Nuclear Receptor, Liver Receptor Homolog 1, Bound to Synthetic Agonists.

    Science.gov (United States)

    Mays, Suzanne G; Okafor, C Denise; Whitby, Richard J; Goswami, Devrishi; Stec, Józef; Flynn, Autumn R; Dugan, Michael C; Jui, Nathan T; Griffin, Patrick R; Ortlund, Eric A

    2016-12-02

    Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.

  18. Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification

    Science.gov (United States)

    Chicote, Javier U.; DeSalle, Rob; García-España, Antonio

    2017-01-01

    Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly Drosophila melanogaster and F44G4.8/DEP-1 from the nematode Caenorhabditis elegans, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins. PMID:28257417

  19. CX717 as a positive allosteric modulator of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor: research advances%AMPA受体正向变构调节剂CX717研究进展

    Institute of Scientific and Technical Information of China (English)

    贺艺超; 肖典; 齐倩倩; 赵国明; 周辛波

    2013-01-01

    α-氨基-3-羟基-5-甲基-4-异噁唑丙酸(AMPA)受体是离子型谷氨酸受体的一种亚型,分布于中枢神经系统的突触后膜,介导大多数快速兴奋性神经传递.CX717是由美国Cortex制药公司研制的苯甲酰胺类AMPA受体正向调节剂,能够降低AMPA受体失活或降敏的速度从而提高突触的活性,与阿尔茨海默病、帕金森病、抑郁症和注意力缺陷多动症等疾病的治疗密切相关.本文主要综述CX717在化学结构、药代动力学、毒理学和药效学方面的研究进展.%α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor,a subtype of ionotropic glutamate receptors in the postsynaptic membrane of the central nervous system (CNS),mediates most of the fast excitatory neurotransmission.CX717 developed by Cortex Pharmaceuticals Company of the USA belongs to the benzamide series of AMPA receptor positive modulators.It can reduce the speed of AMPA receptor inactivation or desensitization,thereby enhancing synaptic activity,and is closely related to the treatment of Alzheimer's disease,Parkinson's disease,depression and attention deficit hyperactivity disorder(ADHD).This article reviews the latest research of CX717 regarding its structure,pharmacokinetics,toxicology and pharmacodynamics.

  20. 丙泊酚对大鼠胶质瘤细胞侵袭和迁移能力的影响及ADAR2-AMPA受体GluR2通路在其中的作用%Effects of propofol on invasion and migration of glioma cells in rats and the role of ADAR2-AMPA receptor GluR2 pathway

    Institute of Scientific and Technical Information of China (English)

    王欣悦; 王海云; 王国林; 杨卓; 张涛

    2016-01-01

    目的 评价丙泊酚对大鼠胶质瘤细胞侵袭和迁移能力的影响及腺苷脱氨酶(ADAR2)-α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体GluR2通路在其中的作用.方法 传代培养大鼠C6胶质瘤细胞,采用随机数字表法分为4组(n=24):对照组(C组)、丙泊酚组(P组)、阴性siRNA转染+丙泊酚组(NP组)和ADAR2-siRNA转染+丙泊酚组(AP组).C组正常培养;NP组和AP组分别将阴性siRNA或ADAR2-siRNA转染至细胞内,48 h后处理同P组;P组加入丙泊酚,终浓度1.2 μg/ml,孵育6h后换为正常培养液,继续培养18h.采用MTT比色分析法检测细胞活力,Transwell侵袭实验测定侵袭细胞数,细胞划痕实验测定迁移率,Western blot法检测胞核ADAR2和胞膜GluR2的表达.结果 与C组比较,P组和NP组细胞活力、侵袭细胞数和迁移率降低,胞核A-DAR2及胞膜GluR2表达上调(P<0.05);与P组比较,AP组细胞活力、侵袭细胞数和迁移率升高,胞核ADAR2及胞膜GluR2表达下调(P<0.05);与NP组比较,AP组细胞活力、侵袭细胞数和迁移率升高,胞核ADAR2及胞膜GluR2表达下调(P<0.05).结论 丙泊酚可抑制大鼠胶质瘤细胞的侵袭和迁移能力,其机制与激活ADAR2-AMPA受体GluR2通路有关.%Objective To evaluate the effects of propofol on the invasion and migration of glioma cells in the rats and the role of adenosine deaminase acting on RNA 2 (ADAR2)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit glutamate 2 (GluR2) pathway.Methods C6 glioma cells were subcuhured and randomly divided into 4 groups (n =24 each) using a random number table:control group (group C);propofol group (group P);negative siRNA transfection + propofol group (group NP);ADAR2-siRNA transfection + propofol group (group AP).The cells were cultured in the common culture medium in group C.In NP and AP groups,negative siRNA and ADAR2-siRNA were transfected into the cells,respectively,and 48 h later the other procedures were similar

  1. New insights into the structural bases of activation of Cys-loop receptors.

    Science.gov (United States)

    Bouzat, Cecilia

    2012-01-01

    Neurotransmitter receptors of the Cys-loop superfamily mediate rapid synaptic transmission throughout the nervous system, and include receptors activated by ACh, GABA, glycine and serotonin. They are involved in physiological processes, including learning and memory, and in neurological disorders, and they are targets for clinically relevant drugs. Cys-loop receptors assemble either from five copies of one type of subunit, giving rise to homomeric receptors, or from several types of subunits, giving rise to heteromeric receptors. Homomeric receptors are invaluable models for probing fundamental relationships between structure and function. Receptors contain a large extracellular domain that carries the binding sites and a transmembrane region that forms the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50Å to the ion channel gate is central to understanding receptor function. Depending on the receptor subtype, occupancy of either two, as in the prototype muscle nicotinic receptor, or three binding sites, as in homomeric receptors, is required for full activation. The conformational changes initiated at the binding sites are propagated to the gate through the interface between the extracellular and transmembrane domains. This region forms a network that relays structural changes from the binding site towards the pore, and also contributes to open channel lifetime and rate of desensitization. Thus, this coupling region controls the beginning and duration of a synaptic response. Here we review recent advances in the molecular mechanism by which Cys-loop receptors are activated with particular emphasis on homomeric receptors.

  2. A combined computational and structural model of the full-length human prolactin receptor

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Papaleo, Elena; Haxholm, Gitte Wolfsberg

    2016-01-01

    for structural biology. Here, we access the molecular architecture of the monomeric human prolactin receptor by combining experimental and computational efforts. We solve the NMR structure of its transmembrane domain in micelles and collect structural data on overlapping fragments of the receptor with small...

  3. Common structural basis for constitutive activity of the ghrelin receptor family

    DEFF Research Database (Denmark)

    Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

    2004-01-01

    -independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

  4. Structural basis of transcobalamin recognition by human CD320 receptor

    Science.gov (United States)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  5. A new pyrrolyl-quinoxalinedione series of non-NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy.

    Science.gov (United States)

    Löscher, W; Lehmann, H; Behl, B; Seemann, D; Teschendorf, H J; Hofmann, H P; Lubisch, W; Höger, T; Lemaire, H G; Gross, G

    1999-01-01

    Antagonists at the ionotropic non-NMDA [AMPA (amino-methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N-methyl-D-aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non-NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl-quinoxalinedione derivatives, we selected six new competitive non-NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non-NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5-GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA-induced cell death in vitro and, except LU 97175, AMPA-induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA

  6. Structure-function study of the fourth transmembrane segment of the GABAρ1 receptor

    Science.gov (United States)

    Estrada-Mondragón, Argel; Reyes-Ruiz, Jorge Mauricio; Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2010-01-01

    The Cys-loop family of receptors mediates synaptic neurotransmission in the central nervous system of vertebrates. These receptors share several structural characteristics and assemble in the plasma membrane as multimers with fivefold symmetry. Of these, the ionotropic GABA receptors are key players in the pathogenesis of diseases like epilepsy, anxiety, and schizophrenia. Different experimental approaches have shed some light on the mechanisms behind the function of these receptors; but little is known about their structure at high resolution. Sequence homology with the nicotinic acetylcholine receptor predicts that ionotropic GABA receptors possess four transmembrane segments (TM1–4) and that TM2 forms the wall of the ion channel. However, the role of the other three segments is unclear. The GABAρ1 receptor plays a fundamental role in the regulation of neurotransmission along the visual pathway, is highly sensitive to GABA, and exhibits little desensitization. In our recent investigations of the role of TM4 in receptor function, a key residue in this domain (W475) was found to be involved in activation of the receptor. Here we have generated a structural model of the GABAρ1 receptor in silico and assessed its validity by electrophysiologically testing nine amino acid substitutions of W475 and deletions of the neighboring residues (Y474 and S476). The results identify a critical linkage between the ligand-binding domain and the TM4 domain and provide a framework for more detailed structure-function analyses of ionotropic GABA receptors. PMID:20876117

  7. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

    Institute of Scientific and Technical Information of China (English)

    Nianshuang Wang; Xuanling Shi; Liwei Jiang; Senyan Zhang; Dongli Wang; Pei Tong; Dongxing Guo

    2013-01-01

    The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor,dipeptidyl peptidase 4 (DPP4).Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike,which mediates this interaction.We report the 3.0 (A)resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4.Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain.The receptor-binding subdomain interacts with DPP4 p-propeller but not its intrinsic hydrolase domain.MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains,but are notably divergent in the receptorbinding subdomain.Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell.The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction,which can guide development of therapeutics and vaccines against MERS-CoV infection.

  8. Eventos pós-receptor na neurotoxicidade do glutamato: Contribuição da subunidade GluR4 dos receptores AMPA para a activação do factor de transcrição AP-1

    OpenAIRE

    Santos, Armanda Emanuela Castro e

    2003-01-01

    Tese de doutoramento em Farmácia (Bioquímica) apresentada à Fac. de Farmácia da Univ. de Coimbra O objectivo deste estudo foi identificar mecanismos intracelulares envolvidos na resposta excitotóxica, e investigar se a sobreestimulação de receptores ionotrópicos do glutamato diferentes pode activar vias de sinalização intracelular distintas. A estimulação dos neurónios da retina de pinto em cultura com glutamato ou cainato diminuiu a viabilidade celular. O cainato foi mais eficaz a causar ...

  9. Structural and functional diversity of native brain neuronal nicotinic receptors.

    Science.gov (United States)

    Gotti, Cecilia; Clementi, Francesco; Fornari, Alice; Gaimarri, Annalisa; Guiducci, Stefania; Manfredi, Irene; Moretti, Milena; Pedrazzi, Patrizia; Pucci, Luca; Zoli, Michele

    2009-10-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels present in the central and peripheral nervous systems, that are permeable to mono- and divalent cations. They share a common basic structure but their pharmacological and functional properties arise from the wide range of different subunit combinations making up distinctive subtypes. nAChRs are involved in many physiological functions in the central and peripheral nervous systems, and are the targets of the widely used drug of abuse nicotine. In addition to tobacco dependence, changes in their number and/or function are associated with neuropsychiatric disorders, ranging from epilepsy to dementia. Although some of the neural circuits involved in the acute and chronic effects of nicotine have been identified, much less is known about which native nAChR subtypes are involved in specific physiological functions and pathophysiological conditions. We briefly review some recent findings concerning the structure and function of native nAChRs, focusing on the subtypes identified in the mesostriatal and habenulo-interpeduncular pathways, two systems involved in nicotine reinforcement and withdrawal. We also discuss recent findings concerning the effect of chronic nicotine on the expression of native subtypes.

  10. Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

    DEFF Research Database (Denmark)

    Frølund, Bente; Greenwood, Jeremy R; Holm, Mai Marie

    2005-01-01

    and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors...

  11. Pengaruh Ampas Tebu sebagai Adsorbent pada Proses Pretreatment Minyak Jelantah terhadap Karakteristik Biodiesel

    Directory of Open Access Journals (Sweden)

    Ratno Ratno

    2013-09-01

    Full Text Available Telah dilakukan penelitian mengenai pengaruh ampas tebu pada proses pretreatment minyak jelantah terhadap karakteristik biodiesel. Proses pretreatment dilakukan sebelum minyak jelantah diolah menjadi biodiesel, yakni ampas tebu dengan ukuran partikel dan massa yang bervariasi direndam pada minyak tersebut selama 2 jam. Ukuran partikel ampas tebu yang digunakan adalah 80, 115, 170, dan 200 mesh, sedangkan massa ampas tebu divariasi untuk tiap ukuran partikel yaitu 25 gram, 37,5 gram, dan 50 gram. Penggunaan ampas tebu sebagai adsorbent dinilai cukup efektif menurunkan kadar asam lemak bebas (FFA minyak jelantah dengan penurunan terbesar 57,3% terjadi pada minyak jelantah yang telah mengalami pretreatment ampas tebu berukuran partikel 200 mesh sebanyak 50 gram. Biodiesel dibuat dengan mereaksikan minyak jelantah yang telah mengalami pretreatment ampas tebu dengan lauratan Methanol dan Kalium Hidroksida (KOH selama 1 jam pada suhu 55oC. Hasil karakterisasi  menunjukkan bahwa massa jenis, titik nyala, titik kabut, dan titik tuang biodiesel telah memenuhi standar SNI-04-7182-2006 kecuali sampel yang mengalami pretreatment dengan ampas tebu 80 mesh sebanyak 25 gram. Sedangkan viskositas kinematik terdapat 5 sampel yang memenuhi untuk standar yang sama.

  12. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  13. Orexin-A differentially modulates AMPA-preferring responses of ganglion cells and amacrine cells in rat retina.

    Science.gov (United States)

    Zheng, Chao; Deng, Qin-Qin; Liu, Lei-Lei; Wang, Meng-Ya; Zhang, Gong; Sheng, Wen-Long; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2015-06-01

    By activating their receptors (OX1R and OX2R) orexin-A/B regulate wake/sleeping states, feeding behaviors, but the function of these peptides in the retina remains unknown. Using patch-clamp recordings and calcium imaging in rat isolated retinal cells, we demonstrated that orexin-A suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-preferring receptor-mediated currents (AMPA-preferring currents) in ganglion cells (GCs) through OX1R, but potentiated those in amacrine cells (ACs) through OX2R. Consistently, in rat retinal slices orexin-A suppressed light-evoked AMPA-preferring receptor-mediated excitatory postsynaptic currents in GCs, but potentiated those in ACs. Intracellular dialysis of GDP-β-S or preincubation with the Gi/o inhibitor pertussis toxin (PTX) abolished both the effects. Either cAMP/the protein kinase A (PKA) inhibitor Rp-cAMP or cGMP/the PKG blocker KT5823 failed to alter the orexin-A effects. Whilst both of them involved activation of protein kinase C (PKC), the effects on GCs and ACs were respectively eliminated by the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor and phosphatidylcholine (PC)-PLC inhibitor. Moreover, in GCs orexin-A increased [Ca(2+)]i and the orexin-A effect was blocked by intracellular Ca(2+)-free solution and by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. In contrast, orexin-A did not change [Ca(2+)]i in ACs and the orexin-A effect remained in intracellular or extracellular Ca(2+)-free solution. We conclude that a distinct Gi/o/PI-PLC/IP3/Ca(2+)-dependent PKC signaling pathway, following the activation of OX1R, is likely responsible for the orexin-A effect on GCs, whereas a Gi/o/PC-PLC/Ca(2+)-independent PKC signaling pathway, following the activation of OX2R, mediates the orexin-A effect on ACs. These two actions of orexin-A, while working in concert, provide a characteristic way for modulating information processing in the inner retina.

  14. Structure-based receptor MIMICS targeted against bacterial superantigen toxins

    Science.gov (United States)

    Gupta, Goutam; Hong-Geller, Elizabeth; Shiflett, Patrick R.; Lehnert, Nancy M.

    2009-08-18

    The invention provides therapeutic compositions useful in the treatment of bacterial superantigen mediated conditions, such as Toxic Shock Syndrome. The compositions comprise genetically engineered bifunctional polypeptides containing a specific T-cell receptor binding domain and a specific MHC class II receptor binding domain, each targeting non-overlapping epitopes on a superantigen molecule against which they are designed. The anti-superantigen "receptor mimetics" or "chimeras" are rationally designed to recreate the modality of superantigen binding directly to both the TCR and the MHC-II receptor, and are capable of acting as decoys for superantigen binding, effectively out-competing the host T-cell and MHC-II receptors, the natural host receptors.

  15. Effect of brain-derived neurotrophic factor on activity-regulated cytoskeleton-associated protein gene expression in primary frontal cortical neurons. Comparison with NMDA and AMPA

    DEFF Research Database (Denmark)

    El-Sayed, Mona; Hofman-Bang, Jacob; Mikkelsen, Jens D

    2011-01-01

    The effect of brain-derived neurotrophic factor (BDNF) on activity-regulated cytoskeleton-associated protein (Arc) mRNA levels in primary neuronal cultures of rat frontal cortex was characterized pharmacologically and compared to the effect on expression of c-fos, bdnf, neuritin, cox-2 as examples...... of other immediate early genes. BDNF induced a very strong increase (around 100 fold) in Arc mRNA and the maximal effect seen at 25 ng/ml. The effect was dose-dependent with EC50 around 1.6 ng/ml. The time profile revealed a significant effect after 25 min. BDNF also increased levels of c-Fos, neuritin...... and BDNF mRNA, but not COX-2 mRNA. The pharmacological profile of NMDA and AMPA-induced arc gene expression in frontal cortical neurons was compared to BDNF. NMDA and AMPA increased Arc mRNA but their maximal effect did not exceed 20-fold. The effect of AMPA was completely blocked by the NMDA receptor...

  16. Structural basis and functions of abscisic acid receptors PYLs

    Science.gov (United States)

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  17. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    -type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational...

  18. Effects of the AMPA antagonist ZK 200775 on visual function: a randomized controlled trial.

    Directory of Open Access Journals (Sweden)

    Richard Bergholz

    Full Text Available BACKGROUND: ZK 200775 is an antagonist at the alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA receptor and had earned attention as a possible neuroprotective agent in cerebral ischemia. Probands receiving the agent within phase I trials reported on an alteration of visual perception. In this trial, the effects of ZK 200775 on the visual system were analyzed in detail. METHODOLOGY: In a randomised controlled trial we examined eyes and vision before and after the intravenous administration of two different doses of ZK 200775 and placebo. There were 3 groups of 6 probands each: Group 1 recieved 0.03 mg/kg/h, group 2 0.75 mg/kg/h of ZK 200775, the control group received 0.9% sodium chloride solution. Probands were healthy males aged between 57 and 69 years. The following methods were applied: clinical examination, visual acuity, ophthalmoscopy, colour vision, rod absolute threshold, central visual field, pattern-reversal visual evoked potentials (pVEP, ON-OFF and full-field electroretinogram (ERG. PRINCIPAL FINDINGS: No effect of ZK 200775 was seen on eye position or motility, stereopsis, pupillary function or central visual field testing. Visual acuity and dark vision deteriorated significantly in both treated groups. Color vision was most remarkably impaired. The dark-adapted ERG revealed a reduction of oscillatory potentials (OP and partly of the a- and b-wave, furthermore an alteration of b-wave morphology and an insignificantly elevated b/a-ratio. Cone-ERG modalities showed decreased amplitudes and delayed implicit times. In the ON-OFF ERG the ON-answer amplitudes increased whereas the peak times of the OFF-answer were reduced. The pattern VEP exhibited lower amplitudes and prolonged peak times. CONCLUSIONS: The AMPA receptor blockade led to a strong impairment of typical OFF-pathway functions like color vision and the cone ERG. On the other hand the ON-pathway as measured by dark vision and the scotopic ERG was affected as well

  19. The selective activation of the glutamate receptor GluR5 by ATPA is controlled by serine 741.

    Science.gov (United States)

    Nielsen, Mai Marie; Liljefors, Tommy; Krogsgaard-Larsen, Povl; Egebjerg, Jan

    2003-01-01

    Only a few agonists exhibit selectivity between the AMPA and the kainate subtypes of the glutamate receptor. The most commonly used kainate receptor preferring agonist, (S)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid [(S)-ATPA], is an (R,S)-2-amino-3-(5-methyl-3-hydroxy-4-isoxazolyl)propionic acid (AMPA) derivative in which the methyl group at the 5-position of the isoxazole ring has been replaced by a tert-butyl group. When characterized by the two-electrode voltage clamp method in Xenopus laevis oocytes, ATPA exhibits at least 50-fold higher potency on the kainate receptor subtype, GluR5, compared with the AMPA receptors. Through mutagenesis studies of GluR5 and the AMPA receptor subtype, GluR1, we demonstrate that this pronounced selectivity for ATPA can be ascribed to Ser741 in GluR5 and Met722 in GluR1. Examination of other aliphatic substitutions at the 5-position of the isoxazole ring revealed that (R,S)-2-amino-3-(5-isopropyl-3-hydroxy-4-isoxazolyl)propionic acid (isopropyl-AMPA) displayed a 6-fold higher potency for GluR5 than for GluR1, whereas the analogs, propyl-AMPA and isobutyl-AMPA, did not exhibit significantly different potencies. Our study suggests that the GluR5 selectivity was a result not only of steric interference between the bulky tert-butyl group in ATPA and the methionine (Met722) in GluR1 but also a serine-dependent stabilization of the active conformation of GluR5 induced by ATPA. The stabilization was agonist-dependent and observed only for ATPA and isopropyl-AMPA, not for other AMPA analogs with bulky substitutions at the 5-position of the isoxazole ring.

  20. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya (Stanford-MED); (Kyoto); (Gakushuin); (Kyushu)

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  1. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting......, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  2. Structure-function relationships for the interleukin 2 receptor system

    Directory of Open Access Journals (Sweden)

    Richard J. Robb

    1987-01-01

    Full Text Available Receptors for interleukin 2 (IL-2 esit in at least three forms which differ in their subunit compositio, their affinity for ligand and their ability to mediate a cellular reponse. Type I receptors occur following cellular acitivation and consist of the 55,000 m. w. glycoprotein Tac. These receptors bind IL-2 with a low affinity, do not internalize ligand and have not been definitively associated with any response. Type II receptors, on the other hand, conssit of one or more glycoproteins of 70,000 m. w. which have been termed "beta ([beta] chains." They bind IL-2 with an intermediate affinity and rapidly internalize the ligand. [Beta] proteins mediate many cellular IL-2-dependent reponses, including the short-term activation of natural killer cells and the induction of Tac protein expression. Type III receptors consist of a ternary complex of the Tac protein, the [beta] chain(s and IL-2. They are characterized by a paricularly high affinity for ligand association. Type III receptors also internalize ligand and mediate IL-2-dependent responses at low factor concentrations. The identification of two independent IL-2-binding molecules, Tac and [beta], thus provides the elusive molecular explanation for the differences in IL-2 receptor affinity and suggests the potential for selective therapeutic manipulation of IL-2 reponses.

  3. [Opioid receptors of the CNS: function, structure and distribution].

    Science.gov (United States)

    Slamberová, R

    2004-01-01

    Even though the alkaloids of opium, such as morphine and codeine, were isolated at the beginning of 19th century, the opioid receptors were not determined until 1970's. The discovery of endogenous opioid peptides, such as endorphins, enkephalins and dynorphins, has helped to differentiate between the specific opioid receptor subtypes, mu, delta and kappa, that are used up to now. Opioid receptors are distributed in the central nervous system unevenly. Each receptor subtype has its own specific and nonspecific agonists and antagonists. Opioides, as exogenous opioid receptor agonists, are drugs that are often used in medicine for their analgesic effects, but they are also some of the most heavily abused drugs in the world. Opioides may also induce long-term changes in the numbers and binding activities of opioid receptors. Some of our studies in fact demonstrate that prenatal morphine exposure can alter opioid receptors of adult rats. This may begin to provide insight into the sources of some of the morphological and behavioral changes in the progeny of mothers that received or abused opioides during pregnancy.

  4. Muscarinic acetylcholine receptor subtypes: localization and structure/function

    DEFF Research Database (Denmark)

    Brann, M R; Ellis, J; Jørgensen, H

    1993-01-01

    Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues....... The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall...

  5. NMDA and non-NMDA receptor gene expression following global brain ischemia in rats: effect of NMDA and non-NMDA receptor antagonists.

    Science.gov (United States)

    Pellegrini-Giampietro, D E; Pulsinelli, W A; Zukin, R S

    1994-03-01

    Transient forebrain or global ischemia in rats induces selective and delayed damage of hippocampal CA1 neurons. In a previous study, we have shown that expression of GluR2, the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit that governs Ca2+ permeability, is preferentially reduced in CA1 at a time point preceding neuronal degeneration. Postischemic administration of the selective AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), protects CA1 neurons against delayed death. In this study we examined the effects of NBQX (at a neuroprotective dose) and of MK-801 (a selective NMDA receptor antagonist, not protective in this model) on kainate/AMPA receptor gene expression changes after global ischemia. We also examined the effects of transient forebrain ischemia on expression of the NMDA receptor subunit NMDAR1. In ischemic rats treated with saline, GluR2 and GluR3 mRNAs were markedly reduced in CA1 but were unchanged in CA3 or dentate gyrus. GluR1 and NMDAR1 mRNAs were not significantly changed in any region examined. Administration of NBQX or MK-801 did not alter the ischemia-induced changes in kainate/AMPA receptor gene expression. These findings suggest that NBQX affords neuroprotection by a direct blockade of kainate/AMPA receptors, rather than by a modification of GluR2 expression changes.

  6. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype.

    Science.gov (United States)

    Ichiyama, Susumu; Oka, Yoshiaki; Haga, Kazuko; Kojima, Shuichi; Tateishi, Yukihiro; Shirakawa, Masahiro; Haga, Tatsuya

    2006-01-09

    We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

  7. The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles.

    Science.gov (United States)

    Kudryashev, Mikhail; Castaño-Díez, Daniel; Deluz, Cédric; Hassaine, Gherici; Grasso, Luigino; Graf-Meyer, Alexandra; Vogel, Horst; Stahlberg, Henning

    2016-01-01

    The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

  8. Positive Allosteric Modulators of 2-Amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic Acid Receptors Belonging to 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-pyridothiadiazine Dioxides and Diversely Chloro-Substituted 4-Cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides

    DEFF Research Database (Denmark)

    Francotte, Pierre; Nørholm, Ann-Beth; Deva, Taru;

    2014-01-01

    Two 4-ethyl-substituted pyridothiadiazine dioxides belonging to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor positive allosteric modulators were cocrystallized with the GluA2 ligand binding domain in order to decipher the impact of the position of the nitrogen atom...... on their binding mode at the AMPA receptors. The latter was found to be very similar to that of previously described benzothiadiazine-type AMPA receptor modulators. The affinity of the two compounds for the receptor was determined by isothermal titration calorimetry. Accordingly, the synthesis and biological...

  9. Selective Negative Allosteric Modulation Of Metabotropic Glutamate Receptors - A Structural Perspective of Ligands and Mutants

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Isberg, Vignir; Tehan, Benjamin G

    2015-01-01

    modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different m...

  10. Structure-activity relationship studies of argiotoxins

    DEFF Research Database (Denmark)

    Poulsen, Mette H; Lucas, Simon; Bach, Tinna B;

    2013-01-01

    Argiotoxin-636 (ArgTX-636), a natural product from the spider Argiope lobata, is a potent but nonselective open-channel blocker of ionotropic glutamate (iGlu) receptors. Here, three series of analogues were designed to exploit selectivity among iGlu receptors, taking advantage of a recently devel......, respectively. Thus, the first structure-activity relationship study of ArgTX-636 has been carried out and has provided lead compounds for probing the ion channel region of iGlu receptors....... developed solid-phase synthetic methodology for the synthesis of ArgTX-636 and analogues. Initially, the importance of secondary amino groups in the polyamine chain was studied by the synthesis of systematically modified ArgTX-636 analogues, which were evaluated for pharmacological activity at NMDA and AMPA...

  11. L-(TH)glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Monaghan, D.T.; Yao, D.; Cotman, C.W.

    1985-08-12

    The anatomical distribution of L-(TH)glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding of L-(TH)glutamate is accounted for by the presence of 3 distinct binding sites when measured in the absence of CaS , Cl and Na ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labelled by D-(TH)2-amino-5-phosphonopentanoate (D-(TH)AP5), (TH)kainate ((TH)KA) and (TH) -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((TH)AMPA) which are thought to be selective ligands for the N-methyl-D-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively. (Auth.). 29 refs.; 1 figure; 1 table.

  12. Structure of the murine constitutive androstane receptor complexed to androstenol: a molecular basis for inverse agonism

    Energy Technology Data Exchange (ETDEWEB)

    Shan, L.; Vincent, J.; Brunzelle, J.S.; Dussault, I.; Lin, M.; Ianculescu, I.; Sherman, M.A.; Forman, B.M.; Fernandez, E. (Tennesse)

    2010-03-08

    The nuclear receptor CAR is a xenobiotic responsive transcription factor that plays a central role in the clearance of drugs and bilirubin while promoting cocaine and acetaminophen toxicity. In addition, CAR has established a 'reverse' paradigm of nuclear receptor action where the receptor is active in the absence of ligand and inactive when bound to inverse agonists. We now report the crystal structure of murine CAR bound to the inverse agonist androstenol. Androstenol binds within the ligand binding pocket, but unlike many nuclear receptor ligands, it makes no contacts with helix H12/AF2. The transition from constitutive to basal activity (androstenol bound) appears to be associated with a ligand-induced kink between helices H10 and H11. This disrupts the previously predicted salt bridge that locks H12 in the transcriptionally active conformation. This mechanism of inverse agonism is distinct from traditional nuclear receptor antagonists thereby offering a new approach to receptor modulation.

  13. Cardiac nuclear receptors: architects of mitochondrial structure and function.

    Science.gov (United States)

    Vega, Rick B; Kelly, Daniel P

    2017-04-03

    The adult heart is uniquely designed and equipped to provide a continuous supply of energy in the form of ATP to support persistent contractile function. This high-capacity energy transduction system is the result of a remarkable surge in mitochondrial biogenesis and maturation during the fetal-to-adult transition in cardiac development. Substantial evidence indicates that nuclear receptor signaling is integral to dynamic changes in the cardiac mitochondrial phenotype in response to developmental cues, in response to diverse postnatal physiologic conditions, and in disease states such as heart failure. A subset of cardiac-enriched nuclear receptors serve to match mitochondrial fuel preferences and capacity for ATP production with changing energy demands of the heart. In this Review, we describe the role of specific nuclear receptors and their coregulators in the dynamic control of mitochondrial biogenesis and energy metabolism in the normal and diseased heart.

  14. Structural Insights into the Interactions between Platelet Receptors and Fibrillar Collagen*

    OpenAIRE

    Herr, Andrew B.; Farndale, Richard W.

    2009-01-01

    Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin α2β1, glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of ...

  15. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Rosenbaum, Daniel M;

    2007-01-01

    Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which...

  16. Glyphosate and AMPA in the estuaries of the Baltic Sea method optimization and field study.

    Science.gov (United States)

    Skeff, Wael; Neumann, Christine; Schulz-Bull, Detlef E

    2015-11-15

    Water samples from ten German Baltic estuaries were collected in 2012 in order to study the presence of the herbicide glyphosate, its primary metabolite AMPA and their potential transport to the marine environment. For the analyses an LC-MS/MS based analytical method after derivatization with FMOC-Cl was optimized and validated for marine water samples. All investigated estuarine stations were contaminated with AMPA and nine of them also with glyphosate. Concentration ranges observed were 28 to 1690ng/L and 45 to 4156ng/L for glyphosate and AMPA, respectively with strong spatial and temporal fluctuations. Both contaminants were found at inbound sampling sites in the stream Muehlenfliess and concentrations decreased along the salinity gradient to the estuaries of the Baltic Sea. The data obtained in this study clearly depict the transport of glyphosate and AMPA to the Baltic Sea. Hence, detailed fate and risk assessment for both contaminants in marine environments are required.

  17. Monitoring glyphosate and AMPA concentrations in wells and drains using the sorbicell passive sampler

    DEFF Research Database (Denmark)

    Vendelboe, Anders Lindblad; de Jonge, Hubert; Møldrup, Per

    2012-01-01

    Glyphosate is one of the world’s most extensively used weed control agents. Glyphosate, and its metabolite aminomethylphosphonic acid (AMPA), are suspected to be hazardous to human health and the aquatic environment. In Denmark, the extensive use has resulted in an increasing number of occurrences......Cell, will decrease the workload and number of samples freeing up funds for larger monitoring programs. When installed in a well the SorbiCell will continuously sample the water giving either a flux-weighed or time-weighted average measurement of the glyphosate/AMPA concentration throughout the sampling period....... It may therefore be possible to measure lower concentrations as the glyphosate/AMPA sorbed in the SorbiCell is an accumulated measurement. Also, glyphosate/AMPA associated with sudden flush events will be detected by the SorbiCells, while such events may pass between two consecutive grab samples...

  18. Novel Functional Properties of Drosophila CNS Glutamate Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yan; Dharkar, Poorva; Han, Tae-Hee; Serpe, Mihaela; Lee, Chi-Hon; Mayer, Mark L.

    2016-12-01

    Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation.

  19. Illuminating the structure and function of Cys-loop receptors

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2008-01-01

    Cys-loop receptors are an important class of ligand-gated ion channels. They mediate fast synaptic neurotransmission, are implicated in various 'channelopathies' and are important pharmacological targets. Recent progress in X-ray crystallography and electron microscopy has provided a considerable...

  20. Structure-based drug design for G protein-coupled receptors.

    Science.gov (United States)

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

  1. Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4*

    Science.gov (United States)

    Butcher, Adrian J.; Hudson, Brian D.; Shimpukade, Bharat; Alvarez-Curto, Elisa; Prihandoko, Rudi; Ulven, Trond; Milligan, Graeme; Tobin, Andrew B.

    2014-01-01

    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr347, Thr349, Ser350, Ser357, and Ser360) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu341, Asp348, and Asp355 located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling. PMID:24817122

  2. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    Science.gov (United States)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, Wout; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-10-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

  3. Structural Characterization of the Hemagglutinin Receptor Specificity from the 2009 H1N1 Influenza Pandemic

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Rui; McBride, Ryan; Nycholat, Corwin M.; Paulson, James C.; Wilson, Ian A. (Scripps)

    2012-02-13

    Influenza virus hemagglutinin (HA) is the viral envelope protein that mediates viral attachment to host cells and elicits membrane fusion. The HA receptor-binding specificity is a key determinant for the host range and transmissibility of influenza viruses. In human pandemics of the 20th century, the HA normally has acquired specificity for human-like receptors before widespread infection. Crystal structures of the H1 HA from the 2009 human pandemic (A/California/04/2009 [CA04]) in complex with human and avian receptor analogs reveal conserved recognition of the terminal sialic acid of the glycan ligands. However, favorable interactions beyond the sialic acid are found only for {alpha}2-6-linked glycans and are mediated by Asp190 and Asp225, which hydrogen bond with Gal-2 and GlcNAc-3. For {alpha}2-3-linked glycan receptors, no specific interactions beyond the terminal sialic acid are observed. Our structural and glycan microarray analyses, in the context of other high-resolution HA structures with {alpha}2-6- and {alpha}2-3-linked glycans, now elucidate the structural basis of receptor-binding specificity for H1 HAs in human and avian viruses and provide a structural explanation for the preference for {alpha}2-6 siaylated glycan receptors for the 2009 pandemic swine flu virus.

  4. Differential effects of glyphosate and aminomethylphosphonic acid (AMPA) on photosynthesis and chlorophyll metabolism in willow plants.

    Science.gov (United States)

    Gomes, Marcelo Pedrosa; Le Manac'h, Sarah Gingras; Maccario, Sophie; Labrecque, Michel; Lucotte, Marc; Juneau, Philippe

    2016-06-01

    We used a willow species (Salix miyabeana cultivar SX64) to examine the differential secondary-effects of glyphosate and aminomethylphosphonic acid (AMPA), the principal glyphosate by-product, on chlorophyll metabolism and photosynthesis. Willow plants were treated with different concentrations of glyphosate (equivalent to 0, 1.4, 2.1 and 2.8kgha(-1)) and AMPA (equivalent to 0, 0.28, 1.4 and 2.8kgha(-1)) and evaluations of pigment contents, chlorophyll fluorescence, and oxidative stress markers (hydrogen peroxide content and antioxidant enzyme activities) in leaves were performed after 12h of exposure. We observed that AMPA and glyphosate trigger different mechanisms leading to decreases in chlorophyll content and photosynthesis rates in willow plants. Both chemicals induced ROS accumulation in willow leaves although only glyphosate-induced oxidative damage through lipid peroxidation. By disturbing chlorophyll biosynthesis, AMPA induced decreases in chlorophyll contents, with consequent effects on photosynthesis. With glyphosate, ROS increases were higher than the ROS-sensitive threshold, provoking chlorophyll degradation (as seen by pheophytin accumulation) and invariable decreases in photosynthesis. Peroxide accumulation in both AMPA and glyphosate-treated plants was due to the inhibition of antioxidant enzyme activities. The different effects of glyphosate on chlorophyll contents and photosynthesis as described in the literature may be due to various glyphosate:AMPA ratios in those plants.

  5. Ceftriaxone attenuates cocaine relapse after abstinence through modulation of nucleus accumbens AMPA subunit expression.

    Science.gov (United States)

    LaCrosse, Amber L; Hill, Kristine; Knackstedt, Lori A

    2016-02-01

    Using the extinction-reinstatement model of cocaine relapse, we and others have demonstrated that the antibiotic ceftriaxone attenuates cue- and cocaine-primed reinstatement of cocaine-seeking. Reinstatement is contingent on the release of glutamate in the nucleus accumbens core (NAc) and manipulations that reduce glutamate efflux or block post-synaptic glutamate receptors attenuate reinstatement. We have demonstrated that the mechanism of action by which ceftriaxone attenuates reinstatement involves increased NAc GLT-1 expression and a reduction in NAc glutamate efflux during reinstatement. Here we investigated the effects of ceftriaxone (100 and 200 mg/kg) on context-primed relapse following abstinence without extinction training and examined the effects of ceftriaxone on GluA1, GluA2 and GLT-1 expression. We conducted microdialysis during relapse to determine if an increase in NAc glutamate accompanies relapse after abstinence and whether ceftriaxone blunts glutamate efflux. We found that both doses of ceftriaxone attenuated relapse. While relapse was accompanied by an increase in NAc glutamate, ceftriaxone (200 mg/kg) was unable to significantly reduce NAc glutamate efflux during relapse despite its ability to upregulate GLT-1. GluA1 was reduced in the NAc by both doses of ceftriaxone while GluA2 expression was unchanged, indicating that ceftriaxone altered AMPA subunit composition following cocaine. Finally, GLT-1 was not altered in the PFC by ceftriaxone. These results indicate that it is possible to attenuate context-primed relapse to cocaine-seeking through modification of post-synaptic receptor properties without attenuating glutamate efflux during relapse. Furthermore, increasing NAc GLT-1 protein expression is not sufficient to attenuate glutamate efflux.

  6. Structural Disorder in the Complex of Human Pregnane X Receptor and the Macrolide Antibiotic Rifampicin

    Energy Technology Data Exchange (ETDEWEB)

    Chrencik, Jill E.; Orans, Jillian; Moore, Linda B.; Xue, Yu; Peng, Li; Collins, Jon L.; Wisely, G. Bruce; Lambert, Millard H.; Kliewer, Steven A.; Redinbo, Matthew R. (U. of Texas-SMED); (UNC)

    2010-07-13

    The human nuclear xenobiotic receptor, pregnane X receptor (PXR), detects a variety of structurally distinct endogenous and xenobiotic compounds and controls expression of genes central to drug and cholesterol metabolism. The macrolide antibiotic rifampicin, a front-line treatment for tuberculosis, is an established PXR agonist and, at 823 Da, is one of the largest known ligands for the receptor. We present the 2.8 {angstrom} crystal structure of the ligand-binding domain of human PXR in complex with rifampicin. We also use structural and mutagenesis data to examine the origins of the directed promiscuity exhibited by the PXRs across species. Three structurally flexible loops adjacent to the ligand-binding pocket of PXR are disordered in this crystal structure, including the 200-210 region that is part of a sequence insert novel to the promiscuous PXRs relative to other members of the nuclear receptor superfamily. The 4-methyl-1-piperazinyl ring of rifampicin, which would lie adjacent to the disordered protein regions, is also disordered and not observed in the structure. Taken together, our results indicate that one wall of the PXR ligand-binding cavity can remain flexible even when the receptor is in complex with an activating ligand. These observations highlight the key role that structural flexibility plays in PXR's promiscuous response to xenobiotics.

  7. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors

    Science.gov (United States)

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo

    2000-01-01

    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  8. Basic fibroblast growth factor increases the number of endogenous neural stem cells and inhibits the expression of amino methyl isoxazole propionic acid receptors in amyotrophic lateral sclerosis mice

    Institute of Scientific and Technical Information of China (English)

    Weihui Huang; Dawei Zang; Yi Lu; Ping Jiang

    2012-01-01

    This study aimed to investigate the number of amino methyl isoxazole propionic acid (AMPA) re-ceptors and production of endogenous neural stem cells in the SOD1G93AG1H transgenic mouse model of amyotrophic lateral sclerosis, at postnatal day 60 following administration of basic fibroblast growth factor (FGF-2). A radioligand binding assay and immunohistochemistry were used to estimate the number of AMPA receptors and endogenous neural stem cells respectively. Results showed that the number of AMPA receptors and endogenous neural stem cells in the brain stem and sensorimotor cortex were significantly increased, while motor function was significantly decreased at postnatal days 90 and 120. After administration of FGF-2 into mice, numbers of endogenous neural stem cells increased, while expression of AMPA receptors decreased, whilst motor functions were recovered. At postnatal day 120, the number of AMPA receptors was negatively correlated with the number of endogenous neural stem cells in model mice and FGF-2-treated mice. Our experimental findings indicate that FGF-2 can inhibit AMPA receptors and increase the number of endogenous neural stem cells, thus repairing neural injury in amyotrophic lateral sclerosis mice.

  9. Molecular dynamics simulation of the structure and dynamics of 5-HT3 serotonin receptor

    Science.gov (United States)

    Antonov, M. Yu.; Popinako, A. V.; Prokopiev, G. A.

    2016-10-01

    In this work, we investigated structure, dynamics and ion transportation in transmembrane domain of the 5-HT3 serotonin receptor. High-resolution (0.35 nm) structure of the 5-HT3 receptor in complex with stabilizing nanobodies was determined by protein crystallography in 2014 (Protein data bank (PDB) code 4PIR). Transmembrane domain of the structure was prepared in complex with explicit membrane environment (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)) and solvent (TIP3P water model). Molecular dynamics protocols for simulation and stabilization of the transmembrane domain of the 5-HT3 receptor model were developed and 60 ns simulation of the structure was conducted in order to explore structural parameters of the system. We estimated the mean force profile for Na+ ions using umbrella sampling method.

  10. Structural and Molecular Evidence Suggesting Coronavirus-driven Evolution of Mouse Receptor.

    Science.gov (United States)

    Peng, Guiqing; Yang, Yang; Pasquarella, Joseph R; Xu, Liqing; Qian, Zhaohui; Holmes, Kathryn V; Li, Fang

    2017-02-10

    Hosts and pathogens are locked in an evolutionary arms race. To infect mice, mouse hepatitis coronavirus (MHV) has evolved to recognize mouse CEACAM1a (mCEACAM1a) as its receptor. To elude MHV infections, mice may have evolved a variant allele from the Ceacam1a gene, called Ceacam1b, producing mCEACAM1b, which is a much poorer MHV receptor than mCEACAM1a. Previous studies showed that sequence differences between mCEACAM1a and mCEACAM1b in a critical MHV-binding CC' loop partially account for the low receptor activity of mCEACAM1b, but detailed structural and molecular mechanisms for the differential MHV receptor activities of mCEACAM1a and mCEACAM1b remained elusive. Here we have determined the crystal structure of mCEACAM1b and identified the structural differences and additional residue differences between mCEACAM1a and mCEACAM1b that affect MHV binding and entry. These differences include conformational alterations of the CC' loop as well as residue variations in other MHV-binding regions, including β-strands C' and C'' and loop C'C''. Using pseudovirus entry and protein-protein binding assays, we show that substituting the structural and residue features from mCEACAM1b into mCEACAM1a reduced the viral receptor activity of mCEACAM1a, whereas substituting the reverse changes from mCEACAM1a into mCEACAM1b increased the viral receptor activity of mCEACAM1b. These results elucidate the detailed molecular mechanism for how mice may have kept pace in the evolutionary arms race with MHV by undergoing structural and residue changes in the MHV receptor, providing insight into this possible example of pathogen-driven evolution of a host receptor protein.

  11. Structural basis for bitter taste receptor activation and its potential role in targeting diabetes

    Directory of Open Access Journals (Sweden)

    Ravinder Abrol

    2015-03-01

    Full Text Available Background: Taste receptors are G protein-coupled receptors that, besides being present in the taste buds, have also been shown to be present in the gastrointestinal (GI system, respiratory system, and brain, though their function at these locations is not well understood. Objective: To understand the nutrient mediated release of gut peptides like GLP-1 from enteroendocrine L-cells of the GI system, we focused on a bitter taste receptor TAS2R38 (based on animal models to investigate the structural basis of its potential role in the release of gut peptides. Methods: The atomic-level structure of bitter taste receptor TAS2R38 was predicted using GEnSeMBLE, a first-principle based GPCR structure prediction method. These structures were obtained for the dominant taster haplotype (PAV as well as for the nontaster haplotype (AVI of the receptor. The known ligands phenylthiocarbamide (PTC and 6-n-propylthiouracil (PTU were docked to these structures to provide a structural basis for the taster and nontaster haplotypes. Results: Docking of known ligands PTU and PTC to taster and nontaster haplotypes of the bitter taste receptor showed a backbone hydrogen bond to residue 262 in taster but not in nontaster haplotype, suggesting a potential mode of action of these molecules in the activation of the bitter taste receptor. Conclusion: These results, combined with the ability of PTC to release gut peptides from in vitro models of the enteroendocrine L-cells, suggest a potential structural basis for TAS2R38 activation that can lead to the release of those peptides. This release has a therapeutic benefit for type 2 diabetes and implies a role for bitter tasting (but safe natural compounds targeting TAS2R38 as potential drug candidates for curing type 2 diabetes.

  12. Glyphosate-Resistant and Conventional Canola (Brassica napus L.) Responses to Glyphosate and Aminomethylphosphonic Acid (AMPA) Treatment.

    Science.gov (United States)

    Corrêa, Elza Alves; Dayan, Franck E; Owens, Daniel K; Rimando, Agnes M; Duke, Stephen O

    2016-05-11

    Glyphosate-resistant (GR) canola contains two transgenes that impart resistance to the herbicide glyphosate: (1) the microbial glyphosate oxidase gene (gox) encoding the glyphosate oxidase enzyme (GOX) that metabolizes glyphosate to aminomethylphosphonic acid (AMPA) and (2) cp4 that encodes a GR form of the glyphosate target enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The objectives of this research were to determine the phytotoxicity of AMPA to canola, the relative metabolism of glyphosate to AMPA in GR and conventional non-GR (NGR) canola, and AMPA pool sizes in glyphosate-treated GR canola. AMPA applied at 1.0 kg ha(-1) was not phytotoxic to GR or NGR. At this AMPA application rate, NGR canola accumulated a higher concentration of AMPA in its tissues than GR canola. At rates of 1 and 3.33 kg ae ha(-1) of glyphosate, GR canola growth was stimulated. This stimulatory effect is similar to that of much lower doses of glyphosate on NGR canola. Both shikimate and AMPA accumulated in tissues of these glyphosate-treated plants. In a separate experiment in which young GR and NGR canola plants were treated with non-phytotoxic levels of [(14)C]-glyphosate, very little glyphosate was metabolized in NGR plants, whereas most of the glyphosate was metabolized to AMPA in GR plants at 7 days after application. Untreated leaves of GR plants accumulated only metabolites (mostly AMPA) of glyphosate, indicating that GOX activity is very high in the youngest leaves. These data indicate that more glyphosate is transformed to AMPA rapidly in GR canola and that the accumulated AMPA is not toxic to the canola plant.

  13. Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Qiuxiang; Zhu, Ya; Li, Jian; Chen, Zhuxi; Han, Gye Won; Kufareva, Irina; Li, Tingting; Ma, Limin; Fenalti, Gustavo; Li, Jing; Zhang, Wenru; Xie, Xin; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Liu, Hong; Stevens, Raymond C.; Zhao, Qiang; Wu, Beili [Scripps; (Chinese Aca. Sci.); (UCSD)

    2013-10-21

    The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom–resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor–gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

  14. Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface.

    Science.gov (United States)

    Saha, Nayanendu; Kolev, Momchil; Nikolov, Dimitar B

    2014-10-01

    Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such as the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind and signal through a single receptor/co-receptor complex that comprises of NgR1/LINGO-1 and either p75 or TROY, triggering intracellular downstream signaling that impedes the re-growth of axons. Structure-function analysis of myelin inhibitors and their neuronal receptors, particularly the NgRs, have provided novel information regarding the molecular details of the inhibitor/receptor/co-receptor interactions. Structural and biochemical studies have revealed the architecture of many of these proteins and identified the molecular regions important for assembly of the inhibitory signaling complexes. It was also recently shown that gangliosides, such as GT1b, mediate receptor/co-receptor binding. In this review, we highlight these studies and summarize our current understanding of the multi-protein cell-surface complexes mediating inhibitory signaling events at the neuron/myelin interface.

  15. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses.

    Science.gov (United States)

    Shi, Yi; Zhang, Wei; Wang, Fei; Qi, Jianxun; Wu, Ying; Song, Hao; Gao, Feng; Bi, Yuhai; Zhang, Yanfang; Fan, Zheng; Qin, Chengfeng; Sun, Honglei; Liu, Jinhua; Haywood, Joel; Liu, Wenjun; Gong, Weimin; Wang, Dayan; Shu, Yuelong; Wang, Yu; Yan, Jinghua; Gao, George F

    2013-10-11

    An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln(226)) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu(226)). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu(226) → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.

  16. Structure of adenovirus bound to cellular receptor car

    Science.gov (United States)

    Freimuth, Paul I.

    2007-01-02

    Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

  17. Paradoxical proepileptic response to NMDA receptor blockade linked to cortical interneuron defect in stargazer mice

    Directory of Open Access Journals (Sweden)

    Atul eMaheshwari

    2013-09-01

    Full Text Available Paradoxical seizure exacerbation by antiepileptic medication is a well-known clinical phenomenon in epilepsy, but the cellular mechanisms remain unclear. One possibility is enhanced network disinhibition by unintended suppression of inhibitory interneurons. We investigated this hypothesis in the stargazer mouse model of absence epilepsy, which bears a mutation in stargazin, an AMPA receptor trafficking protein. If AMPA signaling onto inhibitory GABAergic neurons is impaired, their activation by glutamate depends critically upon NMDA receptors. Indeed, we find that stargazer seizures are exacerbated by NMDA receptor blockade with CPP and MK-801, whereas other genetic absence epilepsy models are sensitive to these antagonists. To determine how an AMPA receptor trafficking defect could lead to paradoxical network activation, we analyzed stargazin and AMPA receptor localization and found that stargazin is detected exclusively in parvalbumin-positive (PV+ fast-spiking interneurons in somatosensory cortex, where it is co-expressed with the AMPA receptor subunit GluA4. PV+ cortical interneurons in stargazer show a near two-fold decrease in the dendrite:soma GluA4 expression ratio compared to wild type littermates. We explored the functional consequence of this trafficking defect on network excitability in neocortical slices. Both NMDA receptor antagonists suppressed 0 Mg2+ induced network discharges in wild type but augmented bursting in stargazer cortex. Interneurons mediate this paradoxical response, since the difference between genotypes was masked by GABA receptor blockade. Our findings provide a cellular locus for AMPA receptor-dependent signaling defects in stargazer cortex and define an interneuron-dependent mechanism for paradoxical seizure exacerbation in absence epilepsy.

  18. Effects of the blood components on the AMPA and NMDA synaptic responses in brain slices in the onset of hemorrhagic stroke.

    Science.gov (United States)

    Mokrushin, Anatoly A; Pavlinova, Larisa I

    2013-12-01

    Blood-borne events play a major role in post bleeding disturbances of the neuronal network. However, very little is known about the early effects of blood plasma, leucocytes, and the red blood cells on the AMPA and NMDA-mediated synaptic responses in the onset of experimental intracranial hemorrhage (ICH). In this study, we used the technique of on-line monitoring of electrophysiological parameters referred to synaptic activity in piriform cortex of SHR rat slice. We exposed the olfactory cortex slices to diluted autologous blood or its components and compared with effects of ferric chloride. Whole blood exerted a total inhibition of synaptic activity in piriform cortex within first 5 min. Dilution of blood induced prolonged epileptic synaptic activation of NMDA receptors. Blood plasma and fraction of leucocytes induced hyperactivation of neurons transforming to epileptiform discharges. Fraction of red blood cells acted biphasic, an initial sharp activity of AMPA- and NMDA-mediated receptors replaced by a following total depression. Our slice-based models of experimental stroke revealed the mechanism of the earliest pathophysiologic events occur in brain tissue during bleeding that may be relevant to the human ICH.

  19. Properties of GluR3 receptors tagged with GFP at the amino or carboxyl terminus

    Science.gov (United States)

    Limon, Agenor; Reyes-Ruiz, Jorge Mauricio; Eusebi, Fabrizio; Miledi, Ricardo

    2007-01-01

    Anatomical visualization of neurotransmitter receptor localization is facilitated by tagging receptors, but this process can alter their functional properties. We have evaluated the distribution and properties of WT glutamate receptor 3 (GluR3) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (WT GluR3) and two receptors in which GFP was tagged to the amino terminus (GFP-GluR3) or to the carboxyl terminus (GluR3-GFP). Although the fluorescence in Xenopus oocytes was stronger in the vegetal hemisphere because of localization of internal structures (probable sites of production, storage or recycling of receptors), the insertion of receptors into the plasma membrane was polarized to the animal hemisphere. The fluorescence intensity of oocytes injected with GluR3-GFP RNA was approximately double that of oocytes injected with GFP-GluR3 RNA. Accordingly, GluR3-GFP oocytes generated larger kainate-induced currents than GFP-GluR3 oocytes, with similar EC50 values. Currents elicited by glutamate, or AMPA coapplied with cyclothiazide, were also larger in GluR3-GFP oocytes. The glutamate- to kainate-current amplitude ratios differed, with GluR3-GFP being activated more efficiently by glutamate than the WT or GFP-GluR3 receptors. This pattern correlates with the slower decay of glutamate-induced currents generated by GluR3-GFP receptors. These changes were not observed when GFP was tagged to the amino terminus, and these receptors behaved like the WT. The antagonistic effects of 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were not altered in any of the tagged receptors. We conclude that GFP is a useful and convenient tag for visualizing these proteins. However, the effects of different sites of tag insertion on receptor characteristics must be taken into account in assessing the roles played by these receptor proteins. PMID:17881566

  20. Effect of Receptor Structure and Length on the Wrapping of a Nanoparticle by a Lipid Membrane

    Directory of Open Access Journals (Sweden)

    Haizhen Zhang

    2014-05-01

    Full Text Available Nanoparticles have been considered as a type of powerful tool to deliver drugs and genes into cells for disease diagnosis and therapies. It has been generally accepted that the internalization of nanoparticles into cells is mostly realized by receptor-mediated endocytosis. However, for the influence of structural factors of receptors on endocytosis, this is still largely unknown. In this paper, computer simulations are applied to investigate the effects of structure (i.e., the number of constituent chains of the receptor and the length of the receptor on the wrapping behavior of nanoparticles by the lipid membrane, which is a key step of receptor-medicated endocytosis. It is found that these structural factors of receptors have strong effects on the nanoparticle’s final interaction configuration with the membrane in the simulations, such as adhering on the membrane surface or being partly or fully wrapped by the membrane. Furthermore, in some cases, the rupture of the lipid membrane occurs. These results are helpful for the understanding of endocytosis and the preparation of advanced nanoscale drug-delivery vectors.

  1. Structural Basis for Antibody Discrimination between Two Hormones That Recognize the Parathyroid Hormone Receptor

    Energy Technology Data Exchange (ETDEWEB)

    McKinstry, William J.; Polekhina, Galina; Diefenbach-Jagger, Hannelore; Ho, Patricia W.M.; Sato, Koh; Onuma, Etsuro; Gillespie, Matthew T.; Martin, T. John; Parker, Michael W.; (SVIMR-A); (Chugai); (Melbourne)

    2009-08-18

    Parathyroid hormone-related protein (PTHrP) plays a vital role in the embryonic development of the skeleton and other tissues. When it is produced in excess by cancers it can cause hypercalcemia, and its local production by breast cancer cells has been implicated in the pathogenesis of bone metastasis formation in that disease. Antibodies have been developed that neutralize the action of PTHrP through its receptor, parathyroid hormone receptor 1, without influencing parathyroid hormone action through the same receptor. Such neutralizing antibodies against PTHrP are therapeutically effective in animal models of the humoral hypercalcemia of malignancy and of bone metastasis formation. We have determined the crystal structure of the complex between PTHrP (residues 1-108) and a neutralizing monoclonal anti-PTHrP antibody that reveals the only point of contact is an {alpha}-helical structure extending from residues 14-29. Another striking feature is that the same residues that interact with the antibody also interact with parathyroid hormone receptor 1, showing that the antibody and the receptor binding site on the hormone closely overlap. The structure explains how the antibody discriminates between the two hormones and provides information that could be used in the development of novel agonists and antagonists of their common receptor.

  2. The genomic structure of the human UFO receptor.

    Science.gov (United States)

    Schulz, A S; Schleithoff, L; Faust, M; Bartram, C R; Janssen, J W

    1993-02-01

    Using a DNA transfection-tumorigenicity assay we have recently identified the UFO oncogene. It encodes a tyrosine kinase receptor characterized by the juxtaposition of two immunoglobulin-like and two fibronectin type III repeats in its extracellular domain. Here we describe the genomic organization of the human UFO locus. The UFO receptor is encoded by 20 exons that are distributed over a region of 44 kb. Different isoforms of UFO mRNA are generated by alternative splicing of exon 10 and differential usage of two imperfect polyadenylation sites resulting in the presence or absence of 1.5-kb 3' untranslated sequences. Primer extension and S1 nuclease analyses revealed multiple transcriptional initiation sites including a major site 169 bp upstream of the translation start site. The promoter region is GC rich, lacks TATA and CAAT boxes, but contains potential recognition sites for a variety of trans-acting factors, including Sp1, AP-2 and the cyclic AMP response element-binding protein. Proto-UFO and its oncogenic counterpart exhibit identical cDNA and promoter regions sequences. Possible modes of UFO activation are discussed.

  3. Glyphosate and AMPA distribution in wind-eroded sediment derived from loess soil.

    Science.gov (United States)

    Bento, Célia P M; Goossens, Dirk; Rezaei, Mahrooz; Riksen, Michel; Mol, Hans G J; Ritsema, Coen J; Geissen, Violette

    2017-01-01

    Glyphosate is one of the most used herbicides in agricultural lands worldwide. Wind-eroded sediment and dust, as an environmental transport pathway of glyphosate and of its main metabolite aminomethylphosphonic acid (AMPA), can result in environmental- and human exposure far beyond the agricultural areas where it has been applied. Therefore, special attention is required to the airborne transport of glyphosate and AMPA. In this study, we investigated the behavior of glyphosate and AMPA in wind-eroded sediment by measuring their content in different size fractions (median diameters between 715 and 8 μm) of a loess soil, during a period of 28 days after glyphosate application. Granulometrical extraction was done using a wind tunnel and a Soil Fine Particle Extractor. Extractions were conducted on days 0, 3, 7, 14, 21 and 28 after glyphosate application. Results indicated that glyphosate and AMPA contents were significantly higher in the finest particle fractions (median diameters between 8 and 18 μm), and lowered significantly with the increase in particle size. However, their content remained constant when aggregates were present in the sample. Glyphosate and AMPA contents correlated positively with clay, organic matter, and silt content. The dissipation of glyphosate over time was very low, which was most probably due to the low soil moisture content of the sediment. Consequently, the formation of AMPA was also very low. The low dissipation of glyphosate in our study indicates that the risk of glyphosate transport in dry sediment to off-target areas by wind can be very high. The highest glyphosate and AMPA contents were found in the smallest soil fractions (PM10 and less), which are easily inhaled and, therefore, contribute to human exposure.

  4. Structure-Function Similarities between a Plant Receptor-like Kinase and the Human Interleukin-1 Receptor-associated Kinase-4*

    OpenAIRE

    2011-01-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology model...

  5. Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor.

    Science.gov (United States)

    Streltsov, V A; Varghese, J N; Carmichael, J A; Irving, R A; Hudson, P J; Nuttall, S D

    2004-08-24

    The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-A structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the "bottom" of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended beta-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

  6. Structural Basis of Intracellular TGF-β Signaling: Receptors and Smads.

    Science.gov (United States)

    Chaikuad, Apirat; Bullock, Alex N

    2016-11-01

    Stimulation of the transforming growth factor β (TGF-β) family receptors activates an intracellular phosphorylation-dependent signaling cascade that culminates in Smad transcriptional activation and turnover. Structural studies have identified a number of allosteric mechanisms that control the localization, conformation, and oligomeric state of the receptors and Smads. Such mechanisms dictate the ordered binding of substrate and adaptor proteins that determine the directionality of the signaling process. Activation of the pathway has been illustrated by the various structures of the receptor-activated Smads (R-Smads) with SARA, Smad4, and YAP, respectively, whereas mechanisms of down-regulation have been elucidated by the structural complexes of FKBP12, Ski, and Smurf1. Interesting parallels have emerged between the R-Smads and the Forkhead-associated (FHA) and interferon regulatory factor (IRF)-associated domains, as well as the Hippo pathway. However, important questions remain as to the mechanism of Smad-independent signaling.

  7. Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM.

    Science.gov (United States)

    Hashiguchi, Takao; Ose, Toyoyuki; Kubota, Marie; Maita, Nobuo; Kamishikiryo, Jun; Maenaka, Katsumi; Yanagi, Yusuke

    2011-02-01

    Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a β-sheet of the membrane-distal ectodomain of SLAM using the side of its β-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their β-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.

  8. Structural mechanism of ligand activation in human calcium-sensing receptor

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

    2016-07-19

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+and PO43-ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ions stabilize the active state, PO43-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

  9. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

  10. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Aaron A.; Liu, Wei; Chun, Eugene; Katritch, Vsevolod; Wu, Huixian; Vardy, Eyal; Huang, Xi-Ping; Trapella, Claudio; Guerrini, Remo; Calo, Girolamo; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (Ferrara); (Scripps); (UNC)

    2012-07-11

    Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, {delta}, {kappa} and {mu} ({delta}-OR, {kappa}-OR and {mu}-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes ({approx}60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors {kappa} (ref. 5) and {mu} (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

  11. Hepatic structural enhancement and insulin resistance amelioration due to AT1 receptor blockade.

    Science.gov (United States)

    Souza-Mello, Vanessa

    2017-01-18

    Over the last decade, the role of renin-angiotensin system (RAS) on the development of obesity and its comorbidities has been extensively addressed. Both circulating and local RAS components are up-regulated in obesity and involved in non-alcoholic fatty liver disease onset. Pharmacological manipulations of RAS are viable strategies to tackle metabolic impairments caused by the excessive body fat mass. Renin inhibitors rescue insulin resistance, but do not have marked effects on hepatic steatosis. However, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (ARB) yield beneficial hepatic remodeling. ARBs elicit body mass loss and normalize insulin levels, tackling insulin resistance. Also, this drug class increases adiponectin levels, besides countering interleukin-6, tumoral necrosis factor-alpha, and transforming growth factor-beta 1. The latter is essential to prevent from liver fibrosis. When conjugated with peroxisome proliferator-activated receptor (PPAR)-alpha activation, ARB fully rescues fatty liver. These effects might be orchestrated by an indirect up-regulation of MAS receptor due to angiotensin II receptor type 1 (AT1R) blockade. These associations of ARB with PPAR activation and ACE2-angiotensin (ANG) (1-7)-MAS receptor axis deserve a better understanding. This editorial provides a brief overview of the current knowledge regarding AT1R blockade effects on sensitivity to insulin and hepatic structural alterations as well as the intersections of AT1R blockade with peroxisome proliferator-activated receptor activation and ACE2-ANG (1-7) - MAS receptor axis.

  12. The effects of AMPA blockade on the spectral profile of human early visual cortex recordings studied with non-invasive MEG.

    Science.gov (United States)

    Muthukumaraswamy, Suresh D; Routley, Bethany; Droog, Wouter; Singh, Krish D; Hamandi, Khalid

    2016-08-01

    The generation of gamma-band (>30 Hz) cortical activity is thought to depend on the reciprocal connections of excitatory glutamatergic principal cells with inhibitory GABAergic interneurons. Both in vitro and in vivo animal studies have shown that blockade of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reduces the amplitude of gamma-band activity. In this registered report, we hypothesised that similar effects would be observed in humans following administration of perampanel, a first in class AMPA antagonist, used in the treatment of epilepsy. In a single-blind placebo-controlled crossover study, 20 healthy male participants completed two study days. On one day participants were given a 6 mg dose of perampanel and on the other an inactive placebo. magnetoencephalography (MEG) recordings of brain activity were taken before and two hours after drug administration, with activity in the visual cortex probed using a stimulation protocol known to induce gamma-band activity in the primary visual cortex. As hypothesised, our results indicated a decrease in gamma-band amplitudes following perampanel administration. The decreases in gamma-band amplitudes observed were temporally restricted to the early time-period of stimulus presentation (up to 400 msec) with no significant effects observed on early evoked responses or alpha rhythms. This suggests that the early time-window of induced visual gamma-band activity, thought to reflect input to the visual cortex from the lateral geniculate nucleus, is most sensitive to AMPA blocking drugs.

  13. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A

    2007-01-01

    crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context...... of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting...

  14. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization.

    Science.gov (United States)

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam

    2014-02-01

    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  15. Transfer of glyphosate and its degradate AMPA to surface waters through urban sewerage systems.

    Science.gov (United States)

    Botta, Fabrizio; Lavison, Gwenaëlle; Couturier, Guillaume; Alliot, Fabrice; Moreau-Guigon, Elodie; Fauchon, Nils; Guery, Bénédicte; Chevreuil, Marc; Blanchoud, Hélène

    2009-09-01

    A study of glyphosate and aminomethyl phosphonic acid (AMPA) transfer in the Orge watershed (France) was carried out during 2007 and 2008. Water samples were collected in surface water, wastewater sewer, storm sewer and wastewater treatment plant (WWTP). These two molecules appeared to be the most frequently detected ones in the rivers and usually exceeded the European quality standard concentrations of 0.1microg L(-1) for drinking water. The annual glyphosate estimated load was 1.9 kg year(-1) upstream (agricultural zone) and 179.5 kg year(-1) at the catchment outlet (urban zone). This result suggests that the contamination of this basin by glyphosate is essentially from urban origin (road and railway applications). Glyphosate reached surface water prevalently through storm sewer during rainfall event. Maximum concentrations were detected in storm sewer just after a rainfall event (75-90 microg L(-1)). High concentrations of glyphosate in surface water during rainfall events reflected urban runoff impact. AMPA was always detected in the sewerage system. This molecule reached surface water mainly via WWTP effluent and also through storm sewer. Variations in concentrations of AMPA during hydrological episodes were minor compared to glyphosate variations. Our study highlights that AMPA and glyphosate origins in urban area are different. During dry period, detergent degradation seemed to be the major AMPA source in wastewater.

  16. The second PGD(2) receptor CRTH2: structure, properties, and functions in leukocytes.

    Science.gov (United States)

    Nagata, Kinya; Hirai, Hiroyuki

    2003-01-01

    Prostaglandin (PG) D(2) plays a broad range of physiological and pathophysiological functions. Until just a few years ago, it was thought that most of the biological actions of PGD(2) are mediated via the classical PGD(2) receptor DP. Recently, we identified a second PGD(2) receptor, chemoattractant receptor-homologous molecule expressed on T helper (Th)2 cells (CRTH2), with different functions relative to DP. Here, we review the recent findings on the structure, tissue distribution, ligand selectivity, signalling pathways, and functions in leukocytes of this receptor. The data suggest that the PGD(2)/CRTH2 system play important roles in allergic inflammation through its stimulatory effects on Th2 cells, eosinophils, and basophils.

  17. Structure-function Aspects of Extracellular Leucine-rich Repeat-containing Cell Surface Receptors in Plants

    Institute of Scientific and Technical Information of China (English)

    Zhao Zhang; Bart PHJ Thomma

    2013-01-01

    Plants exploit several types of cell surface receptors for perception of extracellular signals, of which the extracellular leucine-rich repeat (eLRR)-containing receptors form the major class. Although the function of most plant eLRR receptors remains unclear, an increasing number of these receptors are shown to play roles in innate immunity and a wide variety of developmental processes. Recent efforts using domain swaps, gene shuffling analyses, site-directed mutagenesis, interaction studies, and crystallographic analyses resulted in the current knowledge on ligand binding and the mechanism of activation of plant eLRR receptors. This review provides an overview of eLRR receptor research, specifically summarizing the recent understanding of interactions among plant eLRR receptors, their co-receptors and corresponding ligands. The functions of distinct eLRR receptor domains, and their role in structure, ligand perception and multimeric complex formation are discussed.

  18. From G Protein-coupled Receptor Structure Resolution to Rational Drug Design.

    Science.gov (United States)

    Jazayeri, Ali; Dias, Joao M; Marshall, Fiona H

    2015-08-07

    A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.

  19. Structure and mechanism for recognition of peptidehormones by Class B G-protein-coupled receptors

    Institute of Scientific and Technical Information of China (English)

    Kuntal PAL; Karsten MELCHER; H Eric XU

    2012-01-01

    Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon,parathyroid hormone,and calcitonin.These receptors are involved in a wide spectrum of physiological activities,from metabolic regulation and stress control to development and maintenance of the skeletal system.As such,they are important drug targets for the treatment of diabetes,osteo-porosis,and stress related disorders.Class B GPCRs are organized into two modular domains:an extracellular domain (ECD) and ahelical bundle that contains seven transmembrane helices (TM domain).The ECD is responsible for the high affinity and specificity of hormone binding,and the TM domain is required for receptor activation and signal coupling to downstream G-proteins.Although the structure of the full-length receptor remains unknown,the ECD structures have been well characterized for a number of Class BGPCRs,revealing a common fold for ligand recognition.This review summarizes the general structural principles that guide hormone binding by Class B ECDs and their implications in the design of peptide hormone analogs for therapeutic purposes.

  20. Scavenging ROS dramatically increase NMDA receptor whole-cell currents in painted turtle cortical neurons.

    Science.gov (United States)

    Dukoff, David James; Hogg, David William; Hawrysh, Peter John; Buck, Leslie Thomas

    2014-09-15

    Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle, which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation, which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents, we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca(2+)]i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca(2+) release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca(2+)]i and ROS levels demonstrated that [Ca(2+)]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA/AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca(2+) release.

  1. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel , Tracy M. (Vertex Pharm); (Leiden-MC); (USC); (BMS); (UCSD)

    2016-12-07

    CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

  2. Structural variation and uniformity among tetraloop-receptor interactions and other loop-helix interactions in RNA crystal structures.

    Directory of Open Access Journals (Sweden)

    Li Wu

    Full Text Available Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48 were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the "standard" GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature.

  3. Structural combination of established 5-HT(2A) receptor ligands: new aspects of the binding mode

    DEFF Research Database (Denmark)

    Kramer, Vasko; Herth, Matthias M; Santini, Martin A;

    2010-01-01

    MH.MZ, MDL 100907, and altanserin are structurally similar 4-benzoyl-piperidine derivatives and are well accommodated to receptor interaction models. We combined structural elements of different high-affinity and selective 5-HT(2A) antagonists, as MH.MZ, altanserin, and SR 46349B, to improve......) with a moderate affinity toward the 5-HT(2A) receptor (K(i) = 57 nm). The remarkably reduced affinity of other compounds (4a), (4b), and (4c) (K(i) = 411, 360 and 356 nm respectively) indicates that MH.MZ can only bind to the 5-HT(2A) receptor with the p-fluorophenylethyl residue in a sterically restricted...

  4. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian;

    2015-01-01

    We have developed a new method for the building of pharmacophores for G protein-coupled receptors, a major drug target family. The method is a combination of the ligand- and target-based pharmacophore methods and founded on the extraction of structural fragments, interacting ligand moiety...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... or ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...

  5. Pengaruh Waktu Perendaman Ampas Tebu Sebagai Biomaterial Adsorbent Pada Proses Pretreatment Terhadap Karakteristik Biodiesel Minyak Jelantah

    Directory of Open Access Journals (Sweden)

    Farid Widi Arfika

    2013-09-01

    Full Text Available Tingginya viskositas minyak jelantah dipengaruhi oleh kandungan asam lemak bebas (FFA yang diakibatkan oleh pemanasan berulang-ulang saat menggoreng. Oleh karena itu perlu diadakan pretreatment terhadap minyak jelantah sebelum diproses menjadi biodiesel agar kandungan FFA nya dapat diturunkan. Salah satu cara untuk menurunkan FFA pada minyak jelantah adalah dengan merendam ampas tebu. Pada tugas akhir ini dilakukan penelitian tentang pengaruh waktu perendaman ampas tebu (baggase sebagai biomaterial adsorbent FFA pada minyak jelantah. Ampas tebu berupa serbuk berukuran 65 mesh seberat 25 gr direndamkan ke dalam minyak jelantah sebanyak 500 ml dengan waktu yang bervariasi, mulai dari 2 jam hingga 15 hari. Hasil yang diperoleh berupa penurunan nilai FFA minyak jelantah paling rendah mencapai 0,041% pada perendaman 15 hari.

  6. Structural insights into the nucleotide base specificity of P2X receptors

    Science.gov (United States)

    Kasuya, Go; Fujiwara, Yuichiro; Tsukamoto, Hisao; Morinaga, Satoshi; Ryu, Satoshi; Touhara, Kazushige; Ishitani, Ryuichiro; Furutani, Yuji; Hattori, Motoyuki; Nureki, Osamu

    2017-01-01

    P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors. PMID:28332633

  7. A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information.

    Directory of Open Access Journals (Sweden)

    Miguel E Rentería

    Full Text Available The insulin receptor (IR, the insulin-like growth factor 1 receptor (IGF1R and the insulin receptor-related receptor (IRR are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight 'twist' rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

  8. Structure-based discovery of selective serotonin 5-HT(1B) receptor ligands.

    Science.gov (United States)

    Rodríguez, David; Brea, José; Loza, María Isabel; Carlsson, Jens

    2014-08-05

    The development of safe and effective drugs relies on the discovery of selective ligands. Serotonin (5-hydroxytryptamine [5-HT]) G protein-coupled receptors are therapeutic targets for CNS disorders but are also associated with adverse drug effects. The determination of crystal structures for the 5-HT1B and 5-HT2B receptors provided an opportunity to identify subtype selective ligands using structure-based methods. From docking screens of 1.3 million compounds, 22 molecules were predicted to be selective for the 5-HT1B receptor over the 5-HT2B subtype, a requirement for safe serotonergic drugs. Nine compounds were experimentally verified as 5-HT1B-selective ligands, with up to 300-fold higher affinities for this subtype. Three of the ligands were agonists of the G protein pathway. Analysis of state-of-the-art homology models of the two 5-HT receptors revealed that the crystal structures were critical for predicting selective ligands. Our results demonstrate that structure-based screening can guide the discovery of ligands with specific selectivity profiles.

  9. X-ray structures define human P2X3 receptor gating cycle and antagonist action

    NARCIS (Netherlands)

    Mansoor, Steven E.; Lü, Wei; Oosterheert, W.; Shekhar, Mrinal; Tajkhorshid, Emad; Gouaux, Eric

    2016-01-01

    P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human

  10. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

    Directory of Open Access Journals (Sweden)

    Siew Lee Cheong

    2011-01-01

    Full Text Available In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3 has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists.

  11. X-ray structure of the mouse serotonin 5-HT3 receptor

    NARCIS (Netherlands)

    Hassaine, Gherici; Deluz, Cedric; Grasso, Luigino; Wyss, Romain; Tol, Menno B.; Hovius, Ruud; Graff, Alexandra; Stahlberg, Henning; Tomizaki, Takashi; Desmyter, Aline; Moreau, Christophe; Li, Xiao-Dan; Poitevin, Frederic; Vogel, Horst; Nury, Hugues

    2014-01-01

    Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structur

  12. Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Schuurman-Wolters, Gea K.; Dunny, Gary; Slotboom, Dirk-Jan; Poolman, Bert

    2012-01-01

    Wepresent the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and Opp

  13. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

    Science.gov (United States)

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen

    2016-05-19

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

  14. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels

    Directory of Open Access Journals (Sweden)

    Go Kasuya

    2016-02-01

    Full Text Available P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

  15. Structure-based, rational design of T cell receptors

    Directory of Open Access Journals (Sweden)

    Vincent eZoete

    2013-09-01

    Full Text Available Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce TCR modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction pMHC. Using the well-characterized 2C TCR/SIYR/H-2K(b structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. 54% of the designed sequence replacements exhibited improved pMHC-binding as compared to the native TCR, with up to 150 fold increase in affinity, while preserving specificity. Genetically-engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes. We have complemented the approach with a simplified rigid method to predict the TCR orientation over pMHC. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of

  16. Excitatory amino acid receptor antagonists

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B

    1997-01-01

    We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation......)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments......, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation...

  17. A structural feature of the non-peptide ligand interactions with mice mu-opioid receptors.

    Science.gov (United States)

    Noori, Hamid R; Mucksch, Christian; Urbassek, Herbert M

    2014-01-01

    By binding to and activating the G-protein coupled μ-, κ- and δ-opioid receptors in the central nervous system, opiates are known to induce analgesic and sedative effects. In particular, non-peptide opioid ligands are often used in clinical applications to induce these therapeutically beneficial effects, due to their superior pharmacokinetics and bioavailability in comparison to endogenous neuropeptides. However, since opioid alkaloids are highly addictive substances, it is necessary to understand the exact mechanisms of their actions, specifically the ligand-binding properties of the target receptors, in order to safely apply opiates for therapeutic purposes. Using an in silico molecular docking approach (AutoDock Vina) combined with two-step cluster analysis, we have computationally obtained the docking scores and the ligand-binding pockets of twelve representative non-peptide nonendogenous agonists and antagonists at the crystallographically identified μ-opioid receptor. Our study predicts the existence of two main binding sites that are congruently present in all opioid receptor types. Interestingly, in terms of the agonist or antagonist properties of the substances on the receptors, the clustering analysis suggests a relationship with the position of the ligand-binding pockets, particularly its depth within the receptor structure. Furthermore, the binding affinity of the substances is directly correlated to the proximity of the binding pockets to the extracellular space. In conclusion, the results provide further insights into the structural features of the functional pharmacology of opioid receptors, suggesting the importance of the binding position of non-peptide agonists and antagonists- specifically the distance and the level of exposure to the extracellular space- to their dissociation kinetics and subsequent potency.

  18. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant

    Science.gov (United States)

    Yin, Jie; Mobarec, Juan Carlos; Kolb, Peter; Rosenbaum, Daniel M.

    2015-03-01

    The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) respond to orexin neuropeptides in the central nervous system to regulate sleep and other behavioural functions in humans. Defects in orexin signalling are responsible for the human diseases of narcolepsy and cataplexy; inhibition of orexin receptors is an effective therapy for insomnia. The human OX2 receptor (OX2R) belongs to the β branch of the rhodopsin family of GPCRs, and can bind to diverse compounds including the native agonist peptides orexin-A and orexin-B and the potent therapeutic inhibitor suvorexant. Here, using lipid-mediated crystallization and protein engineering with a novel fusion chimaera, we solved the structure of the human OX2R bound to suvorexant at 2.5 Å resolution. The structure reveals how suvorexant adopts a π-stacked horseshoe-like conformation and binds to the receptor deep in the orthosteric pocket, stabilizing a network of extracellular salt bridges and blocking transmembrane helix motions necessary for activation. Computational docking suggests how other classes of synthetic antagonists may interact with the receptor at a similar position in an analogous π-stacked fashion. Elucidation of the molecular architecture of the human OX2R expands our understanding of peptidergic GPCR ligand recognition and will aid further efforts to modulate orexin signalling for therapeutic ends.

  19. Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

    Directory of Open Access Journals (Sweden)

    Makiko Suwa

    2011-04-01

    Full Text Available An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS http://sevens.cbrc.jp/ that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

  20. Hydrazone based luminescent receptors for fluorescent sensing of Cu{sup 2+}: Structure and spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Soma, E-mail: sommukh445@yahoo.co.in [Department of Environmental Science, University of Kalyani, Kalyani, Nadia, 741235 West Bengal (India); Mal, Palash [Department of Environmental Science, University of Kalyani, Kalyani, Nadia, 741235 West Bengal (India); Stoeckli-Evans, Helen [Institute of Physics, University of Neuchâtel, rue Emile-Argand 11, CH-2000 Neuchâtel (Switzerland)

    2014-11-15

    Two new luminescent hydrazones, HL1 and HL2 were investigated for selective and sensitive fluorescent recognition of Cu{sup 2+} in aqueous medium (CH{sub 3}CN/H{sub 2}O (1:4, v/v) solvent system) with a 1:1 binding stoichiometry. The emission peak of HL (λ{sub em}=405 nm), undergoes significant quenching upon complexation with Cu{sup 2+}. The quantum yields for the receptors and in situ formed Cu{sup 2+} complexes were determined. The absorption ratiometric analysis was carried out in presence of various metal ions to confirm the selectivity of the receptors towards Cu{sup 2+}. They were able to detect Cu{sup 2+} with a ∼0.9 µM detection limit as indicated by fluorimetric measurements. The molecular structures of the receptors were determined by single crystal X-ray diffraction analysis. - Highlights: • Small molecule luminescent hydrazones were developed for recognition of Cu{sup 2+}. • Selectivity and sensitivity were studied spectroscopically in aqueous medium. • Binding stoichiometry, association constant, and quantum yields were calculated. • Receptors have low detection limit for Cu{sup 2+}. • Crystal structures of the receptors were solved by X-ray diffractometry.

  1. Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.; Miller, Laurence J.; Xu, H. Eric (Van Andel); (Mayo)

    2010-06-25

    The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

  2. Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor*

    Science.gov (United States)

    Dolino, Drew M.; Cooper, David; Ramaswamy, Swarna; Jaurich, Henriette; Landes, Christy F.; Jayaraman, Vasanthi

    2015-01-01

    N-Methyl-d-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and l-alanine, and full agonists glycine and d-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-l-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation. PMID:25404733

  3. FERMENTASI CAIR AMPAS KELAPA SAWIT DAN KAPANG RHIZOPUS OLIGOSPORUS UNTUK MENGHASILKAN ASAM LEMAK OMEGA-3

    Directory of Open Access Journals (Sweden)

    Erwin Affandi

    2012-11-01

    :1, linoleic acid (18:2 and linolenic (18:3 increased. However, all fatty acid in low-carbon treatment decreased, except the linolenic-acid. The conclusion: The fermentation of palm-oil waste with Rhizopus oligosporus mold could increase the content of fat and produce fatty acid omega-3.   In addition, the high-carbon substrat could increase the production of unsaturated-fatty acid.  Submit : 19-12-2011  Review : 08-03-2012 Review : 12 -03-2012 revisi : 17–4-2012 56 Keywords: liquefied-fermentation, waste product of palm oil, R.oligosporus, fatty acid 0mega-3 Abstrak Latar belakang: Pemanfaatan kapang Rhizopus. oligosporus untuk menghasilkan asam lemak omega-3 pada substrat cair telah banyak dilakukan. Kandungan lemak ampas kelapa sawit 5,56 gram/100 gram masih berpotensi untuk menghasilkan asam lemak omega-3. Fermentasi padat pada substrat ampas tahu dan ampas kelapa sawit dengan kapang Rhizopus. oligosporus dapat meningkatkan kadar lemak: ampas tahu 34,4%, sedangkan pada substrat ampas kelapa sawit dengan formula tinggi karbon, kadar lemak meningkat 61,57%. Metoda: Sampel ampas sawit diambil dari pabrik industri minyak sawit. Pada penelitian ini ampas sawit dipakai sebagai substrat fermentasi dan kapang yang digunakan adalah R.oligosporus. Untuk bahan suplemen digunakan urea dan sukrosa Kontrol adalah ampas-sawit tanpa suplemen, sedangkan perlakuan ampas sawit ditambahkan urea sebagai sumber Nitrogen(N dan ampas sawit ditambah sukrosa sebagai sumber Karbon(C. Penambahan sumber N sebagai substrat rendah karbon dan sumber C sebagai substrat tinggi karbon. Fermentasi dilakukan selama 7 hari diatas shaker pada suhu ruang.  Produk hasil fermentasi dilakukan analisis: kadar air; abu, lemak, dan asam lemak omega-3. Hasil penelitian: Hasil menunjukkan bahwa kadar air  produk hasil fermentasi menurun pada kontrol dan semua perlakuan. Kadar abu meningkat untuk semua perlakuan. Kandungan lemak pada ampas kontrol dan ampas-sukrosa  meningkat 6,43% dan 31,67%, sedang substrat

  4. Studies decode crystal structure of a neurotrophic factor and the p75 neurotrophin receptor complex

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ CAS biophysicists have made new discoveries about the p75 neurotrophin receptor (p75NTR).Under the guidance of Prof.JIANG Tao of the CAS Institute of Biophysics (IBP),Dr.GONG Yong and Dr.CAO Peng revealed crystal structure of a symmetrical complex of the neurotrophin-3 and p75NTR,bringing to light the mode of their interaction and their structural basis.The work was published by the ioumal Nature on 7 August.

  5. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)

    2012-02-21

    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  6. Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957

    OpenAIRE

    Liu, Junfeng; Stevens, David J.; Lesley F Haire; Walker, Philip A.; Coombs, Peter J.; Russell, Rupert J.; Gamblin, Steven J.; John J Skehel

    2009-01-01

    The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the “Asian Influenza” of 1957. We compare it with the 1918 “Spanish Influenza” hemagglutinin, H1, and the 1968 “Hong Kong Influenza” hemagglutinin, H3, and show that despite its close over...

  7. CLE Peptides in Plants: Proteolytic Processing,Structure-Activity Relationship, and Ligand-Receptor Interaction

    Institute of Scientific and Technical Information of China (English)

    Xiaoming Gao; Yongfeng Guo

    2012-01-01

    Ligand-receptor signaling initiated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) family peptides is critical in regulating cell division and differentiation in meristematic tissues in plants.Biologically active CLE peptides are released from precursor proteins via proteolytic processing.The mature form of CLE ligands consists of 12-13 amino acids with several post-translational modifications.This review summarizes recent progress toward understanding the proteolytic activities that cleave precursor proteins to release CLE peptides,the molecular structure and function of mature CLE ligands,and interactions between CLE ligands and corresponding leucine-rich repeat (LRR) receptor-like kinases (RLKs).

  8. Structure and function of florigen and the receptor complex.

    Science.gov (United States)

    Taoka, Ken-ichiro; Ohki, Izuru; Tsuji, Hiroyuki; Kojima, Chojiro; Shimamoto, Ko

    2013-05-01

    In the 1930s, the flowering hormone, florigen, was proposed to be synthesized in leaves under inductive day length and transported to the shoot apex, where it induces flowering. More recently, generated genetic and biochemical data suggest that florigen is a protein encoded by the gene, FLOWERING LOCUS T (FT). A rice (Oryza sativa) FT homolog, Hd3a, interacts with the rice FD homolog, OsFD1, via a 14-3-3 protein. Formation of this tri-protein complex is essential for flowering promotion by Hd3a in rice. In addition, the multifunctionality of FT homologs, other than for flowering promotion, is an emerging concept. Here we review the structural and biochemical features of the florigen protein complex and discuss the molecular basis for the multifunctionality of FT proteins.

  9. Structural modeling of G-protein coupled receptors: An overview on automatic web-servers.

    Science.gov (United States)

    Busato, Mirko; Giorgetti, Alejandro

    2016-08-01

    Despite the significant efforts and discoveries during the last few years in G protein-coupled receptor (GPCR) expression and crystallization, the receptors with known structures to date are limited only to a small fraction of human GPCRs. The lack of experimental three-dimensional structures of the receptors represents a strong limitation that hampers a deep understanding of their function. Computational techniques are thus a valid alternative strategy to model three-dimensional structures. Indeed, recent advances in the field, together with extraordinary developments in crystallography, in particular due to its ability to capture GPCRs in different activation states, have led to encouraging results in the generation of accurate models. This, prompted the community of modelers to render their methods publicly available through dedicated databases and web-servers. Here, we present an extensive overview on these services, focusing on their advantages, drawbacks and their role in successful applications. Future challenges in the field of GPCR modeling, such as the predictions of long loop regions and the modeling of receptor activation states are presented as well.

  10. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

    Science.gov (United States)

    Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E

    2013-09-17

    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

  11. Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene

    DEFF Research Database (Denmark)

    Blagoev, Blagoy; Nielsen, Mogens M; Angrist, Misha

    2002-01-01

    , a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely...... is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine...

  12. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    Science.gov (United States)

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  13. Aspects of dopamine and acetylcholine release induced by glutamate receptors; Aspectos das liberacoes de dopamina e acetilcolina mediadas por receptores de glutamato

    Energy Technology Data Exchange (ETDEWEB)

    Paes, Paulo Cesar de Arruda

    2002-07-01

    The basal ganglia play an important role in the motor control of rats and humans. This control involves different neurotransmitters and the mutual control of these key elements has been subject to several studies. In this work we determined the role of glutamate on the release of radioactively labelled dopamine and acetylcholine from chopped striatal tissue in vitro. The values of Effective Concentration 50% for glutamate, NMDA, kainic, quisqualic acids and AMPA on the release of dopamine and acetylcholine were obtained. The inhibitory effects of magnesium, tetrodotoxin, MK-801, AP5 and MCPG, as well as the effects of glycin were evaluated. The results suggested that dopamine is influenced by the NMDA type glutamate receptor while acetylcholine seems to be influenced by NMDA, kainate and AMPA receptors. Tetrodotoxin experiments suggested that kainate receptors are both present in cholinergic terminals and cell bodies while AMPA and NMDA receptors are preferentially distributed in cell bodies. Magnesium effectively blocked the NMDA stimulation and unexpectedly also AMPA- and quisqualate-induced acetylcholine release. The latter could not be blocked by MCPG ruling out the participation of methabotropic receptors. MK-801 also blocked NMDA-receptors. Results point out the importance of the glutamic acid control of dopamine and acetylcholine release in striatal tissue. (author)

  14. Mitochondrial superoxide production and MnSOD activity following exposure to an agonist and antagonists of ionotropic receptors in rat brain

    Directory of Open Access Journals (Sweden)

    Radenović Lidija Lj.

    2005-01-01

    Full Text Available The involvement of NMDA and AMPA/kainate receptors in the induction of superoxide production in the rat brain was examined after intrahippocampal injection of kainate, a non-NMDA receptor agonist; kainate plus CNQX, a selective AMPA/kainate receptor antagonist; or kainate plus APV, a selective NMDA receptor antagonist. The measurements took place at different times in the ipsi- and contralateral hippocampus, forebrain cortex, striatum, and cerebellum homogenates. The used glutamate antagonists both ensured sufficient neuroprotection in the sense of lowering superoxide production and raising MnSOD levels, but in the mechanisms and time dynamics of their effects were different. Our findings suggest that NMDA and AMPA/kainate receptors are differentially involved in superoxide production. UDC 612.815 612.82.

  15. Pharmacological analysis of ionotropic glutamate receptor function in neuronal circuits of the zebrafish olfactory bulb.

    Directory of Open Access Journals (Sweden)

    Rico Tabor

    Full Text Available Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca(2+ imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1 interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2 interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3 AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4 ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb.

  16. Structure and Notch receptor binding of the tandem WWE domain of Deltex.

    Science.gov (United States)

    Zweifel, Mark E; Leahy, Daniel J; Barrick, Doug

    2005-11-01

    Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.

  17. Recent advances in structure of progestins and their binding to progesterone receptors.

    Science.gov (United States)

    Cabeza, Marisa; Heuze, Yvonne; Sánchez, Araceli; Garrido, Mariana; Bratoeff, Eugene

    2015-02-01

    The role of progesterone in women's cancers as well as the knowledge of the progesterone receptor (PR) structure has prompted the design of different therapies. The aim of this review is to describe the basic structure of PR agonists and antagonists as well as the recent treatments for illness associated with the progesterone receptor. The rational design for potent and effective drugs for the treatment of female cancer must consider the structural changes of the androgen and progestogen skeleton which are an indicator of their activity as progestins or antiprogestins. The presence of a hydroxyl group at C-17 in the progesterone skeleton brings about a loss of progestational activity whereas acetylation induces a progestational effect. The incorporation of an ethynyl functional group to the testosterone framework results in a loss of androgenic activity with a concomitant enhancement of the progestational effect. On the other hand, an ester function at C-3 of dehydroepiandrosterone skeleton induces partial antagonism to the PR.

  18. Phytoestrogens and Mycoestrogens Induce Signature Structure Dynamics Changes on Estrogen Receptor α

    Directory of Open Access Journals (Sweden)

    Xueyan Chen

    2016-08-01

    Full Text Available Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER and provided rich data in the literature. However, limited assays can offer structure information with regard to the ligand/ER complex. Our current study surveys the global structure dynamics changes for ERα ligand binding domain (LBD when phytoestrogens and mycoestrogens bind. The assay is based on the structure dynamics information probed by hydrogen deuterium exchange mass spectrometry and offers a unique viewpoint to elucidate the mechanism how phytoestrogens and mycoestrogens interact with estrogen receptor. The cluster analysis based on the hydrogen deuterium exchange (HDX assay data reveals a unique pattern when phytoestrogens and mycoestrogens bind with ERα LBD compared to that of estradiol and synthetic estrogen modulators. Our study highlights that structure dynamics could play an important role in the structure function relationship when endocrine disrupters interact with estrogen receptors.

  19. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Directory of Open Access Journals (Sweden)

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  20. A Structural Investigation into Oct4 Regulation by Orphan Nuclear Receptors, Germ Cell Nuclear Factor (GCNF) and Liver Receptor Homolog-1 (LRH-1).

    Science.gov (United States)

    Weikum, Emily R; Tuntland, Micheal L; Murphy, Michael N; Ortlund, Eric A

    2016-10-27

    Oct4 is a transcription factor required for maintaining pluripotency and self-renewal in stem cells. Prior to differentiation, Oct4 must be silenced to allow for the development of the three germ layers in the developing embryo. This fine-tuning is controlled by the nuclear receptors, liver receptor homolog-1 and germ cell nuclear factor. Liver receptor homolog-1 is responsible for driving the expression of Oct4 where germ cell nuclear factor represses its expression upon differentiation. Both receptors bind to a DR0 motif located within the Oct4 promoter. Here, we present the first structure of mouse germ cell nuclear factor DNA binding domain in complex with the Oct4 DR0. The overall structure revealed two molecules bound in a head-to-tail fashion on opposite sides of the DNA. Additionally, we solved the structure of the human liver receptor homolog-1 DNA binding domain bound to the same element. We explore the structural elements that govern Oct4 recognition by these two nuclear receptors.

  1. Structure-Driven Pharmacology of Transient Receptor Potential Channel Vanilloid 1.

    Science.gov (United States)

    Díaz-Franulic, Ignacio; Caceres-Molina, Javier; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2016-09-01

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of "hot" chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies.

  2. Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.

    Science.gov (United States)

    Manni, Sandro; Mineev, Konstantin S; Usmanova, Dinara; Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kirpichnikov, Mikhail P; Winter, Jonas; Matkovic, Milos; Deupi, Xavier; Arseniev, Alexander S; Ballmer-Hofer, Kurt

    2014-08-05

    Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

  3. Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site.

    Science.gov (United States)

    Chen, Ligong; Durkin, Kathleen A; Casida, John E

    2006-03-28

    Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

  4. Structural comparison of phospholipase-A2-binding regions in phospholipase-A2 receptors from various mammals.

    Science.gov (United States)

    Higashino, K; Ishizaki, J; Kishino, J; Ohara, O; Arita, H

    1994-10-01

    We determined the nucleotide sequence of a mouse cDNA encoding the receptor for pancreatic group I phospholipase A2 (PLA2-I). Interspecies structural comparison of the mouse receptor with bovine PLA2-I receptor, whose structure had been clarified, revealed that the fourth carbohydrate-recognition domain (CRD)-like domain (CRD-like 4) was the most conserved among the domains in the PLA2-I receptor, suggesting the functional importance of CRD-like 4. A transient expression experiment with a truncated form of the receptor consisting of three CRD-like domains, from the third to the fifth, demonstrated that the PLA2-I-binding site of the receptor is constituted from these three CRD-like domains, supporting the functional indispensability of CRD-like 4 in the receptor. Since the PLA2-I-binding region was thus assigned to be CRD-like domains 3-5, we further analyzed the structures of the PLA2-I-binding regions in the PLA2-I receptors from the rat, rabbit and human. Furthermore, the obtained PLA2-I receptor cDNA fragments from these animals made it possible to examine the tissue expression patterns of this receptor in various mammals. The results, together with the results of the genomic structural analysis of this gene, indicated that a PLA2 receptor recently characterized by Lambeau et al. [Lambeau, G., Ancian, P., Barhanin, J. & Lazdunski, M. (1994) J. Biol. Chem. 269, 1575-1578] is a rabbit counterpart of the PLA2-I receptor although these two PLA2 receptors have distinctive PLA2-binding specificities.

  5. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  6. Anticonvulsant activity of artificial sweeteners: a structural link between sweet-taste receptor T1R3 and brain glutamate receptors.

    Science.gov (United States)

    Talevi, Alan; Enrique, Andrea V; Bruno-Blanch, Luis E

    2012-06-15

    A virtual screening campaign based on application of a topological discriminant function capable of identifying novel anticonvulsant agents indicated several widely-used artificial sweeteners as potential anticonvulsant candidates. Acesulfame potassium, cyclamate and saccharin were tested in the Maximal Electroshock Seizure model (mice, ip), showing moderate anticonvulsant activity. We hypothesized a probable structural link between the receptor responsible of sweet taste and anticonvulsant molecular targets. Bioinformatic tools confirmed a highly significant sequence-similarity between taste-related protein T1R3 and several metabotropic glutamate receptors from different species, including glutamate receptors upregulated in epileptogenesis and certain types of epilepsy.

  7. New insights for drug design from the X-ray crystallographic structures of G-protein-coupled receptors.

    Science.gov (United States)

    Jacobson, Kenneth A; Costanzi, Stefano

    2012-09-01

    Methodological advances in X-ray crystallography have made possible the recent solution of X-ray structures of pharmaceutically important G protein-coupled receptors (GPCRs), including receptors for biogenic amines, peptides, a nucleoside, and a sphingolipid. These high-resolution structures have greatly increased our understanding of ligand recognition and receptor activation. Conformational changes associated with activation common to several receptors entail outward movements of the intracellular side of transmembrane helix 6 (TM6) and movements of TM5 toward TM6. Movements associated with specific agonists or receptors have also been described [e.g., extracellular loop (EL) 3 in the A(2A) adenosine receptor]. The binding sites of different receptors partly overlap but differ significantly in ligand orientation, depth, and breadth of contact areas in TM regions and the involvement of the ELs. A current challenge is how to use this structural information for the rational design of novel potent and selective ligands. For example, new chemotypes were discovered as antagonists of various GPCRs by subjecting chemical libraries to in silico docking in the X-ray structures. The vast majority of GPCR structures and their ligand complexes are still unsolved, and no structures are known outside of family A GPCRs. Molecular modeling, informed by supporting information from site-directed mutagenesis and structure-activity relationships, has been validated as a useful tool to extend structural insights to related GPCRs and to analyze docking of other ligands in already crystallized GPCRs.

  8. Glyphosate and AMPA distribution in wind-eroded sediment derived from loess soil

    NARCIS (Netherlands)

    Martins Bento, Celia; Goossens, Dirk; Rezaei, Mahrooz; Riksen, M.J.P.M.; Mol, J.G.J.; Ritsema, C.J.; Geissen, V.

    2017-01-01

    Glyphosate is one of the most used herbicides in agricultural lands worldwide. Wind-eroded sediment and dust, as an environmental transport pathway of glyphosate and of its main metabolite aminomethylphosphonic acid (AMPA), can result in environmental- and human exposure far beyond the agricultural

  9. Beyond small-molecule SAR: using the dopamine D3 receptor crystal structure to guide drug design.

    Science.gov (United States)

    Keck, Thomas M; Burzynski, Caitlin; Shi, Lei; Newman, Amy Hauck

    2014-01-01

    The dopamine D3 receptor is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, restless leg syndrome, and drug addiction. The high protein sequence homology between the D3 and D2 receptors has posed a challenge to developing D3 receptor-selective ligands whose behavioral actions can be attributed to D3 receptor engagement, in vivo. However, through primarily small-molecule structure-activity relationship (SAR) studies, a variety of chemical scaffolds have been discovered over the past two decades that have resulted in several D3 receptor-selective ligands with high affinity and in vivo activity. Nevertheless, viable clinical candidates remain limited. The recent determination of the high-resolution crystal structure of the D3 receptor has invigorated structure-based drug design, providing refinements to the molecular dynamic models and testable predictions about receptor-ligand interactions. This chapter will highlight recent preclinical and clinical studies demonstrating potential utility of D3 receptor-selective ligands in the treatment of addiction. In addition, new structure-based rational drug design strategies for D3 receptor-selective ligands that complement traditional small-molecule SAR to improve the selectivity and directed efficacy profiles are examined.

  10. Artonin E and Structural Analogs from Artocarpus Species Abrogates Estrogen Receptor Signaling in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Imaobong Etti

    2016-06-01

    Full Text Available The increasing rate of mortality ensued from breast cancer has encouraged research into safer and efficient therapy. The human Estrogen receptor α has been implicated in the majority of reported breast cancer cases. Molecular docking employing Glide, Schrodinger suite 2015, was used to study the binding affinities of small molecules from the Artocarpus species after their drug-like properties were ascertained. The structure of the ligand-binding domain of human Estrogen receptor α was retrieved from Protein Data Bank while the structures of compounds were collected from PubChem database. The binding interactions of the studied compounds were reported as well as their glide scores. The best glide scored ligand, was Artonin E with a score of −12.72 Kcal when compared to other studied phytomolecules and it evoked growth inhibition of an estrogen receptor positive breast cancer cells in submicromolar concentration (3.8–6.9 µM in comparison to a reference standard Tamoxifen (18.9–24.1 µM within the tested time point (24–72 h. The studied ligands, which had good interactions with the target receptor, were also drug-like when compared with 95% of orally available drugs with the exception of Artoelastin, whose predicted physicochemical properties rendered it less drug-like. The in silico physicochemical properties, docking interactions and growth inhibition of the best glide scorer are indications of the anti-breast cancer relevance of the studied molecules.

  11. [Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors].

    Science.gov (United States)

    Fruchart-Gaillard, Carole; Ménez, André; Servent, Denis

    2005-01-01

    Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthetized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.

  12. Structural and energetic effects of A2A adenosine receptor mutations on agonist and antagonist binding.

    Directory of Open Access Journals (Sweden)

    Henrik Keränen

    Full Text Available To predict structural and energetic effects of point mutations on ligand binding is of considerable interest in biochemistry and pharmacology. This is not only useful in connection with site-directed mutagenesis experiments, but could also allow interpretation and prediction of individual responses to drug treatment. For G-protein coupled receptors systematic mutagenesis has provided the major part of functional data as structural information until recently has been very limited. For the pharmacologically important A(2A adenosine receptor, extensive site-directed mutagenesis data on agonist and antagonist binding is available and crystal structures of both types of complexes have been determined. Here, we employ a computational strategy, based on molecular dynamics free energy simulations, to rationalize and interpret available alanine-scanning experiments for both agonist and antagonist binding to this receptor. These computer simulations show excellent agreement with the experimental data and, most importantly, reveal the molecular details behind the observed effects which are often not immediately evident from the crystal structures. The work further provides a distinct validation of the computational strategy used to assess effects of point-mutations on ligand binding. It also highlights the importance of considering not only protein-ligand interactions but also those mediated by solvent water molecules, in ligand design projects.

  13. Nuclear receptor engineering based on novel structure activity relationships revealed by farnesyl pyrophosphate.

    Science.gov (United States)

    Goyanka, Ritu; Das, Sharmistha; Samuels, Herbert H; Cardozo, Timothy

    2010-11-01

    Nuclear receptors (NRs) comprise the second largest protein family targeted by currently available drugs, acting via specific ligand interactions within the ligand binding domain (LBD). Recently, farnesyl pyrophosphate (FPP) was shown to be a unique promiscuous NR ligand, activating a subset of NR family members and inhibiting wound healing in skin. The current study aimed at visualizing the unique basis of FPP interaction with multiple receptors in order to identify general structure-activity relationships that operate across the NR family. Docking of FPP to the 3D structures of the LBDs of a diverse set of NRs consistently revealed an electrostatic FPP pyrophosphate contact with an NR arginine conserved in the NR family, a hydrophobic farnesyl contact with NR helix-12 and a ligand binding pocket volume between 300 and 430 Å(3) as the minimal requirements for FPP activation of any NR. Lack of any of these structural features appears to render a given NR resistant to FPP activation. We used these structure-activity relationships to rationally design and successfully engineer several mutant human estrogen receptors that retain responsiveness to estradiol but no longer respond to FPP.

  14. Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

    DEFF Research Database (Denmark)

    Nielsen, B S; Banke, T G; Schousboe, A

    1998-01-01

    Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after...

  15. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

    OpenAIRE

    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  16. Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.

    Science.gov (United States)

    Yule, David I; Betzenhauser, Matthew J; Joseph, Suresh K

    2010-06-01

    Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

  17. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    M Franklin; E Navarro; Y Wang; S Patel; P Singh; Y Zhang; K Persaud; A Bari; H Griffith; et al.

    2011-12-31

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  18. The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Franklin, Matthew C.; Navarro, Elizabeth C.; Wang, Yujie; Patel, Sheetal; Singh, Pinki; Zhang, Yi; Persaud, Kris; Bari, Amtul; Griffith, Heather; Shen, Leyi; Balderes, Paul; Kussie, Paul (ImClone)

    2011-10-28

    The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

  19. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A. (UPENN-MED)

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  20. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors.

    Science.gov (United States)

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane-solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane-solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor.

  1. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1.

    Science.gov (United States)

    Fay, Jonathan F; Farrens, David L

    2015-07-07

    G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

  2. Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception.

    Science.gov (United States)

    Roudnitzky, Natacha; Behrens, Maik; Engel, Anika; Kohl, Susann; Thalmann, Sophie; Hübner, Sandra; Lossow, Kristina; Wooding, Stephen P; Meyerhof, Wolfgang

    2015-01-01

    The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen

  3. Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

    Directory of Open Access Journals (Sweden)

    Daniel eClayton

    2015-01-01

    Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

  4. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien (Michigan-Med); (Stanford-MED); (UAB, Spain)

    2012-06-27

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  5. A Hybrid Approach to Structure and Function Modeling of G Protein-Coupled Receptors.

    Science.gov (United States)

    Latek, Dorota; Bajda, Marek; Filipek, Sławomir

    2016-04-25

    The recent GPCR Dock 2013 assessment of serotonin receptor 5-HT1B and 5-HT2B, and smoothened receptor SMO targets, exposed the strengths and weaknesses of the currently used computational approaches. The test cases of 5-HT1B and 5-HT2B demonstrated that both the receptor structure and the ligand binding mode can be predicted with the atomic-detail accuracy, as long as the target-template sequence similarity is relatively high. On the other hand, the observation of a low target-template sequence similarity, e.g., between SMO from the frizzled GPCR family and members of the rhodopsin family, hampers the GPCR structure prediction and ligand docking. Indeed, in GPCR Dock 2013, accurate prediction of the SMO target was still beyond the capabilities of most research groups. Another bottleneck in the current GPCR research, as demonstrated by the 5-HT2B target, is the reliable prediction of global conformational changes induced by activation of GPCRs. In this work, we report details of our protocol used during GPCR Dock 2013. Our structure prediction and ligand docking protocol was especially successful in the case of 5-HT1B and 5-HT2B-ergotamine complexes for which we provide one of the most accurate predictions. In addition to a description of the GPCR Dock 2013 results, we propose a novel hybrid computational methodology to improve GPCR structure and function prediction. This computational methodology employs two separate rankings for filtering GPCR models. The first ranking is ligand-based while the second is based on the scoring scheme of the recently published BCL method. In this work, we prove that the use of knowledge-based potentials implemented in BCL is an efficient way to cope with major bottlenecks in the GPCR structure prediction. Thereby, we also demonstrate that the knowledge-based potentials for membrane proteins were significantly improved, because of the recent surge in available experimental structures.

  6. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity

    NARCIS (Netherlands)

    Blaazer, A.R.; Lange, J.H.M.; van der Neut, M.A.W.; Mulder, A.; den Boon, F.S.; Werkman, T.R.; Kruse, C.G.; Wadman, W.J.

    2011-01-01

    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl

  7. Structural determinants of diphenethylamines for interaction with the κ opioid receptor: Synthesis, pharmacology and molecular modeling studies.

    Science.gov (United States)

    Guerrieri, Elena; Bermudez, Marcel; Wolber, Gerhard; Berzetei-Gurske, Ilona P; Schmidhammer, Helmut; Spetea, Mariana

    2016-10-01

    The κ opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calculations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the KOP receptor attained by molecular dynamics simulations. The structure-activity relationships derived from computational studies was in accordance with pharmacological activities of targeted diphenethylamines at the KOP receptor established by competition binding and G protein activation in vitro assays. Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond, which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed. Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists. Thus, combination of experimental and molecular modeling strategies provides an initial framework for understanding the structural features of diphenethylamines that are essential to promote binding affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding event into molecular changes, ultimately leading to receptor activation.

  8. Evidence for homogeneity of thromboxane A2 receptor using structurally different antagonists.

    Science.gov (United States)

    Swayne, G T; Maguire, J; Dolan, J; Raval, P; Dane, G; Greener, M; Owen, D A

    1988-08-01

    Nine structurally dissimilar thromboxane antagonists (SQ 29548, ICI 185282, AH 23848, BM 13505 (Daltroban), BM 13177 (Sulotroban), SK&F 88046, L-636499, L-640035 and a Bayer compound SK&F 47821) were studied for activity as thromboxane A2 receptor antagonists. The assays used were inhibition of responses induced by the thromboxane mimetic, U46619, on human washed platelet aggregation, rabbit platelet aggregation, rabbit aortic strip contraction, anaesthetised guinea-pig bronchoconstriction, and a radio-labelled ligand (125I-PTA-OH) binding assay as a measure of affinity for the human platelet receptor. The results of the present study, with activities spanning at least four orders of magnitude along with statistically significant correlations (at least P less than 0.01), strongly suggests that between assays, antagonists and species a homogenous population of thromboxane A2 receptors exists. This finding is in contrast to those of a close series of 13-azapinane antagonists studied by other workers which have suggested receptor heterogeneity.

  9. Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists

    Science.gov (United States)

    Barron, Mace G.

    2017-01-01

    The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicable to other nuclear receptors. PMID:28061508

  10. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor

  11. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins

    Directory of Open Access Journals (Sweden)

    Ralf eEnz

    2012-04-01

    Full Text Available Metabotropic glutamate receptors (mGluRs regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g. night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson´s disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors´ C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  12. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    Science.gov (United States)

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  13. Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein

    Directory of Open Access Journals (Sweden)

    Danilo G. Moriel

    2016-03-01

    Full Text Available Urinary tract infection (UTI is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA. We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R, interleukin-4 receptor (IL-4R, and interleukin-10 receptor (IL-10R binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection.

  14. Excitatory amino acid receptor ligands: resolution, absolute stereochemistry, and enantiopharmacology of 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid

    DEFF Research Database (Denmark)

    Johansen, T N; Ebert, B; Bräuner-Osborne, Hans

    1998-01-01

    in mice, 7 (ED50 = 44 mumol/kg) was slightly more potent than AMPA (1) (ED50 = 55 mumol/kg) and twice as potent as Bu-HIBO (6) (ED50 = 94 mumol/kg) as a convulsant, whereas 8 was inactive. After subcutaneous administration in mice, Bu-HIBO (ED50 = 110 mumol/kg) was twice as potent as AMPA (ED50 = 220...... mumol/kg) as a convulsant. Since 7 and Bu-HIBO (EC50 = 37 microM) are much weaker than AMPA (EC50 = 3.5 microM) as AMPA receptor agonists in vitro, the presence of a butyl group in the molecules of Bu-HIBO and 7 seems to facilitate the penetration of these compounds through the blood-brain barrier....

  15. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-26

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due to difficulties in crystallization, experimental structure determination remains extremely difficult for human GPCRs, which have been a major barrier in modern structure-based drug discovery. We proposed a new hybrid protocol, GPCR-I-TASSER, to construct GPCR structure models by integrating experimental mutagenesis data with ab initio transmembrane-helix assembly simulations, assisted by the predicted transmembrane-helix interaction networks. The method was tested in recent community-wide GPCRDock experiments and constructed models with a root mean square deviation 1.26 Å for Dopamine-3 and 2.08 Å for Chemokine-4 receptors in the transmembrane domain regions, which were significantly closer to the native than the best templates available in the PDB. GPCR-I-TASSER has been applied to model all 1,026 putative GPCRs in the human genome, where 923 are found to have correct folds based on the confidence score analysis and mutagenesis data comparison. The successfully modeled GPCRs contain many pharmaceutically important families that do not have previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. All the human GPCR models have been made publicly available through the GPCR-HGmod database at http://zhanglab.ccmb.med.umich.edu/GPCR-HGmod/ The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins which should bring useful impact on the effort of GPCR-targeted drug discovery.

  16. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  17. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  18. Molecular And Structural Basis of Cytokine Receptor Pleiotropy in the Interleukin-4/13 System

    Energy Technology Data Exchange (ETDEWEB)

    LaPorte, S.L.; Juo, Z.S.; Vaclavikova, J.; Colf, L.A.; Qi, X.; Heller, N.M.; Keegan, A.D.; Garcia, K.C.

    2009-05-20

    Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R{alpha}/{gamma}{sub c}/IL-4) and type II (IL-4R/IL-13R{alpha}1/IL-4, IL-4R{alpha}/IL-13R{alpha}1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for {gamma}{sub c}'s ability to recognize six different {gamma}{sub c}-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R{alpha}1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.

  19. The crystal structure of a bimorphinan with highly selective kappa opioid receptor antagonist activity

    Science.gov (United States)

    Urbańczyk-Lipkowska, Zofia; Etter, Margaret C.; Lipkowski, Andrzej W.; Portoghese, Philip S.

    1987-07-01

    The crystal structure of the dihydrobromide heptahydrate of nor-binaltorphimine (17, 17'-bis(cyclopropylmethyl)-6,6',7,7'-tetrahydro-4,5α: 4',5'α-diepoxy-6,6'-imino[7,7' bimorphinan]-3,3',14,14'-tetraol)is presented. This structure is the first reported structure of a rigid bivalent opioid ligand. Two morphinan pharmacophores are connected by a rigid spacer, the pyrrole ring. The nor-binaltorphimine structure itself shows unique, high selectivity as a kappa opioid receptor antagonist. Crystal data: P3 2, Z = 3, a = b = 20.223 (4), c = 9.541(7) Å, α = β = 90°, γ = 120°; R = 0.079 (1765 reflections, Fobs > 1σ( F)).

  20. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  1. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    Science.gov (United States)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-11-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  2. Novel highly potent serotonin 5-HT7 receptor ligands: structural modifications to improve pharmacokinetic properties.

    Science.gov (United States)

    Lacivita, Enza; Di Pilato, Pantaleo; Stama, Madia Letizia; Colabufo, Nicola Antonio; Berardi, Francesco; Perrone, Roberto; De Filippis, Bianca; Laviola, Giovanni; Adriani, Walter; Niso, Mauro; Leopoldo, Marcello

    2013-11-15

    Here we report the synthesis, pharmacological and pharmacokinetic evaluation of a pilot set of compounds structurally related to the potent and selective 5-HT7 ligand LP-211. Among the studied compounds, N-pyridin-3-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (4b) showed high affinity for 5-HT7 receptors (K(i)=23.8 nM), selectivity over 5-HT1A receptors (>50-fold), in vitro metabolic stability (82%) and weak interaction with P-glycoprotein (BA/AB=3.3). Compound 4b was injected ip in mice to preliminarily evaluate its distribution between blood and brain.

  3. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  4. Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

    Science.gov (United States)

    Davis, Jennifer; Correll, Robert N.; Trappanese, Danielle M.; Hoffman, Nicholas E.; Troupes, Constantine D.; Berretta, Remus M.; Kubo, Hajime; Madesh, Muniswamy; Chen, Xiongwen; Gao, Erhe; Molkentin, Jeffery D.; Houser, Steven R.

    2014-01-01

    Rationale The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood. Objective To determine if Ca2+ influx through transient receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca2+ leak. Conclusions Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function. PMID:25047165

  5. Structure of the human [kappa]-opioid receptor in complex with JDTic

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Huixian; Wacker, Daniel; Mileni, Mauro; Katritch, Vsevolod; Han, Gye Won; Vardy, Eyal; Liu, Wei; Thompson, Aaron A.; Huang, Xi-Ping; Carroll, F. Ivy; Mascarella, S. Wayne; Westkaemper, Richard B.; Mosier, Philip D.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C. (VCU); (Scripps); (UNC); (Res. Tri. Inst.)

    2013-04-25

    Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.

  6. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A;

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound...... to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair...

  7. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Directory of Open Access Journals (Sweden)

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  8. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H...... neuronal cell death. We conclude that glutamatergic synaptic activity modulates sPLA -induced neuronal cell death....

  9. Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

    DEFF Research Database (Denmark)

    Chan, K Y; Gupta, S; de Vries, R;

    2010-01-01

    studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by alpha-CGRP, capsaicin......During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical...

  10. Structure-based virtual screening of the nociceptin receptor: hybrid docking and shape-based approaches for improved hit identification.

    Science.gov (United States)

    Daga, Pankaj R; Polgar, Willma E; Zaveri, Nurulain T

    2014-10-27

    The antagonist-bound crystal structure of the nociceptin receptor (NOP), from the opioid receptor family, was recently reported along with those of the other opioid receptors bound to opioid antagonists. We recently reported the first homology model of the 'active-state' of the NOP receptor, which when docked with 'agonist' ligands showed differences in the TM helices and residues, consistent with GPCR activation after agonist binding. In this study, we explored the use of the active-state NOP homology model for structure-based virtual screening to discover NOP ligands containing new chemical scaffolds. Several NOP agonist and antagonist ligands previously reported are based on a common piperidine scaffold. Given the structure-activity relationships for known NOP ligands, we developed a hybrid method that combines a structure-based and ligand-based approach, utilizing the active-state NOP receptor as well as the pharmacophoric features of known NOP ligands, to identify novel NOP binding scaffolds by virtual screening. Multiple conformations of the NOP active site including the flexible second extracellular loop (EL2) loop were generated by simulated annealing and ranked using enrichment factor (EF) analysis and a ligand-decoy dataset containing known NOP agonist ligands. The enrichment factors were further improved by combining shape-based screening of this ligand-decoy dataset and calculation of consensus scores. This combined structure-based and ligand-based EF analysis yielded higher enrichment factors than the individual methods, suggesting the effectiveness of the hybrid approach. Virtual screening of the CNS Permeable subset of the ZINC database was carried out using the above-mentioned hybrid approach in a tiered fashion utilizing a ligand pharmacophore-based filtering step, followed by structure-based virtual screening using the refined NOP active-state models from the enrichment analysis. Determination of the NOP receptor binding affinity of a selected set

  11. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    Science.gov (United States)

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  12. Structure and Mechanism of Receptor Sharing by the IL-10R2 Common Chain

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Sung-il; Jones, Brandi C.; Logsdon, Naomi J.; Harris, Bethany D.; Deshpande, Ashlesha; Radaeva, Svetlana; Halloran, Brian A.; Gao, Bin; Walter, Mark R. (NIH); (UAB)

    2010-07-19

    IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ectodomain at 2.14 {angstrom} resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.

  13. Development of calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in cultured neocortical neurons visualized by cobalt staining

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S

    1998-01-01

    The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non-N-methyl......The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non......-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 microM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7%+/-0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 microM cyclothiazide......, the developmental profile of cobalt uptake mediated by 25 microM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4%+/-0.2% at 5 DIV to 21.7%+/-1.6% at 12 DIV. The effect of 200 microM kainate was similar to that seen with 25...

  14. Glyphosate and AMPA contents in sediments produced by wind erosion of agricultural soils in Argentina

    Science.gov (United States)

    Aparicio, Virginia; Aimar, Silvia; De Gerónimo, Eduardo; Buschiazzo, Daniel; Mendez, Mariano; Costa, José Luis

    2014-05-01

    Wind erosion of soils is an important event in arid and semiarid regions of Argentina. The magnitude of wind erosion occurring under different management practices is relatively well known in this region but less information is available on the quality of the eroded material. Considering that the intensification of agriculture may increase the concentrations of substances in the eroded material, producing potential negative effects on the environment, we analyzed the amount of glyphosate and AMPA in sediments produced by wind erosion of agricultural soils of Argentina. Wind eroded materials were collected by means of BSNE samplers in two loess sites of the semiarid region of Argentina: Chaco and La Pampa. Samples were collected from 1 ha square fields at 13.5, 50 and 150 cm height. Results showed that at higher heights the concentrations of glyphosate and AMPA were mostly higher. The glyphosate concentration was more variable and higher in Chaco (0.66 to 313 µg kg-1) than in La Pampa (4.17 to 114 µg kg-1). These results may be due to the higher use of herbicides in Chaco, where the predominant crops are soybeans and corn, produced under no-tillage. Under these conditions the use of glyphosate for weeds control is a common practice. Conversely, AMPA concentrations were higher in La Pampa (13.1 to 101.3 µg kg-1) than in Chaco (1.3 to 83 µg kg-1). These preliminary results show high concentrations of glyphosate and AMPA in wind eroded materials of agricultural soils of Argentina. More research is needed to confirm these high concentrations in other conditions in order to detect the temporal and spatial distribution patterns of the herbicide.

  15. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes**

    Institute of Scientific and Technical Information of China (English)

    Farfán-García Eunice Dalet; Soriano-Ursúa Marvin Antonio

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that al osteric binding sites are involved in the affinity and selec-tivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifical y, new possibilities are explored in relation to al osteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson’s disease, and on muscarinic receptors for Alzheimer’s disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopa-mine receptor holds promise as a relevant therapeutic strategy for Parkinson’s disease. Regarding the treatment of Alzheimer’s disease, the design of dualsteric ligands for mono-oligomeric musca-rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway.

  16. Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors.

    Science.gov (United States)

    Yeh, Brian K; Igarashi, Makoto; Eliseenkova, Anna V; Plotnikov, Alexander N; Sher, Ifat; Ron, Dina; Aaronson, Stuart A; Mohammadi, Moosa

    2003-03-04

    Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ("b") and IIIc ("c"), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.

  17. Local and global ligand-induced changes in the structure of the GABA(A) receptor.

    Science.gov (United States)

    Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B

    2006-06-13

    Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.

  18. Macrophage Receptor with Collagenous Structure (MARCO Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway.

    Directory of Open Access Journals (Sweden)

    Seishiro Hirano

    Full Text Available The Macrophage Receptor with COllagenous structure (MARCO protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2-3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.

  19. Structural analysis of the evolution of steroid specificity in the mineralocorticoid and glucocorticoid receptors

    Directory of Open Access Journals (Sweden)

    Ollikainen Noah

    2007-02-01

    Full Text Available Abstract Background The glucocorticoid receptor (GR and mineralocorticoid receptor (MR evolved from a common ancestor. Still not completely understood is how specificity for glucocorticoids (e.g. cortisol and mineralocorticoids (e.g. aldosterone evolved in these receptors. Results Our analysis of several vertebrate GRs and MRs in the context of 3D structures of human GR and MR indicates that with the exception of skate GR, a cartilaginous fish, there is a deletion in all GRs, at the position corresponding to Ser-949 in human MR. This deletion occurs in a loop before helix 12, which contains the activation function 2 (AF2 domain, which binds coactivator proteins and influences transcriptional activity of steroids. Unexpectedly, we find that His-950 in human MR, which is conserved in the MR in chimpanzee, orangutan and macaque, is glutamine in all teleost and land vertebrate MRs, in