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Sample records for receptor nr2b subunits

  1. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

    PubMed Central

    Tu, Weihong; Xu, Xin; Peng, Lisheng; Zhong, Xiaofen; Zhang, Wenfeng; Soundarapandian, Mangala M.; Balel, Cherine; Wang, Manqi; Jia, Nali; Zhang, Wen; Lew, Frank; Chan, Sic Lung; Chen, Yanfang; Lu, Youming

    2010-01-01

    SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. PMID:20141836

  2. Role for the NR2B Subunit of the NMDA Receptor in Mediating Light Input to the Circadian System

    PubMed Central

    Wang, LM; Schroeder, A; Loh, D; Smith, D; Lin, K; Han, JH; Michel, S; Hummer, DL; Ehlen, JC; Albers, HE; Colwell, CS

    2008-01-01

    Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N-methyl-Daspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region inhibits the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B levels vary with time of day in SCN tissue using semi-quantitative real-time PCR and Western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced calcium changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system. PMID:18380671

  3. Setdb1 histone methyltransferase regulates mood-related behaviors and expression of the NMDA receptor subunit NR2B.

    PubMed

    Jiang, Yan; Jakovcevski, Mira; Bharadwaj, Rahul; Connor, Caroline; Schroeder, Frederick A; Lin, Cong L; Straubhaar, Juerg; Martin, Gilles; Akbarian, Schahram

    2010-05-26

    Histone methyltransferases specific for the histone H3-lysine 9 residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair, and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to <1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30 kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wild-type mice, systemic treatment with the NR2B antagonist, Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol], and hippocampal small interfering RNA-mediated NR2B/Grin2b knockdown resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

  4. Novel approach to probe subunit-specific contributions to N-methyl-D-aspartate (NMDA) receptor trafficking reveals a dominant role for NR2B in receptor recycling.

    PubMed

    Tang, Tina Tze-Tsang; Badger, John D; Roche, Paul A; Roche, Katherine W

    2010-07-01

    N-Methyl-d-aspartate (NMDA) receptors are expressed at excitatory synapses throughout the brain and are essential for neuronal development and synaptic plasticity. Functional NMDA receptors are tetramers, typically composed of NR1 and NR2 subunits (NR2A-D). NR2A and NR2B are expressed in the forebrain and are thought to assemble as diheteromers (NR1/NR2A, NR1/NR2B) and triheteromers (NR1/NR2A/NR2B). NR2A and NR2B contain cytosolic domains that regulate distinct postendocytic sorting events, with NR2A sorting predominantly into the degradation pathway, and NR2B preferentially trafficking through the recycling pathway. However, the interplay between these two subunits remains an open question. We have now developed a novel approach based on the dimeric feature of the alpha- and beta-chains of the human major histocompatibility complex class II molecule. We created chimeras of alpha- and beta-chains with the NR2A and NR2B C termini and evaluated endocytosis of dimers. Like chimeric proteins containing only a single NR2A or NR2B C-terminal domain, major histocompatibility complex class II-NR2A homodimers sort predominantly to late endosomes, whereas NR2B homodimers traffic to recycling endosomes. Interestingly, NR2A/NR2B heterodimers traffic preferentially through the recycling pathway, and NR2B is dominant in regulating dimer trafficking in both heterologous cells and neurons. In addition, the recycling of NR2B-containing NMDARs in wild-type neurons is not significantly different from NR2A(-/-) neurons. These data support a dominant role for NR2B in regulating the trafficking of triheteromeric NMDARs in vivo. Furthermore, our molecular approach allows for the direct and selective evaluation of dimeric assemblies and can be used to define dominant trafficking domains in other multisubunit protein complexes. PMID:20427279

  5. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

    PubMed

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H; Prorok, Mary; Castellino, Francis J

    2009-08-01

    The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes. PMID:19427876

  6. Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS

    PubMed Central

    Miwa, Hideki; Fukaya, Masahiro; Watabe, Ayako M; Watanabe, Masahiko; Manabe, Toshiya

    2008-01-01

    The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala. PMID:18372311

  7. Structure of the Zinc-Bound Amino-Terminal Domain of the NMDA Receptor NR2B Subunit

    SciTech Connect

    Karakas, E.; Simorowski, N; Furukawa, H

    2009-01-01

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.

  8. SNARE Protein Syntaxin-1 Colocalizes Closely with NMDA Receptor Subunit NR2B in Postsynaptic Spines in the Hippocampus

    PubMed Central

    Hussain, Suleman; Ringsevjen, Håvard; Egbenya, Daniel L.; Skjervold, Torstein L.; Davanger, Svend

    2016-01-01

    Syntaxins are a family of membrane-integrated proteins that are instrumental in exocytosis of vesicles. Syntaxin-1 is an essential component of the presynaptic exocytotic fusion machinery in the brain and interacts with several other proteins. Syntaxin-1 forms a four-helical bundle complex with proteins SNAP-25 and VAMP2 that drives fusion of vesicles with the plasma membrane in the active zone (AZ). Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analyzed the intrasynaptic expression of syntaxin-1 in glutamatergic hippocampal synapses in detail by using quantitative postembedding immunogold labeling. Syntaxin-1 was present in highest concentrations at the presynaptic AZ, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the AZ, as well as presynaptic cytoplasmic (PreCy) vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density (PSD), at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the N-methyl-D-aspartate (NMDA) receptor subunit NR2B, but only weakly with the AMPA receptor subunits GluA2/3. This observation points to the possibility that syntaxin-1 may be involved with NR2B vesicular trafficking from cytoplasmic stores to the postsynaptic plasma membrane, thus facilitating synaptic plasticity. Confocal immunofluorescence double labeling with PSD-95 and ultrastructural fractionation of synaptosomes also confirm localization of syntaxin-1 at the PSD. PMID:26903802

  9. Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat

    PubMed Central

    Maffucci, J. A.; Noel, M. L.; Gillette, R.; Wu, D.; Gore, A. C.

    2009-01-01

    Glutamate, acting through its N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin-releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b-expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3-5 months), middle-aged (9-12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b-immunoreactive cell numbers in the AVPV showed an age-associated decrease in the density of NR2b-immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi-quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1-colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co-expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in

  10. Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation through activating the NR2B subunits of NMDA receptors

    SciTech Connect

    Shi, Wen-Zhu; Miao, Yu-Liang; Guo, Wen-Zhi; Wu, Wei; Li, Bao-Wei; An, Li-Na; Fang, Wei-Wu; Mi, Wei-Dong

    2014-04-25

    Highlights: • Leptin promotes the proliferation of neural stem cells isolated from embryonic mouse hippocampus. • Leptin reverses corticosterone-induced inhibition of neural stem cell proliferation. • The effects of leptin are partially mediated by upregulating NR2B subunits. - Abstract: Corticosterone inhibits the proliferation of hippocampal neural stem cells (NSCs). The removal of corticosterone-induced inhibition of NSCs proliferation has been reported to contribute to neural regeneration. Leptin has been shown to regulate brain development, improve angiogenesis, and promote neural regeneration; however, its effects on corticosterone-induced inhibition of NSCs proliferation remain unclear. Here we reported that leptin significantly promoted the proliferation of hippocampal NSCs in a concentration-dependent pattern. Also, leptin efficiently reversed the inhibition of NSCs proliferation induced by corticosterone. Interestingly, pre-treatment with non-specific NMDA antagonist MK-801, specific NR2B antagonist Ro 25-6981, or small interfering RNA (siRNA) targeting NR2B, significantly blocked the effect of leptin on corticosterone-induced inhibition of NSCs proliferation. Furthermore, corticosterone significantly reduced the protein expression of NR2B, whereas pre-treatment with leptin greatly reversed the attenuation of NR2B expression caused by corticosterone in cultured hippocampal NSCs. Our findings demonstrate that leptin reverses the corticosterone-induced inhibition of NSCs proliferation. This process is, at least partially mediated by increased expression of NR2B subunits of NMDA receptors.

  11. Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

    SciTech Connect

    Xu Xiaohong Ye Yinping; Li Tao; Chen Lei; Tian Dong; Luo Qingqing; Lu Mei

    2010-12-01

    Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) {beta} and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ER{beta} were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17{beta}-estradiol (17{beta}-E{sub 2}) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17{beta}-E{sub 2}. The present results suggest that BPA, like 17{beta}-E{sub 2}, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17{beta}-E{sub 2} when it coexists with 17{beta}-E{sub 2}. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

  12. Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression

    PubMed Central

    Feyissa, Anteneh M.; Zyga, Agata; Stockmeier, Craig A.; Karolewicz, Beata

    2009-01-01

    Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants. PMID:18992785

  13. Conantokins Derived from the Asprella Clade Impart ConRl-B, an NMDA Receptor Antagonist with a Unique Selectivity Profile for NR2B Subunits

    PubMed Central

    Gowd, Konkallu Hanumae; Han, Tiffany S.; Twede, Vernon; Gajewiak, Joanna; Smith, Misty D.; Watkins, Maren; Platt, Randall J.; Toledo, Gabriela; White, H. Steve; Olivera, Baldomero M.; Bulaj, Grzegorz

    2014-01-01

    Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity to the most studied NR2B-selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, conRl-B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products. PMID:22594498

  14. Acute hypoxia differentially affects the NMDA receptor NR1, NR2A and NR2B subunit mRNA levels in the developing chick optic tectum: stage-dependent plasticity in the 2B-2A ratio.

    PubMed

    Vacotto, Marina; Rapacioli, Melina; Flores, Vladimir; de Plazas, Sara Fiszer

    2010-10-01

    It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis. PMID:20596770

  15. Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

    PubMed

    Moraga-Amaro, Rodrigo; González, Hugo; Ugalde, Valentina; Donoso-Ramos, Juan Pablo; Quintana-Donoso, Daisy; Lara, Marcelo; Morales, Bernardo; Rojas, Patricio; Pacheco, Rodrigo; Stehberg, Jimmy

    2016-04-01

    Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. PMID:26714288

  16. Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo.

    PubMed

    Arias, Clorinda; Montiel, Teresa; Peña, Fernando; Ferrera, Patricia; Tapia, Ricardo

    2002-09-01

    Overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors is closely related to epilepsy and excitotoxicity, and the phosphorylation of these receptors may facilitate glutamate-mediated synaptic transmission. Here we show that in awake rats the microinjection into the hippocampus of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, induces in about 20 min intense electroencephalographic and behavioral limbic-type seizures, which are suppressed by the systemic administration of the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate and by the intrahippocampal administration of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinases. Two hours after okadaic acid, when the EEG seizures were intense, an increased serine phosphorylation of some hippocampal proteins, including an enhancement of the serine phosphorylation of the NMDA receptor subunit NR2B, was detected by immunoblotting. Twenty-four hours after okadaic acid a marked destruction of hippocampal CA1 region was observed, which was not prevented by the receptor antagonists. These findings suggest that hyperphosphorylation of glutamate receptors in vivo may result in an increased sensitivity to the endogenous transmitter and therefore induce neuronal hyperexcitability and epilepsy. PMID:12429230

  17. Dual contribution of NR2B subunit of NMDA receptor and SK3 Ca(2+)-activated K+ channel to genetic predisposition to anorexia nervosa.

    PubMed

    Koronyo-Hamaoui, Maya; Frisch, Amos; Stein, Daniel; Denziger, Yardena; Leor, Shani; Michaelovsky, Elena; Laufer, Neil; Carel, Cynthia; Fennig, Silvana; Mimouni, Mark; Ram, Anca; Zubery, Eynat; Jeczmien, Pablo; Apter, Alan; Weizman, Abraham; Gak, Eva

    2007-01-01

    Since identification of the genetic component in anorexia nervosa (AN), genes that partake in serotonergic and dopaminergic systems and in hormonal and weight regulation have been suggested as potential candidates for AN susceptibility. We propose another set of candidate genes. Those are genes that are involved in the signaling pathway using NMDA-R and SK channels and have been suggested as possible effectors of NMDA-R driven signaling. The role of NMDA-R in the etiology of schizophrenia has already been substantiated on various levels. Several studies based on population and family groups have implicated SK3 in schizophrenia and more recently in AN as well. Our study group consisted of 90 AN family trios. We examined the transmission of two potentially functional polymorphisms, 5073T>G polymorphism in the gene encoding the NR2B subunit of NMDA-R and CAG repeats in the coding region of SK3 channel gene. Using HHRR and TDT approaches, we found that both polymorphisms were preferentially transmitted to AN offspring (TDT yielded chi(2)=5.01, p=0.025 for NR2B 5073G alleles and chi(2)=11.75, p<0.001 for SK3 L alleles including >19 repeats). Distribution analysis of the combined NR2B/SK3 genotypes suggests that the contribution of both polymorphisms to AN risk is independent and cumulative (OR=2.44 for NR2B GG genotype and OR=3.01 for SK3 SL and LL genotypes, and OR=6.8 for the combined NR2B/SK3 genotypes including high-risk alleles). These findings point to the contribution of genes associated with the NMDA-R signaling pathway to predisposition and development of AN. PMID:16157352

  18. Co-activation of NR2A and NR2B subunits induces resistance to fear extinction.

    PubMed

    Leaderbrand, Katherine; Corcoran, Kevin A; Radulovic, Jelena

    2014-09-01

    Unpredictable stress is known to profoundly enhance susceptibility to fear and anxiety while reducing the ability to extinguish fear when threat is no longer present. Accordingly, partial aversive reinforcement, via random exposure to footshocks, induces fear that is resistant to extinction. Here we sought to determine the hippocampal mechanisms underlying susceptibility versus resistance to context fear extinction as a result of continuous (CR) and partial (PR) reinforcement, respectively. We focused on N-methyl-D-aspartate receptor (NMDAR) subunits 2A and B (NR2A and NR2B) as well as their downstream signaling effector, extracellular signal-regulated kinase (ERK), based on their critical role in the acquisition and extinction of fear. Pharmacological inactivation of NR2A, but not NR2B, blocked extinction after CR, whereas inactivation of NR2A, NR2B, or both subunits facilitated extinction after PR. The latter finding suggests that co-activation of NR2A and NR2B contributes to persistent fear following PR. In contrast to CR, PR increased membrane levels of ERK and NR2 subunits after the conditioning and extinction sessions, respectively. In parallel, nuclear activation of ERK was significantly reduced after the extinction session. Thus, co-activation and increased surface expression of NR2A and NR2B, possibly mediated by ERK, may cause persistent fear. These findings suggest that patients with post-traumatic stress disorder (PTSD) may benefit from antagonism of specific NR2 subunits. PMID:24055686

  19. Effects of diazoxide on Aβ1-42-induced expression of the NR2B subunit in cultured cholinergic neurons.

    PubMed

    Zhu, Jin; Fu, Qingxi; Xia, Chunfeng; Ma, Guozhao

    2015-12-01

    The accumulation of amyloid-β protein (Aβ) is significant in the pathogenesis of Alzheimer's disease. Several previous studies indicate that the NR2B‑containing N‑methyl‑D‑aspartate receptors are critically involved in the Aβ mediated disruption of neuronal function. Diazoxide (DZ), a highly selective drug capable of opening mitochondrial ATP‑sensitive potassium channels, has neuroprotective effects against neuronal cell death. However, the mechanism by which DZ protects cholinergic neurons against Aβ‑induced cytotoxicity remains to be elucidated. The present study was designed to investigate the effects of DZ pretreatment against Aβ1‑42‑induced expression of NR2B in order to gain novel insights into the neuroprotective mechanisms. Following exposure to Aβ1‑42 for 24 h, the expression of the NR2B subunit remained unchanged compared with the control group. However, a significant increase in the expression of the NR2B subunit was observed following treatment with Aβ1‑42 for 72 h (P<0.05); and the upregulation of the expression of the NR2B subunit was reversed by pretreatment with DZ (P<0.05). These results suggested that DZ may counteract Aβ1‑42‑mediated cytotoxicity by alleviating the expression of NR2B. PMID:26496862

  20. Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology.

    PubMed

    Rodrigues, Marília Danyelle Nunes; Seminotti, Bianca; Amaral, Alexandre Umpierrez; Leipnitz, Guilhian; Goodman, Stephen Irwin; Woontner, Michael; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2015-12-15

    Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients. PMID:26671102

  1. NR2B subunit in the prefrontal cortex: A double-edged sword for working memory function and psychiatric disorders.

    PubMed

    Monaco, Sarah A; Gulchina, Yelena; Gao, Wen-Jun

    2015-09-01

    The prefrontal cortex (PFC) is a brain region featured with working memory function. The exact mechanism of how working memory operates within the PFC circuitry is unknown, but persistent neuronal firing recorded from prefrontal neurons during a working memory task is proposed to be the neural correlate of this mnemonic encoding. The PFC appears to be specialized for sustaining persistent firing, with N-methyl-D-aspartate (NMDA) receptors, especially slow-decay NR2B subunits, playing an essential role in the maintenance of sustained activity and normal working memory function. However, the NR2B subunit serves as a double-edged sword for PFC function. Because of its slow kinetics, NR2B endows the PFC with not only "neural psychic" properties, but also susceptibilities for neuroexcitotoxicity and psychiatric disorders. This review aims to clarify the interplay among working memory, the PFC, and NMDA receptors; demonstrate the importance of NR2B in the maintenance of persistent activity; understand the risks and vulnerabilities of how NR2B is related to the development of neuropsychiatric disorders; identify gaps that currently exist in our understanding of these processes; and provide insights regarding future directions that may clarify these issues. We conclude that the PFC is a specialized brain region with distinct delayed maturation, unique neuronal circuitry, and characteristic NMDA receptor function. The unique properties and development of NMDA receptors, especially enrichment of NR2B subunits, endow the PFC with not only the capability to generate sustained activity for working memory, but also serves as a major vulnerability to environmental insults and risk factors for psychiatric disorders. PMID:26143512

  2. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    SciTech Connect

    Li, Mei; Zhang, Dong-Qing; Wang, Xiang-Zhen; Xu, Tie-Jun

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  3. Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential.

    PubMed

    Mony, Laetitia; Kew, James N C; Gunthorpe, Martin J; Paoletti, Pierre

    2009-08-01

    N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson's and Alzheimer's disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H+, Mg2+, Zn2+, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives ('prodils') are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity. PMID:19594762

  4. Both NR2A and NR2B Subunits of the NMDA Receptor Are Critical for Long-Term Potentiation and Long-Term Depression in the Lateral Amygdala of Horizontal Slices of Adult Mice

    ERIC Educational Resources Information Center

    Muller, Tobias; Albrecht, Doris; Gebhardt, Christine

    2009-01-01

    The lateral nucleus of the amygdala (LA) is implicated in emotional and social behaviors. We recently showed that in horizontal brain slices, activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations in the LA, such as long-term potentiation (LTP) and long-term depression (LTD). In the LA, NR2A- and NR2B-type NMDRs…

  5. Kynurenic acid amides as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, István; Kolok, Sándor; Galgóczy, Kornél; Gere, Anikó; Horváth, Csilla; Farkas, Sándor; Greiner, István; Domány, György

    2007-01-15

    A novel series of kynurenic acid amides, ring-enlarged derivatives of indole-2-carboxamides, was prepared and identified as in vivo active NR2B subtype selective NMDA receptor antagonists. The synthesis and SAR studies are discussed. PMID:17074483

  6. Traxoprodil, a selective antagonist of the NR2B subunit of the NMDA receptor, potentiates the antidepressant-like effects of certain antidepressant drugs in the forced swim test in mice.

    PubMed

    Poleszak, Ewa; Stasiuk, Weronika; Szopa, Aleksandra; Wyska, Elżbieta; Serefko, Anna; Oniszczuk, Anna; Wośko, Sylwia; Świąder, Katarzyna; Wlaź, Piotr

    2016-08-01

    One of the newest substances, whose antidepressant activity was shown is traxoprodil, which is a selective antagonist of the NR2B subunit of the NMDA receptor. The main goal of the present study was to evaluate the effect of traxoprodil on animals' behavior using the forced swim test (FST), as well as the effect of traxoprodil (10 mg/kg) on the activity of antidepressants, such as imipramine (15 mg/kg), fluoxetine (5 mg/kg), escitalopram (2 mg/kg) and reboxetine (2.5 mg/kg). Serotonergic lesion and experiment using the selective agonists of serotonin receptors 5-HT1A and 5-HT2 was conducted to evaluate the role of the serotonergic system in the antidepressant action of traxoprodil. Brain concentrations of tested agents were determined using HPLC. The results showed that traxoprodil at a dose of 20 and 40 mg/kg exhibited antidepressant activity in the FST and it was not related to changes in animals' locomotor activity. Co-administration of traxoprodil with imipramine, fluoxetine or escitalopram, each in subtherapeutic doses, significantly affected the animals' behavior in the FST and, what is important, these changes were not due to the severity of locomotor activity. The observed effect of traxoprodil is only partially associated with serotonergic system and is independent of the effect on the 5-HT1A and 5-HT2 serotonin receptors. The results of an attempt to assess the nature of the interaction between traxoprodil and the tested drugs show that in the case of joint administration of traxoprodil and fluoxetine, imipramine or escitalopram, there were interactions in the pharmacokinetic phase. PMID:26924124

  7. Phosphorylation of NR2B NMDA subunits by protein kinase C in arcuate nucleus contributes to inflammatory pain in rats

    PubMed Central

    Bu, Fan; Tian, Huiyu; Gong, Shan; Zhu, Qi; Xu, Guang-Yin; Tao, Jin; Jiang, Xinghong

    2015-01-01

    The arcuate nucleus (ARC) of the hypothalamus plays a key role in pain processing. Although it is well known that inhibition of NMDA receptor (NMDAR) in ARC attenuates hyperalgesia induced by peripheral inflammation, the underlying mechanism of NMDAR activation in ARC remains unclear. Protein kinase C (PKC) is involved in several signalling cascades activated in physiological and pathological conditions. Therefore, we hypothesised that upregulation of PKC activates NMDARs in the ARC, thus contributing to inflammatory hyperalgesia. Intra-ARC injection of chelerythrine (CC), a specific PKC inhibitor, attenuated complete Freund’s adjuvant (CFA) induced thermal and mechanical hyperalgesia in a dose-dependent manner. In vivo extracellular recordings showed that microelectrophoresis of CC or MK-801 (a NMDAR antagonist) significantly reduced the enhancement of spontaneous discharges and pain-evoked discharges of ARC neurons. In addition, CFA injection greatly enhanced the expression of total and phosphorylated PKCγ in the ARC. Interestingly, CFA injection also remarkably elevated the level of phosphorylated NR2B (Tyr1472) without affecting the expression of total NR2B. Importantly, intra-ARC injection of CC reversed the upregulation of phosphorylated NR2B subunits in the ARC. Taken together, peripheral inflammation leads to an activation of NMDARs mediated by PKC activation in the ARC, thus producing thermal and mechanical hyperalgesia. PMID:26515544

  8. NR2A- and NR2B-Containing NMDA Receptors in the Prelimbic Medial Prefrontal Cortex Differentially Mediate Trace, Delay, and Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Gilmartin, Marieke R.; Kwapis, Janine L.; Helmstetter, Fred J.

    2013-01-01

    Activation of "N"-methyl-D-aspartate receptors (NMDAR) in the prelimbic medial prefrontal cortex (PL mPFC) is necessary for the acquisition of both trace and contextual fear memories, but it is not known how specific NR2 subunits support each association. The NR2B subunit confers unique properties to the NMDAR and may differentially…

  9. New benzoyl urea derivatives as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, I; Greiner, I; Kolok, S; Galgóczy, K; Ignácz-Szendrei, Gy; Horváth, Cs; Farkas, S; Gáti, T; Háda, V; Domány, Gy

    2006-09-01

    A novel series of benzoyl urea derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of the substitution of the piperidine ring on the biological activity of the compounds was studied. Compound 9 was active in the formalin test in mice. PMID:17020160

  10. Benzimidazole-2-carboxamides as novel NR2B selective NMDA receptor antagonists.

    PubMed

    Borza, István; Kolok, Sándor; Gere, Anikó; Nagy, József; Fodor, László; Galgóczy, Kornél; Fetter, József; Bertha, Ferenc; Agai, Béla; Horváth, Csilla; Farkas, Sándor; Domány, György

    2006-09-01

    A novel series of benzimidazole-2-carboxamide derivatives was prepared and identified as NR2B selective NMDA receptor antagonists. The influence of some structural elements, like H-bond donor groups placed on the benzimidazole skeleton and the substitution pattern of the piperidine ring, on the biological activity was studied. Compound 6a showed excellent analgetic activity in the mouse formalin test following po administration. PMID:16782335

  11. Dexmedetomidine protects against learning and memory impairments caused by electroconvulsive shock in depressed rats: Involvement of the NMDA receptor subunit 2B (NR2B)-ERK signaling pathway.

    PubMed

    Gao, Xin; Zhuang, Fu-Zhi; Qin, Shou-Jun; Zhou, Li; Wang, Yun; Shen, Qing-Feng; Li, Mei; Villarreal, Michelle; Benefield, Lauren; Gu, Shu-Ling; Ma, Teng-Fei

    2016-09-30

    Cognitive impairment is a common adverse effect of electroconvulsive therapy (ECT) during treatment for severe depression. Dexmedetomidine (DEX), a sedative-anesthetic drug, is used to treat post-ECT agitation. However, it is not known if DEX can protect against ECT-induced cognitive impairments. To address this, we used chronic unpredictable mild stress (CUMS) to establish a model of depression for ECT treatment. Our Morris water maze and sucrose preference test results suggest that DEX alleviates ECT-induced learning and memory impairments without altering the antidepressant efficacy of ECT. To further investigate the underlying mechanisms of DEX, hippocampal expression of NR2B, p-ERK/ERK, p-CREB/CREB, and BDNF were quantified by western blotting. These results show that DEX suppresses over-activation of NR2B and enhances phosphorylation of ERK1/2 in the hippocampus of ECT-treated depressed rats. Furthermore, DEX had no significant effect on ECT-induced increases in p-CREB and BDNF. Overall, our findings suggest that DEX ameliorates ECT-induced learning and memory impairments in depressed rats via the NR2B-ERK signaling cascade. Moreover, CREB/BDNF seems not appear to participate in the cognitive protective mechanisms of DEX during ECT treatment. PMID:27455425

  12. Amygdala Infusions of an NR2B-Selective or an NR2A-Preferring NMDA Receptor Antagonist Differentially Influence Fear Conditioning and Expression in the Fear-Potentiated Startle Test

    ERIC Educational Resources Information Center

    Walker, David L.; Davis, Michael

    2008-01-01

    Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the…

  13. Overactivation of NR2B-containing NMDA receptors through entorhinal-hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion.

    PubMed

    Xu, Cheng-Shi; Liu, An-Chun; Chen, Juan; Pan, Zhi-Yong; Wan, Qi; Li, Zhi-Qiang; Wang, Ze-Fen

    2015-08-01

    Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase-3β at Ser 9 in the ipsilateral hippocampus. These MCAO-induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N-methyl-d-aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B-containing NMDARs through entorhinal-hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase-3β is an important protein kinase involved in NMDARs-mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B-containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post-stroke dementia. Middle cerebral artery occlusion induces secondary damage in the hippocampus that is remote from primary ischemic regions. We propose that excessive activation of NR2B-containing NMDA receptors through entorhinal-hippocampal connection initiated the accumulation of hyperphosphorylated tau in the hippocampus, which subsequently induced cognitive deficit. This study provides new insights into the prospects of NR2B inhibition in stoke therapy. PMID:25903928

  14. Distinct roles of NR2A and NR2B cytoplasmic tails in long term potentiation

    PubMed Central

    Foster, Kelly A.; McLaughlin, Nathan; Edbauer, Dieter; Phillips, Marnie; Bolton, Andrew; Constantine-Paton, Martha; Sheng, Morgan

    2010-01-01

    NMDA receptors (NMDARs) are critical mediators of activity-dependent synaptic plasticity, but the differential roles of NR2A- versus NR2B-containing NMDARs have been controversial. Here, we investigate the roles of NR2A and NR2B in LTP in organotypic hippocampal slice cultures using RNAi and overexpression, to complement pharmacological approaches. In young slices, when NR2B is the predominant subunit expressed, LTP is blocked by the NR2B-selective antagonist Ro25-6981. As slices mature, and NR2A expression rises, activation of NR2B receptors became no longer necessary for LTP induction. LTP was blocked, however, by RNAi knockdown of NR2B, and this was rescued by coexpression of an RNAi-resistant NR2B (NR2B*) cDNA. Interestingly, a chimeric NR2B subunit in which the C-terminal cytoplasmic tail was replaced by that of NR2A failed to rescue LTP while the reverse chimera, NR2A channel with NR2B tail, was able to restore LTP. Thus expression of NR2B with its intact cytoplasmic tail is required for LTP induction, at an age when channel activity of NR2B-NMDARs is not required for LTP. Overexpression of wildtype NR2A failed to rescue LTP in neurons transfected with NR2B-RNAi construct, despite restoring NMDA-EPSC amplitude to a similar level as NR2B*. Surprisingly, an NR2A construct lacking its entire C-terminal cytoplasmic tail regained its ability to restore LTP. Together these data suggest that the NR2B subunit plays a critical role for LTP, presumably by recruiting relevant molecules important for LTP via its cytoplasmic tail. By contrast, NR2A is not essential for LTP and its cytoplasmic tail seems to carry inhibitory factors for LTP. PMID:20164351

  15. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains.

    PubMed

    Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

    2016-01-01

    Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

  16. A Novel Protein Complex in Membrane Rafts Linking the NR2B Glutamate Receptor and Autophagy Is Disrupted following Traumatic Brain Injury

    PubMed Central

    Bigford, Gregory E.; Alonso, Ofelia F.; Dietrich, W. Dalton

    2009-01-01

    Abstract Hyperactivation of N-methyl-d-aspartate receptors (NRs) is associated with neuronal cell death induced by traumatic brain injury (TBI) and many neurodegenerative conditions. NR signaling efficiency is dependent on receptor localization in membrane raft microdomains. Recently, excitotoxicity has been linked to autophagy, but mechanisms governing signal transduction remain unclear. Here we have identified protein interactions between NR2B signaling intermediates and the autophagic protein Beclin-1 in membrane rafts of the normal rat cerebral cortex. Moderate TBI induced rapid recruitment and association of NR2B and pCaMKII to membrane rafts, and translocation of Beclin-1 out of membrane microdomains. Furthermore, TBI caused significant increases in expression of key autophagic proteins and morphological hallmarks of autophagy that were significantly attenuated by treatment with the NR2B antagonist Ro 25-6981. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin-1 in membrane rafts after TBI. PMID:19335206

  17. Activation of spinal MrgC-Gi-NR2B-nNOS signaling pathway by Mas oncogene-related gene C receptor agonist bovine adrenal medulla 8-22 attenuates bone cancer pain in mice

    PubMed Central

    Sun, Yu’e; Zhang, Juan; Lei, Yishan; Lu, Cui’e; Hou, Bailing; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objectives: In the present study, we investigate the effects of Mas oncogene-related gene (Mrg) C receptors (MrgC) on the expression and activation of spinal Gi protein, N-methyl-D-aspartate receptor subunit 2B (NR2B), and neuronal nitric oxide synthase (nNOS) in mouse model of bone cancer pain. Methods: The number of spontaneous foot lift (NSF) and paw withdrawal mechanical threshold (PWMT) were measured after inoculation of tumor cells and intrathecal injection of MrgC agonist bovine adrenal medulla 8-22 (BAM8-22) or MrgC antagonist anti-MrgC for 14 days after operation. Expression of spinal MrgC, Gi protein, NR2B and nNOS and their phosphorylated forms after inoculation was examined by immunohistochemistry and Western blotting. Double labeling was used to identify the co-localization of NR2B or nNOS with MrgC in spinal cord dorsal horn (SCDH) neurons. The effects of intrathecal injection of BAM8-22 or anti-MrgC on nociceptive behaviors and the corresponding expression of spinal MrgC, Gi protein, NR2B and nNOS were also investigated. Results: The expression of spinal MrgC, Gi protein, NR2B, and nNOS was higher in tumor-bearing mice in comparison to sham mice or normal mice. Intrathecal injection of MrgC agonist BAM8-22 significantly alleviated bone cancer pain, up-regulated MrgC and Gi protein expression, and down-regulated the expression of spinal p-NR2B, t-nNOS and p-nNOS in SCDH on day 14 after operation, whereas administration of anti-MrgC produced the opposite effect. Meanwhile, MrgC-like immunoreactivity (IR) co-localizes with NR2B-IR or nNOS-IR in SCDH neurons. Conclusions: The present study demonstrates that MrgC-activated spinal Gi-NR2B-nNOS signaling pathway plays important roles in the development of bone cancer pain. These findings may provide a novel strategy for the treatment of bone cancer pain. PMID:27158400

  18. Inhibition of NR2B-Containing N-methyl-D-Aspartate Receptors (NMDARs) in Experimental Autoimmune Encephalomyelitis, a Model of Multiple Sclerosis

    PubMed Central

    Farjam, Mojtaba; Beigi Zarandi, Faegheh Baha'addini; Farjadian, Shirin; Geramizadeh, Bita; Nikseresht, Ali Reza; Panjehshahin, Mohammad Reza

    2014-01-01

    Neurodegeneration is the pathophysiological basis for permanent neurological disabilities in multiple sclerosis (MS); thus neuroprotection is emerging as a therapeutic approach in MS research. Modulation of excitotoxicity by inhibition of NMDARs has been suggested for neuroprotection, but selective antagonisation of the NR2B subtype of these receptors, a subtype believed to play a more pivotal role in neurodegeneration, has not been tested in MS. In this study inhibition of NR2B-containing NMDAR was evaluated on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). EAE induction was done using MOG in C57BL/6 mice. Therapeutic administration of different doses of highly selective NR2B-containing NMDAR inhibitor (RO25-6981) was compared with memantine (non-selective NMDAR antagonist) and vehicle. Neurological deficits in EAE animals were more efficiently decreased by selective inhibition of NR2B-containing NMDARs. Histological studies of the spinal cords also showed decreased inflammation, myelin degradation and neuro-axonal degeneration when RO25-6981was administered with higher doses. The effects were dose dependent. Regarding the role of NR2B-containing NMDARs in excitotoxicity, selective inhibition of these receptor subtypes seems to modulate the neurological disabilities and pathological changes in EAE. Further elucidation of the exact mechanism of action as well as more experimental studies can suggest NR2B-containing NMDAR inhibition as a potentially effective treatment strategy for slowing down the clinical deterioration of disability in MS. PMID:25237366

  19. DIFFERENTIAL ROLE OF NR2A AND NR2B NMDA RECEPTORS IN MEDIATING PHENCYCLIDINE-INDUCED PERINATAL NEURONAL APOPTOSIS AND BEHAVIORAL DEFICITS

    PubMed Central

    Anastasio, Noelle C.; Xia, Yan; O’Connor, Zoe R.; Johnson, Kenneth M.

    2009-01-01

    The mechanism underlying PCP-induced apoptosis in perinatal rats and the development of schizophrenic-like behaviors is incompletely understood. We used antagonists for NR2A- and NR2B-containing NMDARs to test the hypothesis that the behavioral and apoptotic effects of PCP are mediated by blockade of NR1/NR2A-containing receptors, rather than NR1/NR2B-containing receptors. Sprague-Dawley rats were treated on PN7, 9, and 11 with PCP (10 mg/kg), PEAQX (NR2A-preferring antagonist, 10, 20, or 40 mg/kg), or ifenprodil (selective NR2B antagonist, 1, 5, or 10 mg/kg) and sacrificed for measurement of caspase-3 activity (an index of apoptosis) or allowed to age and tested for locomotor sensitization to PCP challenge on PN28-35. PCP or PEAQX on PN7, 9, and 11 markedly elevated caspase-3 activity in the cortex; ifenprodil showed no effect. Striatal apoptosis was evident only after sub-chronic treatment with a high dose of PEAQX (20 mg/kg). Animals treated with PCP or PEAQX on PN7, 9 and 11 showed a sensitized locomotor response to PCP challenge on PN28-35. Ifenprodil treatment had no effect on either measure. Therefore, PCP blockade of cortical NR1/NR2A, rather than NR1/NR2B, appears to be responsible for PCP-induced apoptosis and the development of long-lasting behavioral deficits. PMID:19654040

  20. Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli.

    PubMed

    Ng, Fui-Mee; Soh, Wanqin; Geballe, Matthew T; Low, Chian-Ming

    2007-10-12

    The amino-terminal domains (ATDs) of N-methyl-d-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1mg/ml) exists as monodispersed species at 25 degrees C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64nM) and presence (116nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies. PMID:17706601

  1. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene.

    PubMed

    Fujita, Yosuke; Morinobu, Shigeru; Takei, Shiro; Fuchikami, Manabu; Matsumoto, Tomoya; Yamamoto, Shigeto; Yamawaki, Shigeto

    2012-05-01

    Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction. PMID:22364833

  2. SRC Inhibition Reduces NR2B Surface Expression and Synaptic Plasticity in the Amygdala

    ERIC Educational Resources Information Center

    Sinai, Laleh; Duffy, Steven; Roder, John C.

    2010-01-01

    The Src protein tyrosine kinase plays a central role in the regulation of N-methyl-d-aspartate receptor (NMDAR) activity by regulating NMDAR subunit 2B (NR2B) surface expression. In the amygdala, NMDA-dependent synaptic plasticity resulting from convergent somatosensory and auditory inputs contributes to emotional memory; however, the role of Src…

  3. The Tau/A152T mutation, a risk factor for frontotemporal-spectrum disorders, leads to NR2B receptor-mediated excitotoxicity.

    PubMed

    Decker, Jochen Martin; Krüger, Lars; Sydow, Astrid; Dennissen, Frank Ja; Siskova, Zuzana; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2016-04-01

    We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate. PMID:26931569

  4. Protective immunity of rAd5/NR2B vaccine against concomitant aversiveness of spontaneous neuropathic pain following spinal nerve ligation injury

    PubMed Central

    Wang, Gong-Ming; Wang, Xiao-Yan; Liu, Guang-Jie; Cheng, Kun; Wang, Hua; Guo, Shou-Gang

    2015-01-01

    Objective: Peripheral nerve injury elicits an aversive state of spontaneous neuropathic pain, and up to now, the modulation of this concomitant aversive state remains a major therapeutic challenge. NMDA receptor subunits NR2B in the rACC are critically involved in the processing of this aversive state and then a strategy targeted at the NR2B subunit might be promising for modulation of the aversive state. Thus, in the present study, using negative reinforcement animal model to reveal spontaneous pain, we investigated the effect of oral immunization with recombinant adenovirus serotype 5-mediated NR2B gene transfer (rAd5/NR2B) on the modulation of the tonic pain. Material and methods: Following oral administration of the rAd5/NR2B vaccine, NR2B-specific antibodies were induced in serum. And the humoral response was involved in the decreased expression of NR2B protein in the rACC. Results: The present study demonstrated that CPP achieved by spinal administration of clonidine in spinal nerve ligation (SNL) rats revealed the presence of aversive state of spontaneous neuropathic pain. Notably, the humoral autoimmune response blocked the CPP by spinal clonidine, suggesting the relief of the concomitant aversive of spontaneous neuropathic pain in the SNL rats. Conclusion: These data proved the feasibility of oral immunization with rAd5/NR2B for modulation of concomitant aversive of spontaneous neuropathic pain due to peripheral nerve injury. PMID:26309509

  5. Unprecedented Therapeutic Potential with a Combination of A2A/NR2B Receptor Antagonists as Observed in the 6-OHDA Lesioned Rat Model of Parkinson's Disease

    PubMed Central

    Michel, Anne; Downey, Patrick; Nicolas, Jean-Marie; Scheller, Dieter

    2014-01-01

    In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients. PMID:25513815

  6. Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

    PubMed

    Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun

    2016-05-01

    Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation. PMID:25917873

  7. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    PubMed Central

    Ba, Maowen; Kong, Min; Ma, Guozhao

    2015-01-01

    Context Abnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated. Methods In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results Levodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the

  8. Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

    PubMed Central

    Gu, Xiaoping; Zheng, Yaguo; Sun, Yu-e; Liang, Ying; Bo, Jinhua; Ma, Zhengliang

    2012-01-01

    Background The root of Polygala tenuifolia, a traditional Chinese medicine, has been used to improve memory and intelligence, while the underlying mechanisms remain largely unknown. In this study, we investigated the protective effects of senegenin, an component of Polygala tenuifolia root extracts, on cognitive dysfunction induced by hepatic ischemia-reperfusion. Methodology/Principal Findings Initially, we constructed a rat model of hepatic ischemia-reperfusion (HIR) and found that the memory retention ability of rats in the step-down and Y maze test was impaired after HIR, paralleled by a decrease of N-methyl-D-aspartate (NMDA) receptor NR2B subunit mRNA and protein expressions in hippocampus. Furthermore, we found that administration of senegenin by gavage attenuated HIR-induced cognitive impairment in a dose and time dependent manner, and its mechanisms might partly due to the increasing expression of NR2B in rat hippocampus. Conclusions/Significance Cognitive dysfunction induced by HIR is associated with reduction of NR2B expression. Senegenin plays a neuroprotective role in HIR via increasing NR2B expression in rat hippocampus. These findings suggest that senegenin might be a potential agent for prevention and treatment of postoperative cognitive dysfunction (POCD) or other neurodegenerative diseases. PMID:23029109

  9. Endogenous ephrinB2 mediates colon-urethra cross-organ sensitization via Src kinase-dependent tyrosine phosphorylation of NR2B.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Hung; Tung, Kwong-Chung; Chang, Junn-Liang; Lin, Tzer-Bin

    2010-01-01

    Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram activity evoked by pelvic nerve stimulation and protein expression in the lumbosacral (L6-S2) dorsal horn were studied before and after intracolonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization along with an upregulation of endogenous ephrinB2 expression as well as phosphorylation of EphB 1/2, Src-family kinase, and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest that EphBR-ephrinB interactions, which provoke Src-family kinase-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level, are involved in cross-organ sensitization, contributing to the development of viscero-visceral referred pain between the bowel and the urethra. PMID:19864302

  10. Chronic Administration of Benzo(a)pyrene Induces Memory Impairment and Anxiety-Like Behavior and Increases of NR2B DNA Methylation

    PubMed Central

    Zhang, Wenping; Tian, Fengjie; Zheng, Jinping; Li, Senlin; Qiang, Mei

    2016-01-01

    Background Recently, an increasing number of human and animal studies have reported that exposure to benzo(a)pyrene (BaP) induces neurological abnormalities and is also associated with adverse effects, such as tumor formation, immunosuppression, teratogenicity, and hormonal disorders. However, the exact mechanisms underlying BaP-induced impairment of neurological function remain unclear. The aim of this study was to examine the regulating mechanisms underlying the impact of chronic BaP exposure on neurobehavioral performance. Methods C57BL mice received either BaP in different doses (1.0, 2.5, 6.25 mg/kg) or olive oil twice a week for 90 days. Memory and emotional behaviors were evaluated using Y-maze and open-field tests, respectively. Furthermore, levels of mRNA expression were measured by using qPCR, and DNA methylation of NMDA receptor 2B subunit (NR2B) was examined using bisulfate pyrosequencing in the prefrontal cortex and hippocampus. Results Compared to controls, mice that received BaP (2.5, 6.25 mg/kg) showed deficits in short-term memory and an anxiety-like behavior. These behavioral alterations were associated with a down-regulation of the NR2B gene and a concomitant increase in the level of DNA methylation in the NR2B promoter in the two brain regions. Conclusions Chronic BaP exposure induces an increase in DNA methylation in the NR2B gene promoter and down-regulates NR2B expression, which may contribute to its neurotoxic effects on behavioral performance. The results suggest that NR2B vulnerability represents a target for environmental toxicants in the brain. PMID:26901155

  11. Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-D-aspartate receptors☆

    PubMed Central

    Mosley, Cara A.; Myers, Scott J.; Murray, Ernest E.; Santangelo, Rose; Tahirovic, Yesim A.; Kurtkaya, Natalie; Mullasseril, Praseeda; Yuan, Hongjie; Lyuboslavsky, Polina; Le, Phuong; Wilson, Lawrence J.; Yepes, Manuel; Dingledine, Ray; Traynelis, Stephen F.; Liotta, Dennis C.

    2010-01-01

    The synthesis and structure–activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modu-lated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and α1-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood–brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency. PMID:19648014

  12. NR2B-dependent cyclophilin D translocation suppresses the recovery of synaptic transmission after oxygen-glucose deprivation.

    PubMed

    Zhang, Zhihua; Wang, Yongfu; Yan, Shijun; Du, Fang; Yan, Shirley Shidu

    2015-10-01

    N-methyl d-aspartate receptor (NMDA) subunit 2B (NR2B)-containing NMDA receptors and mitochondrial protein cyclophilin D (CypD) are well characterized in mediating neuronal death after ischemia, respectively. However, whether and how NR2B and CypD work together in mediating synaptic injury after ischemia remains elusive. Using an ex vivo ischemia model of oxygen-glucose deprivation (OGD) in hippocampal slices, we identified a NR2B-dependent mechanism for CypD translocation onto the mitochondrial inner membrane. CypD depletion (CypD null mice) prevented OGD-induced impairment in synaptic transmission recovery. Overexpression of neuronal CypD mice (CypD+) exacerbated OGD-induced loss of synaptic transmission. Inhibition of CypD-dependent mitochondrial permeability transition pore (mPTP) opening by cyclosporine A (CSA) attenuated ischemia-induced synaptic perturbation in CypD+ and non-transgenic (non-Tg) mice. The treatment of antioxidant EUK134 to suppress mitochondrial oxidative stress rescued CypD-mediated synaptic dysfunction following OGD in CypD+ slices. Furthermore, OGD provoked the interaction of CypD with P53, which was enhanced in slices overexpressing CypD but was diminished in CypD-null slices. Inhibition of p53 using a specific inhibitor of p53 (pifithrin-μ) attenuated the CypD/p53 interaction following OGD, along with a restored synaptic transmission in both non-Tg and CypD+ hippocampal slices. Our results indicate that OGD-induced CypD translocation potentiates CypD/P53 interaction in a NR2B dependent manner, promoting oxidative stress and loss of synaptic transmission. We also evaluate a new ex vivo chronic OGD-induced ischemia model for studying the effect of oxidative stress on synaptic damage. PMID:26232180

  13. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

    SciTech Connect

    Xu Xiaohong Li Tao; Luo Qingqing; Hong Xing; Xie Lingdan; Tian Dong

    2011-09-01

    Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: > BPA rapidly extended the latency to step down from platform 1 h after footshock. > BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. > ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

  14. Early auditory enrichment with music enhances auditory discrimination learning and alters NR2B protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Cai, Rui; Zhang, Jiping; Sun, Xinde

    2009-01-01

    Previous studies have shown that the functional development of auditory system is substantially influenced by the structure of environmental acoustic inputs in early life. In our present study, we investigated the effects of early auditory enrichment with music on rat auditory discrimination learning. We found that early auditory enrichment with music from postnatal day (PND) 14 enhanced learning ability in auditory signal-detection task and in sound duration-discrimination task. In parallel, a significant increase was noted in NMDA receptor subunit NR2B protein expression in the auditory cortex. Furthermore, we found that auditory enrichment with music starting from PND 28 or 56 did not influence NR2B expression in the auditory cortex. No difference was found in the NR2B expression in the inferior colliculus (IC) between music-exposed and normal rats, regardless of when the auditory enrichment with music was initiated. Our findings suggest that early auditory enrichment with music influences NMDA-mediated neural plasticity, which results in enhanced auditory discrimination learning. PMID:18706452

  15. Cloning, expression, and purification of a recombinant Tat-HA-NR2B9c peptide.

    PubMed

    Zhou, Hai-Hui; Zhang, Ai-Xia; Zhang, Yu; Zhu, Dong-Ya

    2012-10-01

    To design a peptide disrupting the interaction between N-methyl-d-aspartate receptors-2B (NR2B) and postsynaptic density protein-95 (PSD-95), a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide contained a fragment of the cell membrane transduction domain of the human immunodeficiency virus type1 (HIV-1) Tat, a influenza virus hemagglutinin (HA) epitope-tag, and the C-terminal 9 amino acids of NR2B (NR2B9c). We named the chimeric peptide Tat-HA-NR2B9c. The expression plasmid contained a gene fragment encoding the Tat-HA-NR2B9c was ligated to the C-terminal fragment of l-asparaginase (AnsB-C) via a unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in inclusion body in Escherichia coli under isopropyl β-d-1-thiogalactopyranoside (IPTG) and purified by washing with 2M urea, solubilizing in 4M urea, and then ethanol precipitation. The target chimeric peptide Tat-HA-NR2B9c was released from the fusion partner following acid hydrolysis and purified by isoelectric point precipitation and ultrafiltration. SDS-PAGE analysis and MALDI-TOF-MS analysis showed that the purified Tat-HA-NR2B9c was highly homogeneous. Furthermore, we investigated the effects of Tat-HA-NR2B9c on ischemia-induced cerebral injury in the rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion, and found that the peptide reduced infarct size and improved neurological functions. PMID:22944204

  16. Estradiol and the Relationship between Dendritic Spines, NR2B Containing NMDA Receptors, and the Magnitude of Long-Term Potentiation at Hippocampal CA3-CA1 Synapses

    PubMed Central

    Smith, Caroline C.; Vedder, Lindsey C.; McMahon, Lori L.

    2009-01-01

    Summary When circulating estrogen levels decline as a natural consequence of menopause and aging in women, there is an increased incidence of deficits in working memory. In many cases, these deficits are rescued by estrogen replacement therapy. These clinical data therefore highlight the importance of defining the biological pathways linking estrogen to the cellular substrates of learning and memory. It has been known for nearly two decades that estrogen enhances dendritic spine density on apical dendrites of CA1 pyramidal cells in hippocampus, a brain region required for learning. Interestingly, at synapses between CA3-CA1 pyramidal cells, estrogen has also been shown to enhance synaptic NMDA receptor current and the magnitude of long term potentiation, a cellular correlate of learning and memory. Given that synapse density, NMDAR function, and long term potentiation at CA3-CA1 synapses in hippocampus are associated with normal learning, it is likely that modulation of these parameters by estrogen facilitates the improvement in learning observed in rats, primates and humans following estrogen replacement. To facilitate the design of clinical strategies to potentially prevent or reverse the age-related decline in learning and memory during menopause, the relationship between the estrogen-induced morphological and functional changes in hippocampus must be defined and the role these changes play in facilitating learning must be elucidated. The aim of this report is to provide a summary of the proposed mechanisms by which this hormone increases synaptic function and in doing so, it briefly addresses potential mechanisms contributing to the estrogen-induced increase in synaptic morphology and plasticity, as well as important future directions. PMID:19596521

  17. Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

    PubMed

    Liverton, Nigel J; Bednar, Rodney A; Bednar, Bohumil; Butcher, John W; Claiborne, Christopher F; Claremon, David A; Cunningham, Michael; DiLella, Anthony G; Gaul, Stanley L; Libby, Brian E; Lyle, Elizabeth A; Lynch, Joseph J; McCauley, John A; Mosser, Scott D; Nguyen, Kevin T; Stump, Gary L; Sun, Hong; Wang, Hao; Yergey, James; Koblan, Kenneth S

    2007-02-22

    The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease. PMID:17249648

  18. Effects of L-3-n-butylphthalide on cognitive dysfunction and NR2B expression in hippocampus of streptozotocin (STZ)-induced diabetic rats.

    PubMed

    Li, Jie; Zhang, Songyun; Zhang, Lihui; Wang, Ruiying; Wang, Mian

    2015-01-01

    Diabetes mellitus is associated with rapid cognitive decline. Currently, there is no effective treatment for cognitive dysfunction induced by diabetes. L-3-n-Butylphthalide (L-NBP) is a nerve protective drug extracted from seeds of celery, which has been proved to improve learning and memory in vascular dementia animal models by improving microcirculation, protecting mitochondria and increasing long-term potentiation (LTP). NR2B, one of the subunits of N-methyl-D-aspartate receptor, has been proved to be an important factor for the formation of LTP. The study aimed to investigate the role of NR2B in cognitive dysfunction in the rats with type 1 diabetes and define the protective effects of L-NBP on cognition. A rat model of type 1 diabetes was established by a single intraperitoneal injection of streptozotocin at 60 mg/kg. Animals were randomly allocated to four groups: normal control (NC); diabetic control (DC); diabetic + low L-NBP (DL, administered L-NBP 60 mg/kg per day for 12 weeks); and diabetic + high L-NBP (DH, administered L-NBP 120 mg/kg per day, for 12 weeks). After 12 weeks, cognitive and memory changes were investigated in the Morris water maze. The expression of NR2B was assessed by real-time polymerase chain reaction, Western blotting, and immunohistochemistry. Our results indicated that the escape latency was significantly increased and the number of crossing platform was significantly decreased in DC group compared to NC group. Also, the expression of NR2B was significantly declined in DC group. However, compared to DC group, the expression of NR2B of the L-NBP-treated groups was significantly increased and the escape latency was shortened with the DH group being the most obvious. Therefore, L-NBP can improve the cognitive function by up-regulating the expression of NR2B in STZ-diabetic rats, which may provide the direction for future diabetic encephalopathy therapy. PMID:25149651

  19. Effect of Histone Acetylation on N-Methyl-D-Aspartate 2B Receptor Subunits and Interleukin-1 Receptors in Association with Nociception-Related Somatosensory Cortex Dysfunction in a Mouse Model of Sepsis.

    PubMed

    Imamura, Yukio; Yoshikawa, Nao; Murkami, Yuki; Mitani, Satoko; Matsumoto, Naoya; Matsumoto, Hisatake; Yamada, Tomoki; Yamakawa, Kazuma; Nakagawa, Junichiro; Ogura, Hiroshi; Shimazu, Takeshi; Jin, Takashi

    2016-06-01

    Whole-body inflammation (i.e., sepsis) often results in brain-related sensory dysfunction. We previously reported that interleukin (IL)-1 resulted in synaptic dysfunction of septic encephalopathy, but the underlying molecular mechanisms remain unknown, as do effective treatments. Using mice, we examined immunohistochemistry, co-immunoprecipitation, enzyme-linked immunosorbent assay, and behavior analyses, and investigated the role of the N-methyl-D-aspartate 2B subunit (NR2B) of NMDA receptor, IL-1 receptor, and histone acetylation in the pathophysiology underlying sensory dysfunction induced by lipopolysaccharide (LPS). Mice groups of sham-operated, LPS, LPS with an NR2B antagonist, or LPS with resveratrol (a histone acetylation activator) were analyzed. We found that LPS increased NR2B and interleukin-1 receptor (IL-1R) immunoreactivity. The expression of Iba1, a marker for microglia and/or macrophages, increased more significantly in the brain than in the spinal cord, implicating NR2B and IL-1R in brain inflammation. Immunoprecipitation with NR2B and IL-1R revealed related antibodies. Blood levels of IL-1β (i.e., the IL-1R ligand) increased, though not significantly, suggesting that inflammation peaked at 20 h. Behavioral assessments of central (CNS) and peripheral sensory (PNS) function indicated that LPS delayed CNS but not PNS escape latency. Finally, NR2B antagonist or resveratrol in the lateral ventricle antagonized the effects of LPS in the brain and improved animal survival. In summary, histone acetylation may control expression of NR2B and IL-1R, alleviating inflammation-induced sensory neuronal dysfunction caused by LPS. PMID:26682951

  20. Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein

    PubMed Central

    Liaw, Wen-Jinn; Zhu, Xu-Guang; Yaster, Myron; Johns, Roger A; Gauda, Estelle B; Tao, Yuan-Xiang

    2008-01-01

    Postsynaptic density (PSD)-93, a neuronal scaffolding protein, binds to and clusters N-methyl-D-aspartate receptor (NMDAR) subunits NR2A and NR2B at cellular membranes in vitro. However, the roles of PSD-93 in synaptic NR2A and NR2B targeting in the central nervous system and NMDAR-dependent physiologic and pathologic processes are still unclear. We report here that PSD-93 deficiency significantly decreased the amount of NR2A and NR2B in the synaptosomal membrane fractions derived from spinal cord dorsal horn and forebrain cortex but did not change their levels in the total soluble fraction from either region. However, PSD-93 deficiency did not markedly change the amounts of NR2A and NR2B in either synaptosomal or total soluble fractions from cerebellum. In mice deficient in PSD-93, morphine dose-dependent curve failed to shift significantly rightward as it did in wild type (WT) mice after acute and chronic morphine challenge. Unlike WT mice, PSD-93 knockout mice also showed marked losses of NMDAR-dependent morphine analgesic tolerance and associated abnormal sensitivity in response to mechanical, noxious thermal, and formalin-induced inflammatory stimuli after repeated morphine injection. In addition, PSD-93 knockout mice displayed dramatic loss of jumping activity, a typical NMDAR-mediated morphine withdrawal abstinence behavior. These findings indicate that impaired NMDAR-dependent neuronal plasticity following repeated morphine injection in PSD-93 knockout mice is attributed to PSD-93 deletion-induced alterations of synaptic NR2A and NR2B expression in dorsal horn and forebrain cortex neurons. The selective effect of PSD-93 deletion on synaptic NMDAR expression in these two major pain-related regions might provide the better strategies for the prevention and treatment of opioid tolerance and physical dependence. PMID:18851757

  1. Bacopa monnieri Extract (CDRI-08) Modulates the NMDA Receptor Subunits and nNOS-Apoptosis Axis in Cerebellum of Hepatic Encephalopathy Rats

    PubMed Central

    Mondal, Papia; Trigun, Surendra Kumar

    2015-01-01

    Hepatic encephalopathy (HE), characterized by impaired cerebellar functions during chronic liver failure (CLF), involves N-methyl-D-aspartate receptor (NMDAR) overactivation in the brain cells. Bacopa monnieri (BM) extract is a known neuroprotectant. The present paper evaluates whether BM extract is able to modulate the two NMDAR subunits (NR2A and NR2B) and its downstream mediators in cerebellum of rats with chronic liver failure (CLF), induced by administration of 50 mg/kg bw thioacetamide (TAA) i.p. for 14 days, and in the TAA group rats orally treated with 200 mg/kg bw BM extract from days 8 to 14. NR2A is known to impart neuroprotection and that of NR2B induces neuronal death during NMDAR activation. Neuronal nitric oxide synthase- (nNOS-) apoptosis pathway is known to mediate NMDAR led excitotoxicity. The level of NR2A was found to be significantly reduced with a concomitant increase of NR2B in cerebellum of the CLF rats. This was consistent with significantly enhanced nNOS expression, nitric oxide level, and reduced Bcl2/Bax ratio. Moreover, treatment with BM extract reversed the NR2A/NR2B ratio and also normalized the levels of nNOS-apoptotic factors in cerebellum of those rats. The findings suggest modulation of NR2A and NR2B expression by BM extract to prevent neurochemical alterations associated with HE. PMID:26413124

  2. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

    PubMed Central

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-01-01

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions. PMID:26460481

  3. Modulation of protein tyrosine phosphatase activity alters the subunit assembly in native N-methyl-D-aspartate receptor complex.

    PubMed

    Ferrani-Kile, Karima; Leslie, Steven W

    2005-07-01

    The N-methyl-D-aspartate (NMDA) receptor is crucial for development and neuroplasticity as well as excitotoxicity. The biochemical basis of the disassembly and reassembly of NMDA receptor has never been reported. Using coimmunoprecipitation, Western blotting, and mass spectrometry, we show that inhibition of tyrosine phosphatases triggers disassembly of NR1, NR2A, and NR2B in cortical NMDA receptor complexes. Furthermore, the disassembly of the NMDA receptor subunits is immediate, dose-dependent, and reversible and seems to occur through mechanisms linked to Src kinases. Together, these results define a novel role for tyrosine phosphatases in the complex mechanism of NMDA receptor regulation. PMID:15837820

  4. The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development

    PubMed Central

    Keavy, Deborah; Bristow, Linda J.; Sivarao, Digavalli V.; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E.; Weed, Michael R.

    2016-01-01

    The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans. PMID:27035340

  5. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.

    PubMed

    Kornau, H C; Schenker, L T; Kennedy, M B; Seeburg, P H

    1995-09-22

    The N-methyl-D-aspartate (NMDA) receptor subserves synaptic glutamate-induced transmission and plasticity in central neurons. The yeast two-hybrid system was used to show that the cytoplasmic tails of NMDA receptor subunits interact with a prominent postsynaptic density protein PSD-95. The second PDZ domain in PSD-95 binds to the seven-amino acid, COOH-terminal domain containing the terminal tSXV motif (where S is serine, X is any amino acid, and V is valine) common to NR2 subunits and certain NR1 splice forms. Transcripts encoding PSD-95 are expressed in a pattern similar to that of NMDA receptors, and the NR2B subunit co-localizes with PSD-95 in cultured rat hippocampal neurons. The interaction of these proteins may affect the plasticity of excitatory synapses. PMID:7569905

  6. Increased NR2A:NR2B ratio compresses long-term depression range and constrains long-term memory.

    PubMed

    Cui, Zhenzhong; Feng, Ruiben; Jacobs, Stephanie; Duan, Yanhong; Wang, Huimin; Cao, Xiaohua; Tsien, Joe Z

    2013-01-01

    The NR2A:NR2B subunit ratio of the NMDA receptors is widely known to increase in the brain from postnatal development to sexual maturity and to aging, yet its impact on memory function remains speculative. We have generated forebrain-specific NR2A overexpression transgenic mice and show that these mice had normal basic behaviors and short-term memory, but exhibited broad long-term memory deficits as revealed by several behavioral paradigms. Surprisingly, increased NR2A expression did not affect 1-Hz-induced long-term depression (LTD) or 100 Hz-induced long-term potentiation (LTP) in the CA1 region of the hippocampus, but selectively abolished LTD responses in the 3-5 Hz frequency range. Our results demonstrate that the increased NR2A:NR2B ratio is a critical genetic factor in constraining long-term memory in the adult brain. We postulate that LTD-like process underlies post-learning information sculpting, a novel and essential consolidation step in transforming new information into long-term memory. PMID:23301157

  7. Dissociable Roles for the Ventromedial Prefrontal Cortex and Amygdala in Fear Extinction: NR2B Contribution

    PubMed Central

    Diaz-Mataix, Llorenç; Bush, David E.A.; LeDoux, Joseph E.

    2009-01-01

    Fear extinction, which involves learning to suppress the expression of previously learned fear, requires N-methyl-D-aspartate receptors (NMDARs) and is mediated by the amygdala and ventromedial prefrontal cortex (vmPFC). Like other types of learning, extinction involves acquisition and consolidation phases. We recently demonstrated that NR2B-containing NMDARs (NR2Bs) in the lateral amygdala (LA) are required for extinction acquisition, but whether they are involved in consolidation is not known. Further, although it has been shown that NMDARs in the vmPFC are required for extinction consolidation, whether NR2Bs in vmPFC are involved in consolidation is not known. In this report, we investigated the possible role of LA and vmPFC NR2Bs in the consolidation of fear extinction using the NR2B-selective antagonist ifenprodil. We show that systemic treatment with ifenprodil immediately after extinction training disrupts extinction consolidation. Ifenprodil infusion into vmPFC, but not the LA, immediately after extinction training also disrupts extinction consolidation. In contrast, we also show pre-extinction training infusions into vmPFC has no effect. These results, together with our previous findings showing that LA NR2Bs are required during the acquisition phase in extinction, indicate a double dissociation for the phase-dependent role of NR2Bs in the LA (acquisition, not consolidation) and vmPFC (consolidation, not acquisition). PMID:18562331

  8. Changes in synaptic plasticity and expression of glutamate receptor subunits in the CA1 and CA3 areas of the hippocampus after transient global ischemia.

    PubMed

    Han, Xin-Jia; Shi, Zhong-Shan; Xia, Luo-Xing; Zhu, Li-Hui; Zeng, Ling; Nie, Jun-Hua; Xu, Zao-Cheng; Ruan, Yi-Wen

    2016-07-01

    Excess glutamate release from the presynaptic membrane has been thought to be the major cause of ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while CA3 neurons remain relatively intact. This suggests that changes in the properties of pyramidal cells may be the main cause related to ischemic neuronal death. Our previous studies have shown that the densities of dendritic spines and asymmetric synapses in the CA1 area are increased at 12h and 24h after ischemia. In the present study, we investigated changes in synaptic structures in the CA3 area and compared the expression of glutamate receptors in the CA1 and CA3 hippocampal regions of rats after ischemia. Our results demonstrated that the NR2B/NR2A ratio became larger after ischemia although the expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival pathway) decreased in the CA1 area from 6h to 48h after reperfusion. Furthermore, expression of the GluR2 subunit (calcium impermeable) of the AMPA receptor class significantly decreased while the GluR1 subunit (calcium permeable) remained unchanged at the same examined reperfusion times, which subsequently caused an increase in the GluR1/GluR2 ratio. Despite these notable differences in subunit expression, there were no obvious changes in the density of synapses or expression of NMDAR and AMPAR subunits in the CA3 area after ischemia. These results suggest that delayed CA1 neuronal death may be related to the dramatic fluctuation in the synaptic structure and relative upregulation of NR2B and GluR1 subunits induced by transient global ischemia. PMID:27090818

  9. Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice

    PubMed Central

    Fukaya, Masahiro; Kato, Akira; Lovett, Chanel; Tonegawa, Susumu; Watanabe, Masahiko

    2003-01-01

    Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-d-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules. PMID:12676993

  10. Extracellular glutamate level and NMDA receptor subunit expression in mouse olfactory bulb following nanoparticle-rich diesel exhaust exposure.

    PubMed

    Win-Shwe, Tin-Tin; Mitsushima, Dai; Yamamoto, Shoji; Fujitani, Yuji; Funabashi, Toshiya; Hirano, Seishiro; Fujimaki, Hidekazu

    2009-08-01

    In this present study, we aimed to investigate the extracellular glutamate level and memory function-related gene expression in the mouse olfactory bulb after exposure of the animals to nanoparticle-rich diesel exhaust (NRDE) with or without bacterial cell wall component. Lipoteichoic acid (LTA), a cell wall component derived from Staphylococcus aureus, was used to induce systemic inflammation. Male BALB/c mice were exposed to clean air (particle concentration, 4.58 microg/m(3)) or NRDE (148.86 microg/m(3)) 5 h per day on 5 consecutive days of the week for 4 wk with or without weekly intraperitoneal injection of LTA. We examined the extracellular glutamate levels in the olfactory bulb using in vivo microdialysis and high-performance liquid chromatography assay. Then, we collected the olfactory bulb to examine the expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A, and NR2B) and calcium/calmodulin-dependent protein kinase (CaMK) IV and cyclic AMP response element binding protein (CREB)-1 using real-time reverse-transcription polymerase chain reaction (RT-PCR). NRDE and/or LTA caused significantly increased extracellular glutamate levels in the olfactory bulb of mice. Moreover, the exposure of mice to NRDE upregulates NR1, NR2A, NR2B, and CaMKIV mRNAs in the olfactory bulb, while LTA upregulates only NR2B and CREB1 mRNAs. These findings suggest that NRDE and LTA cause glutamate-induced neurotoxicity separately and accompanied by changes in the expression of NMDA receptor subunits and related kinase and transcription factor in the mouse olfactory bulb. This is the first study to show the correlation between glutamate toxicity and memory function-related gene expressions in the mouse olfactory bulb following exposure to NRDE. PMID:19653804

  11. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  12. Fragile X Mental Retardation Protein Interactions with a G quadruplex structure in the 3′-Untranslated Region of NR2B mRNA

    PubMed Central

    Stefanovic, Snezana; DeMarco, Brett A.; Underwood, Ayana; Williams, Kathryn R.; Bassell, Gary J.; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the Post Synaptic Density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3′-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3′-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-D-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets. PMID:26412477

  13. Role of Fyn-mediated NMDA receptor function in prediabetic neuropathy in mice.

    PubMed

    Suo, Meng; Wang, Ping; Zhang, Mengyuan

    2016-08-01

    Diabetic neuropathy is a common complication of diabetes. This study evaluated the role of Fyn kinase and N-methyl-d-aspartate receptors (NMDARs) in the spinal cord in diabetic neuropathy using an animal model of high-fat diet-induced prediabetes. We found that prediabetic wild-type mice exhibited tactile allodynia and thermal hypoalgesia after a 16-wk high-fat diet, relative to normal diet-fed wild-type mice. Furthermore, prediabetic wild-type mice exhibited increased tactile allodynia and thermal hypoalgesia at 24 wk relative to 16 wk. Such phenomena were correlated with increased expression and activation of NR2B subunit of NMDARs, as well as Fyn-NR2B interaction in the spinal cord. Fyn(-/-) mice developed prediabetes after 16-wk high-fat diet treatment and exhibited thermal hypoalgesia, without showing tactile allodynia or altered expression and activation of NR2B subunit, relative to normal diet-fed Fyn(-/-) mice. Finally, intrathecal administrations of Ro 25-6981 (selective NR2B subunit-containing NMDAR antagonist) dose-dependently alleviated tactile allodynia, but not thermal hypoalgesia, at 16 and 24 wk in prediabetic wild-type mice. Our results suggested that Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy and that the increased expression/function of NR2B subunit-containing NMDARs may contribute to the progression of neuropathy in type 2 diabetes. PMID:27146985

  14. Developmental Expression of N-Methyl-d-Aspartate (NMDA) Receptor Subunits in Human White and Gray Matter: Potential Mechanism of Increased Vulnerability in the Immature Brain

    PubMed Central

    Jantzie, Lauren L.; Talos, Delia M.; Jackson, Michele C.; Park, Hyun-Kyung; Graham, Dionne A.; Lechpammer, Mirna; Folkerth, Rebecca D.; Volpe, Joseph J.; Jensen, Frances E.

    2015-01-01

    The pathophysiology of perinatal brain injury is multifactorial and involves hypoxia-ischemia (HI) and inflammation. N-methyl-d-aspartate receptors (NMDAR) are present on neurons and glia in immature rodents, and NMDAR antagonists are protective in HI models. To enhance clinical translation of rodent data, we examined protein expression of 6 NMDAR subunits in postmortem human brains without injury from 20 postconceptional weeks through adulthood and in cases of periventricular leukomalacia (PVL). We hypothesized that the developing brain is intrinsically vulnerable to excitotoxicity via maturation-specific NMDAR levels and subunit composition. In normal white matter, NR1 and NR2B levels were highest in the preterm period compared with adult. In gray matter, NR2A and NR3A expression were highest near term. NR2A was significantly elevated in PVL white matter, with reduced NR1 and NR3A in gray matter compared with uninjured controls. These data suggest increased NMDAR-mediated vulnerability during early brain development due to an overall upregulation of individual receptors subunits, in particular, the presence of highly calcium permeable NR2B-containing and magnesium-insensitive NR3A NMDARs. These data improve understanding of molecular diversity and heterogeneity of NMDAR subunit expression in human brain development and supports an intrinsic prenatal vulnerability to glutamate-mediated injury; validating NMDAR subunit-specific targeted therapies for PVL. PMID:24046081

  15. Developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in human white and gray matter: potential mechanism of increased vulnerability in the immature brain.

    PubMed

    Jantzie, Lauren L; Talos, Delia M; Jackson, Michele C; Park, Hyun-Kyung; Graham, Dionne A; Lechpammer, Mirna; Folkerth, Rebecca D; Volpe, Joseph J; Jensen, Frances E

    2015-02-01

    The pathophysiology of perinatal brain injury is multifactorial and involves hypoxia-ischemia (HI) and inflammation. N-methyl-d-aspartate receptors (NMDAR) are present on neurons and glia in immature rodents, and NMDAR antagonists are protective in HI models. To enhance clinical translation of rodent data, we examined protein expression of 6 NMDAR subunits in postmortem human brains without injury from 20 postconceptional weeks through adulthood and in cases of periventricular leukomalacia (PVL). We hypothesized that the developing brain is intrinsically vulnerable to excitotoxicity via maturation-specific NMDAR levels and subunit composition. In normal white matter, NR1 and NR2B levels were highest in the preterm period compared with adult. In gray matter, NR2A and NR3A expression were highest near term. NR2A was significantly elevated in PVL white matter, with reduced NR1 and NR3A in gray matter compared with uninjured controls. These data suggest increased NMDAR-mediated vulnerability during early brain development due to an overall upregulation of individual receptors subunits, in particular, the presence of highly calcium permeable NR2B-containing and magnesium-insensitive NR3A NMDARs. These data improve understanding of molecular diversity and heterogeneity of NMDAR subunit expression in human brain development and supports an intrinsic prenatal vulnerability to glutamate-mediated injury; validating NMDAR subunit-specific targeted therapies for PVL. PMID:24046081

  16. Intrahippocampal Administration of Ibotenic Acid Induced Cholinergic Dysfunction via NR2A/NR2B Expression: Implications of Resveratrol against Alzheimer Disease Pathophysiology

    PubMed Central

    Karthick, Chennakesavan; Periyasamy, Sabapathy; Jayachandran, Kesavan S.; Anusuyadevi, Muthuswamy

    2016-01-01

    Although several drugs revealed moderate amelioration of symptoms, none of them have sufficient potency to prevent or reverse the progression toward Alzheimer's disease (AD) pathology. Resveratrol (RSV), a polyphenolic compound has shown an outstanding therapeutic effect on a broad spectrum of diseases like age-associated neurodegeneration, inflammation etc. The present study was thus conducted to assess the therapeutic efficacy of RSV in ameliorating the deleterious effects of Ibotenic acid (IBO) in male Wistar rats. Stereotactic intrahippocampal administration of IBO (5 μg/μl) lesioned rats impairs cholinergic transmission, learning and memory performance that is rather related to AD and thus chosen as a suitable model to understand the drug efficacy in preventing AD pathophysiology. Since IBO is an agonist of glutamate, it is expected to exhibit an excitotoxic effect by altering glutamatergic receptors like NMDA receptor. The current study displayed significant alterations in the mRNA expression of NR2A and NR2B subunits of NMDA receptors, and further it is surprising to note that cholinergic receptors decreased in expression particularly α7-nAChR with increased m1AChR. RSV administration (20 mg/kg body weight, i.p.) significantly reduced these changes in IBO induced rats. Glutamatergic and cholinergic receptor alterations were associated with significant changes in the behavioral parameters of rats induced by IBO. While RSV improved spatial learning performance, attenuated immobility, and improvised open field activity in IBO induced rats. NR2B activation in the present study might mediate cell death through oxidative stress that form the basis of abnormal behavioral pattern in IBO induced rats. Interestingly, RSV that could efficiently encounter oxidative stress have significantly decreased stress markers viz., nitrite, PCO, and MDA levels by enhancing antioxidant status. Histopathological analysis displayed significant reduction in the hippocampal

  17. Intrahippocampal Administration of Ibotenic Acid Induced Cholinergic Dysfunction via NR2A/NR2B Expression: Implications of Resveratrol against Alzheimer Disease Pathophysiology.

    PubMed

    Karthick, Chennakesavan; Periyasamy, Sabapathy; Jayachandran, Kesavan S; Anusuyadevi, Muthuswamy

    2016-01-01

    Although several drugs revealed moderate amelioration of symptoms, none of them have sufficient potency to prevent or reverse the progression toward Alzheimer's disease (AD) pathology. Resveratrol (RSV), a polyphenolic compound has shown an outstanding therapeutic effect on a broad spectrum of diseases like age-associated neurodegeneration, inflammation etc. The present study was thus conducted to assess the therapeutic efficacy of RSV in ameliorating the deleterious effects of Ibotenic acid (IBO) in male Wistar rats. Stereotactic intrahippocampal administration of IBO (5 μg/μl) lesioned rats impairs cholinergic transmission, learning and memory performance that is rather related to AD and thus chosen as a suitable model to understand the drug efficacy in preventing AD pathophysiology. Since IBO is an agonist of glutamate, it is expected to exhibit an excitotoxic effect by altering glutamatergic receptors like NMDA receptor. The current study displayed significant alterations in the mRNA expression of NR2A and NR2B subunits of NMDA receptors, and further it is surprising to note that cholinergic receptors decreased in expression particularly α7-nAChR with increased m1AChR. RSV administration (20 mg/kg body weight, i.p.) significantly reduced these changes in IBO induced rats. Glutamatergic and cholinergic receptor alterations were associated with significant changes in the behavioral parameters of rats induced by IBO. While RSV improved spatial learning performance, attenuated immobility, and improvised open field activity in IBO induced rats. NR2B activation in the present study might mediate cell death through oxidative stress that form the basis of abnormal behavioral pattern in IBO induced rats. Interestingly, RSV that could efficiently encounter oxidative stress have significantly decreased stress markers viz., nitrite, PCO, and MDA levels by enhancing antioxidant status. Histopathological analysis displayed significant reduction in the hippocampal

  18. Control of N-methyl-D-aspartate Receptor Function by the NR2 Subunit Amino-Terminal Domain

    PubMed Central

    Yuan, Hongjie; Hansen, Kasper B.; Vance, Katie M.; Ogden, Kevin K.; Traynelis, Stephen F.

    2009-01-01

    NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here we report that the extracellular amino-terminal domain (ATD) of the NR2 subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (POPEN), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D and chimeras of NR2A and NR2D with interchanged ATD (NR2A-(2D-ATD) and NR2D-(2A-ATD)), we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases POPEN in single channel recordings from outside-out patches of HEK 293 cells. By contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases POPEN and mean open duration. These data demonstrate the modular nature of NMDA receptors and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes. PMID:19793963

  19. The N-terminal domains of both NR1 and NR2 subunits determine allosteric Zn2+ inhibition and glycine affinity of N-methyl-D-aspartate receptors.

    PubMed

    Madry, Christian; Mesic, Ivana; Betz, Heinrich; Laube, Bodo

    2007-12-01

    The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1- and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn2+ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly. PMID:17878266

  20. Brain-derived neurotrophic factor acutely enhances tyrosine phosphorylation of the AMPA receptor subunit GluR1 via NMDA receptor-dependent mechanisms.

    PubMed

    Wu, Kuo; Len, Guo-Wei; McAuliffe, Geoff; Ma, Chia; Tai, Jessica P; Xu, Fei; Black, Ira B

    2004-11-01

    Brain-derived growth factor (BDNF) acutely regulates synaptic transmission and modulates hippocampal long-term potentiation (LTP) and long-term depression (LTD), cellular models of plasticity associated with learning and memory. Our previous studies revealed that BDNF rapidly increases phosphorylation of NMDA receptor subunits NR1 and NR2B in the postsynaptic density (PSD), potentially linking receptor phosphorylation to synaptic plasticity. To further define molecular mechanisms governing BDNF actions, we examined tyrosine phosphorylation of GluR1, the most well-characterized subunit of AMPA receptors. Initially, we investigated synaptoneurosomes that contain intact pre- and postsynaptic elements. Incubation of synaptoneurosomes with BDNF for 5 min increased tyrosine phosphorylation of GluR1 in a dose-dependent manner, with a maximal, 4-fold enhancement at 10 ng/ml BDNF. NGF had no effects, suggesting the specificity of BDNF actions. Subsequently, we found that BDNF elicited a maximal, 2.5-fold increase in GluR1 phosphorylation in the PSD at 250 ng/ml BDNF within 5 min, suggesting that BDNF enhances the phosphorylation through postsynaptic mechanisms. Activation of trkB receptors was critical as k252-a, an inhibitor of trk receptor tyrosine kinase, blocked the BDNF-activated GluR1 phosphorylation. In addition, AP-5 and MK 801, NMDA receptor antagonists, blocked BDNF enhancement of phosphorylation in synaptoneurosomes or PSDs. Conversely, NMDA, the specific receptor agonist, evoked respective 3.8- and 2-fold increases in phosphorylation in synaptoneurosomes and PSDs within 5 min, mimicking the effects of BDNF. These findings raise the possibility that BDNF modulates GluR1 activity via changes in NMDA receptor function. Moreover, incubation of synaptoneurosomes or PSDs with BDNF and ifenprodil, a specific NR2B antagonist, reproduced the results of AP-5 and MK-801. Finally, coexposure of synaptoneurosomes or PSDs to BDNF and NMDA was not additive, suggesting that

  1. Expression of N-methyl-D-aspartate receptor subunits in the bovine ovum: ova as a potential source of autoantigens causing anti-NMDAR encephalitis.

    PubMed

    Tachibana, Naoko; Kinoshita, Michiaki; Kametani, Fuyuki; Tanaka, Keiko; Une, Yumi; Komatsu, Yotaro; Kobayashi, Yukihiro; Ikeda, Shu-ichi

    2015-01-01

    Autoimmune synaptic encephalitis is characterized by the presence of autoantibodies against synaptic constituent receptors and manifests as neurological and psychiatric disorders. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is such an autoimmune disorder that predominantly affects young women. It is associated with antibodies against the extracellular region of the NR1 subunit of postsynaptic NMDAR. Each NMDAR functions as a heterotetrameric complex that is composed of four subunits, including NR1 and NR2A, NR2B, or NR2C. Importantly, ovarian teratoma is a typical complication of anti-NMDAR encephalitis in female patients and may contain antigenic neural tissue; however, antigenic sites remain unknown in female patients without ovarian teratoma. The purpose of this study was to investigate the expression of NMDARs in the ovum. We detected NR1 and NR2B immunoreactivity in protein fractions extracted from the bovine ovary and ova by SDS-polyacrylamide gel electrophoresis and immunoblotting analysis. Immunoprecipitates digested with trypsin were analyzed by reverse phase liquid chromatography coupled to tandem mass spectrometry. We obtained the following five peptides: SPFGRFK and KNLQDR, which are consistent with partial sequences of human NR1, and GVEDALVSLK, QPTVAGAPK, and NEVMSSK, which correspond to those of NR2A, NR2B and NR2C, respectively. Immunocytochemical analysis revealed that the bovine ovum was stained with the immunoglobulin G purified from the serum of a patient with anti-NMDAR encephalitis. Taken together, we propose that the normal ovum expresses NMDARs that have strong affinity for the disease-specific IgG. The presence of NMDARs in ova may help explain why young females without ovarian teratomas are also affected by anti-NMDAR encephalitis. PMID:25786541

  2. N-Methyl-d-aspartate (NMDA) Receptor NR2 Subunit Selectivity of a Series of Novel Piperazine-2,3-dicarboxylate Derivatives: Preferential Blockade of Extrasynaptic NMDA Receptors in the Rat Hippocampal CA3-CA1 Synapse

    PubMed Central

    Feng, Bihua; Tsintsadze, Timur S.; Morley, Richard M.; Irvine, Mark W.; Tsintsadze, Vera; Lozovaya, Natasha A.; Jane, David E.; Monaghan, Daniel T.

    2009-01-01

    N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (−) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 μM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor

  3. Experience-Dependent Changes in Excitatory and Inhibitory Receptor Subunit Expression in Visual Cortex

    PubMed Central

    Beston, Brett R.; Jones, David G.; Murphy, Kathryn M.

    2010-01-01

    Experience-dependent development of visual cortex depends on the balance between excitatory and inhibitory activity. This activity is regulated by key excitatory (NMDA, AMPA) and inhibitory (GABAA) receptors. The composition of these receptors changes developmentally, affecting the excitatory–inhibitory (E/I) balance and synaptic plasticity. Until now, it has been unclear how abnormal visual experience affects this balance. To examine this question, we measured developmental changes in excitatory and inhibitory receptor subunits in visual cortex following normal visual experience and monocular deprivation. We used Western blot analysis to quantify expression of excitatory (NR1, NR2A, NR2B, GluR2) and inhibitory (GABAAα1, GABAAα3) receptor subunits. Monocular deprivation promoted a complex pattern of changes in receptor subunit expression that varied with age and was most severe in the region of visual cortex representing the central visual field. To characterize the multidimensional pattern of experience-dependent change in these synaptic mechanisms, we applied a neuroinformatics approach using principal component analysis. We found that monocular deprivation (i) causes a large portion of the normal developmental trajectory to be bypassed, (ii) shifts the E/I balance in favor of more inhibition, and (iii) accelerates the maturation of receptor subunits. Taken together, these results show that monocularly deprived animals have an abnormal balance of the synaptic machinery needed for functional maturation of cortical circuits and for developmental plasticity. This raises the possibility that interventions intended to treat amblyopia may need to address multiple synaptic mechanisms to produce optimal recovery. PMID:21423524

  4. The Role of NMDA Receptor Subtypes in Short-Term Plasticity in the Rat Entorhinal Cortex

    PubMed Central

    Chamberlain, Sophie E. L.; Yang, Jian; Jones, Roland S. G.

    2008-01-01

    We have previously shown that spontaneous release of glutamate in the entorhinal cortex (EC) is tonically facilitated via activation of presynaptic NMDA receptors (NMDAr) containing the NR2B subunit. Here we show that the same receptors mediate short-term plasticity manifested by frequency-dependent facilitation of evoked glutamate release at these synapses. Whole-cell patch-clamp recordings were made from layer V pyramidal neurones in rat EC slices. Evoked excitatory postsynaptic currents showed strong facilitation at relatively low frequencies (3 Hz) of activation. Facilitation was abolished by an NR2B-selective blocker (Ro 25-6981), but unaffected by NR2A-selective antagonists (Zn2+, NVP-AAM077). In contrast, postsynaptic NMDAr-mediated responses could be reduced by subunit-selective concentrations of all three antagonists. The data suggest that NMDAr involved in presynaptic plasticity in layer V are exclusively NR1/NR2B diheteromers, whilst postsynaptically they are probably a mixture of NR1/NR2A, NR1/NR2B diheteromers and NR1/NR2A/NR2B triheteromeric receptors. PMID:18989370

  5. Regulation of NMDA receptor subunit mRNA expression in the guinea pig vestibular nuclei following unilateral labyrinthectomy.

    PubMed

    Sans, N; Sans, A; Raymond, J

    1997-10-01

    The localization of neurons expressing mRNAs for the NR1 and NR2A-D subunits of the glutamatergic NMDA receptor was examined by non-radioactive in situ hybridization throughout the guinea pig vestibular nuclei. After deafferentation of the vestibular nuclei by unilateral labyrinthectomy, modifications of the mRNA distributions were followed for 30 days. A quantitative analysis was performed in the medial vestibular nucleus by comparison of the labelled neurons in the ipsi- and contra-lateral nuclei. In vestibular nuclei, the NR1 subunit mRNA was found in various populations of neurons. The NR2A and NR2C subunit mRNAs were less widely distributed, whereas little NR2D mRNA was detected and only rare cells contained NR2B mRNA. NR1 and NR2A-D mRNAs were colocalized in some but not other neuronal types. Twenty hours after the lesion, there was a transient ipsilateral increase of NR1 mRNA level in the medial vestibular nucleus, followed by a decrease 48 h after the lesion and, at 3 days, by recovery to the control level. An ipsilateral increase in the mRNA level of NR2C subunit was detected 20 h after lesion and maintained at 48 h. No significant changes were apparent in NR2A, NR2B and NR2D mRNA levels. The distributions and the differential signal intensities of NR2A-D mRNAs suggest various subunit organizations of the NMDA receptors in different neurons of the vestibular nuclei. Neuronal plasticity reorganizations in the vestibular nuclei following unilateral labyrinthectomy appear to include only changes in NR1 and NR2C mRNA levels modifying the functional diversity of the NMDA receptor in the ipsilateral medial vestibular nucleus neurons. The transient changes in NR1 and the NR2C subunit mRNA expressions in response to sensory deprivation are consistent with an active role for NMDA receptors in the appearance and development of the vestibular compensatory process. PMID:9421163

  6. Cisplatin induces neuronal activation and increases central AMPA and NMDA receptor subunit gene expression in mice.

    PubMed

    Holland, Ruby A; Leonard, John J; Kensey, Nicholas A; Hannikainen, Paavali A; De Jonghe, Bart C

    2014-09-01

    Although rats and mice do not vomit, these species are widely studied as models of energy balance and sickness behavior. Previous work has shown that rats exhibit similar neuroanatomical activation of brain and visceral afferent pathways following cisplatin chemotherapy compared to vomiting species. However, the neural response to cisplatin in mice is understudied. Here, food intake, body weight, and central c-Fos immunofluorescence were analyzed in the hindbrains of male C57BL/6 mice following IP saline or cisplatin (5mg/kg, and 20mg/kg doses). As glutamate receptor signaling is classically linked to inhibitory feeding pathways in the rodent, gene expression of selected α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor subunits were assessed in the dorsal vagal complex (DVC), parabrachial nucleus (PBN), amygdala, and bed nucleus of the stria terminalis (BNST). Our results show dose-dependent reductions in food intake and body weight following cisplatin treatment, as well as increases in cisplatin-induced c-Fos in the PBN and throughout the DVC. Quantitative PCR analysis shows cisplatin-induced increases in NMDA receptor subunit expression, particularly NR2B, in the DVC, PBN, BNST, and amygdala. In addition, upregulation of AMPA receptor subunits (GluA1 and/or GluA2) were observed in all regions examined except the amygdala. Taken together, these results suggest similar neural pathways mediating cisplatin effects in mice compared to other well-studied species, which are likely mediated by central upregulation of AMPA and NMDA receptors. PMID:24582677

  7. Glucocorticoid mediates water avoidance stress-sensitized colon-bladder cross-talk via RSK2/PSD-95/NR2B in rats.

    PubMed

    Peng, Hsien-Yu; Hsieh, Ming-Chun; Lai, Cheng-Yuan; Chen, Gin-Den; Huang, Yi-Ping; Lin, Tzer-Bin

    2012-11-01

    Unexpected environmental and social stimuli could trigger stress. Although coping with stress is essential for survival, long-term stress impacts visceral functions, and therefore, it plays a role in the development and exacerbation of symptoms of gastrointestinal/urogenital disorders. The aim of this study is to characterize the role of corticosterone in stress-sensitized colon-bladder cross-talk, a phenomenon presumed to underlie the comorbidity of functional bowel and bladder disorders. Cystometry and protein/mRNA expression in the lumbosacral dorsal horn (L6-S1) in response to intracolonic mustard oil (MO) instillation were analyzed in female Wistar-Kyoto rats subjected to water avoidance stress (WAS; 1 h/day for 10 days) or sham stress (WAsham). Whereas it had no effect on baseline-voiding function, chronic stress upregulated plasma corticosterone concentration and dorsal horn spinal p90 ribosomal S6 kinase 2 (RSK2) protein/mRNA levels, and RSK2 immunoreactivity colocalized with NeuN-positive neurons. Intracolonic MO dose-dependently decreased intrercontraction intervals and threshold pressure, provoked spinal RSK2 and NR2B phosphorylation, and enhanced PSD-95-RSK2 and PSD-95-NR2B coupling. Intrathecal kaempferol (a RSK2 activation antagonist; 30 min before MO instillation), bilateral adrenalectomy (7 days prior the stress paradigm), and subcutaneous RU-38486 (a glucocorticoid receptor antagonist; 30 min daily before stress sessions), but not RU-28318 (a mineralocorticoid receptor antagonist), attenuated MO-induced bladder hyperactivity, protein phosphorylation, and protein-protein interactions in the WAS group. Our results suggest that stress-associated glucocorticoid release mediates WAS-dependent sensitization of colon-bladder cross-talk via the spinal RSK2/PSD-95/NR2B cascade and offer a possibility for developing pharmacological strategies for the treatment of stress-related pelvic pain. PMID:23125098

  8. Inhibiting effects of rhynchophylline on zebrafish methamphetamine dependence are associated with amelioration of neurotransmitters content and down-regulation of TH and NR2B expression.

    PubMed

    Jiang, Mingjin; Chen, Yifei; Li, Chan; Peng, Qiuxian; Fang, Miao; Liu, Wei; Kang, Qunzhao; Lin, Yingbo; Yung, Ken Kin Lam; Mo, Zhixian

    2016-07-01

    Others and we have reported that rhynchophylline reverses amphetamine-induced conditioned place preference (CPP) effect which may be partly mediated by amelioration of central neurotransmitters and N-methyl-d-aspartate receptor 2B (NR2B) levels in the rat brains. The current study investigated the inhibiting effects of rhynchophylline on methamphetamine-induced (METH-induced) CPP in adult zebrafish and METH-induced locomotor activity in tyrosine hydroxylase-green fluorescent protein (TH-GFP) transgenic zebrafish larvae and attempted to confirm the hypothesis that these effects were mediated via regulation of neurotransmitters and dopaminergic and glutamatergic systems. After baseline preference test (on days 1-3), zebrafish were injected intraperitoneally METH (on days 4, 6 and 8) or the same volume of fish physiological saline (on days 5 and 7) and were immediately conditioned. Rhynchophylline was administered at 12h after injection of METH. On day 9, zebrafish were tested for METH-induced CPP. Results revealed that rhynchophylline (100mg/kg) significantly inhibited the acquisition of METH-induced CPP, reduced the content of dopamine and glutamate and down-regulated the expression of TH and NR2B in the CPP zebrafish brains. Furthermore, the influence of rhynchophylline on METH-induced locomotor activity was also observed in TH-GFP transgenic zebrafish larvae. Results showed that rhynchophylline (50mg/L) treatment led to a significant reduction on the locomotor activity and TH expression in TH-GFP transgenic zebrafish larvae. Taken together, these data indicate that the inhibition of the formation of METH dependence by rhynchophylline in zebrafish is associated with amelioration of the neurotransmitters dopamine and glutamate content and down-regulation of TH and NR2B expression. PMID:27009763

  9. The receptor subunits generating NMDA receptor mediated currents in oligodendrocytes

    PubMed Central

    Burzomato, Valeria; Frugier, Guillaume; Pérez-Otaño, Isabel; Kittler, Josef T; Attwell, David

    2010-01-01

    NMDA receptors have been shown to contribute to glutamate-evoked currents in oligodendrocytes. Activation of these receptors damages myelin in ischaemia, in part because they are more weakly blocked by Mg2+ than are most neuronal NMDA receptors. This weak Mg2+ block was suggested to reflect an unusual subunit composition including the NR2C and NR3A subunits. Here we expressed NR1/NR2C and triplet NR1/NR2C/NR3A recombinant receptors in HEK cells and compared their currents with those of NMDA-evoked currents in rat cerebellar oligodendrocytes. NR1/NR2C/3A receptors were less blocked by 2 mm Mg2+ than were NR1/NR2C receptors (the remaining current was 30% and 18%, respectively, of that seen without added Mg2+) and showed less channel noise, suggesting a smaller single channel conductance. NMDA-evoked currents in oligodendrocytes showed a Mg2+ block (to 32%) similar to that observed for NR1/NR2C/NR3A and significantly different from that for NR1/NR2C receptors. Co-immunoprecipitation revealed interactions between NR1, NR2C and NR3A subunits in a purified myelin preparation from rat brain. These data are consistent with NMDA-evoked currents in oligodendrocytes reflecting the activation of receptors containing NR1, NR2C and NR3A subunits. PMID:20660562

  10. Stargazin is an AMPA receptor auxiliary subunit.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-01-11

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors mediate fast excitatory synaptic transmission in brain and underlie aspects of synaptic plasticity. Numerous AMPA receptor-binding proteins have been implicated in AMPA receptor trafficking and anchoring. However, the relative contributions of these proteins to the composition of native AMPA receptor complexes in brain remain uncertain. Here, we use blue native gel electrophoresis to analyze the composition of native AMPA receptor complexes in cerebellar extracts. We identify two receptor populations: a functional form that contains the transmembrane AMPA receptor-regulatory protein stargazin and an apo-form that lacks stargazin. Limited proteolysis confirms assembly of stargazin with a large proportion of native AMPA receptors. In contrast, other AMPA receptor-interacting proteins, such as synapse-associated protein 97, glutamate receptor-interacting protein 1, protein kinase Calpha binding protein, N-ethylmaleimide-sensitive fusion protein, AP2, and protein 4.1N, do not show significant association with AMPA receptor complexes on native gels. These data identify stargazin as an auxiliary subunit for a neurotransmitter-gated ion channel. PMID:15630087

  11. Amyloid β peptide oligomers directly activate NMDA receptors.

    PubMed

    Texidó, Laura; Martín-Satué, Mireia; Alberdi, Elena; Solsona, Carles; Matute, Carlos

    2011-03-01

    Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimeŕs disease. PMID:21349580

  12. Mas-Related Gene (Mrg) C Activation Attenuates Bone Cancer Pain via Modulating Gi and NR2B

    PubMed Central

    Lu, Cui’e; Lei, Yishan; Ma, Zhengliang; Gu, Xiaoping

    2016-01-01

    Objective This study is to investigate the role of Mas-related gene (Mrg) C in the pathogenesis and treatment of bone cancer pain (BCP). Methods BCP mouse model was established by osteosarcoma cell inoculation. Pain-related behaviors were assessed with the spontaneous lifting behavior test and mechanical allodynia test. Expression levels of MrgC, Gi, and NR2B in the spinal cord were detected with Western blot analysis and immunohistochemistry. Results Pain-related behavior tests showed significantly increased spontaneous flinches (NSF) and decreased paw withdrawal mechanical threshold (PWMT) in mouse models of BCP. Western blot analysis showed that, compared with the control group and before modeling, all the expression levels of MrgC, Gi, and NR2B in the spinal cord of BCP mice were dramatically elevated, which were especially increased at day 7 after operation and thereafter, in a time-dependent manner. Moreover, the treatment of MrgC agonist BAM8-22 significantly up-regulated Gi and down-regulated NR2B expression levels, in the spinal cord of BCP mice, in a time-dependent manner. On the other hand, anti-MrgC significantly down-regulated Gi expression, while dramatically up-regulated NR2B expression, in the BCP mice. Similar results were obtained from the immunohistochemical detection. Importantly, BAM8-22 significantly attenuated the nociceptive behaviors in the BCP mice. Conclusion Our results indicated the MrgC-mediated Gi and NR2B expression alterations in the BCP mice, which might contribute to the pain hypersensitivity. These findings may provide a novel strategy for the treatment of BCP in clinic. PMID:27152740

  13. PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat

    PubMed Central

    Losi, Gabriele; Prybylowski, Kate; Fu, Zhanyan; Luo, Jianhong; Wenthold, Robert J; Vicini, Stefano

    2003-01-01

    We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro. Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro (DIV) 7–8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for flag-tagged NR2A subunit clusters than for flag-tagged NR2B subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits. PMID:12576494

  14. PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors.

    PubMed

    Cui, Hong; Hayashi, Amy; Sun, Hong-Shuo; Belmares, Michael P; Cobey, Carolyn; Phan, Thuymy; Schweizer, Johannes; Salter, Michael W; Wang, Yu Tian; Tasker, R Andrew; Garman, David; Rabinowitz, Joshua; Lu, Peter S; Tymianski, Michael

    2007-09-12

    In neuronal synapses, PDZ domains [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] of PSD-95 proteins interact with C termini of NMDA receptor [NMDAR (NR)] subunits, linking them to downstream neurotoxic signaling molecules. Perturbing NMDAR/PSD-95 interactions with a Tat peptide comprising the nine C-terminal residues of the NR2B subunit (Tat-NR2B9c) reduces neurons' vulnerability to excitotoxicity and ischemia. However, NR subunit C termini may bind many of >240 cellular PDZs, any of which could mediate neurotoxic signaling independently of PSD-95. Here, we performed a proteomic and biochemical analysis of the interactions of all known human PDZs with synaptic signaling proteins including NR1, NR2A-NR2D, and neuronal nitric oxide synthase (nNOS). Tat-NR2B9c, whose interactions define PDZs involved in neurotoxic signaling, was also used. NR2A-NR2D subunits and Tat-NR2B9c had similar, highly specific, PDZ protein interactions, of which the strongest were with the PSD-95 family members (PSD-95, PSD-93, SAP97, and SAP102) and Tax interaction protein 1 (TIP1). The PSD-95 PDZ2 domain bound NR2A-NR2C subunits most strongly (EC50, approximately 1 microM), and fusing the NR2B C terminus to Tat enhanced its affinity for PSD-95 PDZ2 by >100-fold (EC50, approximately 7 nM). IC50 values for Tat-NR2B9c inhibiting NR2A-NR2C/PSD-95 interactions (approximately 1-10 microM) and nNOS/PSD-95 interactions (200 nM) confirmed the feasibility of such inhibition. To determine which of the PDZ interactions of Tat-NR2B9c mediate neuroprotection, one of PSD-95, PSD-93, SAP97, SAP102, TIP1, or nNOS expression was inhibited in cortical neurons exposed to NMDA toxicity. Only neurons lacking PSD-95 or nNOS but not PSD-93, SAP97, SAP102, or TIP1 exhibited reduced excitotoxic vulnerability. Thus, despite the ubiquitousness of PDZ domain-containing proteins, PSD-95 and nNOS above any other PDZ proteins are keys in effecting NMDAR-dependent excitotoxicity. Consequently, PSD-95

  15. Subunit Arrangement and Function in NMDA Receptors

    SciTech Connect

    Furukawa,H.; Singh, S.; Mancusso, R.; Gouaux, E.

    2005-01-01

    Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

  16. Coupling of energy metabolism and synaptic transmission at the transcriptional level: Role of nuclear respiratory factor 1 in regulating both cytochrome c oxidase and NMDA glutamate receptor subunit genes

    PubMed Central

    Dhar, Shilpa S.; Wong-Riley, Margaret T. T.

    2009-01-01

    Neuronal activity and energy metabolism are tightly coupled processes. Regions high in neuronal activity, especially of the glutamatergic type, have high levels of cytochrome c oxidase (COX). Perturbations in neuronal activity affect the expressions of COX and glutamatergic N-methyl-D-aspartate receptor subunit 1 (NR1). The present study sought to test our hypothesis that the coupling extends to the transcriptional level, whereby NR1 and possibly other NR subunits and COX are co-regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), which regulates all COX subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutations, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Grin 1 (NR1), Grin 2b (NR2b) and COX subunit genes, but not of Grin2a and Grin3a genes. These transcripts were up-regulated by KCl and down-regulated by TTX in cultured primary neurons. However, silencing of NRF-1 with small interference RNA blocked the up-regulation of Grin1, Grin2b, and COX induced by KCl, and over-expression of NRF-1 rescued these transcripts that were suppressed by TTX. NRF-1 binding sites on Grin1 and Grin2b genes are also highly conserved among mice, rats, and humans. Thus, NRF-1 is an essential transcription factor critical in the co-regulation of NR1, NR2b, and COX, and coupling exists at the transcriptional level to ensure coordinated expressions of proteins important for synaptic transmission and energy metabolism. PMID:19144849

  17. Potencies and unblocking kinetic properties of antagonists at recombinant human NMDA receptors in a Xenopus oocytes model.

    PubMed

    Heusler, Peter; Tourette, Amélie; Cussac, Didier

    2015-05-01

    N-methyl-D-aspartate (NMDA) receptor channels are implicated in a wide range of physiological and pathophysiological processes, and a large number of pharmacological agents have been introduced that target the receptor via diverse mechanisms of action. Amongst others, subunit selectivity (in particular for the NR2B receptor subunit) and rapid unblocking kinetics have been put forward as favourable pharmacological properties of NMDA receptor-targeting drugs. Here, we describe a pharmacological characterization of human recombinant NMDA receptors expressed in Xenopus oocytes in an electrophysiological set-up. Using this approach, we compare inhibitor potencies of several known NMDA receptor ligands as well as unblocking kinetic properties of selected compounds. All compounds tested had similar potencies at receptors containing NR2A or NR2B receptors with the exception of traxoprodil, which was selective for NR2B. The rank order of potency was (+)MK-801 > phencyclidine (PCP) ≈ traxoprodil > memantine ≈ ketamine > duloxetine. In line with its proposed rapid dissociation properties, the relatively well-tolerated drug memantine exhibits markedly faster unblocking than ketamine and PCP, similar to the low-affinity compound, duloxetine. Electrophysiological recording in Xenopus oocytes thus allows a relatively convenient comparison of key pharmacological parameters at recombinant human NMDA receptors. PMID:25604077

  18. Behavioural Assessment of the A2a/NR2B Combination in the Unilateral 6-OHDA-Lesioned Rat Model: A New Method to Examine the Therapeutic Potential of Non-Dopaminergic Drugs.

    PubMed

    Michel, Anne; Downey, Patrick; Van Damme, Xavier; De Wolf, Catherine; Schwarting, Rainer; Scheller, Dieter

    2015-01-01

    In Parkinson's disease (PD), dopaminergic therapies are often associated with the development of motor complications. Attention has therefore been focused on the use of non-dopaminergic drugs. This study developed a new behavioural method capable of demonstrating the added value of combining adenosinergic and glutamatergic receptor antagonists in unilateral 6-OHDA lesioned rats. Rats were dosed orally with Tozadenant, a selective A2A receptor antagonist, and three different doses of Radiprodil, an NR2B-selective NMDA receptor antagonist. The drugs were given alone or in combination and rats were placed in an open-field for behavioural monitoring. Video recordings were automatically analysed. Five different behaviours were scored: distance traveled, ipsi- and contraversive turns, body position, and space occupancy. The results show that A2A or NR2B receptor antagonists given alone or in combination did not produce enhanced turning as observed with an active dose of L-Dopa/benserazide. Instead the treated rats maintained a straight body position, were able to shift from one direction to the other and occupied a significantly larger space in the arena. The highest "Tozadenant/Radiprodil" dose combination significantly increased all five behavioural parameters recorded compared to rats treated with vehicle or the same doses of the drugs alone. Our data suggest that the A2A/NR2B antagonist combination may be able to stimulate motor activity to a similar level as that achieved by L-Dopa but in the absence of the side-effects that are associated with dopaminergic hyperstimulation. If these results translate into the clinic, this combination could represent an alternative symptomatic treatment option for PD. PMID:26322641

  19. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    PubMed

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  20. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  1. Effects of sex and chronic neonatal nicotine treatment on Na²⁺/K⁺/Cl⁻ co-transporter 1, K⁺/Cl⁻ co-transporter 2, brain-derived neurotrophic factor, NMDA receptor subunit 2A and NMDA receptor subunit 2B mRNA expression in the postnatal rat hippocampus.

    PubMed

    Damborsky, J C; Winzer-Serhan, U H

    2012-12-01

    Chronic exposure to nicotine during the first postnatal week in rats, a developmental period that corresponds to the third trimester of human gestation, results in sexually dimorphic long-term functional defects in the adult hippocampus. One potential cause could be the sex-specific differences in the maturation of GABA(A) receptor-mediated responses from excitatory to inhibitory, which depends on the expression of the Na(2+)/K(+)/Cl(-) co-transporter 1 (NKCC1) and the K(+)/Cl(-) co-transporter 2 (KCC2). In the rat hippocampus, this switch occurs during the first and second postnatal week in females and males, respectively, and is regulated by nicotinic receptor activation. Excitatory GABAergic signaling can increase brain-derived neurotrophic factor (BDNF) expression, which might exacerbate sex differences by impacting synaptogenesis. We hypothesized that chronic neonatal nicotine (CNN) exposure differentially regulates the expression of these co-transporters and BDNF in males and females. We use quantitative isotopic in situ hybridization to examine the expression of mRNAs for NKCC1, KCC2, BDNF, and NMDA receptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) in the postnatal day (P) 5 and 8 rat hippocampi in both sexes that were either control-treated or with 6mg/kg/day nicotine in milk formula (CNN) via gastric intubation starting at P1. In line with prolonged GABAergic excitation, we found that at P5 males had significantly higher mRNA expression of NKCC1 and BDNF than females. CNN treatment resulted in a significant increase in KCC2 and BDNF mRNA expression in male but not female hippocampus (p<0.05). Males also had higher expression of NR2A and lower expression of NR2B at P5 compared to females (p<0.05). At P8, there were neither sex nor treatment effects on mRNA expression, indicating the end of a critical period for sensitivity to nicotine. These results suggest that differential maturation of GABA(A)R-mediated responses result in sex

  2. Increased glutamate receptor and transporter expression in the cerebral cortex and striatum of gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I.

    PubMed

    Lagranha, Valeska Lizzi; Matte, Ursula; de Carvalho, Talita Giacomet; Seminotti, Bianca; Pereira, Carolina Coffi; Koeller, David M; Woontner, Michael; Goodman, Stephen I; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2014-01-01

    We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I. PMID:24594605

  3. Increased Glutamate Receptor and Transporter Expression in the Cerebral Cortex and Striatum of Gcdh-/- Mice: Possible Implications for the Neuropathology of Glutaric Acidemia Type I

    PubMed Central

    Lagranha, Valeska Lizzi; Matte, Ursula; de Carvalho, Talita Giacomet; Seminotti, Bianca; Pereira, Carolina Coffi; Koeller, David M.; Woontner, Michael; Goodman, Stephen I.; de Souza, Diogo Onofre Gomes; Wajner, Moacir

    2014-01-01

    We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh-/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh-/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh-/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh-/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh-/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh-/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh-/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh-/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I. PMID:24594605

  4. PSD-93 MEDIATES TYROSINE-PHOSPHORYLATION OF THE N-METHYL-D-ASPARTATE RECEPTORS

    PubMed Central

    Sato, Yuko; Tao, Yuan-Xiang; Su, Qingning; Johns, Roger A

    2009-01-01

    Src family protein kinases (SFKs)-mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that PSD-93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4β-PMA in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn. PMID:18423999

  5. Expression of the NMDA receptor subunit GluN3A (NR3A) in the olfactory system and its regulatory role on olfaction in the adult mouse.

    PubMed

    Lee, Jin Hwan; Wei, Ling; Deveau, Todd C; Gu, Xiaohuan; Yu, Shan Ping

    2016-07-01

    Glutamate is an excitatory neurotransmitter in the olfactory system and its N-methyl-D-aspartate-(NMDA) receptor subunits [GluN1 (NR1), GluN2A (NR2A), and GluN2B (NR2B)] are expressed at synapses in the olfactory bulb and olfactory epithelium. Thus, glutamatergic neurons and NMDA receptors play key roles in olfaction. GluN3A (NR3A) is a unique inhibitory subunit in the NMDA receptor complex; however, the expression and functional role of GluN3A in the olfactory bulb and epithelium remain unclear. The present study examined the expression patterns of GluN3A in the olfactory bulb and epithelium and explored its functional role in the olfactory system. Immunohistochemical and Western blot analyses revealed that GluN3A is abundantly expressed in different cellular layers of the olfactory bulb and epithelium of the adult wild type (WT) mice. In littermate GluN3A knockout (GluN3A(-/-); KO) mice, the expression of olfactory marker protein normally found in mature olfactory sensory neurons was significantly reduced in the olfactory bulb and epithelium. A butyl alcohol stimulus increased immediate-early gene c-Fos expression in the olfactory system of WT mice, while this response was absent in GluN3A KO mice. The level of phosphorylated Ca(2+)/calmodulin-dependent kinase II was significantly lower in GluN3A KO mice compared to WT mice. In buried food finding test, GluN3A mice took significantly longer time to find food compared to WT mice. Consistently, impaired odor distinguishing ability was seen in GluN3A KO mice. These findings suggest that GluN3A, expressed in the adult olfactory system, plays a significant regulatory role in olfactory development and functional activity. PMID:26334321

  6. Tandem Subunits Effectively Constrain GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA Receptor-Associated Protein

    PubMed Central

    Boileau, Andrew J.; Pearce, Robert A.; Czajkowski, Cynthia

    2008-01-01

    GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of α, β, and γ subunits can be confounded by receptors expressed from α and β subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a “tandem” α1 subunit linked to a β2 subunit. Cotransfection of the γ2 subunit with αβ-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the γ subunit with nonlinked α1 and β2 subunits. Similarly, expression of α subunits together with αβ-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of α with β subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of αβ-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of α1β2 and α1β2γ2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in α1β2γ2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of αβγ receptors. PMID:16339017

  7. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  8. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    PubMed

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

  9. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  10. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    SciTech Connect

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J.

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  11. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective.

    PubMed

    Rapoport, Basil; McLachlan, Sandra M

    2016-04-01

    The TSH receptor (TSHR) on the surface of thyrocytes is unique among the glycoprotein hormone receptors in comprising two subunits: an extracellular A-subunit, and a largely transmembrane and cytosolic B-subunit. Unlike its ligand TSH, whose subunits are encoded by two genes, the TSHR is expressed as a single polypeptide that subsequently undergoes intramolecular cleavage into disulfide-linked subunits. Cleavage is associated with removal of a C-peptide region, a mechanism similar in some respects to insulin cleavage into disulfide linked A- and B-subunits with loss of a C-peptide region. The potential pathophysiological importance of TSHR cleavage into A- and B-subunits is that some A-subunits are shed from the cell surface. Considerable experimental evidence supports the concept that A-subunit shedding in genetically susceptible individuals is a factor contributing to the induction and/or affinity maturation of pathogenic thyroid-stimulating autoantibodies, the direct cause of Graves' disease. The noncleaving gonadotropin receptors are not associated with autoantibodies that induce a "Graves' disease of the gonads." We also review herein current information on the location of the cleavage sites, the enzyme(s) responsible for cleavage, the mechanism by which A-subunits are shed, and the effects of cleavage on receptor signaling. PMID:26799472

  12. Dicholine succinate, the neuronal insulin sensitizer, normalizes behavior, REM sleep, hippocampal pGSK3 beta and mRNAs of NMDA receptor subunits in mouse models of depression.

    PubMed

    Cline, Brandon H; Costa-Nunes, Joao P; Cespuglio, Raymond; Markova, Natalyia; Santos, Ana I; Bukhman, Yury V; Kubatiev, Aslan; Steinbusch, Harry W M; Lesch, Klaus-Peter; Strekalova, Tatyana

    2015-01-01

    Central insulin receptor-mediated signaling is attracting the growing attention of researchers because of rapidly accumulating evidence implicating it in the mechanisms of plasticity, stress response, and neuropsychiatric disorders including depression. Dicholine succinate (DS), a mitochondrial complex II substrate, was shown to enhance insulin-receptor mediated signaling in neurons and is regarded as a sensitizer of the neuronal insulin receptor. Compounds enhancing neuronal insulin receptor-mediated transmission exert an antidepressant-like effect in several pre-clinical paradigms of depression; similarly, such properties for DS were found with a stress-induced anhedonia model. Here, we additionally studied the effects of DS on several variables which were ameliorated by other insulin receptor sensitizers in mice. Pre-treatment with DS of chronically stressed C57BL6 mice rescued normal contextual fear conditioning, hippocampal gene expression of NMDA receptor subunit NR2A, the NR2A/NR2B ratio and increased REM sleep rebound after acute predation. In 18-month-old C57BL6 mice, a model of elderly depression, DS restored normal sucrose preference and activated the expression of neural plasticity factors in the hippocampus as shown by Illumina microarray. Finally, young naïve DS-treated C57BL6 mice had reduced depressive- and anxiety-like behaviors and, similarly to imipramine-treated mice, preserved hippocampal levels of the phosphorylated (inactive) form of GSK3 beta that was lowered by forced swimming in pharmacologically naïve animals. Thus, DS can ameliorate behavioral and molecular outcomes under a variety of stress- and depression-related conditions. This further highlights neuronal insulin signaling as a new factor of pathogenesis and a potential pharmacotherapy of affective pathologies. PMID:25767439

  13. Dicholine succinate, the neuronal insulin sensitizer, normalizes behavior, REM sleep, hippocampal pGSK3 beta and mRNAs of NMDA receptor subunits in mouse models of depression

    PubMed Central

    Cline, Brandon H.; Costa-Nunes, Joao P.; Cespuglio, Raymond; Markova, Natalyia; Santos, Ana I.; Bukhman, Yury V.; Kubatiev, Aslan; Steinbusch, Harry W. M.; Lesch, Klaus-Peter; Strekalova, Tatyana

    2015-01-01

    Central insulin receptor-mediated signaling is attracting the growing attention of researchers because of rapidly accumulating evidence implicating it in the mechanisms of plasticity, stress response, and neuropsychiatric disorders including depression. Dicholine succinate (DS), a mitochondrial complex II substrate, was shown to enhance insulin-receptor mediated signaling in neurons and is regarded as a sensitizer of the neuronal insulin receptor. Compounds enhancing neuronal insulin receptor-mediated transmission exert an antidepressant-like effect in several pre-clinical paradigms of depression; similarly, such properties for DS were found with a stress-induced anhedonia model. Here, we additionally studied the effects of DS on several variables which were ameliorated by other insulin receptor sensitizers in mice. Pre-treatment with DS of chronically stressed C57BL6 mice rescued normal contextual fear conditioning, hippocampal gene expression of NMDA receptor subunit NR2A, the NR2A/NR2B ratio and increased REM sleep rebound after acute predation. In 18-month-old C57BL6 mice, a model of elderly depression, DS restored normal sucrose preference and activated the expression of neural plasticity factors in the hippocampus as shown by Illumina microarray. Finally, young naïve DS-treated C57BL6 mice had reduced depressive- and anxiety-like behaviors and, similarly to imipramine-treated mice, preserved hippocampal levels of the phosphorylated (inactive) form of GSK3 beta that was lowered by forced swimming in pharmacologically naïve animals. Thus, DS can ameliorate behavioral and molecular outcomes under a variety of stress- and depression-related conditions. This further highlights neuronal insulin signaling as a new factor of pathogenesis and a potential pharmacotherapy of affective pathologies. PMID:25767439

  14. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  15. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  16. Roles of subunit phosphorylation in regulating glutamate receptor function

    PubMed Central

    Wang, John Q.; Guo, Ming-Lei; Jin, Dao-Zhong; Xue, Bing; Fibuch, Eugene E.; Mao, Li-Min

    2014-01-01

    Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders. PMID:24291102

  17. Effects of C-phycocyanin and Spirulina on Salicylate-Induced Tinnitus, Expression of NMDA Receptor and Inflammatory Genes

    PubMed Central

    Hwang, Juen-Haur; Chen, Jin-Cherng; Chan, Yin-Ching

    2013-01-01

    Effects of C-phycocyanin (C-PC), the active component of Spirulina platensis water extract on the expressions of N-methyl D-aspartate receptor subunit 2B (NR2B), tumor necrosis factor–α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase type 2 (COX-2) genes in the cochlea and inferior colliculus (IC) of mice were evaluated after tinnitus was induced by intraperitoneal injection of salicylate. The results showed that 4-day salicylate treatment (unlike 4-day saline treatment) caused a significant increase in NR2B, TNF-α, and IL-1β mRNAs expression in the cochlea and IC. On the other hand, dietary supplementation with C-PC or Spirulina platensis water extract significantly reduced the salicylate-induced tinnitus and down-regulated the mRNAs expression of NR2B, TNF-α, IL-1β mRNAs, and COX-2 genes in the cochlea and IC of mice. The changes of protein expression levels were generally correlated with those of mRNAs expression levels in the IC for above genes. PMID:23533584

  18. Atomic force microscopy of ionotropic receptors bearing subunit-specific tags provides a method for determining receptor architecture

    NASA Astrophysics Data System (ADS)

    Neish, Calum S.; Martin, Ian L.; Davies, Martin; Henderson, Robert M.; Edwardson, J. Michael

    2003-08-01

    We have developed an atomic force microscopy (AFM)-based method for the determination of the subunit architecture of ionotropic receptors, and tested the method using the GABAA receptor as a model system. The most common form of the GABAA receptor probably consists of 2alpha1-, 2beta2- and 1gamma2-subunits. We show here that the arrangement of subunits around the central Cl- ion channel can be deduced by AFM of receptors tagged with subunit-specific antibodies. Transfection of cells with DNA encoding alpha1-, beta2- and gamma2-subunits resulted in the production of receptors containing all three subunits, as judged by both immunoblot analysis and the binding of [3H]-Ro15-1788, a specific radioligand for the GABAA receptor. A His6-tag on the alpha1-subunit was used to purify the receptor from membrane fractions of transfected cells. After incubation with anti-His6 immunoglobulin G, some receptors became tagged with either one or two antibody molecules. AFM analysis of complexes containing two bound antibodies showed that the most common angle between the two tags was 135°, close to the value of 144° expected if the two alpha-subunits are separated by a third subunit. This method is applicable to the complete elucidation of the subunit arrangement around the GABAA receptor rosette, and can also be applied to other ionotropic receptors.

  19. Antibodies to GABAA receptor α1 and γ2 subunits

    PubMed Central

    Pettingill, Philippa; Kramer, Holger B.; Coebergh, Jan Adriaan; Pettingill, Rosie; Maxwell, Susan; Nibber, Anjan; Malaspina, Andrea; Jacob, Anu; Irani, Sarosh R.; Buckley, Camilla; Beeson, David; Lang, Bethan; Waters, Patrick

    2015-01-01

    Objective: To search for antibodies against neuronal cell surface proteins. Methods: Using immunoprecipitation from neuronal cultures and tandem mass spectrometry, we identified antibodies against the α1 subunit of the γ-aminobutyric acid A receptor (GABAAR) in a patient whose immunoglobulin G (IgG) antibodies bound to hippocampal neurons. We searched 2,548 sera for antibodies binding to GABAAR α, β, and γ subunits on live HEK293 cells and identified the class, subclass, and GABAAR subunit specificities of the positive samples. Results: GABAAR-Abs were identified in 40 of 2,046 (2%) referred sera previously found negative for neuronal antibodies, in 5/502 (1%) previously positive for other neuronal surface antibodies, but not in 92 healthy individuals. The antibodies in 40% bound to either the α1 (9/45, 20%) or the γ2 subunits (9/45, 20%) and were of IgG1 (94%) or IgG3 (6%) subclass. The remaining 60% had lower antibody titers (p = 0.0005), which were mainly immunoglobulin M (IgM) (p = 0.0025), and showed no defined subunit specificity. Incubation of primary hippocampal neurons with GABAAR IgG1 sera reduced surface GABAAR membrane expression. The clinical features of 15 patients (GABAAR α1 n = 6, γ2 n = 5, undefined n = 4) included seizures (47%), memory impairment (47%), hallucinations (33%), or anxiety (20%). Most patients had not been given immunotherapies, but one with new-onset treatment-resistant catatonia made substantial improvement after plasma exchange. Conclusions: The GABAAR α1 and γ2 are new targets for antibodies in autoimmune neurologic disease. The full spectrum of clinical features, treatment responses, correlation with antibody specificity, and in particular the role of the IgM antibodies will need to be assessed in future studies. PMID:25636713

  20. NMDA receptor structures reveal subunit arrangement and pore architecture

    PubMed Central

    Lee, Chia-Hsueh; Lü, Wei; Michel, Jennifer Carlisle; Goehring, April; Du, Juan; Song, Xianqiang; Gouaux, Eric

    2014-01-01

    Summary N-methyl-d-aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present x-ray crystal structures of the GluN1/GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino terminal and ligand binding domains. The transmembrane domains harbor a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ~2-fold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors. PMID:25008524

  1. Essential involvement of the NMDA receptor in ethanol preconditioning-dependent neuroprotection from amyloid-betain vitro.

    PubMed

    Mitchell, Robert M; Neafsey, Edward J; Collins, Michael A

    2009-10-01

    In several epidemiological studies, moderate ethanol consumption has been associated with reduced risks of cognitive decline or Alzheimer's dementia. Of potential relevance is that brain cultures preconditioned with moderate ethanol concentrations are resistant to neurotoxic Alzheimer's amyloid-beta (Abeta) peptides. Using rat cerebellar mixed cultures we investigated whether certain membrane receptors were early 'sensors' in moderate ethanol preconditioning (MEP). In a 6-day MEP protocol (30 mM ethanol), neuroprotection from Abeta25-35 was undiminished by antagonism during the first 3 days of either adenosine A(1) or Galpha(i/o) protein-coupled receptors. However, similar cotreatment with memantine or DL-2-amino-5-phosphono-pentanoic acid (AP-5), antagonists of NMDA receptors (NMDAR), abolished neuroprotection, indicating key early involvement of this ionotropic glutamate receptor. Also in these cultures, directly activating NMDAR using subexcitotoxic NMDA preconditioning prevented Abeta neurotoxicity. By day 2 of MEP, we observed increased levels of NMDAR subunits NR1, NR2B, and NR2C that persisted through day 6. Interestingly, memantine co-exposure blocked elevations in the obligatory NR1 subunit. Furthermore, 2 days of MEP significantly increased two indicators of synaptic NMDAR localization, NR2B phospho-Tyr1472, and post-synaptic density 95 scaffolding protein. The results indicate that ethanol preconditioning-dependent neuroprotection is associated with early increases in NR subunits concomitant with enhancement of synaptic localization and activity of NMDAR. PMID:19694907

  2. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs.

    PubMed Central

    Ymer, S; Schofield, P R; Draguhn, A; Werner, P; Köhler, M; Seeburg, P H

    1989-01-01

    Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Images PMID:2548852

  3. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain.

    PubMed

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs. PMID:26241350

  4. Abnormal subcellular localization of GABAA receptor subunits in schizophrenia brain

    PubMed Central

    Mueller, T M; Remedies, C E; Haroutunian, V; Meador-Woodruff, J H

    2015-01-01

    Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABAAR). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1–6, β1–3 or γ2 GABAAR subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABAAR subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABAARs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABAAR subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N=16) and comparison (N=14) subjects and found evidence of abnormal localization of the β1 and β2 GABAAR subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β252 kDa), 50 kDa (β250 kDa) and 48 kDa (β248 kDa). In the ER, we found increased total β2 GABAAR subunit (β2ALL) expression driven by increased β250 kDa, a decreased ratio of β248 kDa:β2ALL and an increased ratio of β250 kDa:β248 kDa. Decreased ratios of β1:β2ALL and β1:β250 kDa in both the ER and SYN fractions and an increased ratio of β252 kDa:β248 kDa at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABAARs. PMID:26241350

  5. Brain regional distribution of GABA(A) receptors exhibiting atypical GABA agonism: roles of receptor subunits.

    PubMed

    Halonen, Lauri M; Sinkkonen, Saku T; Chandra, Dev; Homanics, Gregg E; Korpi, Esa R

    2009-11-01

    The major inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABA(A) receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding to GABA(A) receptors in brain sections and compared the displacing capacities of 10mM GABA and 1mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABA(A) receptor alpha1, alpha4, delta, and alpha4+delta subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to "GABA-insensitive" (GIS) [(35)S]TBPS binding. THIP displaced more [(35)S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of delta KO and alpha4+delta KO mice, being only slightly diminished in that of alpha1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in alpha4 KO mice. However, it was fully abolished in alpha1 KO mice, indicating that the alpha1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABA(A) receptors in brain sections showing reduced displacing capacity of [(35)S]TBPS binding by GABA (partial agonism) minimally require the assembly of alpha1 and beta subunits in the forebrain and of alpha6 and beta subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABA(A) receptors. PMID:19397945

  6. Brain regional distribution of GABAA receptors exhibiting atypical GABA agonism: roles of receptor subunits

    PubMed Central

    Halonen, Lauri M.; Sinkkonen, Saku T.; Chandra, Dev; Homanics, Gregg E.; Korpi, Esa R.

    2009-01-01

    The major inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA), has only partial efficacy at certain subtypes of GABAA receptors. To characterize these minor receptor populations in rat and mouse brains, we used autoradiographic imaging of t-butylbicyclophosphoro[35S]thionate ([35S]TBPS) binding to GABAA receptors in brain sections and compared the displacing capacities of 10 mM GABA and 1 mM 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), a competitive GABA-site agonist. Brains from GABAA receptor α1, α4, δ, and α4 + δ subunit knockout (KO) mouse lines were used to understand the contribution of these particular receptor subunits to “GABA-insensitive” (GIS) [35S]TBPS binding. THIP displaced more [35S]TBPS binding than GABA in several brain regions, indicating that THIP also inhibited GIS-binding. In these regions, GABA prevented the effect of THIP on GIS-binding. GIS-binding was increased in the cerebellar granule cell layer of δ KO and α4 + δ KO mice, being only slightly diminished in that of α1 KO mice. In the thalamus and some other forebrain regions of wild-type mice, a significant amount of GIS-binding was detected. This GIS-binding was higher in α4 KO mice. However, it was fully abolished in α1 KO mice, indicating that the α1 subunit was obligatory for the GIS-binding in the forebrain. Our results suggest that native GABAA receptors in brain sections showing reduced displacing capacity of [35S]TBPS binding by GABA (partial agonism) minimally require the assembly of α1 and β subunits in the forebrain and of α6 and β subunits in the cerebellar granule cell layer. These receptors may function as extrasynaptic GABAA receptors. PMID:19397945

  7. Alcohol- and alcohol antagonist-sensitive human GABAA receptors: tracking δ subunit incorporation into functional receptors.

    PubMed

    Meera, Pratap; Olsen, Richard W; Otis, Thomas S; Wallner, Martin

    2010-11-01

    GABA(A) receptors (GABA(A)Rs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABA(A)Rs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn(2+) block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABA(A)Rs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABA(A)Rs. PMID:20699325

  8. Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

    PubMed Central

    Meera, Pratap; Olsen, Richard W.; Otis, Thomas S.

    2010-01-01

    GABAA receptors (GABAARs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABAARs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn2+ block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABAARs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABAARs. PMID:20699325

  9. Selective vulnerability of hippocampal cornu ammonis 1 pyramidal cells to excitotoxic insult is associated with the expression of polyamine-sensitive N-methyl-D-asparate-type glutamate receptors

    PubMed Central

    Butler, Tracy R.; Self, Rachel L.; Smith, Katherine J.; Sharrett-Field, Lynda J.; Berry, Jennifer N.; Littleton, John M.; Pauly, James R.; Mulholland, Patrick J.; Prendergast, Mark A.

    2009-01-01

    Excess glutamate release and stimulation of post-synaptic glutamatergic receptors have been implicated in the pathophysiology of many neurological diseases. The hippocampus, and the pyramidal cell layer of the cornu ammonus 1 (CA1) region in particular, has been noted for its selective sensitivity to excitotoxic insults. The current studies examined the role of N-methyl-D-aspartate (NMDA) receptor subunit composition and sensitivity to stimulatory effects of the polyamine spermidine, an allosteric modulator of NMDA NR2 subunit activity, in hippocampal CA1 region sensitivity to excitotoxic insult. Organotypic hippocampal slice cultures of 8 day-old neonatal rat were obtained and maintained in vitro for 5 days. At this time, immunohistochemical analysis of mature neuron density (NeuN); microtubule associated protein-2(a,b) density (MAP-2); and NMDA receptor NR1 and NR2B subunit density in the primary cell layers of the dentate gyrus (DG), CA3, and CA1 regions, was conducted. Further, autoradiographic analysis of NMDA receptor distribution and density (i.e. [125I]MK-801 binding) and spermidine (100 μM)-potentiated [125I]MK-801 binding in the primary cell layers of these regions was examined. A final series of studies examined effects of prolonged exposure to NMDA (0.1–10 μM) on neurodegeneration in the primary cell layers of the DG, CA3, and CA1 regions, in the absence and presence of spermidine (100 μM) or ifenprodil (100 μM), an allosteric inhibitor of NR2B polypeptide subunit activity. The pyramidal cell layer of the CA1 region demonstrated significantly greater density of mature neurons, MAP-2, NR1 and NR2B subunits, and [125I]MK-801 binding than the CA3 region or DG. Twenty-four hour NMDA (10 μM) exposure produced marked neurodegeneration (~350% of control cultures) in the CA1 pyramidal cell region that was significantly reduced by co-exposure to ifenprodil or APV. The addition of spermidine significantly potentiated [125I]MK-801 binding and

  10. IONOTROPIC GLUTAMATE RECEPTORS mRNA EXPRESSION IN THE HUMAN THALAMUS: ABSENCE OF CHANGE IN SCHIZOPHRENIA

    PubMed Central

    Dracheva, Stella; Byne, William; Chin, Benjamin; Haroutunian, Vahram

    2009-01-01

    Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventrolateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of NMDA receptor; GluR1-4 subunits of AMPA receptor; GluR5-7 and KA1-2 subunits of kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1≫NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia. PMID:18462708

  11. Developmental and cell-selective variations in N-methyl-d-aspartate receptor degradation by calpain

    PubMed Central

    Dong, Yi Na; Wu, Hai-Yan; Hsu, Fu-Chun; Coulter, Douglas A.; Lynch, David R.

    2008-01-01

    NMDA receptors play critical roles in synaptic modulation and neurological disorders. In this study, we investigated the developmental changes in NR2 cleavage by NMDA receptor-activated calpain in cultured cortical and hippocampal neurons. Calpain activity increased with development, associated with increased expression of NMDA receptors but not of calpain I. The activation of calpain in immature and mature cortical cultures was inhibited by antagonists of NR1/2B and NR1/2A/2B receptors, whereas the inhibition of NR1/2B receptors did not alter calpain activation in mature hippocampal cultures. The degradation of NR2 subunits by calpain differed with developmental age. NR2A was not a substrate of calpain in mature hippocampal cultures, but was cleaved in immature cortical and hippocampal cultures. NR2B degradation by calpain in cortical cultures decreased with development, but the level of degradation of NR2B in hippocampal cultures did not change. The kinetics of NMDA receptor-gated whole cell currents were also modulated by calpain activation in a manner that varied with developmental stage in vitro. In early (but not later) developmental stages, calpain activation altered the NMDA-evoked current rise time and time constants for both desensitization and deactivation. Our data suggest that the susceptibility of the NMDA receptor to cleavage by calpain varies with neuronal maturity in a manner that may alter its electrophysiological properties. PMID:16899064

  12. PSD-95 and Calcineurin Control the Sensitivity of NMDA Receptors to Calpain Cleavage in Cortical Neurons

    PubMed Central

    Yuen, Eunice Y.; Ren, Yi; Yan, Zhen

    2010-01-01

    The N-methyl-D-aspartate receptor (NMDAR) is a Ca2+-permeable glutamate receptor mediating many neuronal functions under normal and pathological conditions. Ca2+-influx via NMDARs activates diverse intracellular targets, including Ca2+-dependent protease calpain. Biochemical studies suggest that NR2A and NR2B subunits of NMDARs are substrates of calpain. Our physiological data showed that calpain, activated by prolonged NMDA treatment (100 µM, 5 min) of cultured cortical neurons, irreversibly decreased the whole-cell currents mediated by extrasynaptic NMDARs. Animals exposed to transient forebrain ischemia, a condition that activates calpain, exhibited the reduced NMDAR current density and the lower full-length NR2A/B level in a calpain-dependent manner. Disruption of the association between NMDARs and the scaffolding protein PSD-95 facilitated the calpain regulation of synaptic NMDAR responses and NR2 cleavage in cortical slices, while inhibition of calcineurin activity blocked the calpain effect on NMDAR currents and NR2 cleavage. Calpain-cleaved NR2B subunits were removed from the cell surface. Moreover, cell viability assays showed that calpain, by targeting NMDARs, provided a negative feedback to dampen neuronal excitability in excitotoxic conditions. These data suggest that calpain activation suppresses NMDAR function via proteolytic cleavage of NR2 subunits in vitro and in vivo, and the susceptibility of NMDARs to calpain cleavage is controlled by PSD-95 and calcineurin. PMID:18445709

  13. Endocytosis and degradative sorting of NMDA receptors by conserved membrane-proximal signals.

    PubMed

    Scott, Derek B; Michailidis, Ioannis; Mu, Yuanyue; Logothetis, Diomedes; Ehlers, Michael D

    2004-08-11

    Regulation of the abundance of NMDA receptors (NMDARs) at excitatory synapses is critical during changes in synaptic efficacy underlying learning and memory as well as during synapse formation throughout neural development. However, the molecular signals that govern NMDAR delivery, maintenance, and internalization remain unclear. In this study, we identify a conserved family of membrane-proximal endocytic signals, two within the NMDAR type 1 (NR1) subunit and one within the NR2A and NR2B subunits, necessary and sufficient to drive the internalization of NMDARs. These endocytic motifs reside in the region of NMDAR subunits immediately after the fourth membrane segment, a region implicated in use-dependent rundown and NMDA channel inactivation. Although endocytosis driven by the distal C-terminal domain of NR2B is followed by rapid recycling, internalization mediated by membrane-proximal motifs selectively targets receptors to late endosomes and accelerates degradation. These results define a novel conserved signature of NMDARs regulating internalization and postendocytic trafficking. PMID:15306643

  14. The R18 Polyarginine Peptide Is More Effective Than the TAT-NR2B9c (NA-1) Peptide When Administered 60 Minutes after Permanent Middle Cerebral Artery Occlusion in the Rat

    PubMed Central

    Milani, D.; Knuckey, N. W.; Anderton, R. S.; Cross, J. L.; Meloni, B. P.

    2016-01-01

    We examined the dose responsiveness of polyarginine R18 (100, 300, and 1000 nmol/kg) when administered 60 minutes after permanent middle cerebral artery occlusion (MCAO). The TAT-NR2B9c peptide, which is known to be neuroprotective in rodent and nonhuman primate stroke models, served as a positive control. At 24 hours after MCAO, there was reduced total infarct volume in R18 treated animals at all doses, but this reduction only reached statistical significance at doses of 100 and 1000 nmol/kg. The TAT-NR2B9c peptide reduced infarct volume at doses of 300 and 1000 nmol/kg, but not to a statistically significant extent, while the 100 nmol/kg dose was ineffective. The reduction in infarct volume with R18 and TAT-NR2B9c peptide treatments was mirrored by improvements in one or more functional outcomes (namely, neurological score, adhesive tape removal, and rota-rod), but not to a statistically significant extent. These findings further confirm the neuroprotective properties of polyarginine peptides and for R18 extend its therapeutic time window and dose range, as well as demonstrating its greater efficacy compared to TAT-NR2B9c in a severe stroke model. The superior neuroprotective efficacy of R18 over TAT-NR2B9c highlights the potential of this polyarginine peptide as a lead candidate for studies in human stroke. PMID:27247825

  15. Gene Expression Switching of Receptor Subunits in Human Brain Development

    PubMed Central

    Bar-Shira, Ossnat; Maor, Ronnie; Chechik, Gal

    2015-01-01

    Synaptic receptors in the human brain consist of multiple protein subunits, many of which have multiple variants, coded by different genes, and are differentially expressed across brain regions and developmental stages. The brain can tune the electrophysiological properties of synapses to regulate plasticity and information processing by switching from one protein variant to another. Such condition-dependent variant switch during development has been demonstrated in several neurotransmitter systems including NMDA and GABA. Here we systematically detect pairs of receptor-subunit variants that switch during the lifetime of the human brain by analyzing postmortem expression data collected in a population of donors at various ages and brain regions measured using microarray and RNA-seq. To further detect variant pairs that co-vary across subjects, we present a method to quantify age-corrected expression correlation in face of strong temporal trends. This is achieved by computing the correlations in the residual expression beyond a cubic-spline model of the population temporal trend, and can be seen as a nonlinear version of partial correlations. Using these methods, we detect multiple new pairs of context dependent variants. For instance, we find a switch from GLRA2 to GLRA3 that differs from the known switch in the rat. We also detect an early switch from HTR1A to HTR5A whose trends are negatively correlated and find that their age-corrected expression is strongly positively correlated. Finally, we observe that GRIN2B switch to GRIN2A occurs mostly during embryonic development, presumably earlier than observed in rodents. These results provide a systematic map of developmental switching in the neurotransmitter systems of the human brain. PMID:26636753

  16. Differential long-term neuroadaptations of glutamate receptors in the basolateral and central amygdala after withdrawal from cocaine self-administration in rats.

    PubMed

    Lu, Lin; Dempsey, Jack; Shaham, Yavin; Hope, Bruce T

    2005-07-01

    Humans and laboratory animals remain highly vulnerable to relapse to cocaine-seeking after prolonged periods of withdrawal from the drug. It has been hypothesized that this persistent cocaine relapse vulnerability involves drug-induced alterations in glutamatergic synapses within the mesolimbic dopamine reward system. Previous studies have shown that cocaine self-administration induces long-lasting neuroadaptations in glutamate neurons of the ventral tegmental area and nucleus accumbens. Here, we determined the effect of cocaine self-administration and subsequent withdrawal on glutamate receptor expression in the amygdala, a component of the mesolimbic dopamine system that is involved in cocaine seeking and craving induced by drug-associated cues. Rats were trained for 10 days to self-administer intravenous cocaine (6 h/day) or saline (a control condition) and were killed after one or 30 withdrawal days. Basolateral and central amygdala tissues were assayed for protein expression of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits (GluR1 and GluR2) and the NMDA receptor subunits (NR1, NR2A and NR2B). In the basolateral amygdala, GluR1 but not GluR2 levels were increased on days 1 and 30, NR2A levels were increased on day 1, and NR2B levels were decreased on day 30 of withdrawal from cocaine. In the central amygdala, GluR2 but not GluR1 levels were increased on days 1 and 30, NR1 levels were increased on day 30 and NR2A or NR2B levels were not altered after withdrawal from cocaine. These results indicate that cocaine self-administration and subsequent withdrawal induces long-lasting and differential neuroadaptations in basolateral and central amygdala glutamate receptors. PMID:15953359

  17. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites.

    PubMed

    Hayashi, Takashi; Rumbaugh, Gavin; Huganir, Richard L

    2005-09-01

    Modification of AMPA receptor function is a major mechanism for the regulation of synaptic transmission and underlies several forms of synaptic plasticity. Post-translational palmitoylation is a reversible modification that regulates localization of many proteins. Here, we report that palmitoylation of the AMPA receptor regulates receptor trafficking. All AMPA receptor subunits are palmitoylated on two cysteine residues in their transmembrane domain (TMD) 2 and in their C-terminal region. Palmitoylation on TMD 2 is upregulated by the palmitoyl acyl transferase GODZ and leads to an accumulation of the receptor in the Golgi and a reduction of receptor surface expression. C-terminal palmitoylation decreases interaction of the AMPA receptor with the 4.1N protein and regulates AMPA- and NMDA-induced AMPA receptor internalization. Moreover, depalmitoylation of the receptor is regulated by activation of glutamate receptors. These data suggest that regulated palmitoylation of AMPA receptor subunits modulates receptor trafficking and may be important for synaptic plasticity. PMID:16129400

  18. H-Ras Modulates N-Methyl-d-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity*

    PubMed Central

    Thornton, Claire; Yaka, Rami; Dinh, Son; Ron, Dorit

    2005-01-01

    Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hip-pocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction. PMID:12695509

  19. Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors.

    PubMed

    Chang, Howard Chia-Hao; Rongo, Christopher

    2005-05-01

    AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the Caenorhabditis elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1, or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex. PMID:15840655

  20. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  1. NR2C and NR2D subunits of NMDA receptors in frog and turtle retina.

    PubMed

    Vitanova, Lily Alexandrova

    2012-12-01

    Glutamate NMDA (N-methyl-D-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron-glia interactions, is discussed. PMID:22386206

  2. N-glycosylation sites on the nicotinic ACh receptor subunits regulate receptor channel desensitization and conductance.

    PubMed

    Nishizaki, Tomoyuki

    2003-06-10

    The present study investigated the effects of N-glycosylation sites on Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents and single-channel currents from excised patches. Receptors with the mutant subunit at the asparagine residue on the conserved N-glycosylation site (mbetaN141D, mgammaN141D, or mdeltaN143D) or the serine/threonine residue (mbetaT143A, mgammaS143A, or mdeltaS145A) delayed the rate of current decay as compared with wild-type receptors, and the most striking effect was found with receptors with mbetaT143A or mgammaS143A. For wild-type receptors, the lectin concanavalin A, that binds to glycosylated membrane proteins with high affinity, mimicked this effect. Receptors with mbetaN141D or mdeltaN143D exhibited lower single-channel conductance, but those with mbetaT143A, mgammaS143A, or mdeltaS145A otherwise revealed higher conductance than wild-type receptors. Mean opening time of single-channel currents was little affected by the mutation. N-glycosylation sites, thus, appear to play a role in the regulation of ACh receptor desensitization and ion permeability. PMID:12829329

  3. Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

    PubMed

    Hussy, N; Lukas, W; Jones, K A

    1994-12-01

    1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis. PMID

  4. 5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems

    PubMed Central

    2015-01-01

    Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined. PMID:25951416

  5. Transcription factor assembly on the nicotinic receptor beta4 subunit gene promoter.

    PubMed

    Scofield, Michael D; Brüschweiler-Li, Lei; Mou, Zhongming; Gardner, Paul D

    2008-04-16

    Nicotinic acetylcholine receptors are involved in a plethora of fundamental biological processes ranging from muscle contraction to formation of memories. The receptors are pentameric proteins whose subunits are encoded by distinct genes. Subunit composition of a mature nicotinic receptor is governed in part by the transcriptional regulation of each subunit gene. Here, using chromatin immunoprecipitation assays, we report the interaction of the transcription factors Sp1, Sp3, c-Jun and Sox10 with the beta4 subunit gene promoter in neuronal-like cell lines and rodent brain tissue. Our results corroborate previous in-vitro data demonstrating that these transcription factors interact with the beta4 promoter. Taken together, these data suggest that Sp1, Sp3, c-Jun and Sox10 regulate expression of the beta4 subunit gene in the mammalian brain. PMID:18382288

  6. A neurosteroid potentiation site can be moved among GABAA receptor subunits.

    PubMed

    Bracamontes, John R; Li, Ping; Akk, Gustav; Steinbach, Joe Henry

    2012-11-15

    Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid. PMID:22988137

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

    PubMed

    Riou, Morgane; Stroebel, David; Edwardson, J Michael; Paoletti, Pierre

    2012-01-01

    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. PMID:22493736

  8. Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

    PubMed

    Zaman, S H; Shingai, R; Harvey, R J; Darlison, M G; Barnard, E A

    1992-04-10

    When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation. PMID:1323476

  9. Molecular determinants of NMDA receptor internalization.

    PubMed

    Roche, K W; Standley, S; McCallum, J; Dune Ly, C; Ehlers, M D; Wenthold, R J

    2001-08-01

    Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites. PMID:11477425

  10. Subunit composition of α5-containing nicotinic receptors in the rodent habenula

    PubMed Central

    Scholze, Petra; Koth, Gabriele; Orr-Urtreger, Avi; Huck, Sigismund

    2012-01-01

    Gene association studies in humans have linked the α5 subunit gene CHRNA5 to an increased risk for nicotine dependence. In the CNS, nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit are expressed at relatively high levels in the habenulo-interpeduncular system. Recent experimental evidence furthermore suggests that α5-containing receptors in the habenula play a key role in controlling the intake of nicotine in rodents. We have now analysed the subunit composition of hetero-oligomeric nAChRs in the habenula of postnatal day 18 (P18) C57Bl/6J control mice and of mice with deletions of the α5, the β2, or the β4 subunit genes. Receptors consisting of α3β4*1 clearly outnumbered α4β2*-containing receptors not only in P18 but also in adult mice. We found low levels of α5-containing receptors in both mice (6%) and rats (2.5% of overall nAChRs). Observations in β2 and β4 null mice indicate that although α5 requires the presence of the β4 subunit for assembling (but not of β2), α5 in wild-type mice assembles into receptors that also contain the subunits α3, β2, and β4. PMID:22380605

  11. Subunit Composition of Neurotransmitter Receptors in the Immature and in the Epileptic Brain

    PubMed Central

    Sánchez Fernández, Iván; Loddenkemper, Tobias

    2014-01-01

    Neuronal activity is critical for synaptogenesis and the development of neuronal networks. In the immature brain excitation predominates over inhibition facilitating the development of normal brain circuits, but also rendering it more susceptible to seizures. In this paper, we review the evolution of the subunit composition of neurotransmitter receptors during development, how it promotes excitation in the immature brain, and how this subunit composition of neurotransmission receptors may be also present in the epileptic brain. During normal brain development, excitatory glutamate receptors peak in function and gamma-aminobutiric acid (GABA) receptors are mainly excitatory rather than inhibitory. A growing body of evidence from animal models of epilepsy and status epilepticus has demonstrated that the brain exposed to repeated seizures presents a subunit composition of neurotransmitter receptors that mirrors that of the immature brain and promotes further seizures and epileptogenesis. Studies performed in samples from the epileptic human brain have also found a subunit composition pattern of neurotransmitter receptors similar to the one found in the immature brain. These findings provide a solid rationale for tailoring antiepileptic treatments to the specific subunit composition of neurotransmitter receptors and they provide potential targets for the development of antiepileptogenic treatments. PMID:25295256

  12. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  13. Roles of nicotinic acetylcholine receptor β subunits in function of human α4-containing nicotinic receptors

    PubMed Central

    Wu, Jie; Liu, Qiang; Yu, Kewei; Hu, Jun; Kuo, Yen-Ping; Segerberg, Marsha; St John, Paul A; Lukas, Ronald J

    2006-01-01

    Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*-nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2- and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2- and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*-nAChR with distinctive physiological and pharmacological features. Diversity in α4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence. PMID:16825297

  14. Identification of subunits of acetylcholine receptor that interact with a cholesterol photoaffinity probe

    SciTech Connect

    Middlemas, D.S.; Raftery, M.A.

    1987-03-10

    All four subunits of the acetylcholine receptor in membrane vesicles isolated from Torpedo californica have been labeled with (/sup 3/H)cholesteryl diazoacetate. As this probe incorporates into lipid bilayers analogously to cholesterol, this result indicates that acetylcholine receptor interacts with cholesterol. This investigation also demonstrates that this probe is a useful reagent for studying the interaction of cholesterol with membrane proteins.

  15. Physiological Roles of Calpain 1 Associated to Multiprotein NMDA Receptor Complex

    PubMed Central

    Averna, Monica; Pellegrini, Matteo; Cervetto, Chiara; Pedrazzi, Marco; Bavestrello, Margherita; De Tullio, Roberta; Salamino, Franca; Pontremoli, Sandro; Melloni, Edon

    2015-01-01

    We have recently demonstrated that in resting conditions calpain 1, but not calpain 2, is specifically associated to the N-Methyl-D-Aspartate receptor (NMDAR) multiprotein complex. We are here reporting that in SKNBE neuroblastoma cells or in freshly isolated nerve terminals from adult rat hippocampus, the proteolytic activity of calpain 1 resident at the NMDAR is very low under basal conditions and greatly increases following NMDAR stimulation. Since the protease resides at the NMDAR in saturating amounts, variations in Ca2+ influx promote an increase in calpain 1 activity without affecting the amount of the protease originally associated to NMDAR. In all the conditions examined, resident calpain 1 specifically cleaves NR2B at the C-terminal region, leading to its internalization together with NR1 subunit. While in basal conditions intracellular membranes include small amounts of NMDAR containing the calpain-digested NR2B, upon NMDAR stimulation nearly all the receptor molecules are internalized. We here propose that resident calpain 1 is involved in NMDAR turnover, and following an increase in Ca2+ influx, the activated protease, by promoting the removal of NMDAR from the plasma membranes, can decrease Ca2+ entrance through this channel. Due to the absence of calpastatin in such cluster, the activity of resident calpain 1 may be under the control of HSP90, whose levels are directly related to the activation of this protease. Observations of different HSP90/calpain 1 ratios in different ultrasynaptic compartments support this conclusion. PMID:26431040

  16. Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function.

    PubMed

    Madry, Christian; Mesic, Ivana; Bartholomäus, Ingo; Nicke, Annette; Betz, Heinrich; Laube, Bodo

    2007-03-01

    Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function. PMID:17214961

  17. Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

    PubMed Central

    Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

    2015-01-01

    Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. PMID:25229716

  18. Both alpha and beta subunits of human choriogonadotropin photoaffinity label the hormone receptor.

    PubMed Central

    Ji, I; Ji, T H

    1981-01-01

    It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor. Images PMID:6272303

  19. Deletion of the 5-HT3A-receptor subunit blunts the induction of cocaine sensitization

    PubMed Central

    Hodge, C. W.; Bratt, A. M.; Kelley, S. P.

    2008-01-01

    Serotonin (5-HT) receptors are classified into seven groups (5-HT1–7), comprising at least 14 structurally and pharmacologically distinct receptor subtypes. Pharma-cological antagonism of ionotropic 5-HT3 receptors has been shown to modulate both behavioral and neuro-chemical aspects of the induction of sensitization to cocaine. It is not known, however, if specific molecular subunits of the 5-HT3 receptor influence the development of cocaine sensitization. To address this question, we studied the effects of acute and chronic intermittent cocaine administration in mice with a targeted deletion of the gene for the 5-HT3A-receptor subunit (5-HT3A −/−). 5-HT3A (−/−) mice showed blunted induction of cocaine-induced locomotor sensitization as compared with wild-type littermate controls. 5-HT3A (−/−) mice did not differ from wild-type littermate controls on measures of basal motor activity or response to acute cocaine treatment. Enhanced locomotor response to saline injection following cocaine sensitization was observed equally in 5-HT3A (−/−) and wild-type mice suggesting similar conditioned effects associated with chronic cocaine treatment. These data show a role for the 5-HT3A-receptor subunit in the induction of behavioral sensitization to cocaine and suggest that the 5-HT3A molecular subunit modulates neurobehavioral adaptations to cocaine, which may underlie aspects of addiction. PMID:17559417

  20. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  1. Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease.

    PubMed

    Calon, Frédéric; Lim, Giselle P; Morihara, Takashi; Yang, Fusheng; Ubeda, Oliver; Salem, Norman; Frautschy, Sally A; Cole, Greg M

    2005-08-01

    Epidemiological data indicate that low n-3 polyunsaturated fatty acids (PFA) intake is a readily manipulated dietary risk factor for Alzheimer's disease (AD). Studies in animals confirm the deleterious effect of n-3 PFA depletion on cognition and on dendritic scaffold proteins. Here, we show that in transgenic mice overexpressing the human AD gene APPswe (Tg2576), safflower oil-induced n-3 PFA deficiency caused a decrease in N-methyl-D-aspartate (NMDA) receptor subunits, NR2A and NR2B, in the cortex and hippocampus with no loss of the presynaptic markers, synaptophysin and synaptosomal-associated protein 25 (SNAP-25). n-3 PFA depletion also decreased the NR1 subunit in the hippocampus and Ca2+/calmodulin-dependent protein kinase (CaMKII) in the cortex of Tg2576 mice. These effects of dietary n-3 PFA deficiency were greatly amplified in Tg2576 mice compared to nontransgenic mice. Loss of the NR2B receptor subunit was not explained by changes in mRNA expression, but correlated with p85alpha phosphatidylinositol 3-kinase levels. Most interestingly, n-3 PFA deficiency dramatically increased levels of protein fragments, corresponding to caspase/calpain-cleaved fodrin and gelsolin in Tg2576 mice. This effect was minimal in nontransgenic mice suggesting that n-3 PFA depletion potentiated caspase activation in the Tg2576 mouse model of AD. Dietary supplementation with docosahexaenoic acid (DHA; 22 : 6n-3) partly protected from NMDA receptor subunit loss and accumulation of fodrin and gelsolin fragments but fully prevented CaMKII decrease. The marked effect of dietary n-3 PFA on NMDA receptors and caspase/calpain activation in the cortex of an animal model of AD provide new insights into how dietary essential fatty acids may influence cognition and AD risk. PMID:16101743

  2. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  3. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  4. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    PubMed

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

  5. Agonist and antagonist effects of nicotine on chick neuronal nicotinic receptors are defined by alpha and beta subunits.

    PubMed

    Hussy, N; Ballivet, M; Bertrand, D

    1994-09-01

    1. Functional neuronal nicotinic receptors were reconstituted in Xenopus oocytes by the nuclear injection of different combinations of chick and rat cDNAs encoding alpha and beta subunits. The pharmacology of these nicotinic receptors was investigated using two-electrode voltage clamp. 2. The sensitivity of the chick alpha 3/beta 2, alpha 3/beta 4, and alpha 4/beta 2 receptors to acetylcholine (ACh) and neuronal bungarotoxin differed markedly, indicating that both subunits contribute to the pharmacological properties of the receptors. 3. Nicotine acted as an agonist on the chick alpha 3/beta 4 and alpha 4/beta 2 receptors and rat alpha 3/beta 2 receptor. In contrast, nicotine (at concentrations > 3 microM) was only a weak partial agonist of the chick alpha 3/beta 2 receptor. Moreover, nicotine coapplied with 3 microM ACh on the chick alpha 3/beta 2 receptor acted as a potent competitive antagonist, with an IC50 of 0.43 microM. No antagonist effect of nicotine could be revealed on the other nicotinic receptors. 4. The effect of nicotine was tested on hybrid receptors obtained by coinjection of chick and rat cDNAs encoding the alpha 3 and beta 2 subunits (yielding the rat alpha 3/chick beta 2 and chick alpha 3/rat beta 2 receptors). Nicotine (10 microM) strongly inhibited both hybrid receptors. 5. Chimeric subunits were constructed by exchanging a segment located in the extracellular N-termini of chick alpha 3 and alpha 4 subunits and chick alpha 3 and rat alpha 3 subunits. These subunits were coexpressed in oocytes with chick or rat beta 2 subunits. The effect of nicotine on these receptors pointed to the importance of a 15 amino acid stretch located 3' of the first transmembrane segment in the determination of the agonist and antagonist action of nicotine. 6. Within this 15 amino acid segment, a single residue differs in chick and rat alpha 3 subunits, at position 198, within the ligand binding site of alpha subunits. Gln198 of the rat alpha 3 subunit was replaced

  6. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  7. A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.

    PubMed

    Sun, Bing; Chen, Linhai; Liu, Lei; Xia, Zhixiong; Pin, Jean-Philippe; Nan, Fajun; Liu, Jianfeng

    2016-03-15

    An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition. PMID:26772870

  8. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.

    PubMed

    Jiménez-Vázquez, Eric N; Díaz-Velásquez, Clara E; Uribe, R M; Arias, Juan M; García, Ubaldo

    2016-02-01

    Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates. PMID:26577600

  9. Prefrontal GABA(A) receptor alpha-subunit expression in normal postnatal human development and schizophrenia.

    PubMed

    Duncan, Carlotta E; Webster, Maree J; Rothmond, Debora A; Bahn, Sabine; Elashoff, Michael; Shannon Weickert, Cynthia

    2010-07-01

    Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABA(A) receptor alpha subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABA(A) receptor alpha-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABA(A) receptor alpha subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6weeks to 49years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABA(A) receptor alpha1 and alpha5 subunits during the first few years of postnatal development, with a 60% decrease in alpha5 mRNA expression and a doubling of alpha1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p=0.0012) and decreased alpha5 mRNA expression (p=0.038) in the DLPFC with no significant change of other alpha subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical alpha1 or alpha2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of alpha5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts. PMID:20100621

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

    ERIC Educational Resources Information Center

    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…

  11. Alpha-7 and alpha-4 nicotinic receptor subunit immunoreactivity in genioglossus muscle motoneurons.

    PubMed

    Dehkordi, Ozra; Millis, Richard M; Dennis, Gary C; Coleman, Bernell R; Johnson, Sheree M; Changizi, Loubat; Ovid Trouth, C

    2005-02-15

    In the present study, immunohistochemistry combined with retrograde labeling techniques were used to determine if hypoglossal motoneurons (HMNs), retrogradely labeled after cholera toxin B subunit (CTB) injection to the genioglossus muscle in rats, show immunoreactivity for alpha-7 and alpha-4 subunits of nicotinic acetylcholine receptors (nAChRs). CTB-positive HMNs projecting to the genioglossus muscle were consistently labeled throughout the rostrocaudal extent of the hypoglossal nuclei with the greatest labeling at and caudal to area postrema. Alpha-7 subunit immunoreactivity was found in 39.44+/-5.10% of 870 CTB-labeled motoneurons and the alpha-4 subunit in 51.01+/-3.71% of 983 CTB-positive neurons. Rostrally, the number of genioglossal motoneurons demonstrating immunoreactivity for the alpha-7 subunit was 45.85+/-10.04% compared to 34.96+/-5.11% at and caudal to area postrema (P>0.1). The number of genioglossal motoneurons that showed immunoreactivity for the alpha-4 subunit was 55.03+/-4.83% at and caudal to area postrema compared to 42.98+/-3.90% in rostral areas (P=0.074). These results demonstrate that nAChR immunoreactivity is present in genioglossal motoneurons and suggest a role for alpha-7 and alpha-4 subunits containing nAChRs in the regulation of upper airway patency. PMID:15705531

  12. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    PubMed

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  13. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    SciTech Connect

    Alcaraz, G.; Kinet, J.P.; Liu, T.Y.; Metzger, H.

    1987-05-05

    The ..cap alpha.., ..beta.., ..gamma.. subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the ..cap alpha.. chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for ..cap alpha.. and ..beta... However, these putative homologies were not apparent when tryptic maps of the biosynthetically ((/sup 3/H)leucine) labeled subunits were analyzed.

  14. Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum

    SciTech Connect

    Hwang, J.; Menon, K.N.J.

    1986-05-29

    The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the /sup 125/I-hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum /sup 125/I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of /sup 125/I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14.

  15. Hco-LGC-38 is novel nematode cys-loop GABA receptor subunit.

    PubMed

    Siddiqui, Salma Z; Brown, David D R; Accardi, Michael V; Forrester, Sean G

    2012-10-01

    We have identified and characterized a novel cys-loop GABA receptor subunit (Hco-LGC-38) from the parasitic nematode Haemonchus contortus. This subunit is present in parasitic and free-living nematodes and shares similarity to both the UNC-49 group of GABA receptor subunits from nematodes and the resistant to dieldrin (RDL) receptors of insects. Expression of the Hco-lgc-38 gene in Xenopus oocytes and subsequent electrophysiological analysis has revealed that the gene encodes a homomeric channel sensitive to GABA (EC(50) 19 μM) and the GABA analogue muscimol. The sensitivity of the Hco-LGC-38 channel to GABA is similar to reported values for the Drosophila RDL receptor whereas its lower sensitivity to muscimol is similar to nematode GABA receptors. Hco-LGC-38 is also highly sensitive to the channel blocker picrotoxin and moderately sensitive to fipronil and dieldrin. Homology modeling of Hco-LGC-38 and subsequent docking of GABA and muscimol into the binding site has uncovered several types of potential interactions with binding-site residues and overall appears to share similarity with models of other invertebrate GABA receptors. PMID:22940478

  16. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    ERIC Educational Resources Information Center

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  17. The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation.

    PubMed

    Kamens, Helen M; Hoft, Nicole R; Cox, Ryan J; Miyamoto, Jill H; Ehringer, Marissa A

    2012-08-01

    Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and β3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses. PMID:22572056

  18. The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation

    PubMed Central

    Kamens, Helen M.; Hoft, Nicole R.; Cox, Ryan J.; Miyamoto, Jill; Ehringer, Marissa A.

    2012-01-01

    Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and β3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses. PMID:22572056

  19. Positioning of the α-subunit isoforms confers a functional signature to γ-aminobutyric acid type A receptors

    PubMed Central

    Minier, Frédéric; Sigel, Erwin

    2004-01-01

    Fast synaptic inhibitory transmission in the CNS is mediated by γ-aminobutyric acid type A (GABAA) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different α-subunit isoforms, α1 and α6, in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized γ2β2α1β2α1, γ2β2α6β2α6, γ2β2α1β2α6, and γ2β2α6β2α1 GABAA receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single α6-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the α-subunit neighboring the γ2-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain α1- and α6-subunits. This method may also be applied to the study of other ion channels. PMID:15136735

  20. Changes in NMDA receptor-induced cyclic nucleotide synthesis regulate the age-dependent increase in PDE4A expression in primary cortical cultures

    PubMed Central

    Hajjhussein, Hassan; Suvarna, Neesha U.; Gremillion, Carmen; Judson Chandler, L.; O’Donnell, James M.

    2007-01-01

    NMDA receptor-induced cAMP and cGMP are selectively hydrolyzed by PDE4 and PDE2, respectively, in rat primary cerebral cortical and hippocampal cultures. Because cAMP levels regulate the expression of PDE4 in rat primary cortical cultures, we examined the manner in which NMDA receptor activity regulates the age-dependent increase in the expression of PDE4A observed in vivo and in vitro. Inhibiting the activity of NR2B subunit with ifenprodil blocked NMDA receptor-induced cGMP synthesis and increased NMDA receptor-induced cAMP levels in a manner that reduced PDE4 activity. Therefore, NR1/NR2B receptor-induced cGMP signaling is involved in an acute cross-talk regulation of NR1/NR2A receptor-induced cAMP levels, mediated by PDE4. Chronic inhibition of NMDA receptor activity with MK-801 reduced PDE4A1 and PDE4A5 expression and activity in a time-dependent manner; this effect was reversed by adding the PKA activator dbr-cAMP. Inhibiting GABA receptors with bicuculline increased NMDA receptor-induced cAMP synthesis and PDE4A expression in cultures treated between DIV 16 and DIV 21 but not in cultures treated between DIV 8 and DIV 13. This effect was due to a high tone of NMDA receptor-induced cGMP in younger cultures, which negatively regulated the expression of PDE4A by a PKG-mediated process. The present results are consistent with behavioral data showing that both PDE4 and PDE2 are involved in NMDA receptor-mediated memory processes. PMID:17407767

  1. Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Effects on the binding activity with receptor sites and interactions between subunits.

    PubMed Central

    Cheng, K W

    1976-01-01

    The reaction of iodoacetic acid with bovine lutropin (luteinizing hormone) at pH 3.0 was specific for methionine residues; it was slow and reached its equilibrium after 12 h at 37 degrees C. The number of modified methionine residues increased proportionately with the amount of the alkylating reagent in the reaction mixture. In the presence of a 20-fold molar excess of iodoacetic acid with respect to methionine, essentially all methionine residues in both subunits of bovine lutropin were carboxymethylated. Studies of various recombinations of modified and native alpha and beta subunits showed that methionine residues in bovine lutropin were not essential for interactions between subunits. Various recombinants were characterized by polyacrylamide-gel electrophoresis and gel filtration of Sephadex G-100. Immunological cross-reactivity by radioimmunoassay of the recombinants of modified alpha and beta subunits was relatively similar to that of the native subunits. However, the biological activity measured by receptor-site binding of the recombinants of alpha and beta chains with a total of three alkylated methionine residues was less than 5% of the activity of native lutropin. It is noteworthy that recombinants of a modified subunit and a native counterpart subunit regenerated 20-30 % of biological activity. These findings suggested that at least 1-2 methionine residues in each subunit are involved in the hormone-receptor interaction for bovine lutropin. Images PLATE 1 PMID:187169

  2. Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

    PubMed Central

    Kazlauskas, A; Durden, D L; Cooper, J A

    1991-01-01

    Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis. Images PMID:1653029

  3. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

  4. Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans.

    PubMed

    Sloan, Megan A; Reaves, Barbara J; Maclean, Mary J; Storey, Bob E; Wolstenholme, Adrian J

    2015-11-01

    The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing. PMID:26747395

  5. Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

    PubMed Central

    Marshall, J; Buckingham, S D; Shingai, R; Lunt, G G; Goosey, M W; Darlison, M G; Sattelle, D B; Barnard, E A

    1990-01-01

    We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA. PMID:1702381

  6. DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

    PubMed Central

    Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

    2011-01-01

    The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

  7. Chronic early postnatal scream sound stress induces learning deficits and NMDA receptor changes in the hippocampus of adult mice.

    PubMed

    Hu, Lili; Han, Bo; Zhao, Xiaoge; Mi, Lihua; Song, Qiang; Wang, Jue; Song, Tusheng; Huang, Chen

    2016-04-13

    Chronic scream sounds during adulthood affect spatial learning and memory, both of which are sexually dimorphic. The long-term effects of chronic early postnatal scream sound stress (SSS) during postnatal days 1-21 (P1-P21) on spatial learning and memory in adult mice as well as whether or not these effects are sexually dimorphic are unknown. Therefore, the present study examines the performance of adult male and female mice in the Morris water maze following exposure to chronic early postnatal SSS. Hippocampal NR2A and NR2B levels as well as NR2A/NR2B subunit ratios were tested using immunohistochemistry. In the Morris water maze, stress males showed greater impairment in spatial learning and memory than background males; by contrast, stress and background females performed equally well. NR2B levels in CA1 and CA3 were upregulated, whereas NR2A/NR2B ratios were downregulated in stressed males, but not in females. These data suggest that chronic early postnatal SSS influences spatial learning and memory ability, levels of hippocampal NR2B, and NR2A/NR2B ratios in adult males. Moreover, chronic early stress-induced alterations exert long-lasting effects and appear to affect performance in a sex-specific manner. PMID:27015584

  8. Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Miledi, R

    1989-01-01

    Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensitized as slowly as cat AcChoR. In contrast, the hybrid AcChoRs expressed by coinjection with the Torpedo gamma subunit mRNA desensitized much more rapidly than cat AcChoR. The AcChoRs expressed in oocytes injected with cat muscle mRNA together with the Torpedo beta, gamma, and delta subunit mRNAs desensitized as rapidly as Torpedo AcChoR, indicating that the cat alpha subunit does not play an important role in determining the slow rate of desensitization. It is concluded that the difference in the rates of desensitization of cat and Torpedo AcChoRs is determined mainly by differences in their respective gamma subunits. Images PMID:2536157

  9. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    PubMed Central

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-01-01

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. Images PMID:1709742

  10. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    PubMed

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-05-15

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID:1709742

  11. The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ.

    PubMed

    Smith, R A; Branca, A A; Reichert, L E

    1985-11-15

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate

  12. Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits

    PubMed Central

    Dehkordi, Ozra; Kc, Prabha; Balan, Kannan V.; Haxhiu, Musa A.

    2007-01-01

    Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the α7 homomeric and α4β2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of α7, α4, and β2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin h subunit (CTb), injected into the upper lung lobe (n = 4) or extrathoracic trachea (n = 4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express α7 and α4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain α7 and β2 subunits, but less than half of them show detectable α4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (α7, α4, and β2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways. PMID:16616705

  13. Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.

    PubMed

    Hsiao, Bernard; Mihalak, Karla B; Repicky, Sarah E; Everhart, Drew; Mederos, Ana H; Malhotra, Arun; Luetje, Charles W

    2006-01-01

    We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors. PMID:16189299

  14. Opposite Effects of KCTD Subunit Domains on GABAB Receptor-mediated Desensitization*

    PubMed Central

    Seddik, Riad; Jungblut, Stefan P.; Silander, Olin K.; Rajalu, Mathieu; Fritzius, Thorsten; Besseyrias, Valérie; Jacquier, Valérie; Fakler, Bernd; Gassmann, Martin; Bettler, Bernhard

    2012-01-01

    GABAB receptors assemble from principle and auxiliary subunits. The principle subunits GABAB1 and GABAB2 form functional heteromeric GABAB(1,2) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K+ channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABAB2, and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif. PMID:23035119

  15. Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport

    PubMed Central

    Bai, Xiaobo; Karasmanis, Eva P.; Spiliotis, Elias T.

    2016-01-01

    Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo. PMID:26823018

  16. Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport.

    PubMed

    Bai, Xiaobo; Karasmanis, Eva P; Spiliotis, Elias T

    2016-03-15

    Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo. PMID:26823018

  17. A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons.

    PubMed

    Kinney, Jefferson W; Davis, Christopher N; Tabarean, Iustin; Conti, Bruno; Bartfai, Tamas; Behrens, M Margarita

    2006-02-01

    Several lines of evidence suggest that a hypoglutamatergic condition may induce a phenotypic loss of cortical parvalbumin (PV)-positive GABAergic interneurons, such as that observed in brain tissue of schizophrenic subjects. However, it is not known whether the loss of PV interneurons is a consequence of the hypoglutamatergic condition or a secondary aspect of the disease. We characterized the signaling and subunit expression of NMDA receptors in cultured cortical PV interneurons and determined whether a hypoglutamatergic condition, created by direct application of sublethal concentrations of ketamine or subunit-selective NMDA receptor antagonists, can affect the expression of the GABAergic markers as observed in vivo. Real-time PCR performed on mRNA isolated from single neurons showed that PV interneurons present a fivefold higher NR2A/NR2B ratio than pyramidal neurons. Brief, nontoxic, exposure to NMDA led to an increase in ERK1/2 (extracellular signal-regulated kinase 1/2) and cAMP response element-binding protein phosphorylation in PV interneurons, and this increase was blocked by the NR2A-selective antagonist NVP-AAM077. Application of the nonselective NMDA receptor antagonist ketamine, at sublethal concentrations, induced a time and dose-dependent decrease in parvalbumin and GAD67 immunoreactivity specifically in PV interneurons. These effects were reversible and were also observed with the NR2A-selective antagonist, whereas the NR2B-selective antagonist Ro-25-6981 only partially reduced GAD67 immunoreactivity. Coexposure to the calcium channel opener BayK, or the group I metabotropic glutamate receptor agonist DHPG [(RS)-3,5-dihydroxyphenylglycine] attenuated the decrease in GAD67 and parvalbumin induced by the NMDA receptor antagonists. These results suggest that the activity of NR2A-containing NMDA receptors play a pivotal role in the maintenance of the GABAergic function of PV interneurons. PMID:16452684

  18. Tonic inhibition in spinal ventral horn interneurons mediated by α5 subunit-containing GABA(A) receptors.

    PubMed

    Castro, Alberto; Aguilar, Justo; González-Ramírez, Ricardo; Loeza-Alcocer, Emanuel; Canto-Bustos, Martha; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2011-08-19

    GABA(A) receptors mediate synaptic and tonic inhibition in many neurons of the central nervous system. These receptors can be constructed from a range of different subunits deriving from seven identified families. Among these subunits, α(5) has been shown to mediate GABAergic tonic inhibitory currents in neurons from supraspinal nuclei. Likewise, immunohistochemical and in situ hybridization studies have shown the presence of the α(5) subunit in spinal cord neurons, though almost nothing is known about its function. In the present report, using slices of the adult turtle spinal cord as a model system we have recorded a tonic inhibitory current in ventral horn interneurons (VHIs) and determined the functional contribution of the α(5) subunit-containing GABA(A) receptors to this current. Patch clamp studies show that the GABAergic tonic inhibitory current in VHIs is not affected by the application of antagonists of the α(4/6) subunit-containing GABA(A) receptors, but is sensitive to L-655708, an antagonist of the GABA(A) receptors containing α(5) subunits. Last, by using RT-PCR and immunohistochemistry we confirmed the expression of the α(5) subunit in the turtle spinal cord. Together, these results suggest that GABA(A) receptors containing the α(5) subunit mediate the tonic inhibitory currents observed in VHIs. PMID:21798246

  19. Association analysis of GABA receptor subunit genes on 5q33 with heroin dependence in a Chinese male population.

    PubMed

    Loh, E W; Tang, N L S; Lee, D T S; Liu, S I; Stadlin, Alfreda

    2007-06-01

    GABAA receptor subunit genes clustered on 5q33 play a role in the development of alcoholism and methamphetamine use disorder without psychosis. The present study explored the possible contribution of the same subunit genes to the development of heroin dependence. Single nucleotide polymorphisms (SNPs) of the GABAA receptor subunits GABRB2, GABRA6, GABRA1, and GABRG2 were examined in 178 male Han Chinese heroin-dependent and 170 male control subjects. A significant difference in allele frequency for the SNP rs211014 in the GABAAgamma2 receptor subunit gene between cases and controls was identified (P = 0.015). A possible mechanism for the involvement of the GABA receptor subunit genes on 5q33 in the development of heroin dependence is discussed. PMID:17440936

  20. Receptor-binding region in human choriogonadotropin/lutropin. beta. subunit

    SciTech Connect

    Keutmann, H.T.; Charlesworth, M.C.; Mason, K.A.; Ostrea, T.; Johnson, L.; Ryan, R.J.

    1987-04-01

    Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) ..beta.. subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG..beta..-(38-57) and hLH..beta..-(38-57) peptides inhibited binding of /sup 125/I-labeled hCG half-maximally at 1.51 x 10/sup -4/ and 2.03 x 10/sup -5/ M, respectively, while other peptide hormones and fragments from elsewhere in the ..beta.. subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10/sup -5/ M (hCG) and 2.18 x 10/sup -5/ M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG..beta..-(38-57) peptide, native hCG and ..beta.. subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of ..beta.. subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.

  1. Escobar Syndrome Is a Prenatal Myasthenia Caused by Disruption of the Acetylcholine Receptor Fetal γ Subunit

    PubMed Central

    Hoffmann, Katrin; Müller, Juliane S.; Stricker, Sigmar; Megarbane, Andre; Rajab, Anna; Lindner, Tom H.; Cohen, Monika; Chouery, Eliane; Adaimy, Lynn; Ghanem, Ismat; Delague, Valerie; Boltshauser, Eugen; Talim, Beril; Horvath, Rita; Robinson, Peter N.; Lochmüller, Hanns; Hübner, Christoph; Mundlos, Stefan

    2006-01-01

    Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the γ-subunit gene (CHRNG) of the AChR. Our functional studies show that γ-subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney–cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two α, one β, and one δ subunit are always present. By switching γ to ɛ subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the γ subunit were thought to be lethal, as they are in γ-knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because γ expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway. PMID:16826520

  2. Identification of domains influencing assembly and ion channel properties in α7 nicotinic receptor and 5-HT3 receptor subunit chimaeras

    PubMed Central

    Gee, V J; Kracun, S; Cooper, S T; Gibb, A J; Millar, N S

    2007-01-01

    Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR α7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing α7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on α7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some α7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. PMID:17721553

  3. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    PubMed Central

    Murray, Teresa A; Liu, Qiang; Whiteaker, Paul; Wu, Jie; Lukas, Ronald J

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion. PMID:19498423

  4. Estimating the efficiency of benzodiazepines on GABAA receptors comprising γ1 or γ2 subunits

    PubMed Central

    Baburin, I; Khom, S; Timin, E; Hohaus, A; Sieghart, W; Hering, S

    2008-01-01

    Background and purpose: Heterologous expression of α1, β2 and γ2S(γ1) subunits produces a mixed population of GABAA receptors containing α1β2 or α1β2γ2S(γ1) subunits. GABA sensitivity (lower in receptors containing γ1 or γ2S subunits) and the potentiation of GABA-activated chloride currents (IGABA) by benzodiazepines (BZDs) are dependent on γ2S(γ1) incorporation. A variable γ subunit incorporation may affect the estimation of IGABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for mixed population of α1β2 and α1β2γ2S(γ1) receptors. Experimental approach: We investigated the relation between GABA sensitivity (EC50) and BZD modulation by analysing triazolam-, clotiazepam- and midazolam-induced potentiation of IGABA in Xenopus oocytes under two-microelectrode voltage clamp. Key results: Plotting EC50 versus BZD-induced shifts of GABA concentration-response curves (ΔEC50(BZD)) of oocytes injected with different amounts of α1, β2 and γ2S(γ1) cRNA (1:1:1–1:1:10) revealed a linear regression between γ2S(γ1)-mediated reduction of GABA sensitivity (EC50) and ΔEC50(BZD). The slope factors of the regression were always higher for oocytes expressing α1β2γ1 subunit receptors (1.8±0.1 (triazolam), 1.6±0.1 (clotiazepam), 2.3±0.2 (midazolam)) than for oocytes expressing α1β2γ2S receptors (1.4±0.1 (triazolam), 1.4±0.1 (clotiazepam), 1.3±0.1 (midazolam)). Mutant GABAA receptors (α1β2-R207Cγ2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors=1.1±0.1 (triazolam), 1.1±0.1 (clotiazepam), 1.2±0.1 (midazolam)). Conclusions and implications: Regression analysis enabled the estimation of BZD efficiency when variable mixtures of α1β2 and α1β2γ2S(γ1) receptors are expressed and provided new insights into the γ2S(γ1) dependency of BZD action. PMID:18604239

  5. Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.

    PubMed

    Melzer, Nima; Villmann, Carmen; Becker, Kristina; Harvey, Kirsten; Harvey, Robert J; Vogel, Nico; Kluck, Christoph J; Kneussel, Matthias; Becker, Cord-Michael

    2010-02-01

    The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs. PMID:19959465

  6. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  7. GluN3A: An NMDA Receptor Subunit with Exquisite Properties and Functions

    PubMed Central

    Kehoe, Laura A.

    2013-01-01

    N-methyl-D-aspartate receptors (NMDAR) are pivotal for synaptic plasticity and memory formation. Conventional NMDAR consist of heterotetrameric structures composed of GluN1 and GluN2 subunits. A third subunit, GluN3, can also assemble with NMDAR subunits giving a remarkable modification of their heteromeric structure, forming a “nonconventional” NMDAR. As a consequence, the stoichiometry and kinetic properties of the receptors are dramatically changed. Among the GluN3 family, the GluN3A subunit has been the focus of a large amount of studies during recent years. These studies reveal that GluN3A is transiently expressed during development and could play a role in the fine tuning of neuronal networks as well as associated diseases. Moreover, GluN3A distribution outside the postsynaptic densities, including perisynaptic astrocytes, places it at a strategic position to play an important role in the interactions between neurons and glial cells. This review highlights GluN3A properties and addresses its role in neurophysiology and associated pathologies. PMID:24386575

  8. Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression

    PubMed Central

    Follesa, Paolo; Floris, Gabriele; Asuni, Gino P.; Ibba, Antonio; Tocco, Maria G.; Zicca, Luca; Mercante, Beniamina; Deriu, Franca; Gorini, Giorgio

    2015-01-01

    Chronic ethanol consumption causes structural and functional reorganization in the hippocampus and induces alterations in the gene expression of gamma-aminobutyric acid type A receptors (GABAARs). Distinct forced intermittent exposure models have been used previously to investigate changes in GABAAR expression, with contrasting results. Here, we used repeated cycles of a Chronic Intermittent Ethanol paradigm to examine the relationship between voluntary, dependence-associated ethanol consumption, and GABAAR gene expression in mouse hippocampus. Adult male C57BL/6J mice were exposed to four 16-h ethanol vapor (or air) cycles in inhalation chambers alternated with limited-access two-bottle choice between ethanol (15%) and water consumption. The mice exposed to ethanol vapor showed significant increases in ethanol consumption compared to their air-matched controls. GABAAR alpha4 and delta subunit gene expression were measured by qRT-PCR at different stages. There were significant changes in GABAAR delta subunit transcript levels at different time points in ethanol-vapor exposed mice, while the alpha4 subunit levels remained unchanged. Correlated concurrent blood ethanol concentrations suggested that GABAAR delta subunit mRNA levels fluctuate depending on ethanol intoxication, dependence, and withdrawal state. Using a vapor-based Chronic Intermittent Ethanol procedure with combined two-bottle choice consumption, we corroborated previous evidences showing that discontinuous ethanol exposure affects GABAAR delta subunit expression but we did not observe changes in alpha4 subunit. These findings indicate that hippocampal GABAAR delta subunit expression changes transiently over the course of a Chronic Intermittent Ethanol paradigm associated with voluntary intake, in response to ethanol-mediated disturbance of GABAergic neurotransmission. PMID:26617492

  9. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

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

    PubMed Central

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

    2016-01-01

    Summary 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. PMID:26924438

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

    PubMed

    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. PMID:26924438

  12. Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated alpha-subunit.

    PubMed Central

    Sawruk, E; Schloss, P; Betz, H; Schmitt, B

    1990-01-01

    Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or alpha 2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR alpha-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila. Images Fig. 3. Fig. 4. PMID:1697262

  13. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

    PubMed Central

    Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

  14. Extrasynaptic α6 Subunit-Containing GABAA Receptors Modulate Excitability in Turtle Spinal Motoneurons

    PubMed Central

    Andres, Carmen; Aguilar, Justo; González-Ramírez, Ricardo; Elias-Viñas, David; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2014-01-01

    Motoneurons are furnished with a vast repertoire of ionotropic and metabotropic receptors as well as ion channels responsible for maintaining the resting membrane potential and involved in the regulation of the mechanisms underlying its membrane excitability and firing properties. Among them, the GABAA receptors, which respond to GABA binding by allowing the flow of Cl− ions across the membrane, mediate two distinct forms of inhibition in the mature nervous system, phasic and tonic, upon activation of synaptic or extrasynaptic receptors, respectively. In a previous work we showed that furosemide facilitates the monosynaptic reflex without affecting the dorsal root potential. Our data also revealed a tonic inhibition mediated by GABAA receptors activated in motoneurons by ambient GABA. These data suggested that the high affinity GABAA extrasynaptic receptors may have an important role in motor control, though the molecular nature of these receptors was not determined. By combining electrophysiological, immunofluorescence and molecular biology techniques with pharmacological tools here we show that GABAA receptors containing the α6 subunit are expressed in adult turtle spinal motoneurons and can function as extrasynaptic receptors responsible for tonic inhibition. These results expand our understanding of the role of GABAA receptors in motoneuron tonic inhibition. PMID:25531288

  15. Extrasynaptic α6 subunit-containing GABAA receptors modulate excitability in turtle spinal motoneurons.

    PubMed

    Andres, Carmen; Aguilar, Justo; González-Ramírez, Ricardo; Elias-Viñas, David; Felix, Ricardo; Delgado-Lezama, Rodolfo

    2014-01-01

    Motoneurons are furnished with a vast repertoire of ionotropic and metabotropic receptors as well as ion channels responsible for maintaining the resting membrane potential and involved in the regulation of the mechanisms underlying its membrane excitability and firing properties. Among them, the GABAA receptors, which respond to GABA binding by allowing the flow of Cl- ions across the membrane, mediate two distinct forms of inhibition in the mature nervous system, phasic and tonic, upon activation of synaptic or extrasynaptic receptors, respectively. In a previous work we showed that furosemide facilitates the monosynaptic reflex without affecting the dorsal root potential. Our data also revealed a tonic inhibition mediated by GABAA receptors activated in motoneurons by ambient GABA. These data suggested that the high affinity GABAA extrasynaptic receptors may have an important role in motor control, though the molecular nature of these receptors was not determined. By combining electrophysiological, immunofluorescence and molecular biology techniques with pharmacological tools here we show that GABAA receptors containing the α6 subunit are expressed in adult turtle spinal motoneurons and can function as extrasynaptic receptors responsible for tonic inhibition. These results expand our understanding of the role of GABAA receptors in motoneuron tonic inhibition. PMID:25531288

  16. Transmembrane Signaling by the Aspartate Receptor: Engineered Disulfides Reveal Static Regions of the Subunit Interface†

    PubMed Central

    Chervitz, Stephen A.; Lin, Christina M.; Falke, Joseph J.

    2010-01-01

    Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18–75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays. PMID:7626643

  17. Early immune response and regulation of IL-2 receptor subunits

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J. B.; Cogoli, Augusto

    2005-01-01

    Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and

  18. Nr3a-containing NMDA receptors promote neurotransmitter release and spike timing-dependent plasticity

    PubMed Central

    Larsen, Rylan S.; Corlew, Rebekah J.; Henson, Maile A.; Roberts, Adam C.; Mishina, Masayoshi; Watanabe, Masahiko; Lipton, Stuart A.; Nakanishi, Nobuki; Pérez-Otaño, Isabel; Weinberg, Richard J.; Philpot, Benjamin D.

    2012-01-01

    Recent evidence suggests that presynaptic-acting NMDA receptors (preNMDARs) are important for neocortical synaptic transmission and plasticity. We found that unique properties of the Nr3a subunit enable preNMDARs to enhance spontaneous and evoked glutamate release and that Nr3a is required for spike timing–dependent long-term depression in the juvenile mouse visual cortex. In the mature cortex, Nr2b-containing preNMDARs enhanced neurotransmission in the absence of magnesium, indicating that presynaptic NMDARs may function under depolarizing conditions throughout life. Our findings indicate that Nr3a relieves preNMDARs from the dual-activation requirement of ligand-binding and depolarization; the developmental removal of Nr3a limits preNMDAR functionality by restoring this associative property. PMID:21297630

  19. Removal of GABAA Receptor γ2 Subunits from Parvalbumin Neurons Causes Wide-Ranging Behavioral Alterations

    PubMed Central

    Leppä, Elli; Linden, Anni-Maija; Vekovischeva, Olga Y.; Swinny, Jerome D.; Rantanen, Ville; Toppila, Esko; Höger, Harald; Sieghart, Werner; Wulff, Peer; Wisden, William; Korpi, Esa R.

    2011-01-01

    We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABAA receptors on parvalbumin (Pv) cells. The GABAA receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[35S]thionate suggested an increased amount of GABAA receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons). This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception. PMID:21912668

  20. Subunit composition of mammalian transient receptor potential channels in living cells.

    PubMed

    Hofmann, Thomas; Schaefer, Michael; Schultz, Günter; Gudermann, Thomas

    2002-05-28

    Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches--i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation--we investigate the combinatorial rules of TRPC assembly. Our data show that (i) TRPC2 does not interact with any known TRPC protein and (ii) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (iii) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies--e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells. PMID:12032305

  1. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    SciTech Connect

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L. )

    1989-09-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-(4-(2-(2-((4- aminophenyl)methylcarbonylamino)ethylaminocarbonyl)- ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-((R)-1-methyl- 2-phenylethyl)adenosine (R-PIA) greater than (+)-N6-((S)-1-methyl-2- phenylethyl)adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-(beta, gamma-imido)triphosphate.

  2. Juvenile stress-induced alteration of maturation of the GABAA receptor alpha subunit in the rat.

    PubMed

    Jacobson-Pick, Shlomit; Elkobi, Alina; Vander, Shelly; Rosenblum, Kobi; Richter-Levin, Gal

    2008-11-01

    Profound evidence indicates that GABAA receptors are important in the control of physiological response to stress and anxiety. The alpha subunit type composition contributes significantly to the functional characterization of the GABAA receptors. The alpha2, alpha3, alpha5 subunits are predominately expressed in the brain during embryonic and early postnatal periods of normal rats, whilst alpha1 are most prominent during later developmental stages. In the present study, we examined the long-term effects of juvenile stress on GABA alpha subunit expression in adulthood in the amygdala and hippocampus. We applied the elevated platform stress paradigm at juvenility and used the open-field and startle response tests to assess anxiety level in adulthood. Juvenile stress effects without behavioural tests in adulthood were also examined since previous studies indicated that the mere exposure to these tests might be stressful for rats, enhancing the effects of the juvenile exposure to stress. In adulthood, we quantitatively determined the level of expression of alpha1, alpha2 and alpha3 in the hippocampus and amygdala. Our results indicate that subjecting juvenile stressed rats to additional challenges in adulthood results in an immature-like expression profile of these subunits. To test for potential functional implications of these alterations we examined the effects of the anxiolytic (diazepam) and the sedative (brotizolam) benzodiazepines on juvenile stressed and control rats following additional challenges in adulthood. Juvenile stressed rats were more sensitive to diazepam and less sensitive to brotizolam, suggesting that the alterations in GABA alpha subunit expression in these animals have functional consequences. PMID:18364065

  3. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-09-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake Ac

  4. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-01-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake Ac

  5. AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

    PubMed

    Uusi-Oukari, Mikko; Kontturi, Leena-Stiina; Kallinen, Sampsa A; Salonen, Virpi

    2010-04-01

    Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum. PMID:20170697

  6. Different glutamate receptors convey feedforward and recurrent processing in macaque V1.

    PubMed

    Self, Matthew W; Kooijmans, Roxana N; Supèr, Hans; Lamme, Victor A; Roelfsema, Pieter R

    2012-07-01

    Neurons in the primary visual cortex (V1) receive feedforward input from the thalamus, which shapes receptive-field properties. They additionally receive recurrent inputs via horizontal connections within V1 and feedback from higher visual areas that are thought to be important for conscious visual perception. Here, we investigated what roles different glutamate receptors play in conveying feedforward and recurrent inputs in macaque V1. As a measure of recurrent processing, we used figure-ground modulation (FGM), the increased activity of neurons representing figures compared with background, which depends on feedback from higher areas. We found that feedforward-driven activity was strongly reduced by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas this drug had no effect on FGM. In contrast, blockers of the NMDA receptor reduced FGM, whereas their effect on visually driven activity varied with the subunit specificity of the drug. The NMDA receptor blocker 2-amino-5-phosphonovalerate (APV) caused a slight reduction of the visual response, whereas ifenprodil, which targets NMDA receptors containing the NMDA receptor NR2B subunit, increased the visual response. These findings demonstrate that glutamate receptors contribute differently to feedforward and recurrent processing in V1 and suggest ways to selectively disrupt recurrent processing so that its role in visual perception can be elucidated. PMID:22615394

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed Central

    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

  9. Desformylflustrabromine: A Novel Positive Allosteric Modulator for beta2 Subunit Containing Nicotinic Receptor Sub-Types.

    PubMed

    Pandya, Anshul A

    2016-01-01

    Nicotinic acetylcholine receptors are ligand-gated transmembrane ion channels that are present at the neuromuscular junction and in different locations in the nervous system. The different subtypes of neuronal nicotinic acetylcholine receptors that are found in the brain are thought to be involved in many neurological processes such as pain, cognitive function and depression, as well as in the pathophysiology of numerous neurological diseases and conditions. While the neurotransmitter acetylcholine is an endogenous agonist for all nicotinic receptors subtypes, many drugs that act as agonists and antagonists have also been identified or developed for these receptors. In addition, a novel class of compounds described as allosteric modulators have also been identified or developed for nicotinic acetylcholine receptors. Allosteric modulators are ligands that bind to nicotinic receptors at sites other than the orthosteric site where acetylcholine binds. One such allosteric modulator is desformylflustrabromine. Five chemical analogs along with desformylflustrabromine act as positive allosteric modulator for nAChRs that contain the beta2 subunit in their pentameric structure. Here the discovery and development, medicinal chemistry and pharmacological actions of desformylflustrabromine have been discussed. Desformylflustrabromine and its chemical analogs have the potential to develop into clinically used drugs for neurological diseases and conditions where nicotinic acetylcholine receptors are involved. PMID:26818864

  10. Lesion of the substantia nigra pars compacta downregulates striatal glutamate receptor subunit mRNA expression.

    PubMed

    Fan, X D; Li, X M; Ashe, P C; Juorio, A V

    1999-12-11

    This is a study of the effect of the unilateral administration of dopamine (DA) in the pars compacta of the substantia nigra (SN) of the rat on striatal glutamate receptor subunit (GluR1, GluR2 and NMDAR1) gene expression determined by in situ hybridization. The location of the nigral lesion was determined by tyrosine hydroxylase (TH) immunohistochemistry and its extent by the striatal DA and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations. The DA-induced lesions produce significant bilateral reductions in the expression of GluR1 and NMDAR1 subunit mRNA in the medio-lateral striatum, whereas the expression of striatal GluR2 receptors was not changed. The reduction in GluR1 and NMDAR1 subunit mRNA may be the consequence of glutamatergic hyperactivity developed in the presence of a damaged nigro-striatal system and these may be associated with the genesis of some neurodegenerative diseases. PMID:10629751

  11. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects

  12. CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE-INDUCED CURRENTS.

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 μM α-bungarotoxin completely inhibited nicotine currents whereas 10 μM d-tubocurarine had a partial effect demonstrating that β1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin. PMID:27357353

  13. Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

    NASA Astrophysics Data System (ADS)

    Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

    1987-10-01

    The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

  14. Nuclear respiratory factor 2 regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

    PubMed

    Priya, Anusha; Johar, Kaid; Nair, Bindu; Wong-Riley, Margaret T T

    2014-12-01

    Neuronal activity is highly dependent on energy metabolism. Nuclear respiratory factor 2 (NRF-2) tightly couples neuronal activity and energy metabolism by transcriptionally co-regulating all 13 subunits of an important energy-generating enzyme, cytochrome c oxidase (COX), as well as critical subunits of excitatory NMDA receptors. AMPA receptors are another major class of excitatory glutamatergic receptors that mediate most of the fast excitatory synaptic transmission in the brain. They are heterotetrameric proteins composed of various combinations of GluA1-4 subunits, with GluA2 being the most common one. We have previously shown that GluA2 (Gria2) is transcriptionally regulated by nuclear respiratory factor 1 (NRF-1) and specificity protein 4 (Sp4), which also regulate all subunits of COX. However, it was not known if NRF-2 also couples neuronal activity and energy metabolism by regulating subunits of the AMPA receptors. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate the expression of Gria2, but not of Gria1, Gria3, or Gria4 genes in neurons. By regulating the GluA2 subunit of the AMPA receptor, NRF-2 couples energy metabolism and neuronal activity at the transcriptional level through a concurrent and parallel mechanism with NRF-1 and Sp4. PMID:25245478

  15. The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells.

    PubMed

    Herd, Murray B; Haythornthwaite, Alison R; Rosahl, Thomas W; Wafford, Keith A; Homanics, Gregg E; Lambert, Jeremy J; Belelli, Delia

    2008-02-15

    The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this

  16. Adolescent Alcohol Exposure Alters GABAA Receptor Subunit Expression in Adult Hippocampus

    PubMed Central

    Centanni, Samuel W.; Teppen, Tara; Risher, Mary-Louise; Fleming, Rebekah L.; Moss, Julia L.; Acheson, Shawn K.; Mulholland, Patrick J.; Pandey, Subhash C.; Chandler, L. Judson; Swartzwelder, H. Scott

    2014-01-01

    Background The long-term consequences of adolescent alcohol abuse that persist into adulthood are poorly understood and have not been widely investigated. We have shown that intermittent exposure to alcohol during adolescence decreased the amplitude of GABAA receptor-mediated tonic currents in hippocampal dentate granule cells in adulthood. The aim of the present study was to investigate the enduring effects of chronic intermittent alcohol exposure during adolescence or adulthood on the expression of hippocampal GABAA receptors (GABAARs). Methods We used a previously characterized tissue fractionation method to isolate detergent resistant membranes and soluble fractions, followed by western blots to measure GABAAR protein expression. We also measured mRNA levels of GABAAR subunits using quantitative real-time PCR. Results Although the protein levels of α1-, α4- and δ-GABAAR subunits remained stable between postnatal day (PD) 30 (early adolescence) and PD71 (adulthood), the α5-GABAAR subunit was reduced across that period. In rats that were subjected to adolescent intermittent ethanol (AIE) exposure between PD30–46, there was a significant reduction in the protein levels of the δ-GABAAR, in the absence of any changes in mRNA levels, at 48 hours and 26 days after the last ethanol exposure. Protein levels of the α4-GABAAR subunit were significantly reduced, but mRNA levels were increased, 26 days (but not 48 hours) after the last AIE exposure. Protein levels of α5-GABAAR were not changed by AIE, but mRNA levels were reduced at 48hrs but normalized 26 days after AIE. In contrast to the effects of AIE, chronic intermittent exposure to ethanol during adulthood (CIE) had no effect on expression of any of the GABAAR subunits examined. Conclusions AIE produced both short- and long-term alterations of GABAAR subunits mRNA and protein expression in the hippocampus, whereas CIE produced no long lasting effects on those measures. The observed reduction of protein

  17. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  18. Generation of Functional Inhibitory Synapses Incorporating Defined Combinations of GABA(A) or Glycine Receptor Subunits

    PubMed Central

    Dixon, Christine L.; Zhang, Yan; Lynch, Joseph W.

    2015-01-01

    Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR) and glycine receptor (GlyR) isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of individual isoforms under synaptic stimulation conditions in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2–5 weeks. PMID:26778954

  19. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  20. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. PMID:3458258

  1. Association between GABAA Receptor Subunit Gene Cluster and Zolpidem-Induced Complex Sleep Behaviors in Han Chinese

    PubMed Central

    Tsai, Jui-Hsiu; Yang, Pinchen; Lin, Hung-Hsun; Cheng, Kuang-hung; Yang, Yi-Hsin; Wu, Ming-Tsang; Chen, Cheng-Chung

    2013-01-01

    Study Objectives: To investigate and elucidate the role of GABAA receptor subunits, specifically the 2 genetic markers at the GABAA α1 and GABAA α6 receptors, in zolpidem-induced complex sleep behaviors (CSBs). Design: Genetic association study. Setting: Kaohsiung Medical University-affiliated hospitals, Kaohsiung, Taiwan. Patients: 30 zolpidem-induced CSB subjects and 37 controls. Interventions: N/A. Measurements and Results: The χ2 test demonstrated an association between the A15G variant at the GABAA α1 receptor subunit gene and zolpidem-induced CSBs (P = 0.007). The adjusted odds ratio of the GABAA α1 receptor subunit genotype for the risk of zolpidem-induced CSBs was approximately 10 (OR = 9.99, 95% CI = 1.82, 74.87; P = 0.013). Conclusions: The finding reveals that the A15G variant at the GABAA α1 receptor subunit gene confers a high risk of zolpidem-induced CSBs and may be considered in clinical services. Citation: Tsai JH; Yang P; Lin HH; Cheng Kh; Yang YH; Wu MT; Chen CC. Association between GABAA receptor subunit gene cluster and zolpidem-induced complex sleep behaviors in Han Chinese. SLEEP 2013;36(2):197–202. PMID:23372267

  2. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor

    PubMed Central

    McCracken, Mandy L.; Gorini, Giorgio; McCracken, Lindsay M.; Mayfield, R. Dayne; Harris, R. Adron; Trudell, James R.

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  3. Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor.

    PubMed

    McCracken, Mandy L; Gorini, Giorgio; McCracken, Lindsay M; Mayfield, R Dayne; Harris, R Adron; Trudell, James R

    2016-01-01

    Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model

  4. The cellular expression of GABA(A) receptor alpha1 subunit during spermatogenesis in the mouse testis.

    PubMed

    Kanbara, Kiyoto; Okamoto, Keiko; Nomura, Sakashi; Kaneko, Takeshi; Watanabe, Masahito; Otsuki, Yoshinori

    2010-10-01

    GABA(A) receptors are pentamers in structure and are mainly composed of alpha, beta and gamma subunits. These receptors are known to function as chloride channels. We observed alpha5, beta1 and gamma3 subunit immunoreactivity in the mouse testes, specifically in the cytoplasm surrounding the nucleus in the spermatocytes and spermatids. In the current study, alpha1 subunit immunoreactivity was located in the nucleus of spermatogonia, spermatocytes and round spermatids. Immunoelectron microscopy revealed that the alpha1 subunit was localized within the nucleus of pachytene and diplotene spermatocytes in the area of condensed chromatin rather than extended chromatin. Protein sequence analysis revealed that the alpha1 subunit included DM DNA binding domains that were related to transcription factors involved in testicular differentiation in adult mice. These findings suggest that the alpha1 subunit may undertake a gene transcription function during the maturation of germ cells. a1 immunoreactivity was also detected within the mitochondria of spermatocytes and in the acrosome of round and elongated spermatids. Although the precise physiological role of the GABA(A) receptor alpha1 subunit in mitochondria remains unknown, we hypothesize that its function in the acrosome may be related to the acrosome reaction during fertilization or during spermatogenesis. PMID:20712007

  5. Complex control of GABA(A) receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    PubMed

    Mulligan, Megan K; Wang, Xusheng; Adler, Adrienne L; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal), even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer 4-100 fold

  6. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    SciTech Connect

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  7. NMDA receptor mediates chronic visceral pain induced by neonatal noxious somatic stimulation

    PubMed Central

    Miranda, Adrian; Mickle, Aaron; Bruckert, Mitchell; Kannampalli, Pradeep; Banerjee, Banani; Sengupta, Jyoti N.

    2014-01-01

    NMDA receptors (NMDAR) are important in the development and maintenance of central sensitization. Our objective was to investigate the role of spinal neurons and NMDAR in the maintenance of chronic visceral pain. Neonatal rats were injected with acidic saline adjusted to pH4.0 in the gastrocnemius muscle every other day for 12 days. In adult rats, NR1 and NR2B subunits were examined in the lumbo-sacral (LS) spinal cord. A baseline, visceromotor response (VMR) to graded colorectal distension (CRD) was recorded before and after administration of the NMDA antagonist, CGS-19755. Extracellular recordings were performed from CRD-sensitive LS spinal neurons and pelvic nerve afferents (PNA) before and after CGS-19755. Rats that received pH 4.0 saline injections demonstrated a significant increase in the expression NR2B subunits and VMR response to CRD >20mmHg. CGS-19755 (i.v. or i.t.) had no effect in naïve rats, but significantly decreased the response to CRD in pH4.0 saline injected rats. CGS-19755 had no effect on the spontaneous firing of SL-A, but decreased that of SL-S. Similarly, CGS-19755 attenuates the responses of SL-S neurons to CRD, but had no effect on SL-A neurons or on the response characteristics of PNA fibers. Neonatal noxious somatic stimulation results in chronic visceral hyperalgesia and sensitizes a specific subpopulation of CRD-sensitive spinal neurons. The sensitization of these SL-S spinal neurons is attenuated by the NMDAR antagonist. The results of this study suggest that spinal NMDARs play an important role in the development of hyperalgesia early in life. PMID:25281204

  8. Selective NR1/2B N-methyl-D-aspartate receptor antagonists among indole-2-carboxamides and benzimidazole-2-carboxamides.

    PubMed

    Borza, István; Bozó, Eva; Barta-Szalai, Gizella; Kiss, Csilla; Tárkányi, Gábor; Demeter, Adám; Gáti, Tamás; Háda, Viktor; Kolok, Sándor; Gere, Anikó; Fodor, László; Nagy, József; Galgóczy, Kornél; Magdó, Ildikó; Agai, Béla; Fetter, József; Bertha, Ferenc; Keserü, György M; Horváth, Csilla; Farkas, Sándor; Greiner, István; Domány, György

    2007-03-01

    (4-Benzylpiperidine-1-yl)-(6-hydroxy-1H-indole-2-yl)-methanone (6a) derived from (E)-1-(4-benzylpiperidin-1-yl)-3-(4-hydroxy-phenyl)-propenone (5) was identified as a potent NR2B subunit-selective antagonist of the NMDA receptor. To establish the structure-activity relationship (SAR) and to attempt the improvement of the ADME properties of the lead, a series of compounds were prepared and tested. Several derivatives showed low nanomolar activity both in the binding and in the functional assay. In a formalin-induced hyperalgesia model in mice, 6a and (4-benzylpiperidine-1-yl)-[5(6)-hydroxy-1H-benzimidazol-2-yl]-methanone (60a) were as active as besonprodil (2) after oral administration. A CoMSIA model was developed based on binding data of a series of indole- and benzimidazole-2-carboxamides. PMID:17290978

  9. Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E

    PubMed Central

    Kapeller, Johannes; Möller, Dorothee; Lasitschka, Felix; Autschbach, Frank; Hovius, Ruud; Rappold, Gudrun; Brüss, Michael; Gershon, Michael D.

    2011-01-01

    Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT3 receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT3 receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT3 receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia. PMID:21192076

  10. Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling

    PubMed Central

    Yocum, Gene T.; Siviski, Matthew E.; Yim, Peter D.; Fu, Xiao Wen; Poe, Michael M.; Cook, James M.; Harrison, Neil; Perez-Zoghbi, Jose; Emala, Charles W.

    2015-01-01

    The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2′F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca2+]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2′F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca2+]i, store-operated Ca2+ entry, and methacholine-induced Ca2+ oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm. PMID:25659897