Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

Science.gov (United States)

Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

2002-04-01

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

International Nuclear Information System (INIS)

Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

2014-01-01

Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

Partial agonists and subunit selectivity at NMDA receptors

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Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

2010-01-01

Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

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

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Zhe eJin

2014-01-01

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

Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

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Sujatha P Koduvayur

Full Text Available 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.

γ-Aminobutyric Acid Type B (GABAB) Receptor Internalization Is Regulated by the R2 Subunit*

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Hannan, Saad; Wilkins, Megan E.; Dehghani-Tafti, Ebrahim; Thomas, Philip; Baddeley, Stuart M.; Smart, Trevor G.

2011-01-01

γ-Aminobutyric acid type B (GABAB) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABAB receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABAB receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABAB receptor. PMID:21724853

Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

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Mellor, J R; Wisden, W; Randall, A D

2000-07-10

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

Deletion of P2X2 and P2X3 receptor subunits does not alter motility of the mouse colon

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Matthew DeVries

2010-03-01

Full Text Available Purinergic P2X receptors contribute to neurotransmission in the gut. P2X receptors are ligand-gated cation channels that mediate synaptic excitation in subsets of enteric neurons. The present study evaluated colonic motility in vitro and in vivo in wild type (WT and P2X2 and P2X3 subunit knockout (KO mice. The muscarinic receptor agonist, bethanechol (0.3-3 micromolar, caused similar contractions of the longitudinal muscle in colon segments from WT, P2X2 and P2X3 subunit KO mice. Nicotine (1-300 micromolar, acting at neuronal nicotinic receptors, caused similar longitudinal muscle relaxations in colonic segments from WT and P2X2 and P2X3 subunit KO mice. Nicotine-induced relaxations were inhibited by nitro-L-arginine (NLA, 100 micromolar and apamin (0.1 micromolar which block inhibitory neuromuscular transmission. ATP (1-1000 micromolar caused contractions only in the presence of NLA and apamin. ATP-induced contractions were similar in colon segments from WT, P2X2 and P2X3 KO mice. The mouse colon generates spontaneous migrating motor complexes (MMCs in vitro. The MMC frequency was higher in P2X2 KO compared to WT tissues; other parameters of the MMC were similar in colon segments from WT, P2X2 and P2X3 KO mice. 5-Hydroxytryptophan-induced fecal output was similar in WT, P2X2 and P2X3 KO mice. These data indicate that nicotinic receptors are located predominately on inhibitory motor neurons supplying the longitudinal muscle in the mouse colon. P2X2 or P2X3 subunit containing receptors are not localized to motorneurons supplying the longitudinal muscle. Synaptic transmission mediated by P2X2 or P2X3 subunit containing receptors is not required for propulsive motility in the mouse colon.

Expression Profile of the Integrin Receptor Subunits in the Guinea Pig Sclera.

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Wang, Kevin K; Metlapally, Ravikanth; Wildsoet, Christine F

2017-06-01

The ocular dimensional changes in myopia reflect increased scleral remodeling, and in high myopia, loss of scleral integrity leads to biomechanical weakening and continued scleral creep. As integrins, a type of cell surface receptors, have been linked to scleral remodeling, they represent potential targets for myopia therapies. As a first step, this study aimed to characterize the integrin subunits at the messenger RNA level in the sclera of the guinea pig, a more recently added but increasingly used animal model for myopia research. Primers for α and β integrin subunits were designed using NCBI/UCSC Genome Browser and Primer3 software tools. Total RNA was extracted from normal scleral tissue and isolated cultured scleral fibroblasts, as well as liver and lung, as reference tissues, all from guinea pig. cDNA was produced by reverse transcription, PCR was used to amplify products of predetermined sizes, and products were sequenced using standard methods. Guinea pig scleral tissue expressed all known integrin alpha subunits except αD and αE. The latter integrin subunits were also not expressed by cultured guinea pig scleral fibroblasts; however, their expression was confirmed in guinea pig liver. In addition, isolated cultured fibroblasts did not express integrin subunits αL, αM, and αX. This difference between results for cultured cells and intact sclera presumably reflects the presence in the latter of additional cell types. Both guinea pig scleral tissue and isolated scleral fibroblasts expressed all known integrin beta subunits. All results were verified through sequencing. The possible contributions of integrins to scleral remodeling make them plausible targets for myopia prevention. Data from this study will help guide future ex vivo and in vitro studies directed at understanding the relationship between scleral integrins and ocular growth regulation in the guinea pig model for myopia.

Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

DEFF Research Database (Denmark)

Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

2002-01-01

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

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Torrão A.S.

1997-01-01

Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

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Holbrook, Joanna D; Gill, Catherine H; Zebda, Noureddine; Spencer, Jon P; Leyland, Rebecca; Rance, Kim H; Trinh, Han; Balmer, Gemma; Kelly, Fiona M; Yusaf, Shahnaz P; Courtenay, Nicola; Luck, Jane; Rhodes, Andrew; Modha, Sundip; Moore, Stephen E; Sanger, Gareth J; Gunthorpe, Martin J

2009-01-01

The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

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Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

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

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Ben-Wen Li

2015-12-01

Full Text Available 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

Sex Hormone Receptor Expression in the Human Vocal Fold Subunits.

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Kirgezen, Tolga; Sunter, Ahmet Volkan; Yigit, Ozgur; Huq, Gulben Erdem

2017-07-01

The study aimed to evaluate the existence of sex hormone receptors in the subunits of vocal fold. This is a cadaver study. The androgen, estrogen, and progesterone receptors were examined in the epithelium (EP), superficial layer of the lamina propria (SLP), vocal ligament (VL), and macula flava (MF) of the vocal folds from 42 human cadavers (21 male, 21 female) by immunohistochemical methods. Their staining ratios were scored and statistically compared. The androgen receptor score was significantly higher for the MF than for the EP and SLP (P vocal fold, mostly in the MF and VLs. Copyright © 2017 The Voice Foundation. Published by Elsevier Inc. All rights reserved.

Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

NARCIS (Netherlands)

Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

Memory Deficits Induced by Inflammation Are Regulated by α5-Subunit-Containing GABAA Receptors

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Dian-Shi Wang

2012-09-01

Full Text Available Systemic inflammation causes learning and memory deficits through mechanisms that remain poorly understood. Here, we studied the pathogenesis of memory loss associated with inflammation and found that we could reverse memory deficits by pharmacologically inhibiting α5-subunit-containing γ-aminobutyric acid type A (α5GABAA receptors and deleting the gene associated with the α5 subunit. Acute inflammation reduces long-term potentiation, a synaptic correlate of memory, in hippocampal slices from wild-type mice, and this reduction was reversed by inhibition of α5GABAA receptor function. A tonic inhibitory current generated by α5GABAA receptors in hippocampal neurons was increased by the key proinflammatory cytokine interleukin-1β through a p38 mitogen-activated protein kinase signaling pathway. Interleukin-1β also increased the surface expression of α5GABAA receptors in the hippocampus. Collectively, these results show that α5GABAA receptor activity increases during inflammation and that this increase is critical for inflammation-induced memory deficits.

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

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

1995-12-01

particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Immunocytochemical and stereological analysis of GABA(B) receptor subunit expression in the rat vestibular nucleus following unilateral vestibular deafferentation.

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Zhang, Rong; Ashton, John; Horii, Arata; Darlington, Cynthia L; Smith, Paul F

2005-03-10

The process of behavioral recovery that occurs following damage to one vestibular labyrinth, vestibular compensation, has been attributed in part to a down-regulation of GABA(B) receptors in the vestibular nucleus complex (VNC) ipsilateral to the lesion, which could potentially reduce commissural inhibition from the contralateral VNC. In this study, we tested the possibility that this occurs through a decrease in the expression of either the GABA(B1) or GABA(B2) subunits of the GABA(B) receptor. We used Western blotting to quantify the expression of these subunits in the VNC at 10 h and 50 h following unilateral vestibular deafferentation (UVD) or sham surgery in rats. We then used immunocytochemistry and stereological counting methods to estimate the number of neurons expressing these subunits in the MVN at 10 h and 2 weeks following UVD or sham surgery. Compared to sham controls, we found no significant changes in either the expression of the two GABA(B) receptor subunits in the VNC or in the number of MVN neurons expressing these GABA(B) receptor subunits post-UVD. These results suggest that GABA(B) receptor expression does not change substantially in the VNC during the process of vestibular compensation.

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

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Amanda Lorraine Wright

2012-04-01

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

Characterisation of the human NMDA receptor subunit NR3A glycine binding site

DEFF Research Database (Denmark)

Nilsson, A; Duan, J; Mo-Boquist, L-L

2007-01-01

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

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Zhao, Lan-Xue; Ge, Yan-Hui; Xiong, Cai-Hong; Tang, Ling; Yan, Ying-Hui; Law, Ping-Yee; Qiu, Yu; Chen, Hong-Zhuan

2018-03-06

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

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Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

DEFF Research Database (Denmark)

Kachel, Hamid S; Patel, Rohit N; Franzyk, Henrik

2016-01-01

-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents...

Adult naked mole-rat brain retains the NMDA receptor subunit GluN2D associated with hypoxia tolerance in neonatal mammals.

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Peterson, Bethany L; Park, Thomas J; Larson, John

2012-01-11

Adult naked mole-rats show a number of systemic adaptations to a crowded underground habitat that is low in oxygen and high in carbon dioxide. Remarkably, brain slice tissue from adult naked mole-rats also is extremely tolerant to oxygen deprivation as indicated by maintenance of synaptic transmission under hypoxic conditions as well as by a delayed neuronal depolarization during anoxia. These characteristics resemble hypoxia tolerance in brain slices from neonates in a variety of mammal species. An important component of neonatal tolerance to hypoxia involves the subunit composition of NMDA receptors. Neonates have a high proportion of NMDA receptors with GluN2D subunits which are protective because they retard calcium entry into neurons during hypoxic episodes. Therefore, we hypothesized that adult naked mole-rats retain a protective, neonatal-like, NMDA receptor subunit profile. We used immunoblotting to assess age-related changes in NMDA receptor subunits in naked mole-rats and mice. The results show that adult naked mole-rat brain retains a much greater proportion of the hypoxia-protective GluN2D subunit compared to adult mice. However, age-related changes in other subunits (GluN2A and GluN2B) from the neonatal period to adulthood were comparable in mice and naked mole-rats. Hence, adult naked mole-rat brain only retains the neonatal NMDA receptor subunit that is associated with hypoxia tolerance. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Double dissociation of spike timing-dependent potentiation and depression by subunit-preferring NMDA receptor antagonists in mouse barrel cortex.

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Banerjee, Abhishek; Meredith, Rhiannon M; Rodríguez-Moreno, Antonio; Mierau, Susanna B; Auberson, Yves P; Paulsen, Ole

2009-12-01

Spike timing-dependent plasticity (STDP) is a strong candidate for an N-methyl-D-aspartate (NMDA) receptor-dependent form of synaptic plasticity that could underlie the development of receptive field properties in sensory neocortices. Whilst induction of timing-dependent long-term potentiation (t-LTP) requires postsynaptic NMDA receptors, timing-dependent long-term depression (t-LTD) requires the activation of presynaptic NMDA receptors at layer 4-to-layer 2/3 synapses in barrel cortex. Here we investigated the developmental profile of t-LTD at layer 4-to-layer 2/3 synapses of mouse barrel cortex and studied their NMDA receptor subunit dependence. Timing-dependent LTD emerged in the first postnatal week, was present during the second week and disappeared in the adult, whereas t-LTP persisted in adulthood. An antagonist at GluN2C/D subunit-containing NMDA receptors blocked t-LTD but not t-LTP. Conversely, a GluN2A subunit-preferring antagonist blocked t-LTP but not t-LTD. The GluN2C/D subunit requirement for t-LTD appears to be synapse specific, as GluN2C/D antagonists did not block t-LTD at horizontal cross-columnar layer 2/3-to-layer 2/3 synapses, which was blocked by a GluN2B antagonist instead. These data demonstrate an NMDA receptor subunit-dependent double dissociation of t-LTD and t-LTP mechanisms at layer 4-to-layer 2/3 synapses, and suggest that t-LTD is mediated by distinct molecular mechanisms at different synapses on the same postsynaptic neuron.

Expression of NMDA receptor subunits in human blood lymphocytes: A peripheral biomarker in online computer game addiction.

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Sadat-Shirazi, Mitra-Sadat; Vousooghi, Nasim; Alizadeh, Bentolhoda; Makki, Seyed Mohammad; Zarei, Seyed Zeinolabedin; Nazari, Shahrzad; Zarrindast, Mohammad Reza

2018-05-23

Background and aims Repeated performance of some behaviors such as playing computer games could result in addiction. The NMDA receptor is critically involved in the development of behavioral and drug addictions. It has been claimed that the expression level of neurotransmitter receptors in the brain may be reflected in peripheral blood lymphocytes (PBLs). Methods Here, using a real-time PCR method, we have investigated the mRNA expression of GluN2A, GluN2D, GluN3A, and GluN3B subunits of the NMDA receptor in PBLs of male online computer game addicts (n = 25) in comparison with normal subjects (n = 26). Results Expression levels of GluN2A, GluN2D, and GluN3B subunits were not statistically different between game addicts and the control group. However, the mRNA expression of the GluN3A subunit was downregulated in PBLs of game addicts. Discussion and conclusions Transcriptional levels of GluN2A and GluN2D subunits in online computer game addicts are similar to our previously reported data of opioid addiction and are not different from the control group. However, unlike our earlier finding of drug addiction, the mRNA expression levels of GluN3A and GluN3B subunits in PBLs of game addicts are reduced and unchanged, respectively, compared with control subjects. It seems that the downregulated state of the GluN3A subunit of NMDA receptor in online computer game addicts is a finding that deserves more studies in the future to see whether it can serve as a peripheral biomarker in addiction studies, where the researcher wants to rule out the confusing effects of abused drugs.

Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

International Nuclear Information System (INIS)

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

1989-01-01

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

The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

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Gurd, J W; Bissoon, N

1997-08-01

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

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Ariel Avila

2013-08-01

Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

Energy Technology Data Exchange (ETDEWEB)

Machaalani, R., E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)

2014-05-01

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

International Nuclear Information System (INIS)

Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

2014-01-01

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta

Differential expression of AMPA-type glutamate receptor subunits during development of the chick optic tectum

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Batista S.S.

2002-01-01

Full Text Available Glutamate receptors have been often associated with developmental processes. We used immunohistochemical techniques to evaluate the expression of the AMPA-type glutamate receptor (GluR subunits in the chick optic tectum (TeO. Chick embryos from the 5th through the 20th embryonic day (E5-E20 and one-day-old (P1 chicks were used. The three types of immunoreactivity evaluated (GluR1, GluR2/3, and GluR4 had different temporal and spatial expression patterns in the several layers of the TeO. The GluR1 subunit first appeared as moderate staining on E7 and then increased on E9. The mature GluR1 pattern included intense staining only in layer 5 of the TeO. The GluR2/3 subunits presented low expression on E5, which became intense on E7. The staining for GluR2/3 changed to very intense on E14 in tectal layer 13. Staining of layer 13 neurons is the most prominent feature of GluR immunoreactivity in the adult TeO. The GluR4 subunit generally presented the lowest expression starting on E7, which was similar to the adult pattern. Some instances of transient expression of GluR subunits were observed in specific cell populations from E9 through E20. These results demonstrate a differential expression of the GluR subunits in the embryonic TeO, adding information about their possible functions in the developmental processes of the visual system.

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

International Nuclear Information System (INIS)

Le Marchand-Brustel, Y.; Ballotti, R.; Gremeaux, T.; Tanti, J.F.; Brandenburg, D.; Van Obberghen, E.

1989-01-01

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form

Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

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Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

2012-01-01

Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

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Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

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Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

2000-05-31

cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

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Gersende Maugars

Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

Nature and regulation of the insulin receptor: structure and function

International Nuclear Information System (INIS)

Czech, M.P.

1985-01-01

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner

Olanzapine Reverses MK-801-Induced Cognitive Deficits and Region-Specific Alterations of NMDA Receptor Subunits

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Liu, Xiao; Li, Jitao; Guo, Chunmei; Wang, Hongli; Sun, Yaxin; Wang, Han; Su, Yun-Ai; Li, Keqing; Si, Tianmei

2018-01-01

Cognitive dysfunction constitutes an essential component in schizophrenia for its early presence in the pathophysiology of the disease and close relatedness to life quality of patients. To develop effective treatment of cognitive deficits, it is important to understand their neurobiological causes and to identify potential therapeutic targets. In this study, adopting repeated MK-801 treatment as an animal model of schizophrenia, we investigated whether antipsychotic drugs, olanzapine and haloperidol, can reverse MK-801-induced cognitive deficits and how the reversal processes recruited proteins involved in glutamate neurotransmission in rat medial prefrontal cortex (mPFC) and hippocampus. We found that low-dose chronic MK-801 treatment impaired object-in-context recognition memory and reversal learning in the Morris water maze, leaving reference memory relatively unaffected, and that these cognitive deficits can be partially reversed by olanzapine, not haloperidol, treatment. At the molecular level, chronic MK-801 treatment resulted in the reduction of multiple N-methyl-D-aspartate (NMDA) receptor subunits in rat mPFC and olanzapine, not haloperidol, treatment restored the levels of GluN1 and phosphorylated GluN2B in this region. Taken together, MK-801-induced cognitive deficits may be associated with region-specific changes in NMDA receptor subunits and the reversal of specific NMDA receptor subunits may underlie the cognition-enhancing effects of olanzapine. PMID:29375333

Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

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

2010-02-15

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

International Nuclear Information System (INIS)

Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr.; O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi; Uhl, G.R.

1991-01-01

Type A γ-aminobutyric acid (GABA A ) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA A subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ 1 , with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

The Barley Magnesium Chelatase 150-kD Subunit Is Not an Abscisic Acid Receptor1[OA

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Müller, André H.; Hansson, Mats

2009-01-01

Magnesium chelatase is the first unique enzyme of the chlorophyll biosynthetic pathway. It is composed of three gene products of which the largest is 150 kD. This protein was recently identified as an abscisic acid receptor in Arabidopsis (Arabidopsis thaliana). We have evaluated whether the barley (Hordeum vulgare) magnesium chelatase large subunit, XanF, could be a receptor for the phytohormone. The study involved analysis of recombinant magnesium chelatase protein as well as several induced chlorophyll-deficient magnesium chelatase mutants with defects identified at the gene and protein levels. Abscisic acid had no effect on magnesium chelatase activity and binding to the barley 150-kD protein could not be shown. Magnesium chelatase mutants showed a wild-type response in respect to postgermination growth and stomatal aperture. Our results question the function of the large magnesium chelatase subunit as an abscisic acid receptor. PMID:19176716

NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition

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María Verónica Baez

2018-01-01

Full Text Available NMDA ionotropic glutamate receptors (NMDARs are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

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

Science.gov (United States)

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

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Maria Veronica Baez

Full Text Available NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1 adult rats that had explored an open field (OF for 5 minutes, a time sufficient to induce habituation, 2 mature rat hippocampal neuron cultures depolarized by KCl and 3 hippocampal slices from adult rats where long term potentiation (LTP was induced by theta-burst stimulation (TBS. GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor or actinomycin D (a transcription inhibitor during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

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Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

GABAA receptor B subunit expression in the superior frontal cortex of human alcoholics

International Nuclear Information System (INIS)

Buckley, S.T.; Dodd, P.R.

2001-01-01

Full text: Changes in GABA A receptor pharmacology can be ascribed to alterations in expression of specific GABA A receptor subunits. Ethanol is known to be a potent agonist of the GABA A receptor. Chronic abuse of alcohol in humans results in damage of selective brain regions such as the superior frontal cortex (SFC), leading to neuronal cell loss. Studies in our laboratory 1 and elsewhere 2 have shown differences in expression of a number of GABA A receptor subunits in chronic human alcoholism. This suggests that alterations in GABA A receptor composition may be involved in the pathogenesis of alcoholic brain damage. We analysed the expression of the β 1 ,β 2 and β 3 isoforms of the GABA A receptor by a competitive reverse transcription polymerase chain reaction (RT-PCR) technique, which utilised an internal standard (IS) for quantitation. 35 S-dATP was incorporated to enable visualisation of the PCR products. Human brain tissue was obtained at autopsy and stored in 0.32 M sucrose at -80 deg C. Total RNA was extracted from pathologically susceptible and spared regions, SFC and motor cortex respectively,of 22 control and 22 alcoholic patients. 1 μg of total RNA from each sample was co-amplified with 0.5 pg of IS and a ratio determined. A standard consisting of known amounts of β 1 cRNA titrated against 0.5 pg of IS enabled a standard curve to be generated for quantitation of each unknown sample. The samples were subjected to polyacrylamide gel electrophoresis and the dried gel exposed to a phosphorimager screen. Data analysis was performed using the ImageQuant program. Initial results indicate that there is a reduction in expression of all the β transcripts in alcoholics when compared with controls, which supports the hypothesis that the GABA A receptor is altered by alcohol abuse. Supported by NHMRC. Copyright (2001) Australian Neuroscience Society

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

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Florian Wegner

Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Synthetic α subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

International Nuclear Information System (INIS)

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

1986-01-01

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) α subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 μg of peptide (T α 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 α subunit. Peptide H α 125-147 differs from T α 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound 125 I-labelled Hα 125-147 antibody bound Hα 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither Hα 125-147 nor T α 125-147. Rats immunized with H α 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 α 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

Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ SubunitS⃞

OpenAIRE

O'Shea, Sean M.; Williams, Carrie A.; Jenkins, Andrew

2009-01-01

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of α and β subunits of type “A” GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the γ subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engi...

Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor γ2 subunit

Science.gov (United States)

Kittler, Josef T.; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R.; McAinsh, Kristina; Arancibia-Carcamo, I. Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J.

2008-01-01

The regulation of the number of γ2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface γ2-subunit-containing GABAARs is regulated. Here, we identify a γ2-subunit-specific Yxxφ-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for γ2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxφ motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that γ2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition. PMID:18305175

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

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McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

International Nuclear Information System (INIS)

Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

1987-01-01

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

Sub-unit Specific Regulation of Type-A GABAergic Receptors during Post-Natal Development of the Auditory Cortex

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Liisa A. Tremere

2011-01-01

Full Text Available The GABA-A receptor has been strongly implicated in the organization and function of cortical sensory circuits in the adult mammal. In the present work, changes in the expression patterns of select GABA-A subunits were examined as a function of development. The RNA expression profiles for three subunit types were studied, α1, β2/3 and δ at four developmental time points, (p0, p15, p30 and p90. The o1, β2/3 subunits were present at birth and following a modest increase early in life; mRNA expression for these subunits were found at stable levels throughout life. The expression pattern for the δ subunit showed the most dramatic changes in the number of positive cells as a function of age. In early life, p0 through p15 expression of mRNA for the δ subunit was quite low but increased in later life, p30 and p90. Together these data suggest that much of the potential for inhibitory connectivity is laid down in the pre and early post-natal periods.

Transcriptional regulators of Na, K-ATPase subunits

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Zhiqin eLi

2015-10-01

Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

Science.gov (United States)

Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

2014-01-01

Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

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

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Sayyed Alireza Talaei

2015-06-01

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

Regulation of synaptic inhibition by phospho-dependent binding of the AP2 complex to a YECL motif in the GABAA receptor gamma2 subunit.

Science.gov (United States)

Kittler, Josef T; Chen, Guojun; Kukhtina, Viktoria; Vahedi-Faridi, Ardeschir; Gu, Zhenglin; Tretter, Verena; Smith, Katharine R; McAinsh, Kristina; Arancibia-Carcamo, I Lorena; Saenger, Wolfram; Haucke, Volker; Yan, Zhen; Moss, Stephen J

2008-03-04

The regulation of the number of gamma2-subunit-containing GABA(A) receptors (GABA(A)Rs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface gamma2-subunit-containing GABA(A)Rs is regulated. Here, we identify a gamma2-subunit-specific Yxxvarphi-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for gamma2-subunit tyrosine phosphorylation. Blocking GABA(A)R-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxxvarphi motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that gamma2-subunit-containing heteromeric GABA(A)Rs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABA(A)R surface levels and synaptic inhibition.

Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

NARCIS (Netherlands)

De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.

2006-01-01

It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the

Fast and Slow Inhibition in the Visual Thalamus Is Influenced by Allocating GABAA Receptors with Different γ Subunits

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Zhiwen Ye

2017-04-01

Full Text Available Cell-type specific differences in the kinetics of inhibitory postsynaptic conductance changes (IPSCs are believed to impact upon network dynamics throughout the brain. Much attention has focused on how GABAA receptor (GABAAR α and β subunit diversity will influence IPSC kinetics, but less is known about the influence of the γ subunit. We have examined whether GABAAR γ subunit heterogeneity influences IPSC properties in the thalamus. The γ2 subunit gene was deleted from GABAARs selectively in the dorsal lateral geniculate nucleus (dLGN. The removal of the γ2 subunit from the dLGN reduced the overall spontaneous IPSC (sIPSC frequency across all relay cells and produced an absence of IPSCs in a subset of relay neurons. The remaining slower IPSCs were both insensitive to diazepam and zinc indicating the absence of the γ2 subunit. Because these slower IPSCs were potentiated by methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, we propose these IPSCs involve γ1 subunit-containing GABAAR activation. Therefore, γ subunit heterogeneity appears to influence the kinetics of GABAAR-mediated synaptic transmission in the visual thalamus in a cell-selective manner. We suggest that activation of γ1 subunit-containing GABAARs give rise to slower IPSCs in general, while faster IPSCs tend to be mediated by γ2 subunit-containing GABAARs.

The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

DEFF Research Database (Denmark)

Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine

2011-01-01

The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive α1-subunit-containing GABA type A receptors (GABA(A)Rs); however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation, we....... Mutating residues 360-375 decreases both the accumulation of α1-containing GABA(A)Rs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of mIPSCs. We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site....... Mutation of Thr375 to a phosphomimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, and therefore receptor accumulation at synapses, and the amplitude of mIPSCs. Finally, single-particle tracking reveals that gephyrin reduces the diffusion of α1-subunit...

Age-Related Differences in NMDA Receptor Subunits of Prenatally Methamphetamine-Exposed Male Rats

Czech Academy of Sciences Publication Activity Database

Vrajová, M.; Schutová, B.; Klaschka, Jan; Štěpánková, H.; Řípová, D.; Šlamberová, R.

2014-01-01

Roč. 39, č. 11 (2014), s. 2040-2046 ISSN 0364-3190 Grant - others:GA ČR(CZ) GAP303/10/0580; Ministerstvo školství(CZ) CSM 7/CRP/2014; Univerzita Karlova(CZ) Prvouk P34; Univerzita Karlova(CZ) 260045/SVV/2014; Prague Psychiatric Center(CZ) MH CZ–DRO: 00023752 Institutional support: RVO:67985807 Keywords : methamphetamine * in-utero * NMDA receptor subunits * hippocampus Subject RIV: FH - Neurology Impact factor: 2.593, year: 2014

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study

NARCIS (Netherlands)

Vandenbranden, C. A.; Kamphuis, W.; Nunes Cardozo, B.; Kamermans, M.

2000-01-01

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and

LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

Science.gov (United States)

Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

2012-01-01

GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit.

Science.gov (United States)

Takehara, Akio; Hosokawa, Masayo; Eguchi, Hidetoshi; Ohigashi, Hiroaki; Ishikawa, Osamu; Nakamura, Yusuke; Nakagawa, Hidewaki

2007-10-15

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

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

Energy Technology Data Exchange (ETDEWEB)

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.

Repeated ketamine administration alters N-methyl-d-aspartic acid receptor subunit gene expression: Implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans

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Lipsky, Robert H

2015-01-01

For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-d-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. PMID:25245072

Repeated ketamine administration alters N-methyl-D-aspartic acid receptor subunit gene expression: implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans.

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Xu, Ke; Lipsky, Robert H

2015-02-01

For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. © 2014 by the Society for Experimental Biology and Medicine.

Genetic ablation of the GluK4 kainate receptor subunit causes anxiolytic and antidepressant-like behavior in mice.

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Catches, Justin S; Xu, Jian; Contractor, Anis

2012-03-17

There is a clear link between dysregulation of glutamatergic signaling and mood disorders. Genetic variants in the glutamate receptor gene GRIK4, which encodes the kainate receptor subunit GluK4, alter the susceptibility for depression, bipolar disorder and schizophrenia. Here we demonstrate that Grik4(-/-) mice have reduced anxiety and an antidepressant-like phenotype. In the elevated zero-maze, a test for anxiety and risk taking behavior, Grik4(-/-) mice spent significantly more time exploring the open areas of the maze. In anxiogenic tests of marble-burying and novelty-induced suppression of feeding, anxiety-like behavior was consistently reduced in knockout animals. In the forced swim test, a test of learned helplessness that is used to determine depression-like behavior, knockout mice demonstrated significantly less immobility suggesting that Grik4 ablation has an antidepressant-like effect. Finally, in the sucrose preference test, a test for anhedonia in rodents, Grik4(-/-) mice demonstrated increased sucrose preference. Expression of the GluK4 receptor subunit in the forebrain is restricted to the CA3 region of the hippocampus and dentate gyrus regions where KARs are known to modulate synaptic plasticity. We tested whether Grik4 ablation had effects on mossy fiber (MF) plasticity and found there to be a significant impairment in LTP likely through a loss of KAR modulation of excitability of the presynaptic MF axons. These studies demonstrate a clear anxiolytic and antidepressant phenotype associated with ablation of Grik4 and a parallel disruption in hippocampal plasticity, providing support for the importance of this receptor subunit in mood disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

Long-term potentiation in the CA1 hippocampus induced by NR2A subunit-containing NMDA glutamate receptors is mediated by Ras-GRF2/Erk map kinase signaling.

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Shan-xue Jin

Full Text Available BACKGROUND: NMDA-type glutamate receptors (NMDARs are major contributors to long-term potentiation (LTP, a form of synaptic plasticity implicated in the process of learning and memory. These receptors consist of calcium-permeating NR1 and multiple regulatory NR2 subunits. A majority of studies show that both NR2A and NR2B-containing NMDARs can contribute to LTP, but their unique contributions to this form of synaptic plasticity remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that NR2A and NR2B-containing receptors promote LTP differently in the CA1 hippocampus of 1-month old mice, with the NR2A receptors functioning through Ras-GRF2 and its downstream effector, Erk Map kinase, and NR2B receptors functioning independently of these signaling molecules. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that NR2A-, but not NR2B, containing NMDA receptors induce LTP in pyramidal neurons of the CA1 hippocampus from 1 month old mice through Ras-GRF2 and Erk. This difference add new significance to the observation that the relative levels of these NMDAR subtypes is regulated in neurons, such that NR2A-containing receptors become more prominent late in postnatal development, after sensory experience and synaptic activity.

Requirement of Nicotinic Acetylcholine Receptor Subunit β2 in the Maintenance of Spiral Ganglion Neurons during Aging

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Bao, Jianxin; Lei, Debin; Du, Yafei; Ohlemiller, Kevin K.; Beaudet, Arthur L.; Role, Lorna W.

2008-01-01

Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express α9 nAChR subunits and on the peripheral projections of SGNs that express α2, α4 –7, and β2–3 nAChR subunits. A significantly decreased expression of the β2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the β2 nAChR subunit (β2−/−), but not mice lacking the α5 nAChR subunit (α5−/−), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for β2 nAChR subunit in the maintenance of SGNs during aging. PMID:15788760

Increased GABA(A receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy.

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Keith B Hengen

Full Text Available GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA(ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA(AR expression on brainstem neurons of the ventral respiratory column (VRC. In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA(AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA(AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA(AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life despite increased neurosteroid levels during pregnancy.

Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze

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Soheil eKeihan Falsafi

2015-10-01

Full Text Available GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM task, a robust paradigm for evaluation of spatial learning.Trained or untrained (yoked control C57BL/6J male mice (n=10/group were subjected to the MTM task and sacrificed 6 hours following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA and a proximity ligation assay (PLA were also used to see if the two GABAB subunits are present in the receptor complex.Single bands were observed on Western blots, each representing GABAB1, GABAB1a or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

The GABAA receptor α and β subunits but not the density of muscimol binding sites are altered in the auditory-linguistic association cortex of subjects with schizophrenia

International Nuclear Information System (INIS)

Farnbach-Pralong, D.; Bradbury, R.; Tomaskovic, E.; Copolov, D.; Dean, B.

1998-01-01

Full text: An increase in the density of postsynaptic GABA A receptors has recently been reported in the prefrontal cortex of subjects with schizophrenia. This increase has been hypothesised to represent an up-regulation in response a decrease in the density of GABAergic interneurons. In order to determine whether the GABA A receptor is also altered in the auditory-linguistic association cortex of the schizophrenic brain, we used quantitative autoradiography to measure the density of that receptor in tissue obtained at autopsy from 20 control subjects and 20 subjects with schizophrenia matched for sex and age. The density of GABA A receptors was measured as the difference in the binding of the specific ligand [ 3 H]muscimol (100 nM) in the presence or in the absence of 10 5 M SR95531. There was no significant difference in the density of [ 3 H]muscimol binding between tissue from schizophrenic (554.9±20,5 fmol/mg TE) and non-schizophrenic (580.1±26.2 fmol/mg TE) subjects. The abundance of the α and β subunits of the GABA A receptor was also measured in particulate membranes prepared from tissue from 6 control and 6 schizophrenic subjects using Western blots. Detection with monoclonal antibodies and chemiluminescence showed that in tissue from control subjects, there was a significant correlation between the levels of α and β subunits (r=0.817, p=0.047). However, there was no such correlation in tissue from schizophrenic subjects (r=0.265, p=0.61), where in 2 subjects large levels of β-subunit were not matched by similar levels of α subunit. These preliminary results suggest mat there may be a failure for up-regulated GABA A receptor subunits to assemble into functional receptors in this brain region for some subjects with schizophrenia. Copyright (1998) Australian Neuroscience Society

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

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Varju, P; Schlett, K; Eisel, U; Madarász, E

2001-06-01

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Deletion of the GluA1 AMPA Receptor Subunit Alters the Expression of Short-Term Memory

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Sanderson, David J.; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

2011-01-01

Deletion of the GluA1 AMPA receptor subunit selectively impairs short-term memory for spatial locations. We further investigated this deficit by examining memory for discrete nonspatial visual stimuli in an operant chamber. Unconditioned suppression of magazine responding to visual stimuli was measured in wild-type and GluA1 knockout mice.…

Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

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Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

2013-04-01

GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits (α1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

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Christine Laura Dixon

2015-12-01

Full Text Available 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 inhibitory postsynaptic currents mediated by individual isoforms 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.

NMDA receptor function during senescence: implication on cognitive performance

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Ashok eKumar

2015-12-01

Full Text Available N-methyl-D-aspartate (NMDA receptors, a family of L-glutamate receptors, play an important role in learning and memory, and are critical for spatial memory. These receptors are tetrameric ion channels composed of a family of related subunits. One of the hallmarks of the aging human population is a decline in cognitive function; studies in the past couple of years have demonstrated deterioration in NMDA receptor subunit expression and function with advancing age. However, a direct relationship between impaired memory function and a decline in NMDA receptors is still ambiguous. Recent studies indicate a link between an age-associated NMDA receptor hypofunction and memory impairment and provide evidence that age-associated enhanced oxidative stress might be contributing to the alterations associated with senescence. However, clear evidence is still deficient in demonstrating the underlying mechanisms and a relationship between age-associated impaired cognitive faculties and NMDA receptor hypofunction. The current review intends to present an overview of the research findings regarding changes in expression of various NMDA receptor subunits and deficits in NMDA receptor function during senescence and its implication in age-associated impaired hippocampal-dependent memory function.

The effect of NMDA-NR2B receptor subunit over-expression on olfactory memory task performance in the mouse.

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White, Theresa L; Youngentob, Steven L

2004-09-17

The N-methyl-D-aspartate (NMDA) receptor in the forebrain is thought to modulate some forms of memory formation, with the NR2B subunit being particularly relevant to this process. Relative to wild-type mice, transgenic animals in which the NR2B subunit was over-expressed demonstrate superior memory in a number of behavioral tasks, including object recognition [Nature 401 (1999) 63]. The purpose of the present study was to explore the generality of such phenomena, interpreted as the effect of increasing NR2B expression on the retention of other types of sensory-related information. To accomplish this, we focused our evaluation on the highly salient sensory modality of olfaction. In the first experiment, mice performed both a novel-object-recognition task identical to that performed by Tang et al. [Nature 401 (1999) 63] and a novel-odor-recognition task analogously constructed. Although the results of the object recognition task were consistent with the previous literature, there was no evidence of an effect of NR2B over-expression on the retention of odor recognition memory in the specific task performed. As it was possible that, unlike object recognition memory, novel odor recognition is not NMDA-receptor-dependent, a second task was designed using the social transmission of food preference paradigm. In contrast to the foregoing olfactory task, there is evidence that the latter procedure is, indeed, NMDA-dependent. The results of the second study demonstrated that transgenic mice with NR2B over-expression had a clear memory advantage in this alternative odor memory paradigm. Taken together, these results suggest the NR2B subunit is an important component in some but not all forms of olfactory memory organization. Moreover, for those functions that are NMDA-receptor-dependent, these data support the growing literature demonstrating the importance of the NR2B subunit.

Lessons from crystal structures of kainate receptors

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

2017-01-01

Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory...... synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

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Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

2015-01-01

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit

Analysis of NR3A receptor subunits in human native NMDA receptors

DEFF Research Database (Denmark)

Nilsson, Anna; Eriksson, Maria; Muly, E Chris

2007-01-01

NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human...... primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only...... very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2...

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

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Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αβγ are linked to hibernating state in hamsters

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Alò Raffaella

2010-09-01

Full Text Available Abstract Background The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB and hibernating (HIB hamsters. Attempts were made to identify the type of αβγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. Results Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p 1 ratio (over total α subunits considered in the present study in the medial preoptic area (MPOA and arcuate nucleus (Arc of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%. Regarding the other two subunits (β and γ, elevated β3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for β3 and γ2 during hibernating conditions. Conclusion We conclude that different αβγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

NARCIS (Netherlands)

Varju, P; Schlett, K; Eisel, U; Madarasz, E

NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits.

Autoinactivation of the stargazin-AMPA receptor complex: subunit-dependency and independence from physical dissociation.

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Artur Semenov

Full Text Available Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

Distinct roles of synaptic and extrasynaptic GABAA receptors in striatal inhibition dynamics

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Ruixi eLuo

2013-11-01

Full Text Available Striatonigral and striatopallidal projecting medium spiny neurons (MSNs express dopamine D1 (D1+ and D2 receptors (D2+, respectively. Both classes receive extensive GABAergic input via expression of synaptic, perisynaptic and extrasynaptic GABAA receptors. The activation patterns of different presynaptic GABAergic neurons produce transient and sustained GABAA receptor-mediated conductance that fulfill distinct physiological roles. We performed single and dual whole cell recordings from striatal neurons in mice expressing fluorescent proteins in interneurons and MSNs. We report specific inhibitory dynamics produced by distinct activation patterns of presynaptic GABAergic neurons as source of synaptic, perisynaptic and extrasynaptic inhibition. Synaptic GABAA receptors in MSNs contain the α2, γ2 and a β subunit. In addition, there is evidence for the developmental increase of the α1 subunit that contributes to faster inhibitory postsynaptic current (IPSC. Tonic GABAergic currents in MSNs from adult mice are carried by extrasynaptic receptors containing the α4 and δ subunit, while in younger mice this current is mediated by receptors that contain the α5 subunit. Both forms of tonic currents are differentially expressed in D1+ and D2+ MSNs. This study extends these findings by relating presynaptic activation with pharmacological analysis of inhibitory conductance in mice where the β3 subunit is conditionally removed in fluorescently labeled D2+ MSNs and in mice with global deletion of the δ subunit. Our results show that responses to low doses of gaboxadol (2μM, a GABAA receptor agonist with preference to δ subunit, are abolished in the δ but not the β3 subunit knock out mice. This suggests that the β3 subunit is not a component of the adult extrasynaptic receptor pool, in contrast to what has been shown for tonic current in young mice. Deletion of the β3 subunit from D2+ MSNs however, removed slow spontaneous IPSCs, implicating its

Editing modifies the GABA(A) receptor subunit alpha3

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Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

2007-01-01

Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

NMDA receptor GluN2A/GluN2B subunit ratio as synaptic trait of levodopa-induced dyskinesias: from experimental models to patients

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Manuela eMellone

2015-07-01

Full Text Available Levodopa-induced dyskinesias (LIDs are major complications in the pharmacological management of Parkinson’s disease (PD. Abnormal glutamatergic transmission in the striatum is considered a key factor in the development of LIDs. This work aims at i. characterizing NMDA receptor GluN2A/GluN2B subunit ratio as a common synaptic trait in rat and primate models of LIDs and in dyskinetic PD patients, and ii. validating the potential therapeutic effect of a cell-permeable peptide interfering with GluN2A synaptic localization on the dyskinetic behavior of these experimental models of LIDs. Here we demonstrate an altered ratio of synaptic GluN2A/GluN2B-containing NMDA receptors in the striatum of levodopa-treated dyskinetic rats and monkeys as well as in post-mortem tissue from dyskinetic PD patients. The modulation of synaptic NMDA receptor composition by a cell-permeable peptide interfering with GluN2A subunit interaction with the scaffolding protein PSD-95 leads to a reduction in the dyskinetic motor behavior in the two animal models of LIDs. Our results indicate that targeting synaptic NMDA receptor subunit composition may represent an intriguing therapeutic approach aimed at ameliorating levodopa motor side effects.

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

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Elster, L; Kristiansen, U; Pickering, D S

2001-01-01

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit

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Floridi Aristide

2008-10-01

Full Text Available Abstract Background Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β2-adrenergic receptor (β2AR and Gαs indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gαs-fused β2AR. Results The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion The covalent linkage between β2AR and Gαs does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes

Propranolol decreases retention of fear memory by modulating the stability of surface glutamate receptor GluA1 subunits in the lateral amygdala.

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Zhou, Jun; Luo, Yi; Zhang, Jie-Ting; Li, Ming-Xing; Wang, Can-Ming; Guan, Xin-Lei; Wu, Peng-Fei; Hu, Zhuang-Li; Jin, You; Ni, Lan; Wang, Fang; Chen, Jian-Guo

2015-11-01

Posttraumatic stress disorder (PTSD) is a mental disorder with enhanced retention of fear memory and has profound impact on quality of life for millions of people worldwide. The β-adrenoceptor antagonist propranolol has been used in preclinical and clinical studies for the treatment of PTSD, but the mechanisms underlying its potential efficacy on fear memory retention remain to be elucidated. We investigated the action of propranolol on the retention of conditioned fear memory, the surface expression of glutamate receptor GluA1 subunits of AMPA receptors and synaptic adaptation in the lateral amygdala (LA) of rats. Propranolol attenuated reactivation-induced strengthening of fear retention while reducing enhanced surface expression of GluA1 subunits and restoring the impaired long-term depression in LA. These effects of propranolol were mediated by antagonizing reactivation-induced enhancement of adrenergic signalling, which activates PKA and calcium/calmodulin-dependent protein kinase II and then regulates the trafficking of AMPA receptors via phosphorylation of GluA1 subunits at the C-terminus. Both i.p. injection and intra-amygdala infusion of propranolol attenuated reactivation-induced enhancement of fear retention. Reactivation strengthens fear retention by increasing the level of noradrenaline and promotes the surface expression of GluA1 subunits and the excitatory synaptic transmission in LA. These findings uncover one mechanism underlying the efficiency of propranolol on retention of fear memories and suggest that β-adrenoceptor antagonists, which act centrally, may be more suitable for the treatment of PTSD. © 2015 The British Pharmacological Society.

Chronic MK-801 Application in Adolescence and Early Adulthood: A Spatial Working Memory Deficit in Adult Long-Evans Rats But No Changes in the Hippocampal NMDA Receptor Subunits

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Uttl, Libor; Petrasek, Tomas; Sengul, Hilal; Svojanovska, Marketa; Lobellova, Veronika; Vales, Karel; Radostova, Dominika; Tsenov, Grygoriy; Kubova, Hana; Mikulecka, Anna; Svoboda, Jan; Stuchlik, Ales

2018-01-01

The role of NMDA receptors in learning, memory and hippocampal function has long been recognized. Post-mortem studies have indicated that the expression or subunit composition of the NMDA glutamate receptor subtype might be related to the impaired cognitive functions found in schizophrenia patients. NMDA receptor antagonists have been used to develop animal models of this disorder. There is accumulating evidence showing that not only the acute but also the chronic application of NMDA receptor antagonists may induce schizophrenia-like alterations in behavior and brain functions. However, limited evidence is available regarding the consequences of NMDA receptor blockage during periods of adolescence and early adulthood. This study tested the hypothesis that a 2-week treatment of male Long-Evans and Wistar rats with dizocilpine (MK-801; 0.5 mg/kg daily) starting at postnatal days (PD) 30 and 60 would cause a long-term cognitive deficit and changes in the levels of NMDA receptor subunits. The working memory version of the Morris water maze (MWM) and active place avoidance with reversal on a rotating arena (Carousel) requiring cognitive coordination and flexibility probed cognitive functions and an elevated-plus maze (EPM) was used to measure anxiety-like behavior. The western blot method was used to determine changes in NMDA receptor subunit levels in the hippocampus. Our results showed no significant changes in behaviors in Wistar rats. Slightly elevated anxiety-like behavior was observed in the EPM in Long-Evans rats with the onset of treatment on PD 30. Furthermore, Long-Evans rats treated from PD 60 displayed impaired working memory in the MWM. There were; however, no significant changes in the levels of NMDA receptor subunits because of MK-801 administration. These findings suggest that a 2-week treatment starting on PD 60 in Long-Evans rats leads to long-term changes in working memory, but this deficit is not paralleled by changes in NMDA receptor subunits. These

[Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

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Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

2011-01-01

Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice.

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Wu, Xiaorong; Tuzun, Erdem; Saini, Shamsher S; Wang, Jun; Li, Jing; Aguilera-Aguirre, Leopoldo; Huda, Ruksana; Christadoss, Premkumar

2015-12-01

Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

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Emiliane Taillebois

Full Text Available Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI, thiamethoxam (TMX and clothianidin (CLT. Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM. Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml and TMX (LC50 = 0.034 µg/ml were more toxic than CLT (LC50 = 0.118 µg/ml. The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

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

International Nuclear Information System (INIS)

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

2011-01-01

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.

Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

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Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

2013-01-01

Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

Epilepsy, E/I balance and GABAA receptor plasticity

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Jean-Marc Fritschy

2008-03-01

Full Text Available GABAA receptors mediate most of the fast inhibitory transmission in the CNS. They form heteromeric complexes assembled from a large family of subunit genes. The existence of multiple GABAA receptor subtypes differing in subunit composition, localization and functional properties underlies their role for fi ne-tuning of neuronal circuits and genesis of network oscillations. The differential regulation of GABAA receptor subtypes represents a major facet of homeostatic synaptic plasticity and contributes to the excitation/inhibition (E/I balance under physiological conditions and upon pathological challenges. The purpose of this review is to discuss recent fi ndings highlighting the signifi cance of GABAA receptor heterogeneity for the concept of E/I balance and its relevance for epilepsy. Specifi cally, we address the following issues: (1 role for tonic inhibition, mediated by extrasynaptic GABAA receptors, for controlling neuronal excitability; (2 signifi cance of chloride ion transport for maintenance of the E/I balance in adult brain; and (3 molecular mechanisms underlying GABAA receptor regulation (traffi cking, posttranslational modifi cation, gene transcription that are important for homoeostatic plasticity. Finally, the relevance of these fi ndings is discussed in light of the involvement of GABAA receptors in epileptic disorders, based on recent experimental studies of temporal lobe epilepsy (TLE and absence seizures and on the identifi cation of mutations in GABAA receptor subunit genes underlying familial forms of epilepsy.

Expression of the Hippocampal NMDA Receptor GluN1 Subunit and Its Splicing Isoforms in Schizophrenia: Postmortem Study

Czech Academy of Sciences Publication Activity Database

Vrajová, M.; Šťastný, František; Horáček, J.; Lochman, J.; Šerý, O.; Peková, S.; Klaschka, Jan; Höschl, C.

2010-01-01

Roč. 35, č. 7 (2010), s. 994-1002 ISSN 0364-3190 Grant - others:GA MZd(CZ) NR9324 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10300504 Keywords : schizophrenia * hippocampus * GluN1 subunit of NMDA receptor * splice variants * laterality Subject RIV: FL - Psychiatry, Sexuology Impact factor: 2.608, year: 2010

Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

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Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

2014-01-01

We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

Transcriptional regulators of Na, K-ATPase subunits

OpenAIRE

Zhiqin eLi; Sigrid A Langhans

2015-01-01

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

SAHA (Vorinostat Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

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Nela Durisic

2018-03-01

Full Text Available The GABAA receptor (GABAAR α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile

The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

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Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

1997-10-01

The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

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Sally M Williamson

2009-07-01

Full Text Available Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29, nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29, levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the

Role of NMDA receptor GluN2D subunit in the antidepressant effects of enantiomers of ketamine

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Soichiro Ide

2017-11-01

Full Text Available We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA receptor GluN2D subunit knockout (GluN2D-KO mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS-ketamine and (S-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R-ketamine.

Anxiogenic properties of an inverse agonist selective for α3 subunit-containing GABAA receptors

OpenAIRE

Atack, John R; Hutson, Peter H; Collinson, Neil; Marshall, George; Bentley, Graham; Moyes, Christopher; Cook, Susan M; Collins, Ian; Wafford, Keith; McKernan, Ruth M; Dawson, Gerard R

2005-01-01

α3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABAA receptors containing an α3 rather than an α1, α2 or α5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of α3IA are most probably mediated by the α3 subtype.α3IA has good CNS penetration in rats and mice as measured using a [3H]Ro 15-1788 in vivo bi...

Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

KAUST Repository

Kiraly, D. D.

2011-08-31

The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

KAUST Repository

Kiraly, D. D.; Lemtiri-Chlieh, Fouad; Levine, E. S.; Mains, R. E.; Eipper, B. A.

2011-01-01

The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

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Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis

2014-01-01

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies. PMID:24801634

Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

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A Mobascher

Full Text Available Variation in genes coding for nicotinic acetylcholine receptor (nAChR subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP, an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.

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

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Shi, Wen-Zhu [Anesthesia and Operation Center, Hainan Branch of Chinese PLA General Hospital, Hainan 572013 (China); Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China); Miao, Yu-Liang [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Guo, Wen-Zhi [Department of Anesthesiology, Beijing Military General Hospital of Chinese People’s Liberation Army, Beijing 100700 (China); Wu, Wei, E-mail: wwzwgk@163.com [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); Li, Bao-Wei [Department of Head and Neck Surgery of Otolaryngology, PLA No. 306 Hospital, Beijing 100101 (China); An, Li-Na [Department of Anesthesiology, Armed Police General Hospital, Beijing 100039 (China); Fang, Wei-Wu [Department of Anesthesiology, PLA No. 306 Hospital, Beijing 100101 (China); Mi, Wei-Dong, E-mail: elite2005gg@163.com [Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing 100853 (China)

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.

Do motor control genes contribute to interindividual variability in decreased movement in patients with pain?

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Mishra Bikash K

2007-07-01

Full Text Available Abstract Background Because excessive reduction in activities after back injury may impair recovery, it is important to understand and address the factors contributing to the variability in motor responses to pain. The current dominant theory is the "fear-avoidance model", in which the some patients' heightened fears of further injury cause them to avoid movement. We propose that in addition to psychological factors, neurochemical variants in the circuits controlling movement and their modification by pain may contribute to this variability. A systematic search of the motor research literature and genetic databases yielded a prioritized list of polymorphic motor control candidate genes. We demonstrate an analytic method that we applied to 14 of these genes in 290 patients with acute sciatica, whose reduction in movement was estimated by items from the Roland-Morris Disability Questionnaire. Results We genotyped a total of 121 single nucleotide polymorphisms (SNPs in 14 of these genes, which code for the dopamine D2 receptor, GTP cyclohydrolase I, glycine receptor α1 subunit, GABA-A receptor α2 subunit, GABA-A receptor β1 subunit, α-adrenergic 1C, 2A, and 2C receptors, serotonin 1A and 2A receptors, cannabinoid CB-1 receptor, M1 muscarinic receptor, and the tyrosine hydroxylase, and tachykinin precursor-1 molecules. No SNP showed a significant association with the movement score after a Bonferroni correction for the 14 genes tested. Haplotype analysis of one of the blocks in the GABA-A receptor β1 subunit showed that a haplotype of 11% frequency was associated with less limitation of movement at a nominal significance level value (p = 0.0025 almost strong enough to correct for testing 22 haplotype blocks. Conclusion If confirmed, the current results may suggest that a common haplotype in the GABA-A β1 subunit acts like an "endogenous muscle relaxant" in an individual with subacute sciatica. Similar methods might be applied a larger set of

Role of NMDA receptor GluN2D subunit in the antidepressant effects of enantiomers of ketamine.

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Ide, Soichiro; Ikekubo, Yuiko; Mishina, Masayoshi; Hashimoto, Kenji; Ikeda, Kazutaka

2017-11-01

We investigated the rapid and sustained antidepressant effects of enantiomers of ketamine in N-methyl-d-aspartate (NMDA) receptor GluN2D subunit knockout (GluN2D-KO) mice. Intraperitoneal administration of ketamine or its enantiomers 10 min before the tail-suspension test exerted significant antidepressant effects on restraint stress-induced depression in both wildtype and GluN2D-KO mice. The antidepressant effects of (RS)-ketamine and (S)-ketamine were sustained 96 h after the injection in both wildtype and GluN2D-KO mice, but such sustained antidepressant effects of (R)-ketamine were only observed in wildtype mice. These data suggest that the GluN2D subunit is critical for the sustained antidepressant effects of (R)-ketamine. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

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

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

2010-12-15

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

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

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V. Baptista

2005-01-01

Full Text Available The nucleus tractus solitarius (NTS plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV. The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

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Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

2015-11-01

Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

A juvenile form of postsynaptic hippocampal long-term potentiation in mice deficient for the AMPA receptor subunit GluR-A

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Jensen, V.; Kaiser, K.M.M.; Borchardt, T.; Adelmann, G.; Rozov, A.; Burnashev, N.; Brix, C.; Frotscher, M.; Anderson, P.; Hvalby, O.; Sakmann, B.; Seeburg, P.H.; Sprengel, R.

2003-01-01

In adult mice, long-term potentiation (LTP) of synaptic transmission at CA3-to-CA1 synapses induced by tetanic stimulation requires L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors containing GluR-A subunits. Here, we report a GluR-A-independent form of LTP, which is comparable in

Neonatal seizures alter NMDA glutamate receptor GluN2A and 3A subunit expression and function in hippocampal CA1 neurons

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Zhou, Chengwen; Sun, Hongyu; Klein, Peter M.; Jensen, Frances E.

2015-01-01

Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS) model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA) in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the N-Methyl-D-aspartate (NMDA) subtype of glutamate receptor (GluN) is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P)10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48–96 h following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs) were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg2+ sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain. PMID:26441533

Time-Dependent Alterations in the Expression of NMDA Receptor Subunits along the Dorsoventral Hippocampal Axis in an Animal Model of Nascent Psychosis.

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Dubovyk, Valentyna; Manahan-Vaughan, Denise

2018-04-10

Psychosis is a mental condition that is characterized by hallucinations, delusions, disordered thought, as well as socio-emotional and cognitive impairments. Once developed, it tends to progress into a chronic psychotic illness. Here, the duration of untreated psychosis plays a crucial role: the earlier the treatment begins, relative to the first episode of the disease, the better the patient's functional prognosis. To what extent the success of early interventions relate to progressive changes at the neurotransmitter receptor level is as yet unclear. In fact, very little is known as to how molecular changes develop, transform, and become established following the first psychotic event. One neurotransmitter receptor for which a specific role in psychosis has been discussed is the N-methyl-d-aspartate receptor (NMDAR). This receptor is especially important for information encoding in the hippocampus. The hippocampus is one of the loci of functional change in psychosis, to which a role in the pathophysiology of psychosis has been ascribed. Here, we examined whether changes in NMDAR subunit expression occur along the dorsoventral axis of the hippocampus 1 week and 3 months after systemic treatment with an NMDAR antagonist (MK801) that initiates a psychosis-like state in adult rats. We found early (1 week) upregulation of the GluN2B levels in the dorso-intermediate hippocampus and late (3 month) downregulation of GluN2A expression across the entire CA1 region. The ventral hippocampus did not exhibit subunit expression changes. These data suggest that a differing vulnerability of the hippocampal longitudinal axis may occur in response to MK801-treatment and provide a time-resolved view of the putative development of pathological changes of NMDAR subunit expression in the hippocampus that initiate with an emulated first episode and progress through to the chronic stabilization of a psychosis-like state in rodents.

Involvement of N-methyl-D-aspartate receptor subunits in zinc-mediated modification of CA1 long-term potentiation in the developing hippocampus.

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Takeda, Atsushi; Itagaki, Kosuke; Ando, Masaki; Oku, Naoto

2012-03-01

Zinc is an endogenous N-methyl-D-aspartate (NMDA) receptor blocker. It is possible that zinc-mediated modification of hippocampal CA1 long-term potentiation (LTP) is linked to the expression of NMDA receptor subunits, which varies with postnatal development. In the present study, the effect of ZnCl(2) and CaEDTA, a membrane-impermeable zinc chelator, on CA1 LTP induction was examined in hippocampal slices from immature (3-week-old) and young (6-week-old) rats. Tetanus (10-100 Hz, 1 sec)-induced CA1 LTP was more greatly enhanced in 3-week-old rats. CA1 LTP was inhibited in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, and CaEDTA in 3-week-old rats, as in the case of 6-week-old rats reported previously. In 3-week-old rats, on the other hand, 5 μM ZnCl(2) attenuated NMDA receptor-mediated EPSPs more than in 6-week-old rats and significantly attenuated CA1 LTP. Moreover, 5 μM ZnCl(2) significantly attenuated CA1 LTP in the presence of (2R,4S)-4-(3-phosphonopropyl)-2-piperidinecarboxylic acid (PPPA), an NR2A antagonist, in 3-week-old rats, but not that in the presence of ifenprodil, an NR2B antagonist, suggesting that zinc-mediated attenuation of CA1 LTP is associated with the preferential expression of NR2B subunit in 3-week-old rats. In 6-week-old rats, however, 5 μM ZnCl(2) significantly potentiated CA1 LTP and also CA1 LTP in the presence of PPPA. The present study demonstrates that endogenous zinc may participate in the induction of CA1 LTP. It is likely that the changes in expression of NMDA receptor subunits are involved in the zinc-mediated modification of CA1 LTP in the developing hippocampus. Copyright © 2011 Wiley Periodicals, Inc.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

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Tzilhav Shem-Ad

Full Text Available The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Inter-subunit interactions across the upper voltage sensing-pore domain interface contribute to the concerted pore opening transition of Kv channels.

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Shem-Ad, Tzilhav; Irit, Orr; Yifrach, Ofer

2013-01-01

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.

Circadian and developmental regulation of N-methyl-d-aspartate-receptor 1 mRNA splice variants and N-methyl-d-aspartate-receptor 3 subunit expression within the rat suprachiasmatic nucleus

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Bendová, Z; Sumová, A; Mikkelsen, Jens D.

2009-01-01

The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase...... shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties...

Loss of GluN2D subunit results in social recognition deficit, social stress, 5-HT2C receptor dysfunction, and anhedonia in mice.

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Yamamoto, Hideko; Kamegaya, Etsuko; Hagino, Yoko; Takamatsu, Yukio; Sawada, Wakako; Matsuzawa, Maaya; Ide, Soichiro; Yamamoto, Toshifumi; Mishina, Masayoshi; Ikeda, Kazutaka

2017-01-01

The N-methyl-d-aspartate (NMDA) receptor channel is involved in various physiological functions, including learning and memory. The GluN2D subunit of the NMDA receptor has low expression in the mature brain, and its role is not fully understood. In the present study, the effects of GluN2D subunit deficiency on emotional and cognitive function were investigated in GluN2D knockout (KO) mice. We found a reduction of motility (i.e., a depressive-like state) in the tail suspension test and a reduction of sucrose preference (i.e., an anhedonic state) in GluN2D KO mice that were group-housed with littermates. Despite apparently normal olfactory function and social interaction, GluN2D KO mice exhibited a decrease in preference for social novelty, suggesting a deficit in social recognition or memory. Golgi-Cox staining revealed a reduction of the complexity of dendritic trees in the accessory olfactory bulb in GluN2D KO mice, suggesting a deficit in pheromone processing pathway activation, which modulates social recognition. The deficit in social recognition may result in social stress in GluN2D KO mice. Isolation housing is a procedure that has been shown to reduce stress in mice. Interestingly, 3-week isolation and treatment with agomelatine or the 5-hydroxytryptamine-2C (5-HT 2C ) receptor antagonist SB242084 reversed the anhedonic-like state in GluN2D KO mice. In contrast, treatment with the 5-HT 2C receptor agonist CP809101 induced depressive- and anhedonic-like states in isolated GluN2D KO mice. These results suggest that social stress that is caused by a deficit in social recognition desensitizes 5-HT 2c receptors, followed by an anhedonic- and depressive-like state, in GluN2D KO mice. The GluN2D subunit of the NMDA receptor appears to be important for the recognition of individuals and development of normal emotionality in mice. 5-HT 2C receptor antagonism may be a therapeutic target for treating social stress-induced anhedonia. This article is part of the Special

The GluN2B subunit represents a major functional determinant of NMDA receptors in human induced pluripotent stem cell-derived cortical neurons

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Ioana Neagoe

2018-04-01

Full Text Available Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However, few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study, we assessed expression, functionality, and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC-derived cortical neurons (iCell Neurons and iCell GlutaNeurons. We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. Keywords: Human induced pluripotent stem cell-derived neurons, iCell Neurons, iCell GlutaNeurons, NMDA receptors, GluN2B, Positive allosteric modulators

The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

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Lansdell Stuart J

2012-06-01

Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3 and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7 with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7 with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in

Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

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van Maanen, Marjolein A.; Stoof, Susanne P.; Larosa, Gregory J.; Vervoordeldonk, Margriet J.; Tak, Paul P.

2010-01-01

BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis

Synaptic Changes in AMPA Receptor Subunit Expression in Cortical Parvalbumin Interneurons in the Stargazer Model of Absence Epilepsy

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Nadia K. Adotevi

2017-12-01

Full Text Available Feedforward inhibition is essential to prevent run away excitation within the brain. Recent evidence suggests that a loss of feed-forward inhibition in the corticothalamocortical circuitry may underlie some absence seizures. However, it is unclear if this aberration is specifically linked to loss of synaptic excitation onto local fast-spiking parvalbumin-containing (PV+ inhibitory interneurons, which are responsible for mediating feedforward inhibition within cortical networks. We recently reported a global tissue loss of AMPA receptors (AMPARs, and a specific mistrafficking of these AMPARs in PV+ interneurons in the stargazer somatosensory cortex. The current study was aimed at investigating if cellular changes in AMPAR expression were translated into deficits in receptors at specific synapses in the feedforward inhibitory microcircuit. Using western blot immunolabeling on biochemically isolated synaptic fractions, we demonstrate a loss of AMPAR GluA1–4 subunits in the somatosensory cortex of stargazers compared to non-epileptic control mice. Furthermore, using double post-embedding immunogold-cytochemistry, we show a loss of GluA1–4-AMPARs at excitatory synapses onto cortical PV+ interneurons. Altogether, these data indicate a loss of synaptic AMPAR-mediated excitation of cortical PV+ inhibitory neurons. As the cortex is considered the site of initiation of spike wave discharges (SWDs within the corticothalamocortical circuitry, loss of AMPARs at cortical PV+ interneurons likely impairs feed-forward inhibitory output, and contributes to the generation of SWDs and absence seizures in stargazers.

Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption

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Marta A Ślimak

2014-01-01

Full Text Available The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3 and β4 nicotinic acetylcholine receptor (nAChR subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN tract is particularly enriched in α3β4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb in mice altered nicotine consumption. Given that β4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of β4 to nicotine receptor activity in the MHb. We screened for missense SNPs with allele frequencies > 0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that β4A90I and β4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant β4D447Y, significantly increased nicotine-evoked current amplitudes, while β4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS, showed reduced nicotine currents. We employed lentiviruses to express β4 or β4 variants in the MHb. Immunoprecipitation studies confirmed that β4 lentiviral-mediated expression leads to specific upregulation of α3β4 but not β2 nAChRs in the Mhb. Mice injected with the β4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the β4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the β4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular β4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine

Stress-induced changes of hippocampal NMDA receptors: modulation by duloxetine treatment.

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Francesca Calabrese

Full Text Available It is now well established that the glutamatergic system contributes to the pathophysiology of depression. Exposure to stress, a major precipitating factor for depression, enhances glutamate release that can contribute to structural abnormalities observed in the brain of depressed subjects. On the other hand, it has been demonstrated that NMDA antagonists, like ketamine, exert an antidepressant effect at preclinical and clinical levels. On these bases, the purpose of our study was to investigate whether chronic mild stress is associated with specific alterations of the NMDA receptor complex, in adult rats, and to establish whether concomitant antidepressant treatment could normalize such deficits. We found that chronic stress increases the expression of the obligatory GluN1 subunit, as well as of the accessory subunits GluN2A and GluN2B at transcriptional and translational levels, particularly in the ventral hippocampus. Concomitant treatment with the antidepressant duloxetine was able to normalize the increase of glutamatergic receptor subunit expression, and correct the changes in receptor phosphorylation produced by stress exposure. Our data suggest that prolonged stress, a condition that has etiologic relevance for depression, may enhance glutamate activity through post-synaptic mechanisms, by regulating NMDA receptors, and that antidepressants may in part normalize such changes. Our results provide support to the notion that antidepressants may exert their activity in the long-term also via modulation of the glutamatergic synapse.

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

International Nuclear Information System (INIS)

Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

1989-01-01

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice.

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Prut, L; Prenosil, G; Willadt, S; Vogt, K; Fritschy, J-M; Crestani, F

2010-07-01

The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce alpha5-GABA(A) receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA(A)alpha5-subunit contents, as occurring in alpha5(H105R) knock-in mice, on the memory for location of objects. This required the behavioral characterization of alpha5(H105R) and wild-type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of alpha5-subunits and retained long-term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T- and eight-arm mazes. Processing of object, position and context memories and object-guided response learning were spared. Genotype difference in object-in-place memory retrieval and in encoding and response learning strategies for object-location combinations manifested as a bias favoring object-based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in alpha5(H105R) mice a behavioral-cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased alpha5-subunit contents.

Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

International Nuclear Information System (INIS)

Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

1990-01-01

Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: Interactions between αβ heterodimeric receptor halves

International Nuclear Information System (INIS)

Wilden, P.A.; Treadway, J.L.; Morrison, B.D.; Pessin, J.E.

1989-01-01

Examination of 125 I-IGF-1 affinity cross-linking and β-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated αβ heterodimeric IGF-1 receptors into an α 2 β 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor. The formation of the α 2 β 2 heterotetrameric IGF-1 receptor complex from the partially purified αβ heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified αβ heterodimers into an α 2 β 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified αβ heterodimeric insulin receptor complex. Incubation of the α 2 β 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125 I-IGF-1 binding or IGF-1 stimulation of protein kinase activity. However, IAN treatment of the αβ heterodimeric IGF-1 receptors inhibited the IGF-1 dependent covalent formation of the disulfide-linked α 2 β 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated αβ heterodimeric IGF-1 receptor complexes into a disulfide-linked α 2 β 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association. Therefore, unlike the insulin receptor in which noncovalent association is sufficient for kinase activation, only the covalent assembly of the IGF-1 receptor αβ heterodimers into the α 2 β 2 heterotetrameric holoreceptor complex is associated with ligand-stimulated protein kinase activation

GABAA receptor subunit expression changes in the human Alzheimer's disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus.

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Kwakowsky, Andrea; Calvo-Flores Guzmán, Beatriz; Pandya, Madhavi; Turner, Clinton; Waldvogel, Henry J; Faull, Richard L

2018-02-27

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. GABA type A receptors (GABA A Rs) are severely affected in Alzheimer's disease (AD). However, the distribution and subunit composition of GABA A Rs in the AD brain are not well understood. This is the first comprehensive study to show brain region- and cell layer-specific alterations in the expression of the GABA A R subunits α1-3, α5, β1-3 and γ2 in the human AD hippocampus, entorhinal cortex and superior temporal gyrus (STG). In late-stage AD tissue samples using immunohistochemistry we found significant alteration of all investigated GABA A Rs subunits except for α3 and β1 that were well preserved. The most prominent changes include an increase in GABA A R α1 expression associated with AD in all layers of the CA3 region, in the stratum (str.) granulare and hilus of the dentate gyrus (DG). We found a significant increase in GABA A R α2 expression in the str. oriens of the CA1-3, str. radiatum of the CA2,3 and decrease in the str. pyramidale of the CA1 region in AD cases. In AD there was a significant increase in GABA A R α5 subunit expression in str. pyramidale, str. oriens of the CA1 region and decrease in the STG. We also found a significant decrease in the GABA A R β3 subunit immunoreactivity in the str. oriens of the CA2, str. granulare and str. moleculare of the DG. In conclusion, these findings indicate that the expression of the GABA A R subunits shows brain region- and layer-specific alterations in AD, and these changes could significantly influence and alter GABA A R function in the disease. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

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Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

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

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Pamela eCantanelli

2014-08-01

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

GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

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Fatemi, S Hossein; Folsom, Timothy D

2015-09-01

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

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Lentes, K.U.; Venter, J.C.

1986-05-01

Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

Effects of ibuprofen on cognition and NMDA receptor subunit expression across aging.

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Márquez Loza, Alejandra; Elias, Valerie; Wong, Carmen P; Ho, Emily; Bermudez, Michelle; Magnusson, Kathy R

2017-03-06

Age-related declines in long- and short-term memory show relationships to decreases in N-methyl-d-aspartate (NMDA) receptor expression, which may involve inflammation. This study was designed to determine effects of an anti-inflammatory drug, ibuprofen, on cognitive function and NMDA receptor expression across aging. Male C57BL/6 mice (ages 5, 14, 20, and 26months) were fed ibuprofen (375ppm) in NIH31 diet or diet alone for 6weeks prior to testing. Behavioral testing using the Morris water maze showed that older mice performed significantly worse than younger in spatial long-term memory, reversal, and short-term memory tasks. Ibuprofen enhanced overall performance in the short-term memory task, but this appeared to be more related to improved executive function than memory. Ibuprofen induced significant decreases over all ages in the mRNA densities for GluN2B subunit, all GluN1 splice variants, and GluN1-1 splice forms in the frontal cortex and in protein expression of GluN2A, GluN2B and GluN1 C2' cassettes in the hippocampus. GluN1-3 splice form mRNA and C2' cassette protein were significantly increased across ages in frontal lobes of ibuprofen-treated mice. Ibuprofen did not alter expression of pro-inflammatory cytokines IL-1β and TNFα, but did reduce the area of reactive astrocyte immunostaining in frontal cortex of aged mice. Enhancement in executive function showed a relationship to increased GluN1-3 mRNA and decreased gliosis. These findings suggest that inflammation may play a role in executive function declines in aged animals, but other effects of ibuprofen on NMDA receptors appeared to be unrelated to aging or inflammation. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

DEFF Research Database (Denmark)

Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

2015-01-01

BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn......BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence...

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

Science.gov (United States)

Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong

2005-10-01

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

Evaluation of peptide designing strategy against subunit reassociation in mucin 1: A steered molecular dynamics approach.

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J Lesitha Jeeva Kumari

Full Text Available Subunit reassociation in mucin 1, a breast cancer tumor marker, is reported as one of the critical factors for its cytoplasmic activation. Inhibition of its heterodimeric association would therefore result in loss of its function and alter disease progression. The present study aimed at evaluating peptide inhibitor designing strategies that may serve as antagonist against this receptor-ligand alliance. Several peptides and their derivatives were designed based on native residues, subunit interface, hydrogen bonding and secondary structure. Docking studies with the peptides were carried on the receptor subunit and their binding affinities were evaluated using steered molecular dynamics simulation and umbrella sampling. Our results showed that among all the different classes of peptides evaluated, the receptor based peptide showed the highest binding affinity. This result was concurrent with the experimental observation that the receptor-ligand alliance in mucin 1 is highly specific. Our results also show that peptide ligand against this subunit association is only stabilized through native residue inter-protein interaction irrespective of the peptide structure, peptide length and number of hydrogen bonds. Consistency in binding affinity, pull force and free energy barrier was observed with only the receptor derived peptides which resulted in favorable interprotein interactions at the interface. Several observations were made and discussed which will eventually lead to designing efficient peptide inhibitors against mucin 1 heterodimeric subunit reassociation.

Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

DEFF Research Database (Denmark)

Meibom, K L; Kallipolitis, B H; Ebright, R H

2000-01-01

At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer......, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP...

Ionotropic crustacean olfactory receptors.

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Elizabeth A Corey

Full Text Available The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs, the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs, as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

NARCIS (Netherlands)

Stojanovic, Tamara; Capo, Ivan; Aronica, Eleonora; Adle-Biassette, Homa; Höger, Harald; Sieghart, Werner; Kovacs, Gabor G.; Milenkovic, Ivan

2016-01-01

γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3) can give rise to multiple receptor subtypes

Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

Science.gov (United States)

McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

2012-01-01

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

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Thorsten Pauly

Full Text Available Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs. Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity.

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Pauly, Thorsten; Ratliff, Miriam; Pietrowski, Eweline; Neugebauer, Rainer; Schlicksupp, Andrea; Kirsch, Joachim; Kuhse, Jochen

2008-07-16

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.

Adult forebrain NMDA receptors gate social motivation and social memory.

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Jacobs, Stephanie; Tsien, Joe Z

2017-02-01

Motivation to engage in social interaction is critical to ensure normal social behaviors, whereas dysregulation in social motivation can contribute to psychiatric diseases such as schizophrenia, autism, social anxiety disorders and post-traumatic stress disorder (PTSD). While dopamine is well known to regulate motivation, its downstream targets are poorly understood. Given the fact that the dopamine 1 (D1) receptors are often physically coupled with the NMDA receptors, we hypothesize that the NMDA receptor activity in the adult forebrain principal neurons are crucial not only for learning and memory, but also for the proper gating of social motivation. Here, we tested this hypothesis by examining sociability and social memory in inducible forebrain-specific NR1 knockout mice. These mice are ideal for exploring the role of the NR1 subunit in social behavior because the NR1 subunit can be selectively knocked out after the critical developmental period, in which NR1 is required for normal development. We found that the inducible deletion of the NMDA receptors prior to behavioral assays impaired, not only object and social recognition memory tests, but also resulted in profound deficits in social motivation. Mice with ablated NR1 subunits in the forebrain demonstrated significant decreases in sociability compared to their wild type counterparts. These results suggest that in addition to its crucial role in learning and memory, the NMDA receptors in the adult forebrain principal neurons gate social motivation, independent of neuronal development. Copyright © 2016 Elsevier Inc. All rights reserved.

Importance of GluA1 subunit-containing AMPA glutamate receptors for morphine state-dependency.

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Teemu Aitta-aho

Full Text Available In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test; in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards, in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

A family of photoswitchable NMDA receptors

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Berlin, Shai; Szobota, Stephanie; Reiner, Andreas; Carroll, Elizabeth C; Kienzler, Michael A; Guyon, Alice; Xiao, Tong; Trauner, Dirk; Isacoff, Ehud Y

2016-01-01

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity. DOI: http://dx.doi.org/10.7554/eLife.12040.001 PMID:26929991

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus.

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Naylor, David E; Liu, Hantao; Niquet, Jerome; Wasterlain, Claude G

2013-06-01

After 1h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE. Copyright © 2013 Elsevier Inc. All rights reserved.

Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator

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Sean C. Piantadosi

2016-11-01

Full Text Available Rationale: Current first-line treatments for stress-related disorders such as Major Depressive Disorder (MDD act on monoaminergic systems and take weeks to achieve a therapeutic effect with poor response and low remission rates. Recent research has implicated the GABAergic system in the pathophysiology of depression, including deficits in interneurons targeting the dendritic compartment of cortical pyramidal cells. Objectives: The present study evaluates whether SH-053-2'F-R-CH3 (denoted α5-PAM, a positive allosteric modulator selective for α5-subunit containing GABAA receptors found predominantly on cortical pyramidal cell dendrites has anti-stress effects. Methods: Female and male C57BL6/J mice were exposed to unpredictable chronic mild stress (UCMS and treated with α5-PAM acutely (30 minutes prior to assessing behavior or chronically before being assessed behaviorally. Results: Acute and chronic α5-PAM treatments produce a pattern of decreased stress-induced behaviors (denoted as behavioral emotionality across various tests in female, but not in male mice. Behavioral Z-scores calculated across a panel of tests designed to best model the range and heterogeneity of human symptomatology confirmed that acute and chronic α5-PAM treatments consistently produce significant decreases in behavioral emotionality in several independent cohorts of females. The behavioral responses to α5-PAM could not be completely accounted for by differences in drug brain disposition between female and male mice. In mice exposed to UCMS, expression of the Gabra5 gene was increased in the frontal cortex after acute treatment and in hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusions: We showed that acute and chronic positive modulation of α5 subunit-containing GABAA receptors elicit anti-stress effects in a sex-dependent manner, suggesting novel therapeutic modalities.

E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

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Saha, Siddhartha S; Caviness, Gary; Yi, Guanghui; Raymond, Ernest L; Mbow, M Lamine; Kao, C Cheng

2018-01-01

Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R. © 2017 S. Karger AG, Basel.

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

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Amanda Clause

2017-06-01

Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

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Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

2017-11-01

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

Identification of the D-1 dopamine receptor subunit in rat striatum after photoaffinity labeling

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Kuno, T; Tanaka, C [Kobe Univ. (Japan). School of Medicine

1982-12-28

When rat striatal membranes, photolabeled with (/sup 3/H)dopamine under assay conditions similar to those used for dopamine-sensitive adenylate cyclase, were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, several radioactively labeled bands appeared. Labeling of these bands was reduced in the presence of non-radioactive dopamine during photolysis, but was unaffected by the presence of sulpiride. Haloperidol preferentially reduced the labeling of the main band which had a molecular weight of about 57,000 rather than the other weakly labeled bands. Labeling of this 57,000 dalton protein was not apparent when rat cerebellar membranes were used and was markedly eliminated by kainic acid-induced lesions that destroyed the intrastriatal nerve cell bodies. These results indicate that this 57,000 dalton protein is the binding subunit of the D-1 dopamine receptor.

Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

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McLachlan, Sandra M.; Aliesky, Holly A.; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

2012-01-01

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

Differential regulation of synaptic and extrasynaptic α4 GABA(A) receptor populations by protein kinase A and protein kinase C in cultured cortical neurons.

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Bohnsack, John Peyton; Carlson, Stephen L; Morrow, A Leslie

2016-06-01

The GABAA α4 subunit exists in two distinct populations of GABAA receptors. Synaptic GABAA α4 receptors are localized at the synapse and mediate phasic inhibitory neurotransmission, while extrasynaptic GABAA receptors are located outside of the synapse and mediate tonic inhibitory transmission. These receptors have distinct pharmacological and biophysical properties that contribute to interest in how these different subtypes are regulated under physiological and pathological states. We utilized subcellular fractionation procedures to separate these populations of receptors in order to investigate their regulation by protein kinases in cortical cultured neurons. Protein kinase A (PKA) activation decreases synaptic α4 expression while protein kinase C (PKC) activation increases α4 subunit expression, and these effects are associated with increased β3 S408/409 or γ2 S327 phosphorylation respectively. In contrast, PKA activation increases extrasynaptic α4 and δ subunit expression, while PKC activation has no effect. Our findings suggest synaptic and extrasynaptic GABAA α4 subunit expression can be modulated by PKA to inform the development of more specific therapeutics for neurological diseases that involve deficits in GABAergic transmission. Copyright © 2016 Elsevier Ltd. All rights reserved.

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

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Wieskopf, Jeffrey S; Mathur, Jayanti; Limapichat, Walrati

2015-01-01

expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated...... with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross...

Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

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Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan

2011-01-01

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3...... subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus...... of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs...

Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

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van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

1995-01-01

Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

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Maestrini, E.; Lai, C.; Marlow, A.

1999-01-01

Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study on...

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

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Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-09-01

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Radial symmetry in a chimeric glutamate receptor pore

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Wilding, Timothy J.; Lopez, Melany N.; Huettner, James E.

2014-02-01

Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analysed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that fourfold pore symmetry persists in the open state.

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

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

2009-01-01

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

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

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Glen eAcosta

2012-01-01

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

The role of striatal NMDA receptors in drug addiction.

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Ma, Yao-Ying; Cepeda, Carlos; Cui, Cai-Lian

2009-01-01

The past decade has witnessed an impressive accumulation of evidence indicating that the excitatory amino acid glutamate and its receptors, in particular the N-methyl-D-aspartate (NMDA) receptor subtype, play an important role in drug addiction. Various lines of research using animal models of drug addiction have demonstrated that drug-induced craving is accompanied by significant upregulation of NR2B subunit expression. Furthermore, selective blockade of NR2B-containing NMDA receptors in the striatum, especially in the nucleus accumbens (NAc) can inhibit drug craving and reinstatement. The purpose of this review is to examine the role of striatal NMDA receptors in drug addiction. After a brief description of glutamatergic innervation and NMDA receptor subunit distribution in the striatum, we discuss potential mechanisms to explain the role of striatal NMDA receptors in drug addiction by elucidating signaling cascades involved in the regulation of subunit expression and redistribution, phosphorylation of receptor subunits, as well as activation of intracellular signals triggered by drug experience. Understanding the mechanisms regulating striatal NMDA receptor changes in drug addiction will provide more specific and rational targets to counteract the deleterious effects of drug addiction.

The GABAA Receptor α2 Subunit Activates a Neuronal TLR4 Signal in the Ventral Tegmental Area that Regulates Alcohol and Nicotine Abuse

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Irina Balan

2018-04-01

Full Text Available Alcoholism initiates with episodes of excessive alcohol drinking, known as binge drinking, which is one form of excessive drinking (NIAAA Newsletter, 2004 that is related to impulsivity and anxiety (Ducci et al., 2007; Edenberg et al., 2004 and is also predictive of smoking status. The predisposition of non-alcohol exposed subjects to initiate binge drinking is controlled by neuroimmune signaling that includes an innately activated neuronal Toll-like receptor 4 (TLR4 signal. This signal also regulates cognitive impulsivity, a heritable trait that defines drug abuse initiation. However, the mechanism of signal activation, its function in dopaminergic (TH+ neurons within the reward circuitry implicated in drug-seeking behavior [viz. the ventral tegmental area (VTA], and its contribution to nicotine co-abuse are still poorly understood. We report that the γ-aminobutyric acidA receptor (GABAAR α2 subunit activates the TLR4 signal in neurons, culminating in the activation (phosphorylation/nuclear translocation of cyclic AMP response element binding (CREB but not NF-kB transcription factors and the upregulation of corticotropin-releasing factor (CRF and tyrosine hydroxylase (TH. The signal is activated through α2/TLR4 interaction, as evidenced by co-immunoprecipitation, and it is present in the VTA from drug-untreated alcohol-preferring P rats. VTA infusion of neurotropic herpes simplex virus (HSV vectors for α2 (pHSVsiLA2 or TLR4 (pHSVsiTLR4 but not scrambled (pHSVsiNC siRNA inhibits signal activation and both binge alcohol drinking and nicotine sensitization, suggesting that the α2-activated TLR4 signal contributes to the regulation of both alcohol and nicotine abuse.

Neto2 Assembles with Kainate Receptors in DRG Neurons during Development and Modulates Neurite Outgrowth in Adult Sensory Neurons.

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Vernon, Claire G; Swanson, Geoffrey T

2017-03-22

Peripheral sensory neurons in the dorsal root ganglia (DRG) are the initial transducers of sensory stimuli, including painful stimuli, from the periphery to central sensory and pain-processing centers. Small- to medium-diameter non-peptidergic neurons in the neonatal DRG express functional kainate receptors (KARs), one of three subfamilies of ionotropic glutamate receptors, as well as the putative KAR auxiliary subunit Neuropilin- and tolloid-like 2 (Neto2). Neto2 alters recombinant KAR function markedly but has yet to be confirmed as an auxiliary subunit that assembles with and alters the function of endogenous KARs. KARs in neonatal DRG require the GluK1 subunit as a necessary constituent, but it is unclear to what extent other KAR subunits contribute to the function and proposed roles of KARs in sensory ganglia, which include promotion of neurite outgrowth and modulation of glutamate release at the DRG-dorsal horn synapse. In addition, KARs containing the GluK1 subunit are implicated in modes of persistent but not acute pain signaling. We show here that the Neto2 protein is highly expressed in neonatal DRG and modifies KAR gating in DRG neurons in a developmentally regulated fashion in mice. Although normally at very low levels in adult DRG neurons, Neto2 protein expression can be upregulated via MEK/ERK signaling and after sciatic nerve crush and Neto2 -/- neurons from adult mice have stunted neurite outgrowth. These data confirm that Neto2 is a bona fide KAR auxiliary subunit that is an important constituent of KARs early in sensory neuron development and suggest that Neto2 assembly is critical to KAR modulation of DRG neuron process outgrowth. SIGNIFICANCE STATEMENT Pain-transducing peripheral sensory neurons of the dorsal root ganglia (DRG) express kainate receptors (KARs), a subfamily of glutamate receptors that modulate neurite outgrowth and regulate glutamate release at the DRG-dorsal horn synapse. The putative KAR auxiliary subunit Neuropilin- and

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

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Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

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Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Uhr, Manfred; Wagner, Eva-Maria; Gilling, Kate E; Parsons, Chris G; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer; Rammes, Gerhard

2013-07-01

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

International Nuclear Information System (INIS)

Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

2004-01-01

The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

Cross-linking of hCG to luteal receptors

Energy Technology Data Exchange (ETDEWEB)

Ji, T.H.; Ji, I.

1985-01-01

Photoaffinity labeling of the lutropin/choriogonadotropin (LH/hCG) receptor system on porcine granulosa cells has demonstrated that both the ..cap alpha.. and ..beta.. subunits of hCG directly photoaffinity label the hormone receptor. Three new bands appear on SDS-PAGE as a consequence of photoaffinity labeling by each subunit: the molecular weights of the three bands (106K, 88K, and 83K) produced by the subunit are larger by approximately 10K than those of the three bands (96K, 76K, and 73K) labeled by the ..cap alpha.. subunit. Although it could be a coincidence that the molecular weight of the ..beta.. subunit is approximately 10K larger than that of the ..cap alpha.. subunit, the similarity in these differences suggests the possibility that both the ..cap alpha.. and ..beta.. subunits have labeled the same polypeptides.

Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

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Cui Yan Lu

2017-01-01

Full Text Available Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD. Gamma-aminobutyric acid (GABA deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5 in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

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Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

2008-09-01

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

Early continuous white noise exposure alters auditory spatial sensitivity and expression of GAD65 and GABAA receptor subunits in rat auditory cortex.

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

2010-04-01

Sensory experiences have important roles in the functional development of the mammalian auditory cortex. Here, we show how early continuous noise rearing influences spatial sensitivity in the rat primary auditory cortex (A1) and its underlying mechanisms. By rearing infant rat pups under conditions of continuous, moderate level white noise, we found that noise rearing markedly attenuated the spatial sensitivity of A1 neurons. Compared with rats reared under normal conditions, spike counts of A1 neurons were more poorly modulated by changes in stimulus location, and their preferred locations were distributed over a larger area. We further show that early continuous noise rearing induced significant decreases in glutamic acid decarboxylase 65 and gamma-aminobutyric acid (GABA)(A) receptor alpha1 subunit expression, and an increase in GABA(A) receptor alpha3 expression, which indicates a returned to the juvenile form of GABA(A) receptor, with no effect on the expression of N-methyl-D-aspartate receptors. These observations indicate that noise rearing has powerful adverse effects on the maturation of cortical GABAergic inhibition, which might be responsible for the reduced spatial sensitivity.

Pharmacological characterisation of α6β4* nicotinic acetylcholine receptors assembled from three different α6/α3 subunit chimeras in tsA201 cells

DEFF Research Database (Denmark)

Jensen, Anne Bjørnskov; Hoestgaard-Jensen, Kirsten; Jensen, Anders A.

2014-01-01

by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L...... should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors....

Caloric restriction increases learning consolidation and facilitates synaptic plasticity through mechanisms dependent on NR2B subunits of the NMDA receptor.

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Fontán-Lozano, Angela; Sáez-Cassanelli, José Luis; Inda, Mari Carmen; de los Santos-Arteaga, Mercedes; Sierra-Domínguez, Sergio Antonio; López-Lluch, Guillermo; Delgado-García, José María; Carrión, Angel Manuel

2007-09-19

One of the main focal points of aging research is the search for treatments that will prevent or ameliorate the learning and memory deficiencies associated with aging. Here we have examined the effects of maintaining mature mice on a long-term intermittent fasting diet (L-IFD). We found that L-IFD enhances learning and consolidation processes. We also assessed the long-term changes in synaptic efficiency in these animals. L-IFD mice showed an increase in low-theta-band oscillations, paired-pulse facilitation, and facilitation of long-term synaptic plasticity in the hippocampus with respect to mice fed ad libitum. In addition, we found an increase in the expression of the NMDA receptor subunit NR2B in some brain areas of L-IFD mice. Specific antagonism of this subunit in the hippocampus reversed the beneficial effects of L-IFD. These data provide a molecular and cellular mechanism by which L-IFD may enhance cognition, ameliorating some aging-associated cognitive deficits.

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

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Dipankar Bhattacharya

Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

International Nuclear Information System (INIS)

He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

2004-01-01

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

Science.gov (United States)

Taupin, J L; Anderson, P

1994-12-01

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.

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Thomas B Duguet

2016-07-01

Full Text Available Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the

Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice.

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Jorratt, Pascal; Delano, Paul H; Delgado, Carolina; Dagnino-Subiabre, Alexies; Terreros, Gonzalo

2017-01-01

The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC) neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs) through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR) is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO) mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT) mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

DEFF Research Database (Denmark)

Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana

2006-01-01

Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

Science.gov (United States)

Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

1991-04-01

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Dopamine Receptor-Specific Contributions to the Computation of Value.

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Burke, Christopher J; Soutschek, Alexander; Weber, Susanna; Raja Beharelle, Anjali; Fehr, Ernst; Haker, Helene; Tobler, Philippe N

2018-05-01

Dopamine is thought to play a crucial role in value-based decision making. However, the specific contributions of different dopamine receptor subtypes to the computation of subjective value remain unknown. Here we demonstrate how the balance between D1 and D2 dopamine receptor subtypes shapes subjective value computation during risky decision making. We administered the D2 receptor antagonist amisulpride or placebo before participants made choices between risky options. Compared with placebo, D2 receptor blockade resulted in more frequent choice of higher risk and higher expected value options. Using a novel model fitting procedure, we concurrently estimated the three parameters that define individual risk attitude according to an influential theoretical account of risky decision making (prospect theory). This analysis revealed that the observed reduction in risk aversion under amisulpride was driven by increased sensitivity to reward magnitude and decreased distortion of outcome probability, resulting in more linear value coding. Our data suggest that different components that govern individual risk attitude are under dopaminergic control, such that D2 receptor blockade facilitates risk taking and expected value processing.

Conantokin probes of NMDA receptors in normal and Alzheimer disease human cerebral cortex

International Nuclear Information System (INIS)

Ragnarsson, L.; Dodd, P.R.; Lewis, R.J.

2002-01-01

Full text: The pharmacology of the N-methyl-D-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer disease cases and matched controls. The affinity and density of the [ 3 H]MK-801 binding site were delineated along with the enhancement of [ 3 H]MK-801 binding by glutamate and spermine. Sites with distinct pharmacologies were distributed regionally through the cortex. The differences could not be explained by variations in the parameters of [ 3 H]MK-801 binding; rather, the data suggest that the subunit composition of NMDA receptors may be locally variable. Selective differences were also found between controls and Alzheimer disease cases in certain brain regions. The interactions of human NMDA sites with the Ala(7) and Lys(7) derivatives of conantokin-G (Con-G) were also characterized. Ala(7)-con-G showed the higher affinity of the two peptides, and also defined two distinct binding sites in controls. In distinction to the Ala(7) peptide, Lys(7)- con-G showed preferential binding to receptor sites in Alzheimer disease cf. control brain. Modified conantokins are useful for identifying differences in subunit composition of the NMDA receptors between brain areas. They may also have potential as protective agents against over-excitation mediated by specific NMDA receptors, which might contribute to localized brain damage in Alzheimer disease. For further characterization of the pharmacology of different NMDA receptor subunits, a mammalian expression system has been developed for the analysis of their responses to selected ligands, including conantokins. Copyright (2002) Australian Neuroscience Society

Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

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Carson Kai-Kwong Ley

Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

Institute of Scientific and Technical Information of China (English)

HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting

2009-01-01

Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

Picosecond dynamics of the glutamate receptor in response to agonist-induced vibrational excitation.

Science.gov (United States)

Kubo, Minoru; Shiomitsu, Eiji; Odai, Kei; Sugimoto, Tohru; Suzuki, Hideo; Ito, Etsuro

2004-02-01

Conformational changes of proteins are dominated by the excitation and relaxation processes of their vibrational states. To elucidate the mechanism of receptor activation, the conformation dynamics of receptors must be analyzed in response to agonist-induced vibrational excitation. In this study, we chose the bending vibrational mode of the guanidinium group of Arg485 of the glutamate receptor subunit GluR2 based on our previous studies, and we investigated picosecond dynamics of the glutamate receptor caused by the vibrational excitation of Arg485 via molecular dynamics simulations. The vibrational excitation energy in Arg485 in the ligand-binding site initially flowed into Lys730, and then into the J-helix at the subunit interface of the ligand-binding domain. Consequently, the atomic displacement in the subunit interface around an intersubunit hydrogen bond was evoked in about 3 ps. This atomic displacement may perturb the subunit packing of the receptor, triggering receptor activation. Copyright 2003 Wiley-Liss, Inc.

Nuclear respiratory factor 2 regulates the expression of the same NMDA receptor subunit genes as NRF-1: both factors act by a concurrent and parallel mechanism to couple energy metabolism and synaptic transmission.

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

2013-01-01

Neuronal activity and energy metabolism are tightly coupled processes. Previously, we found that nuclear respiratory factor 1 (NRF-1) transcriptionally co-regulates energy metabolism and neuronal activity by regulating all 13 subunits of the critical energy generating enzyme, cytochrome c oxidase (COX), as well as N-methyl-d-aspartate (NMDA) receptor subunits 1 and 2B, GluN1 (Grin1) and GluN2B (Grin2b). We also found that another transcription factor, nuclear respiratory factor 2 (NRF-2 or GA-binding protein) regulates all subunits of COX as well. The goal of the present study was to test our hypothesis that NRF-2 also regulates specific subunits of NMDA receptors, and that it functions with NRF-1 via one of three mechanisms: complementary, concurrent and parallel, or a combination of complementary and concurrent/parallel. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation of mouse neuroblastoma cells and rat visual cortical tissue, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate Grin1 and Grin2b genes, but not any other NMDA subunit genes. Grin1 and Grin2b transcripts were up-regulated by depolarizing KCl, but silencing of NRF-2 prevented this up-regulation. On the other hand, over-expression of NRF-2 rescued the down-regulation of these subunits by the impulse blocker TTX. NRF-2 binding sites on Grin1 and Grin2b are conserved among species. Our data indicate that NRF-2 and NRF-1 operate in a concurrent and parallel manner in mediating the tight coupling between energy metabolism and neuronal activity at the molecular level. Copyright © 2012 Elsevier B.V. All rights reserved.

Correlated cone noise decreases rod signal contributions to the post-receptoral pathways.

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Hathibelagal, Amithavikram R; Feigl, Beatrix; Zele, Andrew J

2018-04-01

This study investigated how invisible extrinsic temporal white noise that correlates with the activity of one of the three [magnocellular (MC), parvocellular (PC), or koniocellular (KC)] post-receptoral pathways alters mesopic rod signaling. A four-primary photostimulator provided independent control of the rod and three cone photoreceptor excitations. The rod contributions to the three post-receptoral pathways were estimated by perceptually matching a 20% contrast rod pulse by independently varying the LMS (MC pathway), +L-M (PC pathway), and S-cone (KC pathway) excitations. We show that extrinsic cone noise caused a predominant decrease in the overall magnitude and ratio of the rod contributions to each pathway. Thus, the relative cone activity in the post-receptoral pathways determines the relative mesopic rod inputs to each pathway.

Role of the Rubisco Small Subunit

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Spreitzer, Robert Joseph [Univ. of Nebraska, Lincoln, NE (United States)

2016-11-05

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. However, it is a slow enzyme, and O₂ competes with CO₂ at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO₂. If carboxylation could be increased or oxygenation decreased, an increase in net CO₂ fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO₂/O₂ specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.

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Maelle Jospin

2009-12-01

Full Text Available In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three alpha-subunits, UNC-38, UNC-63 and ACR-12, and two non-alpha-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.

Qualitative variation of photolabelled benzodiazepine receptors in different species.

Science.gov (United States)

Hebebrand, J; Friedl, W; Lentes, K U; Propping, P

1986-01-01

In order to examine whether species differences of benzodiazepine receptor subunits exist, we compared the fluorographic pattern of photoaffinity labelled subunits after SDS-PAGE in five species: fish, frog, chicken, mouse and calf. Each species showed a distinct pattern of specifically labelled proteins. We conclude that species variation of benzodiazepine receptor does indeed exist.

The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

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Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

1996-02-01

Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

The different effects of over-expressing murine NMDA receptor 2B subunit in the forebrain on conditioned taste aversion.

Science.gov (United States)

Li, Shijia; Gu, Yiran; Meng, Bo; Mei, Bing; Li, Fei

2010-09-10

The glutamate transmission system and the N-methyl-D-aspartate receptor (NMDA-R), in particular its 2B subunit (NR2B), have been reported to be possibly related to taste memory as a result of treatment with NMDA antagonists and agonists. In order to further study the role of the NR2B subunit in gustation memory, we applied four different taste aversive tasks to observe the behavior of a transgenic mice model in which the NR2B subunit was specifically over-expressed in the forebrain. We found that in both short- and long-term conditioned taste aversion (CTA) experiments, mice with forebrain expression of the NR2B transgene (Tg) showed significantly enhanced CTA 2 days after training. However, both the Tg and the wild-type (Wt) mice shared the same level of aversive memory on the 30th day after training. In both fast and slow extinction experiments, Tg mice maintained a higher CTA memory than that of control mice in most extinction trials. The third experiment, which involved testing the memory for familiar taste, demonstrated that NR2B augmentation had no benefit on the latent inhibition (LI) of CTA. In addition, the last experiment (two-taste LI) showed a suppression of enhanced CTA in Tg mice when the mice were exposed to both novel and familiar tastes. These data suggested that forebrain NR2B over-expression had different effects on gustatory learning and memory. The transgenic animals were only sensitive to novel but not familiar tastes, and up-regulation of NR2B resulted in enhanced CTA function for only a short period of time. 2010 Elsevier B.V. All rights reserved.

Heterotrimeric G protein subunits are located on rat liver endosomes

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Van Dyke Rebecca W

2004-01-01

Full Text Available Abstract Background Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. Results By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM and basolateral (BLM membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC > compartment for uncoupling of receptor and ligand (CURL > multivesicular bodies (MVB >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. Conclusion We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s of internalized insulin receptors.

Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits.

Science.gov (United States)

Zhang, Yueliang; Han, Yangchun; Yang, Qiong; Wang, Lihua; He, Peng; Liu, Zewen; Li, Zhong; Guo, Huifang; Fang, Jichao

2018-04-01

Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

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Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

2008-01-01

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

Science.gov (United States)

O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

DEFF Research Database (Denmark)

Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek

2015-01-01

neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

DEFF Research Database (Denmark)

Kostenis, Evi; Martini, Lene; Ellis, James

2004-01-01

Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

GABA regulates the rat hypothalamic-pituitary-adrenocortical axis via different GABA-A receptor alpha-subtypes

DEFF Research Database (Denmark)

Mikkelsen, Jens D; Bundzikova, Jana; Larsen, Marianne Hald

2008-01-01

dependent on the composition of the GABA-A receptor subunits through which they act. We show here that positive modulators of alpha(1)-subtype containing GABA-A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas...... after positive modulation of GABA-A receptors composed of alpha(1)-subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non alpha(1)-containing GABA-A receptors....

In silico comparative genomic analysis of GABAA receptor transcriptional regulation

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Joyce Christopher J

2007-06-01

Full Text Available Abstract Background Subtypes of the GABAA receptor subunit exhibit diverse temporal and spatial expression patterns. In silico comparative analysis was used to predict transcriptional regulatory features in individual mammalian GABAA receptor subunit genes, and to identify potential transcriptional regulatory components involved in the coordinate regulation of the GABAA receptor gene clusters. Results Previously unreported putative promoters were identified for the β2, γ1, γ3, ε, θ and π subunit genes. Putative core elements and proximal transcriptional factors were identified within these predicted promoters, and within the experimentally determined promoters of other subunit genes. Conserved intergenic regions of sequence in the mammalian GABAA receptor gene cluster comprising the α1, β2, γ2 and α6 subunits were identified as potential long range transcriptional regulatory components involved in the coordinate regulation of these genes. A region of predicted DNase I hypersensitive sites within the cluster may contain transcriptional regulatory features coordinating gene expression. A novel model is proposed for the coordinate control of the gene cluster and parallel expression of the α1 and β2 subunits, based upon the selective action of putative Scaffold/Matrix Attachment Regions (S/MARs. Conclusion The putative regulatory features identified by genomic analysis of GABAA receptor genes were substantiated by cross-species comparative analysis and now require experimental verification. The proposed model for the coordinate regulation of genes in the cluster accounts for the head-to-head orientation and parallel expression of the α1 and β2 subunit genes, and for the disruption of transcription caused by insertion of a neomycin gene in the close vicinity of the α6 gene, which is proximal to a putative critical S/MAR.

Difference in Perseverative Errors during a Visual Attention Task with Auditory Distractors in Alpha-9 Nicotinic Receptor Subunit Wild Type and Knock-Out Mice

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Pascal Jorratt

2017-11-01

Full Text Available The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through olivocochlear (OC neurons. Medial OC neurons make cholinergic synapses with outer hair cells (OHCs through nicotinic receptors constituted by α9 and α10 subunits. One of the physiological functions of the α9 nicotinic receptor subunit (α9-nAChR is the suppression of auditory distractors during selective attention to visual stimuli. In a recent study we demonstrated that the behavioral performance of alpha-9 nicotinic receptor knock-out (KO mice is altered during selective attention to visual stimuli with auditory distractors since they made less correct responses and more omissions than wild type (WT mice. As the inhibition of the behavioral responses to irrelevant stimuli is an important mechanism of the selective attention processes, behavioral errors are relevant measures that can reflect altered inhibitory control. Errors produced during a cued attention task can be classified as premature, target and perseverative errors. Perseverative responses can be considered as an inability to inhibit the repetition of an action already planned, while premature responses can be considered as an index of the ability to wait or retain an action. Here, we studied premature, target and perseverative errors during a visual attention task with auditory distractors in WT and KO mice. We found that α9-KO mice make fewer perseverative errors with longer latencies than WT mice in the presence of auditory distractors. In addition, although we found no significant difference in the number of target error between genotypes, KO mice made more short-latency target errors than WT mice during the presentation of auditory distractors. The fewer perseverative error made by α9-KO mice could be explained by a reduced motivation for reward and an increased impulsivity during decision making with auditory distraction in KO mice.

T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

OpenAIRE

Roberts, Joseph L.; Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

2007-01-01

CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD...

Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis

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Chien-I Chang

2014-03-01

Full Text Available This study investigates endocytosis of Saccharomyces cerevisiae α-factor receptor and the role that receptor oligomerization plays in this process. α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, we found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, recognition of the endocytosis signal sequence and recognition of the ligand-induced conformational change are likely to be two independent events.

Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor

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Igor E. Deyev

2017-11-01

Full Text Available The orphan insulin receptor-related receptor (IRR, in contrast to its close homologs, the insulin receptor (IR and insulin-like growth factor receptor (IGF-IR can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646–716 within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

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Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

2008-10-01

GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

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Üner, Aykut; Gonçalves, Gabriel H M; Li, Wenjing; Porceban, Matheus; Caron, Nicole; Schönke, Milena; Delpire, Eric; Sakimura, Kenji; Bjørbæk, Christian

2015-10-01

Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

Binding Mode of Insulin Receptor and Agonist Peptide

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

Insulin is a protein hormone secreted by pancreatic β cells. One of its main functions is to keep the balance of glucose inside the body by regulating the absorption and metabolism of glucose in the periphery tissue, as well as the production and storage of hepatic glycogen. The insulin receptor is a transmembrane glycoprotein in which two α subunits with a molecular weight of 135 kD and twoβ subunits with a molecular weight of 95 kD are joined by a disulfide bond to form a β-α-α-β structure. The extracellular α subunit, especially, its three domains near the N-terminal are partially responsible for signal transduction or ligand-binding, as indicated by the experiments. The extracellular α subunits are involved in binding the ligands. The experimental results indicate that the three domains of the N-terminal of the α subunits are the main determinative parts of the insulin receptor to bind the insulin or mimetic peptide.We employed the extracellular domain (PDBID: 1IGR) of the insulin-like growth factor-1 receptor (IGF-1 R ) as the template to simulate and optimize the spatial structures of the three domains in the extracellular domain of the insulin receptor, which includes 468 residues. The work was accomplished by making use of the homology program in the Insight Ⅱ package on an Origin3800 server. The docking calculations of the insulin receptor obtained by homology with hexapeptides were carried out by means of the program Affinity. The analysis indicated that there were hydrogen bonding, and electrostatic and hydrophobic effects in the docking complex of the insulin receptor with hexapeptides.Moreover, we described the spatial orientation of a mimetic peptide with agonist activity in the docking complex. We obtained a rough model of binding of DLAPSQ or STIVYS with the insulin receptor, which provides the powerful theoretical support for designing the minimal insulin mimetic peptide with agonist activity, making it possible to develop oral small

GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study.

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Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Stefanis, C N

2001-05-08

There is accumulated evidence that the genes coding for the receptor of gamma aminobutyric acid (GABA), the most important inhibitory neurotransmitter in the CNS, may be involved in the pathogenesis of affective disorders. In a previous study, we have found a genetic association between the GABA-A receptor alpha5 subunit gene locus (GABRA5) on chromosome 15q11-of 13 and bipolar affective disorder. The aim of the present study was to examine the same subjects to see if there exists a genetic association between bipolar affective disorder and the GABA receptor beta3 subunit gene (GABRB3), which is located within 100 kb from GABRA5. The sample consisted of 48 bipolar patients compared to 44 controls (blood donors). All subjects were Greek, unrelated, and personally interviewed. Diagnosis was based on DSM-IV and ICD-10 criteria. The marker used was a dinucleotide (CA) repeat polymorphism with 12 alleles 179 to 201 bp long; genotyping was successful in all patients and 43 controls. The distribution of GABRB3 genotypes among the controls did not deviate significantly from the Hardy-Weinberg equilibrium. No differences in allelic frequencies between bipolar patients and controls were found for GABRB3, while this locus and GABRA5 did not seem to be in significant linkage disequilibrium. In conclusion, the GABRB3 CA-repeat polymorphism we investigated does not present the observed association between bipolar affective illness and GABRA5. This could be due to higher mutation rate in the GABRB3 CA-repeat polymorphism, but it might also signify that GABRA5 is the gene actually associated with the disease. Copyright 2001 Wiley-Liss, Inc.

INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

Science.gov (United States)

Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

2012-01-01

SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

Serotonin 2A receptors contribute to the regulation of risk-averse decisions

DEFF Research Database (Denmark)

Macoveanu, Julian; Rowe, James B; Hornboll, Bettina

2013-01-01

Pharmacological studies point to a role of the neurotransmitter serotonin (5-HT) in regulating the preference for risky decisions, yet the functional contribution of specific 5-HT receptors remains to be clarified. We used pharmacological fMRI to investigate the role of the 5-HT2A receptors...... in processing negative outcomes and regulating risk-averse behavior. During fMRI, twenty healthy volunteers performed a gambling task under two conditions: with or without blocking the 5-HT2A receptors. The volunteers repeatedly chose between small, likely rewards and large, unlikely rewards. Choices were...

GluN2C/GluN2D subunit-selective NMDA receptor potentiator CIQ reverses MK-801-induced impairment in prepulse inhibition and working memory in Y-maze test in mice

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Suryavanshi, P S; Ugale, R R; Yilmazer-Hanke, D; Stairs, D J; Dravid, S M

2014-01-01

Background and Purpose Despite ample evidence supporting the N-methyl-d-aspartate receptor (NMDAR) hypofunction hypothesis of schizophrenia, progress in the development of effective therapeutics based on this hypothesis has been limited. Facilitation of NMDA receptor function by co-agonists (d-serine or glycine) only partially alleviates the symptoms in schizophrenia; other means to facilitate NMDA receptors are required. NMDA receptor sub-types differ in their subunit composition, with varied GluN2 subunits (GluN2A-GluN2D) imparting different physiological, biochemical and pharmacological properties. CIQ is a positive allosteric modulator that is selective for GluN2C/GluN2D-containing NMDA receptors (Mullasseril et al.). Experimental Approach The effect of systemic administration of CIQ was tested on impairment in prepulse inhibition (PPI), hyperlocomotion and stereotypy induced by i.p. administration of MK-801 and methamphetamine. The effect of CIQ was also tested on MK-801-induced impairment in working memory in Y-maze spontaneous alternation test. Key Results We found that systemic administration of CIQ (20 mg·kg−1, i.p.) in mice reversed MK-801 (0.15 mg·kg−1, i.p.)-induced, but not methamphetamine (3 mg·kg−1, i.p.)-induced, deficit in PPI. MK-801 increased the startle amplitude to pulse alone, which was not reversed by CIQ. In contrast, methamphetamine reduced the startle amplitude to pulse alone, which was reversed by CIQ. CIQ also partially attenuated MK-801- and methamphetamine-induced hyperlocomotion and stereotyped behaviours. Additionally, CIQ reversed the MK-801-induced working memory deficit in spontaneous alternation in a Y-maze. Conclusion and Implications Together, these results suggest that facilitation of GluN2C/GluN2D-containing receptors may serve as an important therapeutic strategy for treating positive and cognitive symptoms in schizophrenia. PMID:24236947

Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

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Signe Tandrup Schmidt

2016-03-01

Full Text Available The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI. Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs, which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the

Chronic prenatal ethanol exposure alters hippocampal GABA(A) receptors and impairs spatial learning in the guinea pig.

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Iqbal, U; Dringenberg, H C; Brien, J F; Reynolds, J N

2004-04-02

Chronic prenatal ethanol exposure (CPEE) can injure the developing brain, and may lead to the fetal alcohol syndrome (FAS). Previous studies have demonstrated that CPEE upregulates gamma-aminobutyric acid type A (GABA(A)) receptor expression in the cerebral cortex, and decreases functional synaptic plasticity in the hippocampus, in the adult guinea pig. This study tested the hypothesis that CPEE increases GABA(A) receptor expression in the hippocampus of guinea pig offspring that exhibit cognitive deficits in a hippocampal-dependent spatial learning task. Timed, pregnant guinea pigs were treated with ethanol (4 g/kg maternal body weight per day), isocaloric-sucrose/pair-feeding, or water throughout gestation. GABA(A) receptor subunit protein expression in the hippocampus was measured at two development ages: near-term fetus and young adult. In young adult guinea pig offspring, CPEE increased spontaneous locomotor activity in the open-field and impaired task acquisition in the Morris water maze. CPEE did not change GABA(A) receptor subunit protein expression in the near-term fetal hippocampus, but increased expression of the beta2/3-subunit of the GABA(A) receptor in the hippocampus of young adult offspring. CPEE did not change either [(3)H]flunitrazepam binding or GABA potentiation of [(3)H]flunitrazepam binding, but decreased the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding, to hippocampal GABA(A) receptors in adult offspring. Correlational analysis revealed a relationship between increased spontaneous locomotor activity and growth restriction in the hippocampus induced by CPEE. Similarly, an inverse relationship was found between performance in the water maze and the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding in the hippocampus. These data suggest that alterations in hippocampal GABA(A) receptor expression and pharmacological properties contribute to hippocampal-related behavioral and cognitive deficits

The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

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Natalia Madrid-López

2017-10-01

Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

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Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

Energy Technology Data Exchange (ETDEWEB)

Gu, Y.

1989-01-01

The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

International Nuclear Information System (INIS)

Gu, Y.

1989-01-01

The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the α-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced 22 Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the γ, δ, and ε subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four (α, β, γ, and δ) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the α, β, and δ subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the γ- and ε- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the γ is replaced by ε

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex

OpenAIRE

1984-01-01

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) show...

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

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Michael Feyder

2007-12-01

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

Reduced tonic inhibition in the dentate gyrus contributes to chronic stress-induced impairments in learning and memory.

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Lee, Vallent; MacKenzie, Georgina; Hooper, Andrew; Maguire, Jamie

2016-10-01

It is well established that stress impacts the underlying processes of learning and memory. The effects of stress on memory are thought to involve, at least in part, effects on the hippocampus, which is particularly vulnerable to stress. Chronic stress induces hippocampal alterations, including but not limited to dendritic atrophy and decreased neurogenesis, which are thought to contribute to chronic stress-induced hippocampal dysfunction and deficits in learning and memory. Changes in synaptic transmission, including changes in GABAergic inhibition, have been documented following chronic stress. Recently, our laboratory demonstrated shifts in EGABA in CA1 pyramidal neurons following chronic stress, compromising GABAergic transmission and increasing excitability of these neurons. Interestingly, here we demonstrate that these alterations are unique to CA1 pyramidal neurons, since we do not observe shifts in EGABA following chronic stress in dentate gyrus granule cells. Following chronic stress, there is a decrease in the expression of the GABAA receptor (GABAA R) δ subunit and tonic GABAergic inhibition in dentate gyrus granule cells, whereas there is an increase in the phasic component of GABAergic inhibition, evident by an increase in the peak amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the numerous changes observed in the hippocampus following stress, it is difficult to pinpoint the pertinent contributing pathophysiological factors. Here we directly assess the impact of a reduction in tonic GABAergic inhibition of dentate gyrus granule cells on learning and memory using a mouse model with a decrease in GABAA R δ subunit expression specifically in dentate gyrus granule cells (Gabrd/Pomc mice). Reduced GABAA R δ subunit expression and function in dentate gyrus granule cells is sufficient to induce deficits in learning and memory. Collectively, these findings suggest that the reduction in GABAA R δ subunit-mediated tonic inhibition

Reduced tonic inhibition in the dentate gyrus contributes to chronic stress-induced impairments in learning and memory

Science.gov (United States)

Hooper, Andrew; Maguire, Jamie

2016-01-01

It is well established that stress impacts the underlying processes of learning and memory. The effects of stress on memory are thought to involve, at least in part, effects on the hippocampus, which is particularly vulnerable to stress. Chronic stress induces hippocampal alterations, including but not limited to dendritic atrophy and decreased neurogenesis, which are thought to contribute to chronic stress-induced hippocampal dysfunction and deficits in learning and memory. Changes in synaptic transmission, including changes in GABAergic inhibition, have been documented following chronic stress. Recently, our laboratory demonstrated shifts in EGABA in CA1 pyramidal neurons following chronic stress, compromising GABAergic transmission and increasing excitability of these neurons. Interestingly, here we demonstrate that these alterations are unique to CA1 pyramidal neurons, since we do not observe shifts in EGABA following chronic stress in dentate gyrus granule cells. Following chronic stress, there is a decrease in the expression of the GABAA receptor (GABAAR) δ subunit and tonic GABAergic inhibition in dentate gyrus granule cells; whereas, there is an increase in the phasic component of GABAergic inhibition, evident by an increase in the peak amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the numerous changes observed in the hippocampus following stress, it is difficult to pinpoint the pertinent contributing pathophysiological factors. Here we directly assess the impact of a reduction in tonic GABAergic inhibition of dentate gyrus granule cells on learning and memory using a mouse model with a decrease in GABAAR δ subunit expression specifically in dentate gyrus granule cells (Gabrd/Pomc mice). Reduced GABAAR δ subunit expression and function in dentate gyrus granule cells is sufficient to induce deficits in learning and memory. Collectively, these findings suggest that the reduction in GABAAR δ subunit-mediated tonic inhibition in

Essential role of NMDA receptor channel ε4 subunit (GluN2D in the effects of phencyclidine, but not methamphetamine.

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Yoko Hagino

Full Text Available Phencyclidine (PCP, a noncompetitive N-methyl-D-aspartate (NMDA receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex in the striatum and prefrontal cortex (PFC using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D] and locomotor activity. PCP significantly increased DA(ex in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.

Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine αV Integrin Utilization by Type A and O Viruses

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Duque, Hernando; Baxt, Barry

2003-01-01

Three members of the αV integrin family of cellular receptors, αVβ1, αVβ3, and αVβ6, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other αV integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the αV integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β1, β5, and β6 integrin subunits. Using these subunits, along with previously cloned bovine αV and β3 subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by αVβ1, αVβ3, αVβ5, and αVβ6 among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used αVβ3 and αVβ6 with relatively high efficiency, while only one virus utilized αVβ1 with moderate efficiency. In contrast, both type O viruses utilized αVβ6 and αVβ1 with higher efficiency than αVβ3. Only low levels of viral replication were detected in αVβ5-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host. PMID:12551988

Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

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Berrout, Liza; Isokawa, Masako

2018-01-01

Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

Therapeutic potential of Mediator complex subunits in metabolic diseases.

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Ranjan, Amol; Ansari, Suraiya A

2018-01-01

The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Does the kappa opioid receptor system contribute to pain aversion?

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Catherine M Cahill

2014-11-01

Full Text Available The kappa opioid receptor (KOR and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor α subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

International Nuclear Information System (INIS)

McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M.

1991-01-01

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) αsubunit forms a binding site for α-bungarotoxin (α-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR α1 subunits were tested for their ability to bind 125 I-α-BTX, and differences in α-BTX affinity were determined by using solution (IC 50 s) and solid-phase (K d s) assays. Panels of overlapping peptides corresponding to the complete α1 subunit of mouse and human were also tested for α-BTX binding, but other sequence segments forming the α-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased α-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/α1-subunit interface a vicinal disulfide bound was not required for α-BTX binding

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

DEFF Research Database (Denmark)

Pandey, A; Podtelejnikov, A V; Blagoev, B

2000-01-01

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

Science.gov (United States)

Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

Site-directed cross-linking: establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis

International Nuclear Information System (INIS)

Milligan, D.L.; Koshland, D.E. Jr.

1988-01-01

Cysteine residues introduced at specific locations in the aspartate receptor of Salmonella typhimurium provide anchor points for cross-linking and serve as chemical markers for structural studies of this oligomeric receptor. These markers have been used to measure the rate of subunit exchange between oligomeric receptors and to show that ligand binding inhibits this exchange. The cysteine-containing receptors can be oxidatively cross-linked to completion within the oligomeric receptor, indicating that the receptor has an even number of subunits. Based on this observation, a technique has been developed that can be used to determine the oligomeric structure of proteins under a variety of experimental conditions. The technique involves the measurement of the effect of dilution by cysteineless receptor subunits on cross-linking and reveals that the aspartate receptor is dimeric in detergent solution, in a mixed-micelle system, and in reconstituted membrane vesicles. Binding of aspartate does not change the oligomeric structure of the receptor, indicating that transmembrane signaling occurs within an oligomeric receptor of constant size

γ1-Containing GABA-A Receptors Cluster at Synapses Where they Mediate Slower Synaptic Currents than γ2-Containing GABA-A Receptors

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Christine L. Dixon

2017-06-01

Full Text Available GABA-A receptors (GABAARs are pentameric ligand-gated ion channels that are assembled mainly from α (α1–6, β (β1–3 and γ (γ1–3 subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In “artificial” synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L, the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.

Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors

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Orth Angela

2009-04-01

Full Text Available Abstract Background N-methyl-D-aspartate receptors (NMDARs are the most complex of ionotropic glutamate receptors (iGluRs. Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity. Results Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants. Conclusion While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.

Delta Subunit-Containing Gamma-Aminobutyric Acid A Receptor Disinhibits Lateral Amygdala and Facilitates Fear Expression in Mice.

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Liu, Zhi-Peng; He, Qing-Hai; Pan, Han-Qing; Xu, Xiao-Bin; Chen, Wen-Bing; He, Ye; Zhou, Jin; Zhang, Wen-Hua; Zhang, Jun-Yu; Ying, Xiao-Ping; Han, Ren-Wen; Li, Bao-Ming; Gao, Tian-Ming; Pan, Bing-Xing

2017-06-15

Maintaining gamma-aminobutyric acidergic (GABAergic) inhibition in the amygdala within a physiological range is critical for the appropriate expression of emotions such as fear and anxiety. The synaptic GABA type A receptor (GABA A R) is generally known to mediate the primary component of amygdala inhibition and prevent inappropriate expression of fear. However, little is known about the contribution of the extrasynaptic GABA A R to amygdala inhibition and fear. By using mice expressing green fluorescent protein in interneurons (INs) and lacking the δ subunit-containing GABA A R (GABA A (δ)R), which is exclusively situated in the extrasynaptic membrane, we systematically investigated the role of GABA A (δ)R in regulating inhibition in the lateral amygdala (LA) and fear learning using the combined approaches of immunohistochemistry, electrophysiology, and behavior. In sharp contrast to the established role of synaptic GABA A R in mediating LA inhibition, we found that either pharmacological or physiological recruitment of GABA A (δ)R resulted in the weakening of GABAergic transmission onto projection neurons in LA while leaving the glutamatergic transmission unaltered, suggesting disinhibition by GABA A (δ)R. The disinhibition arose from IN-specific expression of GABA A (δ)R with its activation decreasing the input resistance of local INs and suppressing their activation. Genetic deletion of GABA A (δ)R attenuated its role in suppressing LA INs and disinhibiting LA. Importantly, the GABA A (δ)R facilitated long-term potentiation in sensory afferents to LA and permitted the expression of learned fear. Our findings suggest that GABA A (δ)R serves as a brake rather than a mediator of GABAergic inhibition in LA. The disinhibition by GABA A (δ)R may help to prevent excessive suppression of amygdala activity and thus ensure the expression of emotion. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

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Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

2017-01-01

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.

Partial Agonism of Taurine at Gamma-Containing Native and Recombinant GABAA Receptors

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Kletke, Olaf; Gisselmann, Guenter; May, Andrea; Hatt, Hanns; A. Sergeeva, Olga

2013-01-01

Taurine is a semi-essential sulfonic acid found at high concentrations in plasma and mammalian tissues which regulates osmolarity, ion channel activity and glucose homeostasis. The structural requirements of GABAA-receptors (GABAAR) gated by taurine are not yet known. We determined taurine potency and efficacy relative to GABA at different types of recombinant GABAAR occurring in central histaminergic neurons of the mouse hypothalamic tuberomamillary nucleus (TMN) which controls arousal. At binary α1/2β1/3 receptors taurine was as efficient as GABA, whereas incorporation of the γ1/2 subunit reduced taurine efficacy to 60–90% of GABA. The mutation γ2F77I, which abolishes zolpidem potentiation, significantly reduced taurine efficacy at recombinant and native receptors compared to the wild type controls. As taurine was a full- or super- agonist at recombinant αxβ1δ-GABAAR, we generated a chimeric γ2 subunit carrying the δ subunit motif around F77 (MTVFLH). At α1/2β1γ2(MTVFLH) receptors taurine became a super-agonist, similar to δ-containing ternary receptors, but remained a partial agonist at β3-containing receptors. In conclusion, using site-directed mutagenesis we found structural determinants of taurine’s partial agonism at γ-containing GABAA receptors. Our study sheds new light on the β1 subunit conferring the widest range of taurine-efficacies modifying GABAAR function under (patho)physiological conditions. PMID:23637894

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit of GS protein

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Kang, Hatan; Lee, Won Kyu; Choi, Yun Hui; Vukoti, Krishna Moorthy; Bang, Won Gi; Yu, Yeon Gyu

2005-01-01

The serotonin type 6 (5-HT 6 ) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G S ). To identify the structural basis for the interaction of the 5-HT 6 receptor with the G S protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT 6 receptor and the α subunit of G S (Gα S ). The direct interaction of iL3 and Gα S was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Gα S were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10 -6 M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Gα S . The critical residues within the iL3 region for the interaction with Gα S were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT 6 receptor mutants

Changes in Glutamate/NMDA Receptor Subunit 1 Expression in Rat Brain after Acute and Subacute Exposure to Methamphetamine

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Walailuk Kerdsan

2009-01-01

Full Text Available Methamphetamine (METH is a psychostimulant drug of abuse that produces long-term behavioral changes including behavioral sensitization, tolerance, and dependence. METH has been reported to induce neurotoxic effects in several areas of the brain via the dopaminergic system. Changes of dopamine function can induce malfunction of the glutamatergic system. Therefore, the aim of the present study was to examine the effects of METH administration on the expression of glutamate N-methyl-D-aspartate receptor subunit 1 (NMDAR1 in frontal cortex, striatum, and hippocampal formation after acute and subacute exposure to METH by western blotting. Male Sprague-Dawley rats were injected intraperitoneally with a single dose of 8 mg/kg METH, 4 mg/kg/day METH for 14 days and saline in acute, subacute, and control groups, respectively. A significant increase in NMDAR1 immunoreactive protein was found in frontal cortex in the subacute group (P=.036 but not in the acute group (P=.580. Moreover, a significant increase in NMDAR1 was also observed in striatum in both acute (P=.025 and subacute groups (P=.023. However, no significant differences in NMDAR1 in hippocampal formation were observed in either acute or subacute group. The results suggest that an upregulation of NMDA receptor expression may be a consequence of glutamatergic dysfunction induced by METH.

Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

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Boughton, Andrew P; Yang, Pei; Tesmer, Valerie M; Ding, Bei; Tesmer, John J G; Chen, Zhan

2011-09-13

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

Vesicular glutamate release from central axons contributes to myelin damage.

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Doyle, Sean; Hansen, Daniel Bloch; Vella, Jasmine; Bond, Peter; Harper, Glenn; Zammit, Christian; Valentino, Mario; Fern, Robert

2018-03-12

The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

International Nuclear Information System (INIS)

Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

1987-01-01

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

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Field Linda M

2011-05-01

Full Text Available Abstract Background Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. Results Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR. Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpβ1 genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T. Conclusion Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also

Oxytocin modulates GABAAR subunits to confer neuroprotection in stroke in vitro.

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Kaneko, Yuji; Pappas, Colleen; Tajiri, Naoki; Borlongan, Cesar V

2016-10-21

Oxytocin protects against ischemia-induced inflammation and oxidative stress, and is associated with GABA (γ-aminobutyric acid, an inhibitory neurotransmitter) signaling transduction in neurons. However, the molecular mechanism by which oxytocin affords neuroprotection, especially the interaction between oxytocin receptor and GABA A receptor (GABA A R), remains to be elucidated. Primary rat neural cells were exposed to oxytocin before induction of experimental acute stroke model via oxygen-glucose deprivation-reperfusion (OGD/R) injury. Pretreatment with oxytocin increased cell viability, decreased the cell damage against oxidative stress, and prevented the release of high mobility group box1 during OGD/R. However, introduction of oxytocin during OGD/R did not induce neuroprotection. Although oxytocin did not affect the glutathione-related cellular metabolism before OGD, oxytocin modulated the expression levels of GABA A R subunits, which function to remove excessive neuronal excitability via chloride ion influx. Oxytocin-pretreated cells significantly increased the chloride ion influx in response to GABA and THIP (δ-GABA A R specific agonist). This study provides evidence that oxytocin regulated GABA A R subunits in affording neuroprotection against OGD/R injury.

Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control.

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Ruiz-Riol, M; Berdnik, D; Llano, A; Mothe, B; Gálvez, C; Pérez-Álvarez, S; Oriol-Tordera, B; Olvera, A; Silva-Arrieta, S; Meulbroek, M; Pujol, F; Coll, J; Martinez-Picado, J; Ganoza, C; Sanchez, J; Gómez, G; Wyss-Coray, T; Brander, C

2017-08-15

Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia ( P HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/β-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor ( IL27RA ) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC. IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins, and

Tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the Middle East respiratory coronavirus (MERS-CoV receptor-binding domain as an antigen.

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Jiaming Lan

Full Text Available The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV pandemic. Some studies have indicated the receptor-binding domain (RBD protein of MERS-CoV spike (S is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m. with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum alone, alum and polyriboinosinic acid (poly I:C or alum and cysteine-phosphate-guanine (CpG oligodeoxynucleotides (ODN. The immune responses of mice vaccinated with RBD, incomplete Freund's adjuvant (IFA and CpG ODN by a subcutaneous (s.c. route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production. Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: implications for angiotensin II-induced vascular dysfunction.

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Liu, Chia-Chi; Karimi Galougahi, Keyvan; Weisbrod, Robert M; Hansen, Thomas; Ravaie, Ramtin; Nunez, Andrea; Liu, Yi B; Fry, Natasha; Garcia, Alvaro; Hamilton, Elisha J; Sweadner, Kathleen J; Cohen, Richard A; Figtree, Gemma A

2013-12-01

Glutathionylation of the Na(+)-K(+) pump's β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na(+)-K(+) pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na(+)-K(+) pump activity is an important determinant of vascular tone through effects on [Ca(2+)]i, we have examined the role of oxidative regulation of the Na(+)-K(+) pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na(+)-K(+)ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K(+)-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na(+)-K(+) ATPase and decrease in K(+)-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K(+)-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K(+)-induced vasorelaxation. Ang II inhibits the Na(+)-K(+) pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump's β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na(+)-K(+) pump and may have an

Enhancing NMDA Receptor Function: Recent Progress on Allosteric Modulators

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Lulu Yao

2017-01-01

Full Text Available The N-methyl-D-aspartate receptors (NMDARs are subtype glutamate receptors that play important roles in excitatory neurotransmission and synaptic plasticity. Their hypo- or hyperactivation are proposed to contribute to the genesis or progression of various brain diseases, including stroke, schizophrenia, depression, and Alzheimer’s disease. Past efforts in targeting NMDARs for therapeutic intervention have largely been on inhibitors of NMDARs. In light of the discovery of NMDAR hypofunction in psychiatric disorders and perhaps Alzheimer’s disease, efforts in boosting NMDAR activity/functions have surged in recent years. In this review, we will focus on enhancing NMDAR functions, especially on the recent progress in the generation of subunit-selective, allosteric positive modulators (PAMs of NMDARs. We shall also discuss the usefulness of these newly developed NMDAR-PAMs.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

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Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Back to the future: Rational maps for exploring acetylcholine receptor space and time.

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Tessier, Christian J G; Emlaw, Johnathon R; Cao, Zhuo Qian; Pérez-Areales, F Javier; Salameh, Jean-Paul J; Prinston, Jethro E; McNulty, Melissa S; daCosta, Corrie J B

2017-11-01

Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties. Copyright © 2017 Elsevier B.V. All rights reserved.

The human epilepsy mutation GABRG2(Q390X) causes chronic subunit accumulation and neurodegeneration.

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Kang, Jing-Qiong; Shen, Wangzhen; Zhou, Chengwen; Xu, Dong; Macdonald, Robert L

2015-07-01

Genetic epilepsy and neurodegenerative diseases are two common neurological disorders that are conventionally viewed as being unrelated. A subset of patients with severe genetic epilepsies who have impaired development and often go on to die of their disease respond poorly to anticonvulsant drug therapy, suggesting a need for new therapeutic targets. Previously, we reported that multiple GABAA receptor epilepsy mutations result in protein misfolding and abnormal receptor trafficking. We have now developed a model of a severe human genetic epileptic encephalopathy, the Gabrg2(+/Q390X) knock-in mouse. We found that, in addition to impairing inhibitory neurotransmission, mutant GABAA receptor γ2(Q390X) subunits accumulated and aggregated intracellularly, activated caspase 3 and caused widespread, age-dependent neurodegeneration. These findings suggest that the fundamental protein metabolism and cellular consequences of the epilepsy-associated mutant γ2(Q390X) ion channel subunit are not fundamentally different from those associated with neurodegeneration. Our results have far-reaching relevance for the identification of conserved pathological cascades and mechanism-based therapies that are shared between genetic epilepsies and neurodegenerative diseases.

Association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) and bipolar affective disorder.

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Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Vassilopoulos, D; Stefanis, C N

1998-02-07

Genetic factors seem to play an important role in the pathogenesis of affective disorder. The candidate gene strategies are being used, among others, to identify the genes conferring vulnerability to the disease. The genes coding for the receptors of gamma-aminobutyric acid (GABA) have been proposed as candidates for affective disorder, since the GABA neurotransmitter system has been implicated in the pathogenesis of the illness. We examined the possible genetic association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) on chromosome 15 and affective disorder, in 48 bipolar patients (BP), 40 unipolar patients (UP), and 50 healthy individuals, age- and sex-matched to the patients. All patients and controls were unrelated Greeks. Diagnoses were made after direct interviews according to the DSM-IV and ICD-10 criteria. For the genotyping, a dinucleotide (CA) repeat marker was used. The polymerase chain reaction (PCR) products found were nine alleles with lengths between 272 and 290 base pairs (bp). The distribution of allelic frequencies of the GABRA5 locus differed significantly between BP patients and controls with the 282-bp allele found to be associated with BP affective disorder, while no such difference was observed between the groups of UP patients and controls nor between the two patient groups. The presence or absence of the 282-bp allele in the genotype of BP patients was not shown to influence the age of onset and the overall clinical severity, but was found to be associated with a preponderance of manic over depressive episodes in the course of the illness.

Nicotinic acetylcholine receptors containing the α7-like subunit mediate contractions of muscles responsible for space positioning of the snail, Helix pomatia L. tentacle.

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Tibor Kiss

Full Text Available Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh responses in muscles to determine the properties of the ACh receptor (AChR, the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium, methyllycaconitine, α-Bungarotoxin (αBgTx and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

Discovery of a novel allosteric modulator of 5-HT3 receptor

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Trattnig, Sarah M; Harpsøe, Kasper; Thygesen, Sarah B

2012-01-01

The ligand-gated ion channels in the Cysloop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA and glycine. Cysloop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel...... receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)subunit and TM1 and TM2 in the (minus)subunit. The Ser248, Leu288, Ile290, Thr294 and Gly306 residues are identified as important...

Medicinal Chemistry of Competitive Kainate Receptor Antagonists

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2010-01-01

Kainic acid (KA) receptors belong to the group of ionotropic glutamate receptors and are expressed throughout in the central nervous system (CNS). The KA receptors have been shown to be involved in neurophysiological functions such as mossy fiber long-term potentiation (LTP) and synaptic plasticity and are thus potential therapeutic targets in CNS diseases such as schizophrenia, major depression, neuropathic pain and epilepsy. Extensive effort has been made to develop subtype-selective KA receptor antagonists in order to elucidate the physiological function of each of the five subunits known (GluK1−5). However, to date only selective antagonists for the GluK1 subunit have been discovered, which underlines the strong need for continued research in this area. The present review describes the structure−activity relationship and pharmacological profile for 10 chemically distinct classes of KA receptor antagonists comprising, in all, 45 compounds. To the medicinal chemist this information will serve as reference guidance as well as an inspiration for future effort in this field. PMID:22778857

Progesterone modulation of alpha5 nAChR subunits influences anxiety-related behavior during estrus cycle.

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Gangitano, D; Salas, R; Teng, Y; Perez, E; De Biasi, M

2009-06-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the alpha5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and alpha5 subunit null mice (alpha5(-/-)) were subjected to the open field activity (OFA), light-dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, alpha5(-/-) behaved like wild-type controls. In the EPM, female alpha5(-/-) mice displayed an anxiolytic-like phenotype, while male alpha5(-/-) mice were undistinguishable from littermate controls. We studied the hypothalamus-pituitary-adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin-releasing factor. Consistent with an anxiolytic-like phenotype, female alpha5(-/-) mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of alpha5, we treated cultured NTera 2 cells with progesterone and found that alpha5 protein levels were upregulated. In addition, brain levels of alpha5 mRNA increased upon progesterone injection into ovariectomized wild-type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic-like in wild-type mice, but no cycle-dependent fluctuations in anxiety levels were found in alpha5(-/-) females. Thus, alpha5-containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone-dependent modulation of alpha5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.

Epac Signaling Is Required for Cocaine-Induced Change in AMPA Receptor Subunit Composition in the Ventral Tegmental Area.

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Liu, Xiaojie; Chen, Yao; Tong, Jiaqing; Reynolds, Ashley M; Proudfoot, Sarah C; Qi, Jinshun; Penzes, Peter; Lu, Youming; Liu, Qing-Song

2016-04-27

Exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) are intracellular receptors for cAMP. Although PKA and its downstream effectors have been studied extensively in the context of drug addiction, whether and how Epac regulates cellular and behavioral effects of drugs of abuse remain essentially unknown. Epac is known to regulate AMPA receptor (AMPAR) trafficking. Previous studies have shown that a single cocaine exposure in vivo leads to an increase in GluA2-lacking AMPARs in dopamine neurons of the ventral tegmental area (VTA). We tested the hypothesis that Epac mediates cocaine-induced changes in AMPAR subunit composition in the VTA. We report that a single cocaine injection in vivo in wild-type mice leads to inward rectification of EPSCs and renders EPSCs sensitive to a GluA2-lacking AMPAR blocker in VTA dopamine neurons. The cocaine-induced increase in GluA2-lacking AMPARs was absent in Epac2-deficient mice but not in Epac1-deficient mice. In addition, activation of Epac with the selective Epac agonist 8-CPT-2Me-cAMP (8-CPT) recapitulated the cocaine-induced increase in GluA2-lacking AMPARs, and the effects of 8-CPT were mediated by Epac2. We also show that conditioned place preference to cocaine was impaired in Epac2-deficient mice and in mice in which Epac2 was knocked down in the VTA but was not significantly altered in Epac1-deficient mice. Together, these results suggest that Epac2 is critically involved in the cocaine-induced change in AMPAR subunit composition and drug-cue associative learning. Addictive drugs, such as cocaine, induce long-lasting adaptions in the reward circuits of the brain. A single intraperitoneal injection of cocaine leads to changes in the composition and property of the AMPAR that carries excitatory inputs to dopamine neurons. Here, we provide evidence that exchange protein directly activated by cAMP (Epac), a cAMP sensor protein, is required for the cocaine-induced changes of the AMPAR. We found that the

3D structure of muscle dihydropyridine receptor

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Montserrat Samsó

2015-01-01

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

Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.

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McIlhinney, R A; Molnár, E

1996-04-01

To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.

The role of GluN2B-containing NMDA receptors in short- and long-term fear recall.

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Mikics, Eva; Toth, Mate; Biro, Laszlo; Bruzsik, Biborka; Nagy, Boglarka; Haller, Jozsef

2017-08-01

N-methyl-d-aspartate (NMDA) receptors are crucial synaptic elements in long-term memory formation, including the associative learning of fearful events. Although NMDA blockers were consistently shown to inhibit fear memory acquisition and recall, the clinical use of general NMDA blockers is hampered by their side effects. Recent studies revealed significant heterogeneity in the distribution and neurophysiological characteristics of NMDA receptors with different GluN2 (NR2) subunit composition, which may have differential role in fear learning and recall. To investigate the specific role of NMDA receptor subpopulations with different GluN2 subunit compositions in the formation of lasting traumatic memories, we contrasted the effects of general NMDA receptor blockade with GluN2A-, GluN2B-, and GluN2C/D subunit selective antagonists (MK-801, PEAQX, Ro25-6981, PPDA, respectively). To investigate acute and lasting consequences, behavioral responses were investigated 1 and 28days after fear conditioning. We found that MK-801 (0.05 and 0.1mg/kg) decreased fear recall at both time points. GluN2B receptor subunit blockade produced highly similar effects, albeit efficacy was somewhat smaller 28days after fear conditioning. Unlike MK-801, Ro25-6981 (3 and 10mg/kg) did not affect locomotor activity in the open-field. In contrast, GluN2A and GluN2C/D blockers (6 and 20mg/kg PEAQX; 3 and 10mg/kg PPDA, respectively) had no effect on conditioned fear recall at any time point and dose. This sharp contrast between GluN2B- and other subunit-containing NMDA receptor function indicates that GluN2B receptor subunits are intimately involved in fear memory formation, and may provide a novel pharmacological target in post-traumatic stress disorder or other fear-related disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of the chicken muscle insulin receptor

International Nuclear Information System (INIS)

Adamo, M.; Simon, J.; Rosebrough, R.W.; McMurtry, J.P.; Steele, N.C.; LeRoith, D.

1987-01-01

Insulin receptors are present in chicken skeletal muscle. Crude membrane preparations demonstrated specific 125 I-insulin binding. The nonspecific binding was high (36-55% of total binding) and slightly lower affinity receptors were found than are typically observed for crude membrane insulin binding in other chicken tissues. Affinity crosslinking of 125 I-insulin to crude membranes revealed insulin receptor alpha-subunits of Mr 128K, intermediate between those of liver (134K) and brain (124K). When solubilized and partially purified on wheat germ agglutinin (WGA) affinity columns, chicken muscle insulin receptors exhibited typical high affinity binding, with approximately 10(-10) M unlabeled insulin producing 50% inhibition of the specific 125 I-insulin binding. WGA purified chicken muscle insulin receptors also exhibited insulin-stimulated autophosphorylation of the beta-subunit, which appeared as phosphorylated bands of 92- and 81K. Both bands were immunoprecipitated by anti-receptor antiserum (B10). WGA purified membranes also demonstrated dose-dependent insulin-stimulated phosphorylation of the exogenous substrate poly(Glu,Tyr)4:1. However, unlike chicken liver, chicken muscle insulin receptor number and tyrosine kinase activity were unaltered by 48 hr of fasting or 48 hr of fasting and 24 hr of refeeding. Thus, despite the presence of insulin receptors in chicken muscle showing normal coupling to receptor tyrosine kinase activity, nutritional alterations modulate these parameters in a tissue-specific manner in chickens

PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTO

Directory of Open Access Journals (Sweden)

Edgar Antonio Reyes Montaño

2011-12-01

Full Text Available Producing polyclonal antibodies (IgY inchickens has advantages over those obtainedin other animal models, since theyhave been used as a tool for studyingdifferent proteins (NMDA glutamate receptorin our case, specifically the NR1subunit. We produced specific antibodiesagainst expression products by thealternative splicing of the gene encodingNMDA receptor NR1 subunit in adult ratbrain. Three peptides corresponding tothe splicing sites (N1, C1 and C2’ cassetteswere designed, synthesised and usedindividually as antigens in hens. Specificimmunoglobulins were purified fromyolks. The antibodies were then used forpurifying the NMDA receptor NR1 subunitusing affinity chromatography couplingthe three antibodies to the support.R

Identification of a tetramerization domain in the C terminus of the vanilloid receptor.

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García-Sanz, Nuria; Fernández-Carvajal, Asia; Morenilla-Palao, Cruz; Planells-Cases, Rosa; Fajardo-Sánchez, Emmanuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

2004-06-09

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

DEFF Research Database (Denmark)

Adelfinger, L; Turecek, R; Ivankova, K

2014-01-01

released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

Heavy subunit of cell surface Gal/GalNAc lectin (Hgl) undergoes degradation via endo-lysosomal compartments in Entamoeba histolytica.

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Verma, Kuldeep; Datta, Sunando

2017-06-14

The human gut parasite Entamoeba histolytica uses a multifunctional virulence factor, Hgl, a cell surface transmembrane receptor subunit of Gal/GalNAc lectin that contributes to adhesion, invasion, cytotoxicity and immune response in the host. At present, the physiologic importance of Hgl receptor is mostly known for pathogenicity of E. histolytica. However, the molecular mechanisms of Hgl trafficking events and their association with the intracellular membrane transport machinery are largely unknown. We used biochemical and microscopy-based assays to understand the Hgl trafficking in the amoebic trophozoites. Our results suggest that the Hgl is constitutively degraded through delivery into amoebic lysosome-like compartments. Further, we also observed that the Hgl was significantly colocalized with amoebic Rab GTPases such as EhRab5, EhRab7A, and EhRab11B. While, we detected association of Hgl with all these Rab GTPases in early vacuolar compartments, only EhRab7A remains associated with Hgl till its transport to amoebic lysosome-like compartments.

Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag*

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Wilkins, Megan E.; Li, Xinyan; Smart, Trevor G.

2008-01-01

GABAB receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABAB receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABAB receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, α-bungarotoxin. By using the α-bungarotoxin binding site-tagged GABAB R1a subunit (R1aBBS), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, α-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABAB receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors. PMID:18812318

Moessbauer spectroscopic studies of hemoglobin and its isolated subunits

International Nuclear Information System (INIS)

Hoy, G.R.; Cook, D.C.; Berger, R.L.; Friedman, F.K.

1986-01-01

Samples of 90% enriched 57Fe hemoglobin and its isolated subunits have been prepared. Moessbauer spectroscopic measurements have been made on three such samples. Sample one contained contributions of oxyhemoglobin, deoxyhemoglobin, and carbonmonoxyhemoglobin. This sample was studied from a temperature of 90 K down to 230 mK. Measurements were also made at 4.2 K using a small applied magnetic field of 1.0 T. In general, the measured quadrupole splittings and isomer shifts for each component agreed with previous measurements on single component samples in the literature, and thus demonstrated that chemically enriched hemoglobin has not been altered. The second and third samples were isolated alpha and beta subunits, respectively. We have found measurable Moessbauer spectral differences between the HbO 2 sites in the alpha subunit sample and the beta subunit sample. The measured Moessbauer spectral areas indicate that the iron ion has the largest mean-square displacement at the deoxy Hb sites as compared to that at the oxy- and carbonmonoxy Hb sites. The mean-square displacement at the HbO 2 sites is the smallest

Insulin-like growth factor-II (IGF II) receptor from rat brain is of lower apparent molecular weight than the IGF II receptor from rat liver

International Nuclear Information System (INIS)

McElduff, A.; Poronnik, P.; Baxter, R.C.

1987-01-01

The binding subunits of the insulin and insulin-like growth factor-I (IGF I) receptors from rat brain are of lower molecular weight than the corresponding receptor in rat liver, possibly due to variations in sialic acid content. We have compared the IGF II receptor from rat brain and rat liver. The brain receptor is of smaller apparent mol wt (about 10 K) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is independent of ligand binding as it persists in iodinated and specifically immunoprecipitated receptors. From studies of wheat germ agglutinin binding and the effect of neuraminidase on receptor mobility, we conclude that this difference is not simply due to variations in sialic acid content. Treatment with endoglycosidase F results in reduction in the molecular size of both liver and brain receptors and after this treatment the aglycoreceptors are of similar size. We conclude that in rat brain tissue the IGF II receptor like the binding subunits of the insulin and IGF I receptors is of lower molecular size than the corresponding receptors in rat liver. This difference is due to differences in N-linked glycosylation

Domain cooperativity in the β1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle.

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Dayal, Anamika; Bhat, Vinayakumar; Franzini-Armstrong, Clara; Grabner, Manfred

2013-04-30

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

Progesterone modulation of α5 nAChR subunits influences anxiety-related behavior during estrus cycle

OpenAIRE

Gangitano, David; Salas, Ramiro; Teng, Yanfen; Perez, Erika; De Biasi, Mariella

2009-01-01

Smokers often report an anxiolytic effect of cigarettes. In addition, stress-related disorders such as anxiety, post-traumatic stress syndrome, and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety-related responses, control and α5 subunit null mice (α5 −/−) were subjected to the open field, light-dark box and elevated plus maze tests. In the open field and light-dark box, α5 −/− behaved like wild type co...

Structure and assembly mechanism for heteromeric kainate receptors.

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Kumar, Janesh; Schuck, Peter; Mayer, Mark L

2011-07-28

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11 nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors. Copyright © 2011 Elsevier Inc. All rights reserved.

Association between GABA-A receptor alpha 5 subunit gene locus and schizophrenia of a later age of onset.

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Papadimitriou, G; Dikeos, D; Daskalopoulou, E; Karadima, G; Avramopoulos, D; Contis, C; Stefanis, C

2001-01-01

Heritability is considered to be a major etiologic factor for schizophrenia. Among the genes considered as candidates for the disease, are those related to GABAergic neurotransmission. Our aim was to test for a genetic association between GABA-A receptor alpha 5 subunit gene locus (GABRA(5)) and schizophrenia. Genotyping of the GABRA(5) locus was performed by the use of a dinucleotide (CA) repeat marker in 46 schizophrenic patients and 50 healthy individuals, all unrelated Greeks. Eight alleles were identified, 276-290 bp long. A nonsignificant excess of the 282-bp allele, which was found in a previous study in a Greek population to be associated with bipolar affective disorder, was observed in schizophrenic patients (33.8 vs. 23.9% in the controls). The frequency of this allele was 43.3% among patients with a later age of onset (over 25 years), differing at a statistically significant level from the controls (p < 0.05). These results suggest that common pathophysiological mechanisms may possibly underlie affective disorders and schizophrenia, at least in a subgroup of patients. Copyright 2001 S. Karger AG, Basel

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[11C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

International Nuclear Information System (INIS)

Christiaans, Johannes A.M.; Klein, Pieter J.; Metaxas, Athanasios; Kooijman, Esther J.M.; Schuit, Robert C.; Leysen, Josée E.; Lammertsma, Adriaan A.; Berckel, Bart N.M. van; Windhorst, Albert D.

2014-01-01

Introduction: The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimer's disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. Methods: N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. Results: N-((5-(4-fluoro-2-[ 11 C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49 ± 3% (decay-corrected) yield, affording 4.1 ± 0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of > 99% and with a specific activity of 78 ± 10 GBq/μmol. Conclusion: A new NR2B PET ligand was developed in high yield. [ 11 C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [ 11 C]4 as an NR2B PET ligand

Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

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Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

2010-09-01

Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

Insulin resistance in uremia: Insulin receptor kinase activity in liver and muscle from chronic uremic rats

International Nuclear Information System (INIS)

Cecchin, F.; Ittoop, O.; Sinha, M.K.; Caro, J.F.

1988-01-01

The authors have studied the structure and function of the partially purified insulin receptors from liver and skeletal muscle in a rat model of severe chronic uremia. 125 I-insulin binding was higher in the liver from uremic rats when compared with ad libitum- and pair-fed controls. Furthermore, the ability of insulin to stimulate the autophosphorylation of the β-subunit and insulin receptor kinase activity using Glu 80 , Tyr 20 as exogenous phosphoacceptor was increased in the liver of the uremic animals. The structural characteristics of the receptors, as determined by electrophoretic mobilities of affinity labeled α-subunit and the phosphorylated β-subunit, were normal in uremia. 125 I-insulin binding and insulin receptor kinase activity were similar in the skeletal muscle from uremic and pair- and ad libitum-fed animals. Thus the data are supportive of the hypothesis that in liver and muscle of chronic uremic rats, insulin resistance is due to a defect(s) distal to the insulin receptor kinase

Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

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Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

Directory of Open Access Journals (Sweden)

Michael S. Parker

2014-03-01

Full Text Available The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY receptors and could apply to many receptors that use large peptidic agonists.

Effects of pilocarpine and kainate-induced seizures on N-methyl-d-aspartate receptor gene expression in the rat hippocampus

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Przewlocka, B.; Labuz, D.; Machelska, H.; Przewlocki, R.; Turchan, J.; Lason, W. [Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow (Poland)

1997-04-14

The effects of pilocarpine- and kainate-induced seizures on N-methyl-d-aspartate receptor subunit-1 messenger RNA and [{sup 3}H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-d-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-d-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [{sup 3}H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [{sup 3}H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-d-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-d-aspartate receptor subunit-1 and [{sup 3}H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate.These data point to some differences in hippocampal N-methyl-d-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-d-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [{sup 3}H]dizocilpine maleate binding in the rat hippocampal formation. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Effects of pilocarpine and kainate-induced seizures on N-methyl-d-aspartate receptor gene expression in the rat hippocampus

International Nuclear Information System (INIS)

Przewlocka, B.; Labuz, D.; Machelska, H.; Przewlocki, R.; Turchan, J.; Lason, W.

1997-01-01

The effects of pilocarpine- and kainate-induced seizures on N-methyl-d-aspartate receptor subunit-1 messenger RNA and [ 3 H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-d-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-d-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [ 3 H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [ 3 H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-d-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-d-aspartate receptor subunit-1 and [ 3 H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate.These data point to some differences in hippocampal N-methyl-d-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-d-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [ 3 H]dizocilpine maleate binding in the rat hippocampal formation. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

Science.gov (United States)

Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

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Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

2008-10-27

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Perez-Ramirez, B.; Martinez-Carrion, M.

1989-01-01

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

Science.gov (United States)

Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

2015-02-05

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag.

Science.gov (United States)

Wilkins, Megan E; Li, Xinyan; Smart, Trevor G

2008-12-12

GABA(B) receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABA(B) receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA(B) receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, alpha-bungarotoxin. By using the alpha-bungarotoxin binding site-tagged GABA(B) R1a subunit (R1a(BBS)), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, alpha-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA(B) receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.

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

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Yoshiaki Shinohara

2009-07-01

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

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

DEFF Research Database (Denmark)

Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan

2010-01-01

lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice...... and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium...

The Fas/Fas ligand death receptor pathway contributes to phenylalanine-induced apoptosis in cortical neurons.

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Xiaodong Huang

Full Text Available Phenylketonuria (PKU, an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe. A recent study showed that the mitochondria-mediated (intrinsic apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic apoptotic pathway and endoplasmic reticulum (ER stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h, suggesting involvement of the Fas receptor (FasR-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

CB2 cannabinoid receptors contribute to bacterial invasion and mortality in polymicrobial sepsis.

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Balázs Csóka

2009-07-01

Full Text Available Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB(2 receptors on immune cells. Here we examined the role of CB(2 receptors in regulating the host's response to sepsis.The role of CB(2 receptors was studied by subjecting CB(2 receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB(2 receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB(2 receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF-kappaB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB(2 receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b(+ and CD19(+ cells during CLP.Taken together, our results establish for the first time that CB(2 receptors are important contributors to septic immune dysfunction and mortality, indicating that CB(2 receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.

Selective Attention to Visual Stimuli Using Auditory Distractors Is Altered in Alpha-9 Nicotinic Receptor Subunit Knock-Out Mice.

Science.gov (United States)

Terreros, Gonzalo; Jorratt, Pascal; Aedo, Cristian; Elgoyhen, Ana Belén; Delano, Paul H

2016-07-06

During selective attention, subjects voluntarily focus their cognitive resources on a specific stimulus while ignoring others. Top-down filtering of peripheral sensory responses by higher structures of the brain has been proposed as one of the mechanisms responsible for selective attention. A prerequisite to accomplish top-down modulation of the activity of peripheral structures is the presence of corticofugal pathways. The mammalian auditory efferent system is a unique neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear bundle, and it has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear neurons in selective attention paradigms. Here, we trained wild-type and α-9 nicotinic receptor subunit knock-out (KO) mice, which lack cholinergic transmission between medial olivocochlear neurons and outer hair cells, in a two-choice visual discrimination task and studied the behavioral consequences of adding different types of auditory distractors. In addition, we evaluated the effects of contralateral noise on auditory nerve responses as a measure of the individual strength of the olivocochlear reflex. We demonstrate that KO mice have a reduced olivocochlear reflex strength and perform poorly in a visual selective attention paradigm. These results confirm that an intact medial olivocochlear transmission aids in ignoring auditory distraction during selective attention to visual stimuli. The auditory efferent system is a neural network that originates in the auditory cortex and projects to the cochlear receptor through the olivocochlear system. It has been proposed to function as a top-down filter of peripheral auditory responses during attention to cross-modal stimuli. However, to date, there is no conclusive evidence of the involvement of olivocochlear

Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

International Nuclear Information System (INIS)

Curtis, B.M.

1986-01-01

Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [ 3 H]nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed α(Mr 135,000), β(Mr 50,000), and γ(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The α and γ subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the α, β, and γ subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel

Bicarbonate Contributes to GABAA Receptor-Mediated Neuronal Excitation in Surgically-Resected Human Hypothalamic Hamartomas

Science.gov (United States)

Do-Young, Kim; Fenoglio, Kristina A.; Kerrigan, John F.; Rho, Jong M.

2009-01-01

SUMMARY The role of bicarbonate (HCO3-) in GABAA receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABAA receptors with muscimol (30 μM) provoked neuronal excitation in over 70% of large (18-22 μM) HH neurons in HCO3- buffer. Subsequent perfusion of HCO3--free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO3--free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO3- efflux through GABAA receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels. PMID:19022626

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes.

Science.gov (United States)

Masschaele, Delphine; De Ceuninck, Leentje; Wauman, Joris; Defever, Dieter; Stenner, Frank; Lievens, Sam; Peelman, Frank; Tavernier, Jan

2017-01-01

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies.

RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes.

Directory of Open Access Journals (Sweden)

Delphine Masschaele

Full Text Available RNF41 (Ring Finger Protein 41 is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein and the EARP (Endosome-Associated Recycling Protein complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies.

ASIC subunit ratio and differential surface trafficking in the brain.

Science.gov (United States)

Wu, Junjun; Xu, Yuanyuan; Jiang, Yu-Qing; Xu, Jiangping; Hu, Youjia; Zha, Xiang-ming

2016-01-08

Acid-sensing ion channels (ASICs) are key mediators of acidosis-induced responses in neurons. However, little is known about the relative abundance of different ASIC subunits in the brain. Such data are fundamental for interpreting the relative contribution of ASIC1a homomers and 1a/2 heteromers to acid signaling, and essential for designing therapeutic interventions to target these channels. We used a simple biochemical approach and semi-quantitatively determined the molar ratio of ASIC1a and 2 subunits in mouse brain. Further, we investigated differential surface trafficking of ASIC1a, ASIC2a, and ASIC2b. ASIC1a subunits outnumber the sum of ASIC2a and ASIC2b. There is a region-specific variation in ASIC2a and 2b expression, with cerebellum and striatum expressing predominantly 2b and 2a, respectively. Further, we performed surface biotinylation and found that surface ASIC1a and ASIC2a ratio correlates with their total expression. In contrast, ASIC2b exhibits little surface presence in the brain. This result is consistent with increased co-localization of ASIC2b with an ER marker in 3T3 cells. Our data are the first semi-quantitative determination of relative subunit ratio of various ASICs in the brain. The differential surface trafficking of ASICs suggests that the main functional ASICs in the brain are ASIC1a homomers and 1a/2a heteromers. This finding provides important insights into the relative contribution of various ASIC complexes to acid signaling in neurons.

SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

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Oliinyk O. S.

2014-02-01

Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

International Nuclear Information System (INIS)

Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

1988-01-01

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor α subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[γ- 32 P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor β subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins

Olfactory Receptors in Non-Chemosensory Organs: The Nervous System in Health and Disease

NARCIS (Netherlands)

Ferrer, Isidro; Garcia-Esparcia, Paula; Carmona, Margarita; Carro, Eva; Aronica, Eleonora; Kovacs, Gabor G.; Grison, Alice; Gustincich, Stefano

2016-01-01

Olfactory receptors (ORs) and down-stream functional signaling molecules adenylyl cyclase 3 (AC3), olfactory G protein α subunit (Gαolf), OR transporters receptor transporter proteins 1 and 2 (RTP1 and RTP2), receptor expression enhancing protein 1 (REEP1), and UDP-glucuronosyltransferases (UGTs)

Deletion of the GluA1 AMPA receptor subunit impairs recency-dependent object recognition memory

Science.gov (United States)

Sanderson, David J.; Hindley, Emma; Smeaton, Emily; Denny, Nick; Taylor, Amy; Barkus, Chris; Sprengel, Rolf; Seeburg, Peter H.; Bannerman, David M.

2011-01-01

Deletion of the GluA1 AMPA receptor subunit impairs short-term spatial recognition memory. It has been suggested that short-term recognition depends upon memory caused by the recent presentation of a stimulus that is independent of contextual–retrieval processes. The aim of the present set of experiments was to test whether the role of GluA1 extends to nonspatial recognition memory. Wild-type and GluA1 knockout mice were tested on the standard object recognition task and a context-independent recognition task that required recency-dependent memory. In a first set of experiments it was found that GluA1 deletion failed to impair performance on either of the object recognition or recency-dependent tasks. However, GluA1 knockout mice displayed increased levels of exploration of the objects in both the sample and test phases compared to controls. In contrast, when the time that GluA1 knockout mice spent exploring the objects was yoked to control mice during the sample phase, it was found that GluA1 deletion now impaired performance on both the object recognition and the recency-dependent tasks. GluA1 deletion failed to impair performance on a context-dependent recognition task regardless of whether object exposure in knockout mice was yoked to controls or not. These results demonstrate that GluA1 is necessary for nonspatial as well as spatial recognition memory and plays an important role in recency-dependent memory processes. PMID:21378100

Changes in glutamate receptor subunits within the medulla in goats after section of the carotid sinus nerves

Science.gov (United States)

Miller, Justin Robert; Neumueller, Suzanne; Muere, Clarissa; Olesiak, Samantha; Pan, Lawrence; Bukowy, John D.; Daghistany, Asem O.; Hodges, Matthew R.

2014-01-01

The mechanisms which contribute to the time-dependent recovery of resting ventilation and the ventilatory CO2 chemoreflex after carotid body denervation (CBD) are poorly understood. Herein we tested the hypothesis that there are time-dependent changes in the expression of specific AMPA, NMDA, and/or neurokinin-1 (NK1R) receptors within respiratory-related brain stem nuclei acutely or chronically after CBD in adult goats. Brain stem tissues were collected acutely (5 days) or chronically (30 days) after sham or bilateral CBD, immunostained with antibodies targeting AMPA (GluA1 or GluA2), NMDA (GluN1), or NK-1 receptors, and optical density (OD) compared. Physiological measurement confirmed categorization of each group and showed ventilatory effects consistent with bilateral CBD (Miller et al. J Appl Physiol 115: 1088–1098, 2013). Acutely after CBD, GluA1 OD was unchanged or slightly increased, but GluA2 and GluN1 OD were reduced 15–30% within the nucleus tractus solitarius (NTS) and in other medullary respiratory nuclei. Chronically after CBD, GluA1 was reduced (P < 0.05) within the caudal NTS and in other nuclei, but there was significant recovery of GluA2 and GluN1 OD. NK1 OD was not significantly different from control after CBD. We conclude that the initial decrease in GluA2 and GluN1 after CBD likely contributes to hypoventilation and the reduced CO2 chemoreflex. The partial recovery of ventilation and the CO2 chemoreflex after CBD parallel a time-dependent return of these receptors to near control levels but likely depend upon additional initiating and maintenance factors for neuroplasticity. PMID:24790015

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

International Nuclear Information System (INIS)

Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang; Xu Zhihan

2006-01-01

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to β 2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively

Dissecting signaling and functions of adhesion G protein-coupled receptors

DEFF Research Database (Denmark)

Araç, Demet; Aust, Gabriela; Calebiro, Davide

2012-01-01

G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

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

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Francy Johanna Moreno-Pedraza

2010-12-01

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

New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

DEFF Research Database (Denmark)

Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G L

2013-01-01

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage...... a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X...... precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated...

Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity

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Douglas Bledsoe

2017-05-01

Full Text Available N-methyl D-aspartate receptors (NMDAR play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D and two GluN3 (A–B subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD and ligand binding domain (LBD, transmembrane domain (TMD and an intracellular C-terminal domain (CTD. Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - Gi complex

International Nuclear Information System (INIS)

Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S.

1991-01-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K d of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the α and β subunits of G i , respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects

Source contribution analysis of surface particulate polycyclic aromatic hydrocarbon concentrations in northeastern Asia by source–receptor relationships

International Nuclear Information System (INIS)

Inomata, Yayoi; Kajino, Mizuo; Sato, Keiichi; Ohara, Toshimasa; Kurokawa, Jun-ichi; Ueda, Hiromasa; Tang, Ning; Hayakawa, Kazuichi; Ohizumi, Tsuyoshi; Akimoto, Hajime

2013-01-01

We analyzed the source–receptor relationships for particulate polycyclic aromatic hydrocarbon (PAH) concentrations in northeastern Asia using an aerosol chemical transport model. The model successfully simulated the observed concentrations. In Beijing (China) benzo[a]pyren (BaP) concentrations are due to emissions from its own domain. In Noto, Oki and Tsushima (Japan), transboundary transport from northern China (>40°N, 40–60%) and central China (30–40°N, 10–40%) largely influences BaP concentrations from winter to spring, whereas the relative contribution from central China is dominant (90%) in Hedo. In the summer, the contribution from Japanese domestic sources increases (40–80%) at the 4 sites. Contributions from Japan and Russia are additional source of BaP over the northwestern Pacific Ocean in summer. The contribution rates for the concentrations from each domain are different among PAH species depending on their particulate phase oxidation rates. Reaction with O 3 on particulate surfaces may be an important component of the PAH oxidation processes. -- Highlights: •Source–receptor analysis was conducted for investigating PAHs in northeast Asia. •In winter, transboundary transport from China is large contribution in leeward. •Relative contribution from Korea, Japan, and eastern Russia is increased in summer. •This seasonal variation is strongly controlled by the meteorological conditions. •The transport distance is different among PAH species. -- Transboundary transport of PAHs in northeast Asia was investigated by source–receptor analysis

Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

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Lin Tong

2011-09-01

Full Text Available Abstract Background shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT™, which expressed siRNA targeting the FMDV receptor, the porcine integrin αv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF and cell enzyme linked immunosorbent assays (cell ELISA, and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Results Our results indicated successful establishment of the iαv U6 RNAi entry vector and the iαv pLenti6/BLOCK -iT expression vector. The functional titer of obtained virus was 1.0 × 106 TU/mL. To compare with the control and mock group, the iαv-PK-15 group αv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV. Conclusions Iαv-PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.

A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila

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Cassar, Marlène; Issa, Abdul-Raouf; Riemensperger, Thomas; Petitgas, Céline; Rival, Thomas; Coulom, Hélène; Iché-Torres, Magali; Han, Kyung-An; Birman, Serge

2015-01-01

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca2+, also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans. PMID:25158689

High and low nightly running behavior associates with nucleus accumbens N-Methyl-d-aspartate receptor (NMDAR) NR1 subunit expression and NMDAR functional differences.

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Grigsby, Kolter B; Kovarik, Cathleen M; Rottinghaus, George E; Booth, Frank W

2018-04-03

The extent to which N-Methyl-d-aspartate (NMDA) receptors facilitate the motivation to voluntarily wheel-run in rodents has yet to be determined. In so, we utilized female Wistar rats selectively bred to voluntarily run high (HVR) and low (LVR) nightly distances in order to examine if endogenous differences in nucleus accumbens (NAc) NMDA receptor expression and function underlies the propensity for high or low motivation to voluntarily wheel-run. 12-14 week old HVR and LVR females were used to examine: 1.) NAc mRNA and protein expression of NMDA subunits NR1, NR2A and NR2B; 2.) NMDA current responses in isolated medium spiny neurons (MSNs) and 3.) NMDA-evoked dopamine release in an ex vivo preparation of NAc punches. Expectedly, there was a large divergence in nightly running distance and time between HVR and LVR rats. We saw a significantly higher mRNA and protein expression of NR1 in HVR compared to LVR rats, while seeing no difference in the expression of NR2A or NR2B. There was a greater current response to a 500 ms application of 300 μM of NMDA in medium-spiny neurons isolated from the NAc HVR compared to LVR animals. On average, NMDA-evoked punches (50 μM of NMDA for 10 min) taken from HVR rats retained ∼54% of the dopamine content compared to their bilateral non-evoked sides, while evoked punches from LVR animals showed no statistical decrease in dopamine content compared to their non-evoked sides. Collectively, these data suggest a potential link between NAc NR1 subunit expression as well as NMDA function and the predisposition for nightly voluntary running behavior in rats. In light of the epidemic rise in physical inactivity, these findings have the potential to explain a neuro-molecular mechanism that regulates the motivation to be physically active. Copyright © 2018 Elsevier B.V. All rights reserved.

Contributions of sex, testosterone, and androgen receptor CAG repeat number to virtual Morris water maze performance.

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Nowak, Nicole T; Diamond, Michael P; Land, Susan J; Moffat, Scott D

2014-03-01

The possibility that androgens contribute to the male advantage typically found on measures of spatial cognition has been investigated using a variety of approaches. To date, evidence to support the notion that androgens affect spatial cognition in healthy young adults is somewhat equivocal. The present study sought to clarify the association between testosterone (T) and spatial performance by extending measurements of androgenicity to include both measures of circulating T as well as an androgen receptor-specific genetic marker. The aims of this study were to assess the contributions of sex, T, and androgen receptor CAG repeat number (CAGr) on virtual Morris water task (vMWT) performance in a group of healthy young men and women. The hypothesis that men would outperform women on vMWT outcomes was supported. Results indicate that CAGr may interact with T to impact navigation performance and suggest that consideration of androgen receptor sensitivity is an important consideration in evaluating hormone-behavior relationships. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pharmacological analysis of the activation and receptor properties of the tonic GABA(CR current in retinal bipolar cell terminals.

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Stefanie M Jones

Full Text Available GABAergic inhibition in the central nervous system (CNS can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABA(A receptors (GABA(ARs respectively. Retinal bipolar cells (BCs exhibit a tonic current mediated by GABA(CRs in their axon terminal, in addition to synaptic GABA(AR and GABA(CR currents, which strongly regulate BC output. The tonic GABA(CR current in BC terminals (BCTs is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABA(CRs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABA(CR current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABA(CRs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABA(CR ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABA(CRs are thought to be ρ1-ρ2 heteromers. To investigate whether GABA(CRs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABA(ARs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABA(CR currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.

Kinin B1 receptors contributes to acute pain following minor surgery in humans

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Brahim Jaime S

2010-02-01

early phase of inflammation and inflammatory pain. The up-regulation of B1 receptors may contribute to acute inflammatory pain through TRPV1 activation.

Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors

DEFF Research Database (Denmark)

Carlson, B X; Belhage, B; Hansen, Gert Helge

1997-01-01

Da (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however...... that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest...

Photo-antagonism of the GABAA receptor.

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Mortensen, Martin; Iqbal, Favaad; Pandurangan, Arun P; Hannan, Saad; Huckvale, Rosemary; Topf, Maya; Baker, James R; Smart, Trevor G

2014-07-29

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation.

Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

Science.gov (United States)

Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

1994-10-25

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

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Sebastian Ströh

Full Text Available In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57, a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99% and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl. In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50% in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less

Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

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Janssen-Bienhold, Ulrike; Schultz, Konrad; Cimiotti, Kerstin; Weiler, Reto; Willecke, Klaus; Dedek, Karin

2013-01-01

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input

Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

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Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

2018-04-01

Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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Cunyou Zhao

Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

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Babinsky, Valerie N.; Head, Rosie A.; Cranston, Treena; Rust, Nigel; Hobbs, Maurine R.; Heath, Hunter; Thakker, Rajesh V.

2013-01-01

BACKGROUND Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein

The membrane-cytoplasm interface of integrin alpha subunits is critical for receptor latency.

OpenAIRE

Briesewitz, R; Kern, A; Smilenov, L B; David, F S; Marcantonio, E E

1996-01-01

Localization of integrin receptors to focal contact sites occurs upon ligand binding. This activity is latent, since unoccupied integrin receptors do not localize to focal contacts. Deletion analysis has revealed that the alpha cytoplasmic domains is required for the maintenance of integrin receptor latency. Our current hypothesis for the mechanism of integrin post-ligand binding events is that there is a change in relationship of alpha and beta cytoplasmic domains, which overcomes receptor l...

The Distribution of Charged Amino Acid Residues and the Ca2+ Permeability of Nicotinic Acetylcholine Receptors: A Predictive Model

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Sergio Fucile

2017-05-01

Full Text Available Nicotinic acetylcholine receptors (nAChRs are cation-selective ligand-gated ion channels exhibiting variable Ca2+ permeability depending on their subunit composition. The Ca2+ permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca2+-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca2+ permeability of nAChRs is lacking. In the last years the Ca2+ permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca2+ current (Pf, i.e., the percentage of the total current carried by Ca2+ ions. In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca2+ influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca2+ permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio to 0.1% (1:4 ratio, much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family.

Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation.

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Wahyu Surya

Full Text Available The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.

Fractalkine/CX3CL1 engages different neuroprotective responses upon selective glutamate receptor overactivation.

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Clotilde eLauro

2015-01-01

Full Text Available Neuronal death induced by overactivation of N-methyl-d-aspartate receptors (NMDARs is implicated in the pathophysiology of many neurodegenerative diseases such as stroke, epilepsy and traumatic brain injury. This toxic effect is mainly mediated by NR2B-containing extrasynaptic NMDARs, while NR2A-containing synaptic NMDARs contribute to cell survival, suggesting the possibility of therapeutic approaches targeting specific receptor subunits. We report that fractalkine/CX3CL1 protects hippocampal neurons from NMDA-induced cell death with a mechanism requiring the adenosine receptors type 2A (A2AR. This is different from CX3CL1-induced protection from glutamate-induced cell death, that fully depends on A1R and requires in part A3R. We show that CX3CL1 neuroprotection against NMDA excitotoxicity involves D-serine, a co-agonist of NR2A/NMDAR, resulting in cyclic AMP-dependent transcription factor (CREB phosphorylation.

The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.

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Matulis, Christina K; Mayo, Kelly E

2012-08-01

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade.

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Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A; Hamilton, Elisha J; Figtree, Gemma A; Rasmussen, Helge H

2015-09-01

Dysregulated nitric oxide (NO)- and superoxide (O2 (·-))-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na(+)-K(+) pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na(+)-K(+) pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47(phox) to p22(phox) with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na(+)-K(+) pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22(phox) with p47(phox), abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na(+)-K(+) pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na(+)-K(+) pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes. Copyright © 2015 the American Physiological Society.

Differential regulation of glutamate receptors in trigeminal ganglia following masseter inflammation

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Lee, Jongseok; Ro, Jin Y.

2007-01-01

The present study examined whether N-methyl-D-aspartate receptor (NMDAR) and 5-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits and group I metabotropic glutamate receptors (mGluRs) are constitutively expressed in trigeminal ganglia (TG) using Western blot analysis in male Sprague Dawley rats. We then investigated whether experimental induction of masseter inflammation influences glutamate receptor expressions by comparing the protein levels from naïve rats to th...

GABA, its receptors, and GABAergic inhibition in mouse taste buds.

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Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

2011-04-13

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

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Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. (Merck Sharp and Dohme Research Lab., Rahway, NJ (United States))

1991-02-01

The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

Role of the Rubisco small subunit. Final report for period May 1, 1997--April 30,2000

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Spreitzer, Robert J.

2000-10-04

CO{sub 2} and O{sub 2} are mutually competitive at the active site of ribulose-1,5-biphosphate (RuBP) carboxylase/oxygenase (Rubisco). Rubisco contains two subunits, each present in eight copies. The 15-kD small subunit is coded by a family of nuclear RbcS genes. Until now, the role of the small subunit in Rubisco structure or catalytic efficiency is not known. Because of other work in eliminating the two RbcS genes in the green algo Chlamydomonas reinhardtii, it is now possible to address questions about the structure-function relationships of the eukaryotic small subunit. There are three specific aims in this project: (1) Alanine scanning mutagenesis is being used to dissect the importance of the {beta}A/{beta}B loop, a feature unique to the eukaryotic small subunit. (2) Random mutagenesis is being used to identify additional residues or regions of the small subunit that are important for holoenzyme assembly and function. (3) Attempts are being made to express foreign small subunits in Chlamydomonas to examine the contribution of small subunits to holoenzyme assembly, catalytic efficiency, and CO{sub 2}/O{sub 2} specificity.

Engineering defined membrane-embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.

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Hawken, Natalie M; Zaika, Elena I; Nakagawa, Terunaga

2017-10-15

The AMPA-type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid-accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex. During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had

Use of Monoclonal Antibodies to Study the Structural Basis of the Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

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1989-09-30

of chicken neurona .4receptor subunits. Sequences of al and a2 are from Net .Ot al. -l Sequences of a3 and a4 were determintl from clones described...Sucrose gradient analysis of neurona & nicotinic receptors was conducted as follows. Chicken ind rat brain receptors were extracted from crude

Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons

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Jong-Woo Sohn

2016-08-01

Full Text Available Objective: The ventromedial hypothalamic nucleus (VMH regulates energy balance and glucose homeostasis. Leptin and insulin exert metabolic effects via their cognate receptors expressed by the steroidogenic factor 1 (SF1 neurons within the VMH. However, detailed cellular mechanisms involved in the regulation of these neurons by leptin and insulin remain to be identified. Methods: We utilized genetically-modified mouse models and performed patch-clamp electrophysiology experiments to resolve this issue. Results: We identified distinct populations of leptin-activated and leptin-inhibited SF1 neurons. In contrast, insulin uniformly inhibited SF1 neurons. Notably, we found that leptin-activated, leptin-inhibited, and insulin-inhibited SF1 neurons are distinct subpopulations within the VMH. Leptin depolarization of SF1 neuron also required the PI3K p110β catalytic subunit. This effect was mediated by the putative transient receptor potential C (TRPC channel. On the other hand, hyperpolarizing responses of SF1 neurons by leptin and insulin required either of the p110α or p110β catalytic subunits, and were mediated by the putative ATP-sensitive K+ (KATP channel. Conclusions: Our results demonstrate that specific PI3K catalytic subunits are responsible for the acute effects of leptin and insulin on VMH SF1 neurons, and provide insights into the cellular mechanisms of leptin and insulin action on VMH SF1 neurons that regulate energy balance and glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: Cellular mechanism, Conditional knockout mouse, Patch clamp technique, Functional heterogeneity, Homeostasis

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

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Suo, Meng; Wang, Ping

2016-01-01

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

NMDA receptors and memory encoding.

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Morris, Richard G M

2013-11-01

It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

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He, Jiuya; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

2017-08-22

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O , had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes

International Nuclear Information System (INIS)

Okamoto, M.; Karasik, A.; White, M.F.; Kahn, C.R.

1991-01-01

To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with 32 P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein

Motor Skills Training Enhances α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor Subunit mRNA Expression in the Ipsilateral Sensorimotor Cortex and Striatum of Rats Following Intracerebral Hemorrhage.

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Tamakoshi, Keigo; Ishida, Kazuto; Kawanaka, Kentaro; Takamatsu, Yasuyuki; Tamaki, Hiroyuki

2017-10-01

We investigated the effects of acrobatic training (AT) on expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits in the sensorimotor cortex and striatum after intracerebral hemorrhage (ICH). Male Wistar rats were divided into 4 groups: ICH without AT (ICH), ICH with AT (ICH + AT), sham operation without AT (SHAM), and sham operation with AT (SHAM + AT). ICH was induced by collagenase injection into the left striatum. The ICH + AT group performed 5 acrobatic tasks daily on days 4-28 post ICH. Forelimb sensorimotor function was evaluated using the forelimb placing test. On days 14 and 29, mRNA expression levels of AMPAR subunits GluR1-4 were measured by real-time reverse transcription-polymerase chain reaction. Forelimb placing test scores were significantly higher in the ICH + AT group than in the ICH group. Expression levels of all AMPAR subunit mRNAs were significantly higher in the ipsilateral sensorimotor cortex of rats in the ICH + AT group than in that of rats in the ICH group on day 29. GluR3 and GluR4 expression levels were reduced in the ipsilateral striatum of rats in the ICH group compared with that of rats in the SHAM group on day 14. These changes may play a critical role in motor skills training-induced recovery after ICH. Copyright © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

International Nuclear Information System (INIS)

Pearson, R.K.; Miller, L.J.; Powers, S.P.; Hadac, E.M.

1987-01-01

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[ 125 I]iodobenzimidate and N-[4-(4'-azido-3'-[ 125 I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125 I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone

Neuron-specific regulation of class I PI3K catalytic subunits and their dysfunction in brain disorders

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Christina eGross

2014-02-01

Full Text Available The PI3K complex plays important roles in virtually all cells of the body. The enzymatic activity of PI3K to phosphorylate phosphoinositides in the membrane is mediated by a group of catalytic and regulatory subunits. Among those, the class I catalytic subunits, p110α, p110β, p110γ and p110δ, have recently drawn attention in the neuroscience field due to their specific dysregulation in diverse brain disorders. While in non-neuronal cells these catalytic subunits may have partially redundant functions, there is increasing evidence that in neurons their roles are more specialized, and confined to distinct receptor-dependent pathways. This review will summarize the emerging role of class I PI3K catalytic subunits in neurotransmitter-regulated neuronal signaling, and their dysfunction in a variety of neurological diseases, including fragile X syndrome, schizophrenia and epilepsy. We will discuss recent literature describing the use of PI3K subunit-selective inhibitors to rescue brain disease-associated phenotypes in in vitro and animal models. These studies give rise to the exciting prospect that these drugs, originally designed for cancer treatment, may be repurposed as therapeutic drugs for brain disorders in the future.

Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats

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Anibal Pacheco

2017-08-01

Full Text Available Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA and N-methyl-D-aspartate (NMDA receptor subunits in dorsal and ventral hippocampus (VH. Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH. By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.

Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively

DEFF Research Database (Denmark)

Collins, C; Duff, C; Duncan, A M

1993-01-01

to human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy......A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor...

Differential Modulation of GABAA and NMDA Receptors by an α7-nicotinic Acetylcholine Receptor Agonist in Chronic Glaucoma

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Xujiao Zhou

2017-12-01

Full Text Available Presynaptic modulation of γ-aminobutyric acid (GABA release by an alpha7 nicotinic acetylcholine receptor (α7-nAChR agonist promotes retinal ganglion cell (RGC survival and function, as suggested by a previous study on a chronic glaucomatous model from our laboratory. However, the role of excitatory and inhibitory amino acid receptors and their interaction with α7-nAChR in physiological and glaucomatous events remains unknown. In this study, we investigated GABAA and N-methyl-D-aspartate (NMDA receptor activity in control and glaucomatous retinal slices and the regulation of amino acid receptor expression and function by α7-nAChR. Whole-cell patch-clamp recordings from RGCs revealed that the α7-nAChR specific agonist PNU-282987 enhanced the amplitude of currents elicited by GABA and reduced the amplitude of currents elicited by NMDA. The positive modulation of GABAA receptor and the negative modulation of NMDA receptor (NMDAR by PNU-282987-evoked were prevented by pre-administration of the α7-nAChR antagonist methyllycaconitine (MLA. The frequency and the amplitude of glutamate receptor-mediated miniature glutamatergic excitatory postsynaptic currents (mEPSCs were not significantly different between the control and glaucomatous RGCs. Additionally, PNU-282987-treated slices showed no alteration in the frequency or amplitude of mEPSCs relative to control RGCs. Moreover, we showed that expression of the α1 subunit of the GABAA receptor was downregulated and the expression of the NMDAR NR2B subunit was upregulated by intraocular pressure (IOP elevation, and the changes of high IOP were blocked by PNU-282987. In conclusion, retina GABAA and NMDARs are modulated positively and negatively, respectively, by activation of α7-nAChR in in vivo chronic glaucomatous models.

[A study on toxic effects of sodium salicylate on rat cochlear spiral ganglion neurons: dopamine receptors mediate expressions of NMDA and GABAA receptors].

Science.gov (United States)

Wei, Ting-Jia; Chen, Hui-Ying; Huang, Xi; Weng, Jing-Jin; Qin, Jiang-Yuan; Su, Ji-Ping

2017-06-25

The aim of the present study was to observe whether dopamine receptor (DR) was involved in the effects of sodium salicylate (SS) on the expressions of N-methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid (GABA) receptors in rat cochlear spiral ganglion neurons (SGNs). Forty-eight hours after primary culture of rat SGNs, immunofluorescence technique was applied to detect expressions of DR1 and DR2, the two subtypes of dopamine receptors. Western blot was performed to assess NMDA receptor NR1 subunit and GABA A receptor subunit α2 (GABRα2) protein expressions in the SGNs after the treatments of SS alone or in combination with DR antagonists. The results demonstrated that: (1) The DR1 and DR2 were expressed in the bodies and axons of the SGN; (2) After the treatment with SS, the surface protein expressions of GABRα2 and NR1 were decreased by 44.69% and 21.57%, respectively, while the total protein expressions showed no significant changes; (3) Neither SS + SCH23390 (DR1 antagonist) group nor SS + Eticlopride (DR2 antagonist) group showed significant differences in GABRα2 and NR1 surface protein expressions compared with the control group. These results suggest that SS regulates the surface GABA A and NMDA receptors trafficking on SGN, and the mechanism may involve DR mediation.

Binding of ATP by pertussis toxin and isolated toxin subunits

International Nuclear Information System (INIS)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-01-01

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [ 3 H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of [ 3 H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [ 3 H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site

Binding of ATP by pertussis toxin and isolated toxin subunits

Energy Technology Data Exchange (ETDEWEB)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L. (Center for Biologics Evaluation and Research, Bethesda, MD (USA))

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

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Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

2008-09-01

We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

Structure and organization of heteromeric AMPA-type glutamate receptors.

Science.gov (United States)

Herguedas, Beatriz; García-Nafría, Javier; Cais, Ondrej; Fernández-Leiro, Rafael; Krieger, James; Ho, Hinze; Greger, Ingo H

2016-04-29

AMPA-type glutamate receptors (AMPARs), which are central mediators of rapid neurotransmission and synaptic plasticity, predominantly exist as heteromers of the subunits GluA1 to GluA4. Here we report the first AMPAR heteromer structures, which deviate substantially from existing GluA2 homomer structures. Crystal structures of the GluA2/3 and GluA2/4 N-terminal domains reveal a novel compact conformation with an alternating arrangement of the four subunits around a central axis. This organization is confirmed by cysteine cross-linking in full-length receptors, and it permitted us to determine the structure of an intact GluA2/3 receptor by cryogenic electron microscopy. Two models in the ligand-free state, at resolutions of 8.25 and 10.3 angstroms, exhibit substantial vertical compression and close associations between domain layers, reminiscent of N-methyl-D-aspartate receptors. Model 1 resembles a resting state and model 2 a desensitized state, thus providing snapshots of gating transitions in the nominal absence of ligand. Our data reveal organizational features of heteromeric AMPARs and provide a framework to decipher AMPAR architecture and signaling. Copyright © 2016, American Association for the Advancement of Science.

Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase

International Nuclear Information System (INIS)

Verkman, A.S.; Skorecki, K.L.; Ausiello, D.A.

1986-01-01

Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone

Mice with subtle reduction of NMDA NR1 receptor subunit expression have a selective decrease in mismatch negativity: Implications for schizophrenia prodromal population.

Science.gov (United States)

Featherstone, Robert E; Shin, Rick; Kogan, Jeffrey H; Liang, Yuling; Matsumoto, Mitsuyuki; Siegel, Steven J

2015-01-01

Reductions in glutamate function are regarded as an important contributory factor in schizophrenia. However, there is a paucity of animal models characterized by developmental and sustained reductions in glutamate function. Pharmacological models using NMDA antagonists have been widely used but these typically produce only transient changes in behavior and brain function. Likewise, mice with homozygous constitutive reductions in glutamate receptor expression show stable brain and behavioral changes, but many of these phenotypes are more severe than the human disease. The current study examines a variety of schizophrenia-related EEG measures in mice with a heterozygous alteration of the NMDA receptor NR1 subunit gene (NR1) that is known to result in reduced NR1 receptor expression in the homozygous mouse (NR1-/-). (NR1+/-) mice showed a 30% reduction in NR1 receptor expression and were reared after weaning in either group or isolated conditions. Outcome measures include the response to paired white noise stimuli, escalating inter-stimulus intervals (ISIs) and deviance-related mismatch negativity (MMN). In contrast to what has been reported in (NR1-/-) mice and mice treated with NMDA antagonists, (NR1+/-) mice showed no change on obligatory Event Related Potential (ERP) measures including the murine P50 and N100 equivalents (P20 and N40), or measures of baseline or evoked gamma power. Alternatively, (NR1+/-) mice showed a marked reduction in response to a deviant auditory tone during MMN task. Data suggest that EEG response to deviant, rather than static, stimuli may be more sensitive for detecting subtle changes in glutamate function. Deficits in these heterozygous NR1 knockdown mice are consistent with data demonstrating MMN deficits among family members of schizophrenia patients and among prodromal patients. Therefore, the current study suggests that (NR1+/-) mice may be among the most sensitive models for increased vulnerability to schizophrenia. Copyright �

Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

Science.gov (United States)

Kokkinos, Petros A; Koutsoukos, Petros G; Deligianni, Despina D

2012-06-01

Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

Targeting of liposomes to cells bearing nerve growth factor receptors mediated by biotinylated NGF

International Nuclear Information System (INIS)

Rosenberg, M.B.

1986-01-01

Previous studies of liposome targeting have concentrated on immunological systems, the use of ligand-receptor interactions has received little attention. The protein hormone beta-nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C-bio-NGF) under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution to produce derivatives with ratios of 1, 2 and 4 biotin moieties per NGF subunit (N-bio-NGF). These derivatives were compared with native NGF for their ability to compete with 125 I-NGF for binding to NGF receptors on rat pheochromocytoma (PC 12) cells at 4 0 C. C-bio-NGF was as effective as native NGF in binding to NGF receptors, while N-bio-NGF containing 1 biotin per NGF subunit was only 28% as active in binding as native NGF. C-bio-NGF, but not N-bio-NGF, mediated the specific binding of 125 I-streptavidin to PC12 cells. Biocytin-NGF, a derivative of C-bio-NGF with an extended spacer chain, was also synthesized and retained full biological and receptor binding activities. C-bio-NGF and biocytin-NGF were as effective as native NGF in a bioassay involving induction of neurite outgrowth from PC12 cells

Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABA(A) receptors.

Science.gov (United States)

Blednov, Y A; Borghese, C M; McCracken, M L; Benavidez, J M; Geil, C R; Osterndorff-Kahanek, E; Werner, D F; Iyer, S; Swihart, A; Harrison, N L; Homanics, G E; Harris, R A

2011-01-01

GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

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Graumann Peter

2009-11-01

Full Text Available Abstract Background The signal recognition particle (SRP receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting. Results In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo. Conclusion The exact function of the SRP receptor (SR in bacteria has so far been enigmatic. Our data show that the bacterial SR is

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

International Nuclear Information System (INIS)

Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

1986-01-01

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125 I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125 I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125 I-labeled human FSH to testis receptor

Piroxicam inhibits NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B subunit: an in silico study elucidating a novel mechanism of action of the drug.

Science.gov (United States)

Mazumder, Muhammed Khairujjaman; Borah, Anupom

2014-12-01

Hyperactivation of GluN2B subunit containing N-methyl-d-aspartate receptors (NMDARs) significantly contributes to the development of several neurodegenerative diseases through a process called excitotoxicity. NMDARs are voltage-gated Ca2+ channels which when activated lead to excessive influx of Ca2+ into neurons thereby exacerbating several calcium-dependent pathways that cause oxidative stress and apoptosis. Several drugs are presently in use to counter the NMDAR-mediated excitotoxic events among which Ifenprodil and its derivatives are GluN2B selective allosteric antagonists. Certain non-steroidal anti-inflammatory drugs (NSAIDs) have also been reported to inhibit NMDARs and the resultant pathologies. Meanwhile, Piroxicam, which is a NSAID, has been reported to be protective in cerebral ischemia-induced neurodegeneration through various pathways. Since Piroxicam has more number of interacting groups as compared to other NSAIDs and also has structural similarities with Ifenprodil, we thought it prudent that Piroxicam may inhibit NMDARs similar to Ifenprodil. By using molecular docking as a tool, we validated the hypothesis and hereby report for the first time that Piroxicam can inhibit GluN2B containing NMDARs through allosteric mode similar to the well known selective antagonist--Ifenprodil; and thus can be a therapeutic drug for the prevention of excitotoxic neurodegeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biochemical study of multiple drug recognition sites on central benzodiazepine receptors

Energy Technology Data Exchange (ETDEWEB)

Trifiletti, R.R.

1986-01-01

The benzodiazepine receptor complex of mammalian brain possesses recognition sites which mediate (at least in part) the pharmacologic actions of the 1,4-benzodiazepines and barbiturates. Evidence is provided suggesting the existence of least seven distinct drug recognition sites on this complex. Interactions between the various recognition sites have been explored using radioligand binding techniques. This information is utilized to provide a comprehensive scheme for characterizing receptor-active drugs on an anxiolytic-anticonvulsant/proconvulsant continuum using radioligand binding techniques, as well as a comprehensive program for identifying potential endogenous receptor-active substances. Further evidence is provided here supporting the notion of benzodiazepine recognition site heterogeneity. Classical 1,4-benzodiazepines do not appear to differentiate two populations of benzodiazepine receptors in an equilibrium sense, but appear to do so in a kinetic sense. An apparent physical separation of the two receptor subtypes can be achieved by differential solubilization. The benzodiazepine binding subunit can be identified by photoaffinity labeling with the benzodiazepine agonist (/sup 3/H)flunitrazepan. Conditions for reproducible partial proteolytic mapping of (/sup 3/H)flunitrazepam photoaffinity labeled receptors are established. From these maps, it is concluded that there are probably no major differences in the primary sequence of the benzodiazepine binding subunit in various regions of the rat central nervous system.

The adjustment of γ-aminobutyric acidA tonic subunits in Huntington's disease: from transcription to translation to synaptic levels into the neostriatum

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Abraham Rosas-Arellano

2018-01-01

Full Text Available γ-Aminobutyric acid (GABA, plays a key role in all stages of life, also is considered the main inhibitory neurotransmitter. GABA activates two kind of membrane receptors known as GABAA and GABAB, the first one is responsible to render tonic inhibition by pentameric receptors containing α4−6, β3, δ, or ρ1−3 subunits, they are located at perisynaptic and/or in extrasynaptic regions. The biophysical properties of GABAA tonic inhibition have been related with cellular protection against excitotoxic injury and cell death in presence of excessive excitation. On this basis, GABAA tonic inhibition has been proposed as a potential target for therapeutic intervention of Huntington's disease. Huntington's disease is a neurodegenerative disorder caused by a genetic mutation of the huntingtin protein. For experimental studies of Huntington's disease mouse models have been developed, such as R6/1, R6/2, HdhQ92, HdhQ150, as well as YAC128. In all of them, some key experimental reports are focused on neostriatum. The neostriatum is considered as the most important connection between cerebral cortex and basal ganglia structures, its cytology display two pathways called direct and indirect constituted by medium sized spiny neurons expressing dopamine D1 and D2 receptors respectively, they display strong expression of many types of GABAA receptors, including tonic subunits. The studies about of GABAA tonic subunits and Huntington's disease into the neostriatum are rising in recent years, suggesting interesting changes in their expression and localization which can be used as a strategy to delay the cellular damage caused by the imbalance between excitation and inhibition, a hallmark of Huntington's disease.

Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

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Alan eNeely

2014-06-01

Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

Science.gov (United States)

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

Science.gov (United States)

Dixon, C I; Rosahl, T W; Stephens, D N

2008-07-01

Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

Science.gov (United States)

Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

2005-01-01

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

Fucosylation and protein glycosylation create functional receptors for cholera toxin

DEFF Research Database (Denmark)

Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

2015-01-01

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...... in normal human intestinal epithelia and could play a role in cholera....

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

DEFF Research Database (Denmark)

Dietrich, J; Geisler, C

1998-01-01

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosph......The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...

Loss of Ethanol Conditioned Taste Aversion and Motor Stimulation in Knockin Mice with Ethanol-Insensitive α2-Containing GABAA Receptors

Science.gov (United States)

Borghese, C. M.; McCracken, M. L.; Benavidez, J. M.; Geil, C. R.; Osterndorff-Kahanek, E.; Werner, D. F.; Iyer, S.; Swihart, A.; Harrison, N. L.; Homanics, G. E.; Harris, R. A.

2011-01-01

GABA type A receptors (GABAA-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABAA-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705–714, 2004; Pharmacol Biochem Behav 90:95–104, 2008; J Psychiatr Res 42:184–191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism. PMID:20876231

Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

DEFF Research Database (Denmark)

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

1999-01-01

) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H...... nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization...

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

International Nuclear Information System (INIS)

Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

1987-01-01

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor.

Science.gov (United States)

Holleran, Brian J; Domazet, Ivana; Beaulieu, Marie-Eve; Yan, Li Ping; Guillemette, Gaétan; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard

2009-04-15

Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256(6.32)-Q283(6.59) fragment of TMD6 and the A295(7.31)-T321(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C(6.44) and W278C(6.54) and TMD7 mutants L298C(7.34), T302C(7.38), and T303C(7.39) to (125)I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C(6.58) in TMD6 and Y300C(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

Science.gov (United States)

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Principles of agonist recognition in Cys-loop receptors

DEFF Research Database (Denmark)

Lynagh, Timothy Peter; Pless, Stephan Alexander

2014-01-01

, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically...

Contributions of conserved residues at the gating interface of glycine receptors

DEFF Research Database (Denmark)

Pless, Stephan Alexander; Leung, Ada W Y; Galpin, Jason D

2011-01-01

and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218......Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial...

Topological dispositions of lysine α380 and lysine γ486 in the acetylcholine receptor from Torpedo californica

International Nuclear Information System (INIS)

Dwyer, B.P.

1991-01-01

The locations have been determined, with respect to the plasma membrane, of lysine α380 and lysine γ486 in the α subunit and the γ subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the α subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the γ subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [ 3 H]-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine α380 and lysine γ486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine α380 is on the inside surface of a vesicle and lysine γ486 is on the outside surface. Because a majority (85%) of the total binding sites for α-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine α380 and lysine γ486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor