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Sample records for receptor g2 subunit

  1. Deletion of the G2A receptor fails to attenuate experimental autoimmune encephalomyelitis

    PubMed Central

    Osmers, Inga; Smith, Sherry S.; Parks, Brian W.; Yu, Shaohua; Srivastava, Roshni; Wohler, Jillian E.; Barnum, Scott R.; Kabarowski, Janusz H.S.

    2009-01-01

    Lysophosphatidylcholine (LPC) is a chemotactic lysolipid produced during inflammation by the hydrolytic action of phospholipase A2 enzymes. LPC stimulates chemotaxis of T cells in vitro through activation of the G protein-coupled receptor, G2A. This has led to the proposition that G2A contributes to the recruitment of T cells to sites of inflammation and thus promotes chronic inflammatory autoimmune diseases associated with the generation and subsequent tissue infiltration of auto-antigen-specific effector T cells. However, one study suggests that G2A may negatively regulate T cell proliferative responses to antigen receptor engagement and thereby attenuates autoimmunity by reducing the generation of autoreactive T cells. To address the relative contribution of these G2A-mediated effects to the pathophysiology of T cell-mediated autoimmune disease, we examined the impact of G2A inactivation on the onset and severity of murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Wild type (G2A+/+) and G2A-deficient (G2A-/-) C57BL/6J mice exhibited a similar incidence and onset of disease following immunization with MOG35-55 peptide. Disease severity was only moderately reduced in G2A-/- mice. Similar numbers of MOG35-55 specific T cells were generated in secondary lymphoid organs of MOG35-55-immunized G2A+/+ and G2A-/- mice. Comparable numbers of T cells were detected in spinal cords of G2A+/+ and G2A-/- mice. We conclude that the proposed anti-proliferative and chemotactic functions of G2A are not manifested in vivo and therefore therapeutic targeting of G2A is unlikely to be beneficial in the treatment of MS. PMID:19135725

  2. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  3. Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

    SciTech Connect

    York, Joanne . E-mail: joanne.york@umontana.edu; Agnihothram, Sudhakar S. . E-mail: sudhakar.agnihothram@umontana.edu; Romanowski, Victor . E-mail: victor@biol.unlp.edu.ar; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2005-12-20

    The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that {alpha}-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.

  4. The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity.

    PubMed

    Hohmann, Stephan W; Angioni, Carlo; Tunaru, Sorin; Lee, Seungkyu; Woolf, Clifford J; Offermanns, Stefan; Geisslinger, Gerd; Scholich, Klaus; Sisignano, Marco

    2017-03-27

    Chemotherapy-induced peripheral neuropathic pain (CIPN) is a common and severe debilitating side effect of many widely used cytostatics. However, there is no approved pharmacological treatment for CIPN available. Among other substances, oxaliplatin causes CIPN in up to 80% of treated patients. Here, we report the involvement of the G-protein coupled receptor G2A (GPR132) in oxaliplatin-induced neuropathic pain in mice. We found that mice deficient in the G2A-receptor show decreased mechanical hypersensitivity after oxaliplatin treatment. Lipid ligands of G2A were found in increased concentrations in the sciatic nerve and dorsal root ganglia of oxaliplatin treated mice. Calcium imaging and patch-clamp experiments show that G2A activation sensitizes the ligand-gated ion channel TRPV1 in sensory neurons via activation of PKC. Based on these findings, we conclude that targeting G2A may be a promising approach to reduce oxaliplatin-induced TRPV1-sensitization and the hyperexcitability of sensory neurons and thereby to reduce pain in patients treated with this chemotherapeutic agent.

  5. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

    Granger, Adam J; Gray, John A; Lu, Wei; Nicoll, Roger A

    2011-09-01

    In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

  6. Estradiol and Estrogen Receptor Agonists Oppose Oncogenic Actions of Leptin in HepG2 Cells

    PubMed Central

    Shen, Minqian; Shi, Haifei

    2016-01-01

    Obesity is a significant risk factor for certain cancers, including hepatocellular carcinoma (HCC). Leptin, a hormone secreted by white adipose tissue, precipitates HCC development. Epidemiology data show that men have a much higher incidence of HCC than women, suggesting that estrogens and its receptors may inhibit HCC development and progression. Whether estrogens antagonize oncogenic action of leptin is uncertain. To investigate potential inhibitory effects of estrogens on leptin-induced HCC development, HCC cell line HepG2 cells were treated with leptin in combination with 17 β-estradiol (E2), estrogen receptor-α (ER-α) selective agonist PPT, ER-β selective agonist DPN, or G protein-coupled ER (GPER) selective agonist G-1. Cell number, proliferation, and apoptosis were determined, and leptin- and estrogen-related intracellular signaling pathways were analyzed. HepG2 cells expressed a low level of ER-β mRNA, and leptin treatment increased ER-β expression. E2 suppressed leptin-induced HepG2 cell proliferation and promoted cell apoptosis in a dose-dependent manner. Additionally E2 reversed leptin-induced STAT3 and leptin-suppressed SOCS3, which was mainly achieved by activation of ER-β. E2 also enhanced ERK via activating ER-α and GPER and activated p38/MAPK via activating ER-β. To conclude, E2 and its receptors antagonize the oncogenic actions of leptin in HepG2 cells by inhibiting cell proliferation and stimulating cell apoptosis, which was associated with reversing leptin-induced changes in SOCS3/STAT3 and increasing p38/MAPK by activating ER-β, and increasing ERK by activating ER-α and GPER. Identifying roles of different estrogen receptors would provide comprehensive understanding of estrogenic mechanisms in HCC development and shed light on potential treatment for HCC patients. PMID:26982332

  7. Analgesic-antitumor peptide inhibits the migration and invasion of HepG2 cells by an upregulated VGSC β1 subunit.

    PubMed

    Guo, Guili; Cui, Yong; Chen, Hong; Zhang, Lili; Zhao, Mingyi; Chen, Bin; Zhang, Jinghai; Liu, Yanfeng

    2016-03-01

    Analgesic-antitumor peptide (AGAP), one of the scorpion toxin polypeptides, has been shown to have an antitumor activity. Recombinant AGAP (rAGAP) was shown to affect the migration and invasion of HepG2 cells via a voltage-gated sodium channel (VGSC) β1 subunit. The VGSC β1 subunit was validated as a cell adhesion molecule (CAM) in human hepatocellular carcinoma (HCC) cell lines. rAGAP suppresses the migration and invasion of HepG2 cells but has no significant effect of human liver HL7702 cells without β1 subunit expression. rAGAP inhibits the migration and invasion of the cells when the VGSC β1 subunit is overexpressed in HL7702 cells. To explain these findings, VGSC β1 subunit messenger RNA (mRNA) and protein levels were measured. The β1 subunit protein level was upregulated in a dose-dependent manner following treatment with rAGAP while there was no significant change in the mRNA level, so rAGAP might be an active component of the VGSC β1 subunit.

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

    PubMed Central

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

    2016-01-01

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

  9. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  10. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

    Both of the ..cap alpha.. and ..beta.. subunits of intact human follitropin (FSH) were radioiodinated with /sup 125/I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH ..cap alpha.. and ..beta.. subunits as well as the ..cap alpha beta.. dimer changed markedly depending on the concentration of reducing agents. /sup 125/I-FSH (Ka = 1.4 x 10/sup 10/ M/sup -1/), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the /sup 125/I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH ..cap alpha beta.. dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized /sup 125/I-FSH (Ka = 1.12 10/sup 10/ M/sup -1/) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa.

  11. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  12. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  13. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  14. Binding interactions with the complementary subunit of nicotinic receptors.

    PubMed

    Blum, Angela P; Van Arnam, Ethan B; German, Laurel A; Lester, Henry A; Dougherty, Dennis A

    2013-03-08

    The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

  15. The Signal Peptide of the Junín Arenavirus Envelope Glycoprotein Is Myristoylated and Forms an Essential Subunit of the Mature G1-G2 Complex

    PubMed Central

    York, Joanne; Romanowski, Victor; Lu, Min; Nunberg, Jack H.

    2004-01-01

    Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking. PMID:15367645

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

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

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

  17. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

    PubMed

    Boylan, Joan M; Salomon, Arthur R; Tantravahi, Umadevi; Gruppuso, Philip A

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.

  18. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  19. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    SciTech Connect

    Boylan, Joan M.; Salomon, Arthur R.; Tantravahi, Umadevi; Gruppuso, Philip A.

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.

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

    SciTech Connect

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

    2010-07-13

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

  1. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  2. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  3. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  4. Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

    PubMed Central

    Zhou, Xiao-Na; Li, Guang-Ming; Xu, Ying-Chen; Zhao, Tuan-Jie; Wu, Ji-Xiang

    2016-01-01

    Background: Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis. The receptor is overexpressed in hepatocellular carcinoma (HCC), and it is associated with the growth and metastatic spread of tumors. DcR3 holds promises as a new target for the treatment of HCC, but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3. The present work, therefore, examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2. Methods: HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3. After the knockdown of DcR3 was confirmed, cell proliferation, clone formation, ability of migrating across transwell membrane, and wound healing were assessed in vitro. Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied. Comparisons between multiple groups were done using one-way analysis of variance (ANOVA), while pairwise comparisons were performed using Student's t test. P < 0.05 was regarded statistically significant. Results: DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2. Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05). The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05). In addition, the messenger RNA levels of MMP 9, VEGF-C, and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05). Conclusions: Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2. Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC. PMID:27779171

  5. Induction of Fas receptor and Fas ligand by nodularin is mediated by NF-{kappa}B in HepG2 cells

    SciTech Connect

    Feng Gong; Li Ying; Bai Yansheng

    2011-03-15

    Nodularin is a natural toxin with multiple features, including inhibitor of protein phosphatases 1 and 2A as well as tumor initiator and promoter. One unique feature of nodularin is that this chemical is a hepatotoxin. It can accumulate into the liver after contact and lead to severe damage to hepatocyte, such as apoptosis. Fas receptor (Fas) and Fas ligand (FasL) system is a critical signaling network triggering apoptosis. In current study, we investigated whether nodularin can induce Fas and FasL expression in HepG2 cell, a well used in vitro model for the study of human hepatocytes. Our data showed nodularin induced Fas and FasL expression, at both mRNA and protein level, in a time- and dose-dependent manner. We also found nodularin induced apoptosis at the concentration and incubation time that Fas and FasL were significantly induced. Neutralizing antibody to FasL reduced nodularin-induced apoptosis. Further studies demonstrated that nodularin promoted nuclear translocation and activation of p65 subunit of NF-{kappa}B. By applying siRNA targeting p65, which knocked down p65 in HepG2 cells, we successfully impaired the activation of NF-{kappa}B by nodularin. In these p65 knockdown cells, we observed that Fas and FasL expression and apoptosis induced by nodularin were significantly reduced. These findings suggest the induction of Fas and FasL expression and thus cell apoptosis in HepG2 cells by nodularin is mediated through NF-{kappa}B pathway.

  6. Galangin suppresses HepG2 cell proliferation by activating the TGF-β receptor/Smad pathway.

    PubMed

    Wang, Yajun; Wu, Jun; Lin, Biyun; Li, Xv; Zhang, Haitao; Ding, Hang; Chen, Xiaoyi; Lan, Liubo; Luo, Hui

    2014-12-04

    Galangin can suppress hepatocellular carcinoma (HCC) cell proliferation. In this study, we demonstrated that galangin induced autophagy by activating the transforming growth factor (TGF)-β receptor/Smad pathway and increased TGF-β receptor I (RI), TGF-βRII, Smad1, Smad2, Smad3 and Smad4 levels but decreased Smad6 and Smad7 levels. Autophagy induced by galangin appears to depend on the TGF-β receptor/Smad signalling pathway because the down-regulation of Smad4 by siRNA or inhibition of TGF-β receptor activation by LY2109761 blocked galangin-induced autophagy. The down-regulation of Beclin1, autophagy-related gene (ATG) 16L, ATG12 and ATG3 restored HepG2 cell proliferation and prevented galangin-induced apoptosis. Our findings indicate a novel mechanism for galangin-induced autophagy via activation of the TGF-β receptor/Smad pathway. The induction of autophagy thus reflects the anti-proliferation effect of galangin on HCC cells.

  7. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  8. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  9. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  10. Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2012-01-01

    Background Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. Principal Findings We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. Conclusions A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species

  11. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    PubMed

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A

    2000-12-01

    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  12. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  13. Mutation of glycine receptor subunit creates beta-alanine receptor responsive to GABA.

    PubMed

    Schmieden, V; Kuhse, J; Betz, H

    1993-10-08

    The amino acid at position 160 of the ligand-binding subunit, alpha 1, is an important determinant of agonist and antagonist binding to the glycine receptor. Exchange of the neighboring residues, phenylalanine at position 159 and tyrosine at position 161, increased the efficacy of amino acid agonists. Whereas wild-type alpha 1 channels expressed in Xenopus oocytes required 0.7 millimolar beta-alanine for a half-maximal response, the doubly mutated (F159Y,Y161F) alpha 1 subunit had an affinity for beta-alanine (which was more potent than glycine) that was 110-fold that of the wild type. Also, gamma-aminobutyric acid and D-serine, amino acids that do not activate wild-type alpha 1 receptors, efficiently gated the mutant channel. Thus, aromatic hydroxyl groups are crucial for ligand discrimination at inhibitory amino acid receptors.

  14. Subunit-dependent effects of nickel on NMDA receptor channels.

    PubMed

    Marchetti, Carla; Gavazzo, Paola

    2003-10-07

    Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.

  15. Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

    Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.

  16. Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting

    PubMed Central

    Durisic, Nela; Godin, Antoine G.; Wever, Claudia M.; Heyes, Colin D.; Lakadamyali, Melike; Dent, Joseph A.

    2012-01-01

    The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations. PMID:22973015

  17. Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells

    SciTech Connect

    McNutt, Markey C.; Kwon, Hyock Joo; Chen, Chiyuan; Chen, Justin R.; Horton, Jay D.; Lagace, Thomas A.

    2009-07-10

    PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

  18. Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells.

    PubMed

    McNutt, Markey C; Kwon, Hyock Joo; Chen, Chiyuan; Chen, Justin R; Horton, Jay D; Lagace, Thomas A

    2009-04-17

    PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

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

    SciTech Connect

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

    1988-03-01

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

  20. Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits.

    PubMed

    Ariano, M A; Larson, E R; Noblett, K L; Sibley, D R; Levine, M S

    1997-08-01

    The striatal cellular coexpression patterns for the D(1A) and D2 dopamine (DA) receptor subtypes and the ionotropic excitatory amino acid (EAA) subunits of the N-methyl-D-aspartate (NMDA-R1) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (GluR1 and GluR2/3) receptor subunits were examined morphologically. Their coincidence was assessed by visualization of mRNA transcripts, localization of encoded receptor proteins, and binding analysis using concurrently paired methods of fluorescence detection. The findings indicated that 1) mRNA transcripts for both receptor systems were detected in the medium-sized neuron population, and the distribution of receptor message closely reflected protein and binding patterns, with the exception of the GluR1 subunit; 2) both DA receptor mRNA transcripts were coexpressed with each ionotropic EAA receptor subunit examined and with each other, and NMDA and AMPA receptor subunits also showed coincident expression; 3) D(1A) DA receptor protein was detected in neurons which coexpressed EAA subunit proteins; and 4) GluR2/3 and NMDA-R1 subunit proteins were coexpressed in medium-sized neurons which also demonstrated D2 DA receptor binding sites. These findings suggest morphological receptor "promiscuity" since the coexpression patterns between DA and EAA receptors were found in all permutations. The results provide a spatial framework for physiological findings describing functional interactions between the two DA receptor types and between specific DA and EAA receptors in the striatum.

  1. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

    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.

  4. Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

    PubMed

    Dominguez-Perrot, C; Feltz, P; Poulter, M O

    1996-11-15

    1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3

  5. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  6. Lateral mobility and anchoring of recombinant GABAA receptors depend on subunit composition.

    PubMed

    Peran, M; Hicks, B W; Peterson, N L; Hooper, H; Salas, R

    2001-10-01

    The clustering of type A gamma-aminobutyric acid receptors (GABA(A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that the subunits of the GABA(A)R play in determining the receptor's cell surface topography and mobility, the alpha1, beta1, beta3, and gamma2s subunits were transfected into COS7, HEK293, and PC12 cells and the distribution and cell surface mobility of these recombinant receptors were examined. Our results show that alpha1 subunits are retained in the endoplasmic reticulum while beta1 and beta3 subunits are sorted to the plasma membrane where they form clusters. Co-expression and co-assembly of alpha1 and beta3 subunits result in the rescue of intracellular alpha1 subunits, which are transported as alphabeta subunit complexes to the cell surface where they formed clusters. Fluorescence photobleach recovery and single particle tracking of recombinant receptors show that, despite clustering, beta3 subunit homooligomers are mobile within a cell surface domain. Inclusion of alpha1 in beta3 or beta3gamma2s complexes, however, dramatically reduces the receptor's lateral mobility in COS 7 and PC12 cells and anchors GABA(A)Rs on the cell surface, suggesting the formation of a direct link to a component of the cytoskeleton. The mobility of recombinant receptors that include the alpha1 subunit mirrors the mobility of GABA(A)Rs on cell bodies and dendrites of cortical and spinal cord neurons. The results suggest that incorporation of alpha1 subunits give rise to a population of GABA(A)Rs that are immobilized on the cell surface.

  7. MMP-7 cleaves the NR1 NMDA receptor subunit and modifies NMDA receptor function

    PubMed Central

    Szklarczyk, Arek; Ewaleifoh, Osefame; Beique, Jean-Claude; Wang, Yue; Knorr, David; Haughey, Norman; Malpica, Tanya; Mattson, Mark P.; Huganir, Richard; Conant, Katherine

    2008-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that play a role in the inflammatory response. These enzymes have been well studied in the context of cancer biology and inflammation. Recent studies, however, suggest that these enzymes also play roles in brain development and neurodegenerative disease. Select MMPs can target proteins critical to synaptic structure and neuronal survival, including integrins and cadherins. Here, we show that one member of the MMP family, MMP-7, which may be released from cells, including microglia, can target a protein critical to synaptic function. Through analysis of extracts from murine cortical slice preparations, we show that MMP-7 cleaves the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor to generate an N-terminal fragment of ∼65 kDa. Moreover, studies with recombinant protein show that MMP-7-mediated cleavage of NR1 occurs at amino acid 517, which is extracellular and just distal to the first transmembrane domain. Data suggest that NR2A, which shares sequence homology with NR1, is also cleaved following treatment of slices with MMP-7, while select AMPA receptor subunits are not. Consistent with a potential effect of MMP-7 on ligand binding, additional experiments demonstrate that NMDA-mediated calcium flux is significantly diminished by MMP-7 pretreatment of cultures. In addition, the AMPA/NMDA ratio is increased by MMP-7 pretreatment. These data suggest that synaptic function may be altered in neurological conditions associated with increased levels of MMP-7.—Szklarczyk, A., Ewaleifoh, O., Beique, J.-C., Wang, Y., Knorr, D., Haughey, N., Malpica, T., Mattson, M. P., Huganir, R., Conant, K. MMP-7 cleaves the NR1 NMDA receptor subunit and modifies NMDA receptor function. PMID:18644839

  8. Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits.

    PubMed

    Criado, Manuel; Valor, Luis M; Mulet, José; Gerber, Susana; Sala, Salvador; Sala, Francisco

    2012-11-01

    Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.

  9. Expression of functional receptors by the human gamma-aminobutyric acid A gamma 2 subunit.

    PubMed

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-03-02

    gamma-Aminobutyric acid A (GABA(A)) receptors are heteromeric membrane proteins formed mainly by various combinations of alpha, beta, and gamma subunits; and it is commonly thought that the gamma 2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the gamma 2L subunit of the human GABA(A) receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a "run-up" of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl(-) ions. The homomeric gamma 2L receptors were also activated by beta-alanine > taurine > glycine, and, like some types of heteromeric GABA(A) receptors, the gamma 2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human gamma 2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABA(A) receptors in situ require further clarification.

  10. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    PubMed Central

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  11. Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine.

    PubMed

    Fenster, C P; Rains, M F; Noerager, B; Quick, M W; Lester, R A

    1997-08-01

    The influence of alpha and beta subunits on the properties of nicotine-induced activation and desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes was examined. Receptors containing alpha4 subunits were more sensitive to activation by nicotine than alpha3-containing receptors. At low concentrations of nicotine, nAChRs containing beta2 subunits reached near-maximal desensitization more rapidly than beta4-containing receptors. The concentration of nicotine producing half-maximal desensitization was influenced by the particular alpha subunit expressed; similar to results for activation, alpha4-containing receptors were more sensitive to desensitizing levels of nicotine than alpha3-containing receptors. The alpha subunit also influenced the rate of recovery from desensitization; this rate was approximately inversely proportional to the apparent nicotine affinity for the desensitized state. The homomeric alpha7 receptor showed the lowest sensitivity to nicotine for both activation and desensitization; alpha7 nAChRs also demonstrated the fastest desensitization kinetics. These subunit-dependent properties remained in the presence of external calcium, although subtle, receptor subtype-specific effects on both the apparent affinities for activation and desensitization and the desensitization kinetics were noted. These data imply that the subunit composition of various nAChRs determines the degree to which receptors are desensitized and/or activated by tobacco-related levels of nicotine. The subtype-specific balance between receptor activation and desensitization should be considered important when the cellular and behavioral actions of nicotine are interpreted.

  12. Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo

    PubMed Central

    Baer, Kristin; Essrich, Christian; Benson, Jack A.; Benke, Dietmar; Bluethmann, Horst; Fritschy, Jean-Marc; Lüscher, Bernhard

    1999-01-01

    Synaptic localization of γ-aminobutyric acid type A (GABAA) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABAA receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABAA receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABAA receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABAA receptors in wild-type mice. PMID:10536013

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

    SciTech Connect

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

    2008-03-07

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

  14. In Vitro Analysis of CsA-Induced Hepatotoxicity in HepG2 Cell Line: Oxidative Stress and α2 and β1 Integrin Subunits Expression

    PubMed Central

    Mostafavi-Pour, Zohreh; Khademi, Fatemeh; Zal, Fatemeh; Sardarian, Ahmad Reza; Amini, Fatemeh

    2013-01-01

    Background Cyclosporine A (CsA)-induced hepatotoxicity could be due to a reduction in α2β1 integrin expression that may either be from the direct effect of CsA itself or from reactive oxygen species (ROS) overproduction. Objectives In this study we aimed to identify the cellular mechanisms underlying CsA-induced hepatic injury by investigating the activation patterns of the antioxidant enzymes, using HepG2 as an in vitro model. Materials and Methods HepG2 cells were cultured with different concentrations of CsA (0, 0.1, 1, 10 μg/ml) for 72 h. Effect of CsA on, 1) cellular integrity, 2) glutathione reductase (GR) and glutathione peroxidase (GPx) activity, 3) cellular levels of glutathione (GSH), 4) intracellular ROS, 5) ALT and AST activities, 6) urea production and 7) α2β1 integrin expression were assayed. Results CsA treatment demonstrated a dose dependent increase in intracellular levels of ROS, GPx activity and decrease in GSH levels (P<0.05). GR activity was mildly attenuated in 1 and 10 µg/ml concentrations of CsA. Alanine aminotranferase (ALT) and aspartate aminotransferase (AST) levels increased in CsA treated cells, while urea synthesis was significantly decreased following treatment with higher concentrations of CsA (P<0.05). Significant down-regulation of β1integrin expression was observed in 1 and 10 µg/ml CsA treated cells while α2 integrin mRNA was significantly down-regulated in all CsA treated cells. Conclusions The observed reduction of α2β1 integrin expression following CsA treatment could be proposed as a possible pathway of CsA-induced hepatotoxicity. Further studies are required to elucidate whether this attenuated expression is due to the direct effect of CsA or caused by overproduction of ROS. PMID:24082890

  15. Dehydroepiandrosterone-induces miR-21 transcription in HepG2 cells through estrogen receptor β and androgen receptor

    PubMed Central

    Teng, Yun; Litchfield, Lacey M.; Ivanova, Margarita M.; Prough, Russell A.; Clark, Barbara J.; Klinge, Carolyn M.

    2014-01-01

    Although oncomiR miR-21 is highly expressed in liver and overexpressed in hepatocellular carcinoma (HCC), its regulation is uncharacterized. We examined the effect of physiologically relevant nanomolar concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEA-S) on miR-21 expression in HepG2 human hepatoma cells. 10 nM DHEA and DHEA-S increase pri-miR-21 transcription in HepG2 cells. Dietary DHEA increased miR-21 in vivo in mouse liver. siRNA and inhibitor studies suggest that DHEA-S requires desulfation for activity and that DHEA-induced pri-miR-21 transcription involves metabolism to androgen and estrogen receptor (AR and ER) ligands. Activation of ERβ and AR by DHEA metabolites androst-5-ene-3,17-dione (ADIONE), androst-5-ene-3β,17β-diol (ADIOL), dihydrotestosterone (DHT), and 5α-androstane-3β,17β-diol (3β-Adiol) increased miR-21 transcription. DHEA-induced miR-21 increased cell proliferation and decreased Pdcd4 protein, a bona fide miR-21. Estradiol (E2) inhibited miR-21 expression via ERα. DHEA increased ERβ and AR recruitment to the miR-21 promoter within the VMP1/TMEM49 gene, with possible significance in hepatocellular carcinoma. PMID:24845419

  16. A Transmembrane Accessory Subunit that Modulates Kainate-Type Glutamate Receptors

    PubMed Central

    Zhang, Wei; St-Gelais, Fannie; Grabner, Chad P.; Trinidad, Jonathan C.; Sumioka, Akio; Morimoto-Tomita, Megumi; Kim, Kwang S.; Straub, Christoph; Burlingame, Alma L.; Howe, James R.; Tomita, Susumu

    2009-01-01

    SUMMARY Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain. PMID:19217376

  17. Kainate receptor subunit diversity underlying response diversity in retinal Off bipolar cells

    PubMed Central

    Lindstrom, Sarah H; Ryan, David G; Shi, Jun; DeVries, Steven H

    2014-01-01

    Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated ∼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1. PMID:24396054

  18. Dancing partners at the synapse: auxiliary subunits that shape kainate receptor function

    PubMed Central

    Copits, Bryan A.; Swanson, Geoffrey T.

    2012-01-01

    Kainate receptors are a family of ionotropic glutamate receptors whose physiological roles differ from those of other subtypes of glutamate receptors in that they predominantly serve as modulators, rather than mediators, of synaptic transmission. Neuronal kainate receptors exhibit unusually slow kinetic properties that have been difficult to reconcile with the behaviour of recombinant kainate receptors. Recently, however, the neuropilin and tolloid-like 1 (NETO1) and NETO2 proteins were identified as auxiliary kainate receptor subunits that shape both the biophysical properties and synaptic localization of these receptors. PMID:22948074

  19. GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity.

    PubMed

    Gerrow, K; Triller, A

    2014-05-01

    GABABRs have a well-established role in controlling neuronal excitability and presynaptic neurotransmitter release. We examined the role of GABABR activity in modulating the number and lateral diffusion of GABAARs at inhibitory synapses. Changes in diffusion of GABAARs at synapses were observed when subunit heterogeneity was taken into account. While α1-GABAARs were unaffected, α2- and α5-GABAARs showed inverse changes in enrichment and diffusion. The intracellular TM3-4 loop of α2 was sufficient to observe the changes in diffusion by GABABR activity, whereas the loop of α5 was not. The opposing effect on α2- and α5-GABAARs was caused by a competition between GABAARs for binding slots at synapses. Receptor immobilization by cross-linking revealed that α5-GABAAR trapping at synapses is regulated by modulation of α2-GABAAR mobility. Finally, PKC activity was determined to be part of the signaling pathway through which GABABR activity modulates α2-GABAAR diffusion at synapses. These results outline a novel mechanism for tuning inhibitory transmission in a subunit-specific manner, and for the first time describe competition between GABAARs with different subunit compositions for binding slots at synapses.

  20. Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

    PubMed

    Werner, David F; Porcu, Patrizia; Boyd, Kevin N; O'Buckley, Todd K; Carter, Jenna M; Kumar, Sandeep; Morrow, A Leslie

    2016-04-01

    GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

  1. Rabies virus binding to an acetylcholine receptor alpha-subunit peptide.

    PubMed

    Lentz, T L

    1990-04-01

    The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.

  2. Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

    PubMed

    Liu, Xiao-cong; Lian, Wei; Zhang, Liang-jun; Feng, Xin-chan; Gao, Yu; Li, Shao-xue; Liu, Chang; Cheng, Ying; Yang, Long; Wang, Xiao-Juan; Chen, Lei; Wang, Rong-quan; Chai, Jin; Chen, Wen-sheng

    2015-01-01

    Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

  3. Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits.

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2008-07-01

    Presynaptic glutamate release elicits brief waves of membrane depolarization in neurons by activating AMPA receptors. Depending on its precise size and shape, current through AMPA receptors gates downstream processes like NMDA receptor activation and action potential generation. Over a decade of research on AMPA receptor structure and function has identified binding sites on AMPA receptors for agonists, antagonists and allosteric modulators as well as key residues underlying differences in the gating behavior of various AMPA receptor subtypes. However, the recent discovery that AMPA receptors are accompanied in the synaptic membrane by a family of auxiliary subunits known as transmembrane AMPA receptor regulatory proteins (TARPs) has revealed that the kinetics and pharmacology of neuronal AMPA receptors differ in many respects from those predicted by classical studies of AMPA receptors in heterologous systems. Here, we summarize recent work and discuss remaining questions concerning the structure and function of native TARP-AMPA receptor complexes.

  4. Cloning and characterization of the ionotropic GABA receptor subunit ρ1 from pig (Sus scrofa).

    PubMed

    Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Miledi, Ricardo

    2014-01-13

    Since human and pig eyes have remarkably anatomical and physiological similitudes swine models have been broadly used for functional studies and therapeutic research. Recently, a GABAρ-mediated relaxation of retinal vascularity suggested that GABAρ signaling may be used to improve retinal blood flow in vascular-driven impaired vision, and a further molecular characterization of GABAρ receptors would be beneficial. However, none of the GABAρ type subunits from pigs has been yet cloned; Among the 19 subunits that compose the family of GABAA receptors, ρ1-3 subunits are capable of forming homomeric channels. These homomeric receptors are particularly interesting because their pharmacological and kinetic properties are notably different from receptors composed by other GABAA subunits. Here we report the cloning of the GABAρ1subunit from the pig and the functional expression of homomeric channels in Xenopus oocytes. The most notable difference found in the pig GABAρ1 receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41-V58) conserved in the rat and the human. This sequence has a higher nucleotidic match with the transcript variant 2 of the human GABAρ1 subunit. Xenopus oocytes injected with cRNA from the receptor generated currents when exposed to GABA that shared all the characteristics of other GABAρ1 subunits in mammals, including its modulation by dopamine. This study will help to increase the knowledge of the genetics of the pig, further the understanding of this important neurotransmitter receptor family and will shed some light in the evolution of these genes among mammals.

  5. Expression and Characterization of a Potent Long-Acting GLP-1 Receptor Agonist, GLP-1-IgG2σ-Fc

    PubMed Central

    Yang, Yi; Chen, Fang; Wan, Deyou; Liu, Yunhui; Yang, Li; Feng, Hongru; Cui, Xinling; Gao, Xin; Song, Haifeng

    2016-01-01

    Human GLP-1 (glucagon-like peptide-1) can produce a remarkable improvement in glycemic control in patients with type 2 diabetes. However, its clinical benefits are limited by its short half-life, which is less than 2 min because of its small size and rapid enzymatic inactivation by dipeptidyl peptidase IV. We engineered GLP-1-IgG2σ-Fc, a 68-kDa fusion protein linking a variant human GLP-1 (A8G/G26E/R36G) to a human IgG2σ constant heavy-chain. A stably transfected Chinese hamster ovary cell line was obtained using electroporation. Western blotting showed that the expressed protein was immunoreactive to both GLP-1 and IgG antibodies. GLP-1-IgG2σ-Fc stimulated insulin secretion from INS-1 cells in a dose- and glucose-dependent manner and increased insulin mRNA expression. The half-life of GLP-1-IgG2σ-Fc in cynomolgus monkeys was approximately 57.1 ± 4.5 h. In the KKAy mouse model of diabetes, one intraperitoneal injection of GLP-1-IgG2σ-Fc (1 mg/kg) reduced blood glucose levels for 5 days. A 4-week repeat-administration study identified sustained effects on blood glucose levels. Oral glucose tolerance tests conducted at the beginning and end of this 4-week period showed that GLP-1-IgG2σ-Fc produced a stable glucose lowering effect. In addition, KKAy mice treated with GLP-1-IgG2σ-Fc showed statistically significant weight loss from day 23. In conclusion, these properties of GLP-1-IgG2σ-Fc demonstrated that it represented a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes. PMID:27232339

  6. Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2014-01-01

    Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants. PMID:25075297

  7. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    PubMed Central

    Matsuda, K; Buckingham, S D; Freeman, J C; Squire, M D; Baylis, H A; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit. PMID:9504393

  8. The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses.

    PubMed

    Schweizer, Claude; Balsiger, Sylvia; Bluethmann, Horst; Mansuy, Isabelle M; Fritschy, Jean-Marc; Mohler, Hanns; Lüscher, Bernhard

    2003-10-01

    The gamma2 subunit of GABA(A) receptor chloride channels is required for normal channel function and for postsynaptic clustering of these receptors during synaptogenesis. In addition, GABA(A) receptor function is thought to contribute to normal postnatal maturation of neurons. Loss of postsynaptic GABA(A) receptors in gamma2-deficient neurons might therefore reflect a deficit in maturation of neurons due to the reduced channel function. Here, we have used the Cre-loxP strategy to examine the clustering function of the gamma2 subunit at mature synapses. Deletion of the gamma2 subunit in the third postnatal week resulted in loss of benzodiazepine-binding sites and parallel loss of punctate immunoreactivity for postsynaptic GABA(A) receptors and gephyrin. Thus, the gamma2 subunit contributes to postsynaptic localization of GABA(A) receptors and gephyrin by a mechanism that is operant in mature neurons and not limited to immature neurons, most likely through interaction with proteins involved in trafficking of synaptic GABA(A) receptors.

  9. Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

    PubMed

    Radley, Jason J; Farb, Claudia R; He, Yong; Janssen, William G M; Rodrigues, Sarina M; Johnson, Luke R; Hof, Patrick R; LeDoux, Joseph E; Morrison, John H

    2007-02-23

    Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

  10. Direct interaction of Ikaros and Foxp1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia

    PubMed Central

    Roman-Trufero, Mónica; Sabbattini, Pierangela; Ferreiros-Vidal, Isabel; Gerrard, Gareth; Asnafi, Vahid; Macintyre, Elizabeth; Merkenschlager, Matthias; Dillon, Niall

    2016-01-01

    Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL. PMID:27588474

  11. Silencing of the scavenger receptor (Class B - Type 1) gene using siRNA-loaded chitosan nanaoparticles in a HepG2 cell model.

    PubMed

    Farid, Mariane M; Hathout, Rania M; Fawzy, Mahmoud; Abou-Aisha, Khaled

    2014-11-01

    Gene silencing mediated by small interfering RNA (siRNA) has gained increasing interest through the past few decades. However, the partial negative charge and the susceptibility to degradation by nucleases have hampered its use in a naked form. In this study, we investigated the use of chitosan nanoparticles as non-viral delivery carriers of siRNA. As a model target, we selected the scavenger receptor (SR-B1), due to its proposed involvement in hepatitis C virus (HCV) internalization. Low molecular weight (LMW) chitosan nanoparticles were prepared by simple ionic gelation using sodium tripolyphosphate (TPP) as a cross-linking agent; a fixed chitosan and TPP concentration of 0.1% was used, and a chitosan to TPP weight ratios of 3:1, 5:1, and 9:1 were investigated. Nanoparticle uptake efficiency was measured using FITC-labeled chitosan nanoparticles and silencing of scavenger receptor class B type 1 (SR-B1) in HepG2 cell line was tested using Western blot analysis. Nanoparticles produced were spherical in shape with an optimum particle size and distribution. The uptake of FITC-labeled nanoparticles by HepG2 cells was found to be both concentration and time dependent. Furthermore, Western Blot analysis showed that SR-B1 siRNA was able to silence the scavenger receptor for up to 96 h of incubation with HepG2 cells.

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

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

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

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

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  15. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors.

    PubMed

    Kowalski, Maria; Hausmann, Ralf; Schmid, Julia; Dopychai, Anke; Stephan, Gabriele; Tang, Yong; Schmalzing, Günther; Illes, Peter; Rubini, Patrizia

    2015-12-01

    The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.

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

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

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

  17. Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

    PubMed

    Banjerdpongchai, Ratana; Wudtiwai, Benjawan; Khaw-On, Patompong; Rachakhom, Wasitta; Duangnil, Natthachai; Kongtawelert, Prachya

    2016-01-01

    Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

  18. Ischaemia differentially regulates GABAB receptor subunits in organotypic hippocampal slice cultures

    PubMed Central

    Cimarosti, Helena; Kantamneni, Sriharsha; Henley, Jeremy M.

    2012-01-01

    Reduced synaptic inhibition due to dysfunction of ionotropic GABAA receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABAB receptors in these pathological processes has not been extensively investigated. We used oxygen–glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABAB1 and GABAB2 receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABAB2 (~75%), while there was no significant change in the levels of GABAB1 after OGD, but an increase after NMDA treatment (~100%). The GABAB receptor agonist baclofen (100 μM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABAB receptors comprise heterodimers of GABAB1 and GABAB2 subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABAB2 is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions. PMID:19328818

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

    PubMed

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

    2016-02-01

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

  20. Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

    PubMed Central

    Akk, Gustav; Eaton, Megan; Li, Ping; Zheng, Steven; Lo, Joshua; Steinbach, Joe Henry

    2013-01-01

    In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit. PMID:24194945

  1. Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

    PubMed Central

    Zhu, Shujia; Riou, Morgane; Yao, C. Andrea; Carvalho, Stéphanie; Rodriguez, Pamela C.; Bensaude, Olivier; Paoletti, Pierre; Ye, Shixin

    2014-01-01

    Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion. PMID:24715733

  2. Ligand-guided homology modelling of the GABAB2 subunit of the GABAB receptor.

    PubMed

    Freyd, Thibaud; Warszycki, Dawid; Mordalski, Stefan; Bojarski, Andrzej J; Sylte, Ingebrigt; Gabrielsen, Mari

    2017-01-01

    γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, and disturbances in the GABAergic system have been implicated in numerous neurological and neuropsychiatric diseases. The GABAB receptor is a heterodimeric class C G protein-coupled receptor (GPCR) consisting of GABAB1a/b and GABAB2 subunits. Two GABAB receptor ligand binding sites have been described, namely the orthosteric GABA binding site located in the extracellular GABAB1 Venus fly trap domain and the allosteric binding site found in the GABAB2 transmembrane domain. To date, the only experimentally solved three-dimensional structures of the GABAB receptor are of the Venus fly trap domain. GABAB receptor allosteric modulators, however, show great therapeutic potential, and elucidating the structure of the GABAB2 transmembrane domain may lead to development of novel drugs and increased understanding of the allosteric mechanism of action. Despite the lack of x-ray crystal structures of the GABAB2 transmembrane domain, multiple crystal structures belonging to other classes of GPCRs than class A have been released within the last years. More closely related template structures are now available for homology modelling of the GABAB receptor. Here, multiple homology models of the GABAB2 subunit of the GABAB receptor have been constructed using templates from class A, B and C GPCRs, and docking of five clusters of positive allosteric modulators and decoys has been undertaken to select models that enrich the active compounds. Using this ligand-guided approach, eight GABAB2 homology models have been chosen as possible structural representatives of the transmembrane domain of the GABAB2 subunit. To the best of our knowledge, the present study is the first to describe homology modelling of the transmembrane domain of the GABAB2 subunit and the docking of positive allosteric modulators in the receptor.

  3. Ligand-guided homology modelling of the GABAB2 subunit of the GABAB receptor

    PubMed Central

    Freyd, Thibaud; Warszycki, Dawid; Mordalski, Stefan; Bojarski, Andrzej J.; Gabrielsen, Mari

    2017-01-01

    γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, and disturbances in the GABAergic system have been implicated in numerous neurological and neuropsychiatric diseases. The GABAB receptor is a heterodimeric class C G protein-coupled receptor (GPCR) consisting of GABAB1a/b and GABAB2 subunits. Two GABAB receptor ligand binding sites have been described, namely the orthosteric GABA binding site located in the extracellular GABAB1 Venus fly trap domain and the allosteric binding site found in the GABAB2 transmembrane domain. To date, the only experimentally solved three-dimensional structures of the GABAB receptor are of the Venus fly trap domain. GABAB receptor allosteric modulators, however, show great therapeutic potential, and elucidating the structure of the GABAB2 transmembrane domain may lead to development of novel drugs and increased understanding of the allosteric mechanism of action. Despite the lack of x-ray crystal structures of the GABAB2 transmembrane domain, multiple crystal structures belonging to other classes of GPCRs than class A have been released within the last years. More closely related template structures are now available for homology modelling of the GABAB receptor. Here, multiple homology models of the GABAB2 subunit of the GABAB receptor have been constructed using templates from class A, B and C GPCRs, and docking of five clusters of positive allosteric modulators and decoys has been undertaken to select models that enrich the active compounds. Using this ligand-guided approach, eight GABAB2 homology models have been chosen as possible structural representatives of the transmembrane domain of the GABAB2 subunit. To the best of our knowledge, the present study is the first to describe homology modelling of the transmembrane domain of the GABAB2 subunit and the docking of positive allosteric modulators in the receptor. PMID:28323850

  4. EM colocalization of AMPA and NMDA receptor subunits at synapses in rat cerebral cortex.

    PubMed

    Kharazia, V N; Phend, K D; Rustioni, A; Weinberg, R J

    1996-05-24

    Electrophysiology and light microscopy suggest that a single excitatory synapse may use both amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Using immunogold electron microscopy, we here provide direct evidence for colocalization at individual synapses in sensorimotor cortex of adult rats. Colocalization was most commonly observed on dendritic spines; subunits of the two classes of receptors seemed to be independently distributed within the synaptic active zone.

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

    ERIC Educational Resources Information Center

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

    2016-01-01

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

  6. Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits.

    PubMed Central

    Qian, H; Ripps, H

    1999-01-01

    Two of the gamma-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of rho-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the rho-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (rho 1B gamma 2) receptors formed by coassembly of the rho 1B-subunit with the gamma 2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA rho and GABAA receptor subunits. It seems highly likely that coassembly of GABAA and rho-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system. PMID:10643085

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

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

    PubMed

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

    2012-08-01

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

  9. LTP requires a reserve pool of glutamate receptors independent of subunit type.

    PubMed

    Granger, Adam J; Shi, Yun; Lu, Wei; Cerpas, Manuel; Nicoll, Roger A

    2013-01-24

    Long-term potentiation (LTP) of synaptic transmission is thought to be an important cellular mechanism underlying memory formation. A widely accepted model posits that LTP requires the cytoplasmic carboxyl tail (C-tail) of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GluA1. To find the minimum necessary requirement of the GluA1 C-tail for LTP in mouse CA1 hippocampal pyramidal neurons, we used a single-cell molecular replacement strategy to replace all endogenous AMPA receptors with transfected subunits. In contrast to the prevailing model, we found no requirement of the GluA1 C-tail for LTP. In fact, replacement with the GluA2 subunit showed normal LTP, as did an artificially expressed kainate receptor not normally found at these synapses. The only conditions under which LTP was impaired were those with markedly decreased AMPA receptor surface expression, indicating a requirement for a reserve pool of receptors. These results demonstrate the synapse's remarkable flexibility to potentiate with a variety of glutamate receptor subtypes, requiring a fundamental change in our thinking with regard to the core molecular events underlying synaptic plasticity.

  10. Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

    PubMed

    Baer, K; Essrich, C; Balsiger, S; Wick, M J; Harris, R A; Fritschy, J M; Lüscher, B

    2000-07-01

    The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

  11. Genetic disruption of the autism spectrum disorder risk gene PLAUR induces GABAA receptor subunit changes

    PubMed Central

    Eagleson, Kathie L.; Gravielle, Maria C.; SchlueterMcFadyen-Ketchum, Lisa J.; Russek, Shelley J.; Farb, David H.; Levitt, Pat

    2010-01-01

    Disruption of the GABAergic system has been implicated in multiple developmental disorders, including epilepsy, autism spectrum disorder and schizophrenia. The human gene encoding uPAR (PLAUR) has been shown recently to be associated with the risk of autism. The uPAR-/- mouse exhibits a regionally selective reduction in GABAergic interneurons in frontal and parietal regions of the cerebral cortex as well as in the CA1 and dentate gyrus subfields of the hippocampus. Behaviorally, these mice exhibit increased sensitivity to pharmacologically-induced seizures, heightened anxiety, and atypical social behavior. Here, we explore potential alterations in GABAergic circuitry that may occur in the context of altered interneuron development. Analysis of gene expression for 13 GABAA receptor subunits using quantitative real-time PCR indicates seven subunit mRNAs (α1, α2, α3, β2, β3, γ2S and γ2L) of interest. Semi-quantitative in situ hybridization analysis focusing on these subunit mRNAs reveals a complex pattern of potential gene regulatory adaptations. The levels of α2 subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of α3 decrease in frontal cortex and CA1. In contrast, α1 subunit mRNAs are unaltered in any region examined. β2 subunit mRNAs are increased in frontal cortex whereas β3 subunit mRNAs are decreased in parietal cortex. Finally, γ2S subunit mRNAs are increased in parietal cortex while γ2L subunit mRNAs are increased in the dentate gyrus, potentially altering the γ2S:γ2L ratio in these two regions. For all subunits, no changes were observed in forebrain regions where GABAergic interneuron numbers are normal. We propose that disrupted differentiation of GABAergic neurons specifically in frontal and parietal cortices leads to regionally-selective alterations in local circuitry and subsequent adaptive changes in receptor subunit composition. Future electrophysiological studies will be useful in determining how alterations in network

  12. Altered GABA(A) receptor subunit expression and pharmacology in human Angelman syndrome cortex.

    PubMed

    Roden, William H; Peugh, Lindsey D; Jansen, Laura A

    2010-10-15

    The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the beta(3)-alpha(5)-gamma(3) GABA(A) receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABA(A) receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of beta(3)/beta(2) and alpha(5)/alpha(1) subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage-clamp analysis of GABA(A) receptor currents was then performed. Studies of GABA(A) receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the alpha(1)-selective benzodiazepine-site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABA(A) receptor affinity and modulation by neurosteroids were unchanged. This shift in GABA(A) receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder.

  13. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  14. Developmental Expression Patterns of GABAA Receptor Subunits in Layer 3 and 5 Pyramidal Cells of Monkey Prefrontal Cortex.

    PubMed

    Datta, Dibyadeep; Arion, Dominique; Lewis, David A

    2015-08-01

    Cortical pyramidal neuron activity is regulated in part through inhibitory inputs mediated by GABAA receptors. The subunit composition of these receptors confers distinct functional properties. Thus, developmental shifts in subunit expression will likely influence the characteristics of pyramidal cell firing and the functional maturation of processes that depend on these neurons. We used laser microdissection and PCR to quantify postnatal developmental changes in the expression of GABAA receptor subunits (α1, α2, α5, β2, γ2, and δ) in layer 3 pyramidal cells of monkey prefrontal cortex, which are critical for working memory. To determine the specificity of these changes, we examined glutamate receptor subunits (AMPA Glur1 and NMDA Grin1) and conducted the same analyses in layer 5 pyramidal cells. Expression of GABAA receptor subunit mRNAs changed substantially, whereas glutamate receptor subunit changes were modest over postnatal development. Some transcripts (e.g., GABAA α1) progressively increased from birth until adulthood, whereas others (e.g., GABAA α2) declined with age. Changes in some transcripts were present in only one layer (e.g., GABAA δ). The development of GABAA receptor subunit expression in primate prefrontal pyramidal neurons is protracted and subunit- and layer-specific. These trajectories might contribute to the molecular basis for the maturation of working memory.

  15. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

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

    PubMed Central

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

    1990-01-01

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

  17. The expression of kainate receptor subunits in hippocampal astrocytes after experimentally induced status epilepticus.

    PubMed

    Vargas, Jay R; Takahashi, D Koji; Thomson, Kyle E; Wilcox, Karen S

    2013-10-01

    Astrocytes have emerged as active participants of synaptic transmission and are increasingly implicated in neurologic disorders including epilepsy. Adult glial fibrillary acidic protein (GFAP)-positive hippocampal astrocytes are not known for ionotropic glutamate receptor expression under basal conditions. Using a chemoconvulsive status epilepticus (SE) model of temporal lobe epilepsy, we show by immunohistochemistry and colocalization analysis that reactive hippocampal astrocytes express kainate receptor (KAR) subunits after SE. In the CA1 region, GluK1, GluK2/3, GluK4, and GluK5 subunit expression was observed in GFAP-positive astrocytes during the seizure-free or "latent" period 1 week after SE. At 8 weeks after SE, a time after SE when spontaneous behavioral seizures occur, the GluK1 and GluK5 subunits remained expressed at significant levels. Kainate receptor subunit expression was found in astrocytes in the hippocampus and surrounding cortex but not in GFAP-positive astrocytes of striatum, olfactory bulb, or brainstem. To examine hippocampal KAR expression more broadly, astroglial-enriched tissue fractions were prepared from dissected hippocampi and were found to have greater GluK4 expression after SE than controls. These results demonstrate that astrocytes begin to express KARs after seizure activity and suggest that their expression may contribute to the pathophysiology of epilepsy.

  18. Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly.

    PubMed

    Ishino, Tetsuya; Harrington, Adrian E; Zaks-Zilberman, Meirav; Scibek, Jeffery J; Chaiken, Irwin

    2008-05-01

    Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.

  19. Nicotine enhances the cyclic AMP-dependent protein kinase-mediated phosphorylation of alpha4 subunits of neuronal nicotinic receptors.

    PubMed

    Hsu, Y N; Edwards, S C; Wecker, L

    1997-12-01

    Studies determined whether alpha4beta2 or alpha3beta2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha4beta2 and 500 nM for alpha3beta2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha4beta2 receptors were incubated with [gamma-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha4 subunit was present. Phosphorylation of alpha4 subunits of alpha4beta2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing alpha3beta2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha4 and not alpha3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.

  20. GABAA receptor epsilon subunit expression in identified peptidergic neurons of the rat hypothalamus.

    PubMed

    Moragues, Nathalie; Ciofi, Philippe; Lafon, Pierrette; Tramu, Gérard; Garret, Maurice

    2003-03-28

    Dual-labeling immunohistochemical or in situ hybridization studies for the recently cloned epsilon-subunit and several neuropeptides were performed in the rat hypothalamus. We revealed an extensive co-expression (>90%) with hypocretin (Hcrt), oxytocin (OT), the gonadotropin-releasing hormone (GnRH), and the melanin-concentrating hormone (MCH) peptides, whereas occasional co-expression (<10%) with cocaine-amphetamine-regulated transcript (CART) was found. Our results suggest that novel GABA(A) receptor subtypes comprising epsilon-subunit are important for metabolic and neuroendocrine functions.

  1. Long chain acyl-CoA synthetase-3 is a molecular target for peroxisome proliferator-activated receptor delta in HepG2 hepatoma cells.

    PubMed

    Cao, Aiqin; Li, Hai; Zhou, Yue; Wu, Minhao; Liu, Jingwen

    2010-05-28

    ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

  2. Electrophysiological properties and subunit composition of GABAA receptors in patients with gelastic seizures and hypothalamic hamartoma.

    PubMed

    Wu, Jie; Chang, Yongchang; Li, Guohui; Xue, Fenqin; DeChon, Jamie; Ellsworth, Kevin; Liu, Qiang; Yang, Kechun; Bahadroani, Nasim; Zheng, Chao; Zhang, Jianliang; Rekate, Harold; Rho, Jong M; Kerrigan, John F

    2007-07-01

    Abnormalities in GABA(A) receptor structure and/or function have been associated with various forms of epilepsy in both humans and animals. Whether this is true for patients with gelastic seizures and hypothalamic hamartoma (HH) is unknown. In this study, we characterized the pharmacological properties and native subunit composition of GABA(A) receptors on acutely dissociated single neurons from surgically resected HH tissues using patch-clamp, immunocytochemical, and RT-PCR techniques. We found that 1) GABA induced an inward current (I(GABA)) at a holding potential of -60 mV; 2) I(GABA) was mimicked by the GABA(A) receptor agonist muscimol and blocked by the GABA(A) receptor antagonist bicuculline, suggesting that I(GABA) was mediated principally through the GABA(A) receptor; 3) the EC(50) and Hill coefficient derived from the I(GABA) concentration-response curve were 6.8 muM and 1.9, respectively; 4) the current-voltage curve was linear at a reversal potential close to zero; and 5) I(GABA) exhibited low sensitivity to zinc and diazepam but higher sensitivity to pentobarbital and pregnanolone. Additionally, using Xenopus oocytes microtransplanted with normal human hypothalamic tissue, we confirmed that the functional properties of GABA(A) receptors were similar to those seen in small isolated HH neurons. Finally, the expression profile of GABA(A) receptor subunits obtained from normal control human hypothalamic tissue was identical to that from surgically resected human HH tissue. Taken together, our data indicate that GABA(A) receptors on small HH neurons exhibit normal pharmacosensitivity and subunit composition. These findings bear relevance to a broader understanding of inhibitory neurotransmission in human HH tissue.

  3. Differences of AMPA and kainate receptor interactomes identify a novel AMPA receptor auxiliary subunit, GSG1L

    PubMed Central

    Shanks, Natalie F.; Savas, Jeffrey N.; Maruo, Tomohiko; Cais, Ondrej; Hirao, Atsushi; Oe, Souichi; Ghosh, Anirvan; Noda, Yasuko; Greger, Ingo H.; Yates, John R.; Nakagawa, Terunaga

    2012-01-01

    AMPA receptor (AMPA-R) complexes consist of channel forming subunits, GluA1–4 and auxiliary proteins including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. Combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors (KA-Rs), we compared the proteins that co-purify with each receptor type in rat brain. This interactome study identified the majority of known interacting proteins and more importantly, provides novel candidates for further studies. We validate the claudin homologue GSG1L as a novel binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function. PMID:22813734

  4. Receptor-binding domain-based subunit vaccines against MERS-CoV.

    PubMed

    Zhang, Naru; Tang, Jian; Lu, Lu; Jiang, Shibo; Du, Lanying

    2015-04-16

    Development of effective vaccines, in particular, subunit-based vaccines, against emerging Middle East respiratory syndrome (MERS) caused by the MERS coronavirus (MERS-CoV) will provide the safest means of preventing the continuous spread of MERS in humans and camels. This review briefly describes the structure of the MERS-CoV spike (S) protein and its receptor-binding domain (RBD), discusses the current status of MERS vaccine development and illustrates the strategies used to develop RBD-based subunit vaccines against MERS. It also summarizes currently available animal models for MERS-CoV and proposes a future direction for MERS vaccines. Taken together, this review will assist researchers working to develop effective and safe subunit vaccines against MERS-CoV and any other emerging coronaviruses that might cause future pandemics.

  5. Voluntary wheel running modulates glutamate receptor subunit gene expression and stress hormone release in Lewis rats.

    PubMed

    Makatsori, A; Duncko, R; Schwendt, M; Moncek, F; Johansson, B B; Jezova, D

    2003-07-01

    Lewis rats that are known to be addiction-prone, develop compulsive running if they have access to running wheels. The present experiments were aimed 1) to evaluate the activation of stress systems following chronic and acute voluntary wheel running in Lewis rats by measurement of hormone release and gene expression of neuropeptides related to hypothalamic-pituitary-adrenocortical (HPA) axis activity and 2) to test the hypothesis that wheel running as a combined model of addictive behavior and stress exposure is associated with modulation of ionotropic glutamate receptor subunits in the ventral tegmental area. Voluntary running for three weeks but not for one night resulted in a rise in plasma corticosterone and adrenocorticotropic hormone (ACTH) levels (p<0.05) compared to those in control rats. Principal component analysis revealed the relation between POMC gene expression in the intermediate pituitary and running rate. Acute exposure of animals to voluntary wheel running induced a significant decrease in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor GluR1 subunit mRNA levels (p<0.01), while repeated voluntary physical activity increased levels of GluR1 mRNA in the ventral tegmentum (p<0.05). Neither acute nor chronic wheel running influenced N-methyl-D-aspartate (NMDA) receptor subunit NR1 mRNA levels in the ventral tegmental area. Thus, the present study revealed changes in AMPA receptor subunit gene expression in a reward-related brain structure as well as an activation of HPA axis in response to compulsive wheel running in Lewis rats. It may be suggested that hormones of HPA axis and glutamate receptors belong to the factors that substantiate higher vulnerability to addictive behavior.

  6. Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

    SciTech Connect

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

    1985-11-15

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

  7. AMPA receptor/TARP stoichiometry visualized by single-molecule subunit counting.

    PubMed

    Hastie, Peter; Ulbrich, Maximilian H; Wang, Hui-Li; Arant, Ryan J; Lau, Anthony G; Zhang, Zhenjie; Isacoff, Ehud Y; Chen, Lu

    2013-03-26

    Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

  8. Effects of aging and caloric restriction on dentate gyrus synapses and glutamate receptor subunits

    PubMed Central

    Newton, Isabel G.; Forbes, M. Elizabeth; Linville, M. Constance; Pang, Hui; Tucker, Elizabeth M.; Riddle, David R.; Brunso-Bechtold, Judy K.

    2009-01-01

    Caloric restriction (CR) attenuates aging-related degenerative processes throughout the body. It is less clear, however, whether CR has a similar effect in the brain, particularly in the hippocampus, an area important for learning and memory processes that often are compromised in aging. In order to evaluate the effect of CR on synapses across lifespan, we quantified synapses stereologically in the middle molecular layer of the dentate gyrus (DG) of young, middle aged, and old Fischer 344 X Brown Norway rats fed ad libitum (AL) or a CR diet from 4 months of age. The results indicate that synapses are maintained across lifespan in both AL and CR rats. In light of this stability, we addressed whether aging and CR influence neurotransmitter receptor levels by measuring subunits of NMDA (NR1, NR2A, and NR2B) and AMPA (GluR1, GluR2) receptors in the DG of a second cohort of AL and CR rats across lifespan. The results reveal that the NR1 and GluR1 subunits decline with age in AL, but not CR rats. The absence of an aging-related decline in these subunits in CR rats, however, does not arise from increased levels in old CR rats. Instead, it is due to subunit decreases in young CR rats to levels that are sustained in CR rats throughout lifespan, but that are reached in AL rats only in old age. PMID:17433502

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

    SciTech Connect

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

    1994-09-01

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

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

    PubMed Central

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

    2016-01-01

    Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  11. Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

    PubMed

    Bass, Chris; Lansdell, Stuart J; Millar, Neil S; Schroeder, Iris; Turberg, Andreas; Field, Linda M; Williamson, Martin S

    2006-01-01

    As part of a program to monitor the susceptibility of cat flea populations to the insecticide imidacloprid we have examined the cat flea nicotinic acetylcholine receptor, the target site protein of the neonicotinoid group of insecticides. Seven nAChR subunits (six alpha-type and one beta-type) were identified in cat flea using a degenerate PCR-based strategy. Five of these were expressed in vitro by creating chimeras containing the N-terminal ligand-binding domain of the cat flea subunits and the C-terminal region of the Drosophila Dalpha2 (SAD) subunit. Two of the five chimeric subunits, Cfalpha1/Dalpha2 and Cfalpha3/Dalpha2, when co-expressed with rat beta2 in Drosophila S2 cells, showed high-affinity binding of both epibatidine (Kd=1.6+/-0.6 and 0.13+/-0.06nM, respectively), and imidacloprid (Ki=142+/-34 and 28.7+/-2.4nM, respectively). It is likely therefore that Cfalpha1 and Cfalpha3 contribute to nAChR populations in vivo that are sensitive to imidacloprid. The identification of cat flea nAChR subunits that have a high affinity for imidacloprid presents candidate genes in which to look for resistance-associated mutations if target-site resistance to imidacloprid arises in domestic pet flea populations.

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

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

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

  13. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

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

    PubMed Central

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

    1990-01-01

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

  15. Identification of a novel nicotinic acetylcholine receptor structural subunit expressed in goldfish retina

    PubMed Central

    1989-01-01

    A new non-alpha (n alpha) member of the nicotinic acetylcholine receptor (nAChR) gene family designated GFn alpha-2 has been identified in goldfish retina by cDNA cloning. This cDNA clone encodes a protein with structural features common to all nAChR subunits sequenced to date; however, unlike all known alpha-subunits of the receptor, it lacks the cysteine residues believed to be involved in acetylcholine binding. Northern blot analysis shows multiple transcripts hybridizing to the GFn alpha-2 cDNA in goldfish retina but undetectable levels of hybridizable RNA in brain, muscle, or liver. S1 nuclease protection experiments indicate that multiple mRNAs are expressed in retina with regions identical or very similar to the GFn alpha-2 sequence. In situ hybridization shows that the gene encoding GFn alpha-2 is expressed predominantly in the ganglion cell layer of the retina. PMID:2465296

  16. Alterations in Purkinje cell GABAA receptor pharmacology following oxygen and glucose deprivation and cerebral ischemia reveal novel contribution of β1-subunit-containing receptors

    PubMed Central

    Kelley, Melissa H.; Ortiz, Justin; Shimizu, Kaori; Grewal, Himmat; Quillinan, Nidia; Herson, Paco S.

    2013-01-01

    Cerebellar Purkinje cells (PCs) are particularly sensitive to cerebral ischemia, and decreased GABAA receptor function following injury is thought to contribute to PC sensitivity to ischemia-induced excitotoxicity. Here we examined the functional properties of the GABAA receptors that are spared following ischemia in cultured Purkinje cells from rat and in vivo ischemia in mouse. Using subunit-specific positive modulators of GABAA receptors, we observed that oxygen and glucose deprivation (OGD) and cardiac arrest-induced cerebral ischemia cause a decrease in sensitivity to the β2/3-subunit-preferring compound, etomidate. However, sensitivity to propofol, a β-subunit-acting compound that modulates β1–3-subunits, was not affected by OGD. The α/γ-subunit-act-ing compounds, diazepam and zolpidem, were also unaffected by OGD. We performed single-cell reverse transcription–polymerase chain reaction on isolated PCs from acutely dissociated cerebellar tissue and observed that PCs expressed the β1-subunit, contrary to previous reports examining GABAA receptor subunit expression in PCs. GABAA receptor β1-subunit protein was also detected in cultured PCs by western blot and by immunohistochemistry in the adult mouse cerebellum and levels remained unaffected by ischemia. High concentrations of loreclezole (30 µm) inhibited PC GABA-mediated currents, as previously demonstrated with β1-subunit-containing GABAA receptors expressed in heterologous systems. From our data we conclude that PCs express the β1-subunit and that there is a greater contribution of β1-subunit-containing GABAA receptors following OGD. PMID:23176253

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

    PubMed Central

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

    2014-01-01

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

  18. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

    PubMed Central

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  19. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  20. Red wine polyphenolics increase LDL receptor expression and activity and suppress the secretion of ApoB100 from human HepG2 cells.

    PubMed

    Pal, Sebely; Ho, Nerissa; Santos, Carlos; Dubois, Paul; Mamo, John; Croft, Kevin; Allister, Emma

    2003-03-01

    Epidemiologic studies suggest that the consumption of red wine may lower the risk of cardiovascular disease. The cardioprotective effect of red wine has been attributed to the polyphenols present in red wine, particularly resveratrol (a stilbene, with estrogen-like activity), and the flavonoids, catechin, epicatechin, quercetin and phenolic acids such as gallic acid. At present, very little is known about the mechanisms by which red wine phenolic compounds benefit the cardiovascular system. Therefore, the aim of this study was to elucidate whether red wine polyphenolics reduce lipoprotein production and clearance by the liver. Cultured HepG2 cells were incubated in the presence of dealcoholized red wine, alcohol-containing red wine and atorvastatin for 24 h. The apolipoprotien B100 (apoB100) protein (marker of hepatic lipoproteins) was quantified on Western blots with an anti-apoB100 antibody and the enhanced chemiluminescence detection system. Apolipoprotein B100 levels in the cells and that secreted into the media were significantly reduced by 50% in liver cells incubated with alcohol-stripped red wine compared with control cells. This effect of dealcoholized red wine on apoB100 production in HepG2 cells was similar to the effect of atorvastatin. Apo B100 production was significantly attenuated by 30% in cells incubated with alcoholized red wine, suggesting that the alcohol was masking the effect of red wine polyphenolics. Apo B100 production was significantly attenuated by 45% with the polyphenolic compounds resveratrol and quercertin. In addition, dealcoholized and alcoholized red wine and atorvastatin significantly increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA and LDL receptor binding activity relative to controls. Dealcoholized red wine also increased LDL receptor gene expression. Collectively, this study suggests that red wine polyphenolics regulate major pathways involved in lipoprotein metabolism.

  1. Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

    SciTech Connect

    Chen, Yue; Zhang, Shunfen; Zhou, Tianyan; Huang, Chaoqun; McLaughlin, Alicia; Chen, Guangping

    2013-04-15

    Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.

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

    PubMed Central

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

    1989-01-01

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

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

    PubMed

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

    1989-09-01

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

  4. AMPA receptor subunits expression and phosphorylation in cingulate cortex in rats following esophageal acid exposure

    PubMed Central

    BANERJEE, B.; MEDDA, B. K.; POCHIRAJU, S.; KANNAMPALLI, P.; LANG, I. M.; SENGUPTA, J. N.; SHAKER, R.

    2014-01-01

    Background We recently reported an increase in N-methyl-d-aspartate (NMDA) receptor subunit expression and CaMKII-dependent phosphorylation of NR2B in the rostral cingulate cortical (rCC) neurons following esophageal acid exposure in rats. As α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors mediate the fast excitatory transmission and play a critical role in synaptic plasticity, in this study, we investigated the effect of esophageal acid exposure in rats on the expression of AMPA receptor subunits and the involvement of these molecular alterations in acid-induced sensitization of neurons in the anterior cingulate (ACC) and midcingulate (MCC) cortices. Methods In molecular study, we examined GluA1 and GluA2 expression and phosphorylation in membrane preparations and in the isolated postsynaptic densities (PSDs) from rats receiving acute esophageal exposure of either saline (control group) or 0.1 NHCl (experimental group). In electrophysiological study, the effect of selective AMPA receptor (Ca2+ permeable) antagonist IEM-1460 and CaMKII inhibitor KN-93 was tested on responses of cortical neurons during acid infusion to address the underlying molecular mechanism of acid-induced sensitization. Key Results The acid exposure significantly increased expression of GluA1, pGluA1Ser831, and phosphorylated CaMKIIThr286, in the cortical membrane preparations. In isolated PSDs, a significant increase in pGluA1Ser831 was observed in acid-treated rats compared with controls. Microinjection of IEM-1460 or KN-93 near the recording site significantly attenuated acid-induced sensitization of cortical neurons. Conclusions & Inferences The underlying mechanism of acid-induced cortical sensitization involves upregulation and CaMKII-mediated phosphorylation of GluA1. These molecular changes of AMPA receptors subunit GluA1 in the cortical neurons might play an important role in acid-induced esophageal hypersensitivity. PMID:24118589

  5. Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.

    PubMed

    McNutt, Markey C; Lagace, Thomas A; Horton, Jay D

    2007-07-20

    Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the proteinase K subfamily of subtilases, promotes internalization and degradation of low density lipoprotein receptors (LDLRs) after binding the receptor on the surface of hepatocytes. PCSK9 has autocatalytic activity that releases the prodomain at the N terminus of the protein. The prodomain remains tightly associated with the catalytic domain as the complex transits the secretory pathway. It is not known whether enzymatic activity is required for the LDLR-reducing effects of PCSK9. Here we expressed the prodomain together with a catalytically inactive protease domain in cells and purified the protein from the medium. The ability of the catalytically inactive PCSK9 to bind and degrade LDLRs when added to culture medium of human hepatoma HepG2 cells at physiological concentrations was similar to that seen using wild-type protein. Similarly, a catalytic-dead version of a gain-of-function mutant, PCSK9(D374Y), showed no loss of activity compared with a catalytically active counterpart; both proteins displayed approximately 10-fold increased activity in degradation of cell surface LDLRs compared with wild-type PCSK9. We conclude that the ability of PCSK9 to degrade LDLRs is independent of catalytic activity and suggest that PCSK9 functions as a chaperone to prevent LDLR recycling and/or to target LDLRs for lysosomal degradation.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  7. Pharmacological properties of GABAA receptors in rat hypothalamic neurons expressing the epsilon-subunit.

    PubMed

    Sergeeva, Olga A; Andreeva, Nadja; Garret, Maurice; Scherer, Annette; Haas, Helmut L

    2005-01-05

    The pharmacological properties and functional role of native GABA(A) receptors (GABA(A)Rs) were investigated in rat hypothalamic neurons expressing the epsilon-subunit with the help of whole-cell patch-clamp recording and single-cell reverse transcription-PCR. Two cell groups were identified: histaminergic tuberomamillary and orexinergic/hypocretinergic neurons. Approximately 25% of histaminergic and 70% of orexinergic neurons contained mRNA encoding for the epsilon-subunit. Double-immunofluorescence staining revealed a somatic localization of this protein in these two neuronal groups. Constitutive activity, diazepam modulation, fast desensitization of maximal currents, and activation by propofol (6-98 microm) of GABA(A)Rs did not correlate with epsilon-subunit expression. Propofol at 3-12 microm potentiated GABA-mediated currents similarly in all neurons. However, noise variance analysis of GABA-mediated currents enhanced by propofol revealed a significant difference between epsilon-positive and epsilon-negative neurons. The former displayed no difference between control and potentiated responses, and, in the latter, noise was decreased in the presence of propofol. Spontaneous IPSCs recorded in cultured hypothalamic neurons were prolonged in the presence of propofol in all epsilon-negative neurons, whereas propofol-resistant IPSCs were recorded in epsilon-positive cells. The infrequent expression of the epsilon-subunit may be a key factor in the recently discovered central role of the tuberomamillary nucleus in anesthesia.

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

    PubMed Central

    McGee, Thomas P.; Bats, Cécile

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects.

    PubMed

    Breer, H; Kleene, R; Hinz, G

    1985-12-01

    The nicotinic acetylcholine receptor as probed by alpha-bungarotoxin binding has been isolated from detergent-solubilized ganglionic membrane preparations from the insect, Locusta migratoria. The isolation and characterization of the receptor protein was achieved by preparation of membrane fragments, extraction by sodium deoxycholate, centrifugation on sucrose density gradient, affinity chromatography, gel electrophoresis, and immunoblotting. The purified receptor protein migrated as a single band on polyacrylamide when native (Mr = 250,000 to 300,000) but also under denaturing conditions (Mr = 65,000) and cross-reacted with some monoclonal antibodies against the Torpedo receptor. In immunohistochemical approaches using polyclonal antibodies the acetylcholine receptor antigenic sites could topochemically be identified at very distinct zones in the neuropil of locust ganglia. The results suggest that the acetylcholine receptor in the central nervous system of insects represents an oligomeric complex composed of four identical or very similar subunits and thus may represent a prototype of the recently proposed homo-oligomeric ancestral acetylcholine receptor.

  11. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block.

    PubMed

    Dravid, Shashank M; Erreger, Kevin; Yuan, Hongjie; Nicholson, Katherine; Le, Phuong; Lyuboslavsky, Polina; Almonte, Antoine; Murray, Ernest; Mosely, Cara; Barber, Jeremy; French, Adam; Balster, Robert; Murray, Thomas F; Traynelis, Stephen F

    2007-05-15

    We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pK(a) values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (-)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

  12. α4β2 Nicotinic Acetylcholine Receptors: RELATIONSHIPS BETWEEN SUBUNIT STOICHIOMETRY AND FUNCTION AT THE SINGLE CHANNEL LEVEL.

    PubMed

    Mazzaferro, Simone; Bermudez, Isabel; Sine, Steven M

    2017-02-17

    Acetylcholine receptors comprising α4 and β2 subunits are the most abundant class of nicotinic acetylcholine receptor in the brain. They contribute to cognition, reward, mood, and nociception and are implicated in a range of neurological disorders. Previous measurements of whole-cell macroscopic currents showed that α4 and β2 subunits assemble in two predominant pentameric stoichiometries, which differ in their sensitivity to agonists, antagonists, and allosteric modulators. Here we compare agonist-elicited single channel currents from receptors assembled with an excess of either the α4 or β2 subunit, forming receptor populations biased toward one or the other stoichiometry, with currents from receptors composed of five concatemeric subunits in which the subunit stoichiometry is predetermined. Our results associate each subunit stoichiometry with a unique single channel conductance, mean open channel lifetime, and sensitivity to the allosteric potentiator 3-[3-(3-pyridinyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS-9283). Receptors with the composition (α4β2)2α4 exhibit high single channel conductance, brief mean open lifetime, and strong potentiation by NS-9283, whereas receptors with the composition (α4β2)2β2 exhibit low single channel conductance and long mean open lifetime and are not potentiated by NS-9283. Thus single channel current measurements reveal bases for the distinct functional and pharmacological properties endowed by different stoichiometries of α4 and β2 subunits and establish pentameric concatemers as a means to delineate interactions between subunits that confer these properties.

  13. Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

    PubMed Central

    1996-01-01

    Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR. PMID:8707823

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

    PubMed Central

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

    2014-01-01

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

  15. The α5 subunit-containing GABAA receptors contribute to chronic pain

    PubMed Central

    Bravo-Hernández, Mariana; Corleto, José A.; Barragán-Iglesias, Paulino; González-Ramírez, Ricardo; Pineda-Farias, Jorge B.; Felix, Ricardo; Calcutt, Nigel A.; Delgado-Lezama, Rodolfo; Marsala, Martin; Granados-Soto, Vinicio

    2016-01-01

    It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-, Complete Freund’s adjuvant (CFA)- and L5/L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5/L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression (RDD) of the Hofmann reflex. Peripheral and intrathecal pre-treatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15–15 nmol) prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptors mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. α5-GABAA receptors were localized in the dorsal spinal cord and DRG co-labeling with NeuN, CGRP and IB4 suggesting their presence in peptidergic and non-peptidergic neurons. These receptors were found mainly in small- and medium-size neurons. Formalin injection enhanced α5-GABAA receptors fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of RDD. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia. PMID:26545088

  16. The α5 subunit containing GABAA receptors contribute to chronic pain.

    PubMed

    Bravo-Hernández, Mariana; Corleto, José A; Barragán-Iglesias, Paulino; González-Ramírez, Ricardo; Pineda-Farias, Jorge B; Felix, Ricardo; Calcutt, Nigel A; Delgado-Lezama, Rodolfo; Marsala, Martin; Granados-Soto, Vinicio

    2016-03-01

    It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-induced, complete Freund's adjuvant (CFA)-induced and L5 and L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5 and L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression of the Hofmann reflex. Peripheral and intrathecal pretreatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15-15 nmol), prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptor mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. The α5-GABAA receptors were localized in the dorsal spinal cord and DRG colabeling with NeuN, CGRP, and IB4 which suggests their presence in peptidergic and nonpeptidergic neurons. These receptors were found mainly in small and medium sized neurons. Formalin injection enhanced α5-GABAA receptor fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of rate-dependent depression. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    1986-05-01

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

  19. Lupin Peptides Modulate the Protein-Protein Interaction of PCSK9 with the Low Density Lipoprotein Receptor in HepG2 Cells

    NASA Astrophysics Data System (ADS)

    Lammi, Carmen; Zanoni, Chiara; Aiello, Gilda; Arnoldi, Anna; Grazioso, Giovanni

    2016-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recently identified as a new useful target for hypercholesterolemia treatment. This work demonstrates that natural peptides, deriving from the hydrolysis of lupin protein and absorbable at intestinal level, are able to inhibit the protein-protein interaction between PCSK9 and the low density lipoprotein receptor (LDLR). In order to sort out the best potential inhibitors among these peptides, a refined in silico model of the PCSK9/LDLR interaction was developed. Docking, molecular dynamics (MD) simulations and peptide binding energy estimations, by MM-GBSA approach, permitted to select the two best candidates among tested peptides that were synthesized and evaluated for their inhibitory activity. The most active was P5 that induced a concentration dependent inhibition of the PCSK9-LDLR binding, with an IC50 value equal to 1.6 ± 0.33 μM. Tested at a 10 μM concentration, this peptide increased by 66 ± 21.4% the ability of HepG2 cells to take up LDL from the extracellular environment.

  20. Lupin Peptides Modulate the Protein-Protein Interaction of PCSK9 with the Low Density Lipoprotein Receptor in HepG2 Cells

    PubMed Central

    Lammi, Carmen; Zanoni, Chiara; Aiello, Gilda; Arnoldi, Anna; Grazioso, Giovanni

    2016-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recently identified as a new useful target for hypercholesterolemia treatment. This work demonstrates that natural peptides, deriving from the hydrolysis of lupin protein and absorbable at intestinal level, are able to inhibit the protein-protein interaction between PCSK9 and the low density lipoprotein receptor (LDLR). In order to sort out the best potential inhibitors among these peptides, a refined in silico model of the PCSK9/LDLR interaction was developed. Docking, molecular dynamics (MD) simulations and peptide binding energy estimations, by MM-GBSA approach, permitted to select the two best candidates among tested peptides that were synthesized and evaluated for their inhibitory activity. The most active was P5 that induced a concentration dependent inhibition of the PCSK9-LDLR binding, with an IC50 value equal to 1.6 ± 0.33 μM. Tested at a 10 μM concentration, this peptide increased by 66 ± 21.4% the ability of HepG2 cells to take up LDL from the extracellular environment. PMID:27424515

  1. NMDA receptor NR2B subunits contribute to PTZ-kindling-induced hippocampal astrocytosis and oxidative stress.

    PubMed

    Zhu, Xinjian; Dong, Jingde; Shen, Kai; Bai, Ying; Zhang, Yuan; Lv, Xuan; Chao, Jie; Yao, Honghong

    2015-05-01

    The N-methyl-d-aspartate (NMDA) receptor plays an important role in the pathophysiology of several neurological diseases, including epilepsy. The present study investigated the effect of NMDA receptor NR2B subunits on pentylenetetrazole (PTZ)-kindling-induced pathological and biochemical events in mice. Our results showed that PTZ-kindling up-regulates the expression of NMDA receptor NR2B subunits in the hippocampus and that kindled mice were characterized by significant astrocytosis and neuron loss in the hippocampus. Oxidative stress, including excessive malondialdehyde (MDA) production and decreased enzymatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), were detected in the hippocampus after the mice were fully kindled. Additionally, expression of brain-derived neurotrophic factor (BDNF) in the hippocampus was found to be up-regulated in PTZ-kindled mice. However, selectively blocking NMDA receptor NR2B subunits by ifenprodil significantly suppressed PTZ-kindling-induced hippocampal astrocytosis, oxidative stress and neuron loss. Furthermore, blocking NMDA receptor NR2B subunits also abolished PTZ-kindling-induced BDNF expression. These results indicate that NMDA receptor NR2B subunits contribute to epilepsy-associated pathological and biochemical events, including hippocampal astrocytosis, oxidative stress and neuron loss, and these events might be correlated with up-regulation of BDNF expression.

  2. Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease

    PubMed Central

    Aliesky, Holly A.; Chen, Chun-Rong; McLachlan, Sandra M.

    2015-01-01

    Context: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. Objective: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. Design: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. Results: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. Conclusions: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers. PMID:25856215

  3. Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster.

    PubMed

    Perry, Trent; Heckel, David G; McKenzie, John A; Batterham, Philip

    2008-05-01

    Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. We focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but we found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel.

  4. Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties.

    PubMed

    Belujon, P; Baufreton, J; Grandoso, L; Boué-Grabot, E; Batten, T F C; Ugedo, L; Garret, M; Taupignon, A I

    2009-10-01

    Fast inhibitory synaptic transmission in the brain relies on ionotropic GABA(A) receptors (GABA(A)R). Eighteen genes code for GABA(A)R subunits, but little is known about the epsilon subunit. Our aim was to identify the synaptic transmission properties displayed by native receptors incorporating epsilon. Immunogold localization detected epsilon at synaptic sites on locus coeruleus (LC) neurons. In situ hybridization revealed prominent signals from epsilon, and mRNAs, some low beta1 and beta3 signals, and no gamma signal. Using in vivo extracellular and in vitro patch-clamp recordings in LC, we established that neuron firing rates, GABA-activated currents, and mIPSC charge were insensitive to the benzodiazepine flunitrazepam (FLU), in agreement with the characteristics of recombinant receptors including an epsilon subunit. Surprisingly, LC provided binding sites for benzodiazepines, and GABA-induced currents were potentiated by diazepam (DZP) in the micromolar range. A number of GABA(A)R ligands significantly potentiated GABA-induced currents, and zinc ions were only active at concentrations above 1 muM, further indicating that receptors were not composed of only alpha and beta subunits, but included an epsilon subunit. In contrast to recombinant receptors including an epsilon subunit, GABA(A)R in LC showed no agonist-independent opening. Finally, we determined that mIPSCs, as well as ensemble currents induced by ultra-fast GABA application, exhibited surprisingly slow rise times. Our work thus defines the signature of native GABA(A)R with a subunit composition including epsilon: differential sensitivity to FLU and DZP and slow rise time of currents. We further propose that alpha(3,) beta(1/3,) and epsilon subunits compose GABA(A)R in LC.

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

    SciTech Connect

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

    1986-03-05

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

  6. Increased phosphorylation of the NR1 subunit of the NMDA receptor following cerebral ischemia.

    PubMed

    Cheung, H H; Teves, L; Wallace, M C; Gurd, J W

    2001-09-01

    The effects of transient cerebral ischemia on phosphorylation of the NR1 subunit of the NMDA receptor by protein kinase C (PKC) and protein kinase A (PKA) were investigated. Adult rats received 15 min of cerebral ischemia followed by various times of recovery. Phosphorylation was examined by immunoblotting hippocampal homogenates with antibodies that recognized NR1 phosphorylated on the PKC phosphorylation sites Ser890 and Ser896, the PKA phosphorylation site Ser897, or dually phosphorylated on Ser896 and Ser897. The phosphorylation of all sites examined increased following ischemia. The increase in phosphorylation by PKC was greater than by PKA. The ischemia-induced increase in phosphorylation was predominantly associated with the population of NR1 that was insoluble in 1% deoxycholate. Enhanced phosphorylation of NR1 by PKC and PKA may contribute to alterations in NMDA receptor function in the postischemic brain.

  7. Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy.

    PubMed

    Peckys, Diana B; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

    2013-01-01

    Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32-56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

  8. Mechanisms of anabolic androgenic steroid inhibition of mammalian ɛ-subunit-containing GABAA receptors

    PubMed Central

    Jones, Brian L; Whiting, Paul J; Henderson, Leslie P

    2006-01-01

    GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ɛ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ɛ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ɛ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50 ≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ɛ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ɛ-subunit significantly alters the profile of AAS

  9. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

    PubMed

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-07

    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  10. Neural regulation of muscle acetylcholine receptor epsilon- and alpha- subunit gene promoters in transgenic mice

    PubMed Central

    1993-01-01

    The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a

  11. Multiple Thyrotropin β-Subunit and Thyrotropin Receptor-Related Genes Arose during Vertebrate Evolution

    PubMed Central

    Maugars, Gersende; Dufour, Sylvie; Cohen-Tannoudji, Joëlle; Quérat, Bruno

    2014-01-01

    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. PMID:25386660

  12. Subunit-dependent postsynaptic expression of kainate receptors on hippocampal interneurons in area CA1.

    PubMed

    Wondolowski, Joyce; Frerking, Matthew

    2009-01-14

    Kainate receptors (KARs) contribute to postsynaptic excitation in only a select subset of neurons. To define the parameters that specify the postsynaptic expression of KARs, we examined the contribution of KARs to EPSCs on hippocampal interneurons in area CA1. Interneurons in stratum radiatum/lacunosum-moleculare express KARs both with and without the GluR5 subunit, but KAR-mediated EPSCs are generated mainly, if not entirely, by GluR5-containing KARs. Extrasynaptic glutamate spillover profoundly recruits AMPA receptors (AMPARs) with little effect on KARs, indicating that KARs are targeted at the synapse more precisely than AMPARs. However, spontaneous EPSCs with a conventional AMPAR component did not have a resolvable contribution of KARs, suggesting that the KARs that contribute to the evoked EPSCs are at a distinct set of synapses. GluR5-containing KARs on interneurons in stratum oriens do not contribute substantially to the EPSC. We conclude that KARs are localized to synapses by cell type-, synapse-, and subunit-selective mechanisms.

  13. NR2B subunit of the NMDA glutamate receptor regulates appetite in the parabrachial nucleus.

    PubMed

    Wu, Qi; Zheng, Ruimao; Srisai, Dollada; McKnight, G Stanley; Palmiter, Richard D

    2013-09-03

    Diphtheria toxin-mediated, acute ablation of hypothalamic neurons expressing agouti-related protein (AgRP) in adult mice leads to anorexia and starvation within 7 d that is caused by hyperactivity of neurons within the parabrachial nucleus (PBN). Because NMDA glutamate receptors are involved in various synaptic plasticity-based behavioral modifications, we hypothesized that modulation of the NR2A and NR2B subunits of the NMDA receptor in PBN neurons could contribute to the anorexia phenotype. We observed by Western blot analyses that ablation of AgRP neurons results in enhanced expression of NR2B along with a modest suppression of NR2A. Interestingly, systemic administration of LiCl in a critical time window before AgRP neuron ablation abolished the anorectic response. LiCl treatment suppressed NR2B levels in the PBN and ameliorated the local Fos induction that is associated with anorexia. This protective role of LiCl on feeding was blunted in vagotomized mice. Chronic infusion of RO25-6981, a selective NR2B inhibitor, into the PBN recapitulated the role of LiCl in maintaining feeding after AgRP neuron ablation. We suggest that the accumulation of NR2B subunits in the PBN contributes to aphagia in response to AgRP neuron ablation and may be involved in other forms of anorexia.

  14. Triangular G2-Splines

    DTIC Science & Technology

    2000-01-01

    energy [1]. Judging the visual ap- pearance of the surfaces by their isophotes we got the best results with the 340 H. Prautzsch and G. Umlauf Fig. 7...An initial control net (left), parameter lines of the resulting G2-surface (middle), top-view of the surface showing isophotes (right). functional F...construction. The initial triangular control net has an irregular vertex of valence 5. The isophotes confirm that the resulting surface is G 2. Fig. 8

  15. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    PubMed Central

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-01-01

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species. PMID:26460032

  16. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    DOE PAGES

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; ...

    2015-10-12

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysismore » reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. In this paper, we hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and finally suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.« less

  17. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

    SciTech Connect

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K.; Mayer, Mark L.

    2015-10-12

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. In this paper, we hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and finally suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  18. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes.

    PubMed

    Alberstein, Robert; Grey, Richard; Zimmet, Austin; Simmons, David K; Mayer, Mark L

    2015-11-03

    Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.

  19. Regulation of the Nicotinic Receptor Alpha7 Subunit by Chronic Stress and Corticosteroids

    PubMed Central

    Hunter, Richard G.; Bloss, Erik B.; McCarthy, Katharine J.; McEwen, Bruce S.

    2010-01-01

    The α7 subunit of the nicotinic acetylcholine receptor (NAchRα7) is one of the principal brain receptors for nicotine and is thought to be a mediator of nicotine’s pro-cognitive effects. While nicotine is known to interact with the stress axis, little is known about the effect of stress or corticosteroids on the expression in the hippocampus, a brain region important to both cognition and stress reactivity. We examined the effects of chronic (21 day) restraint stress (CRS) and adrenalectomy with hormone replacement with the selective mineralocorticoid receptor (MR) agonist aldosterone, the selective glucocorticoid receptor (GR) agonist RU28,362 or corticosterone for 7 days, on the hippocampal expression of NAchRα7 mRNA and protein, as measured by 125I α-Bungarotoxin autoradiography. We found that CRS increase the levels of NAchRα7 mRNA in the CA1, CA3 and Dentate gyrus while levels of the protein were lowered by the same treatment. Corticosteroid replacement showed a GR specific increase in NAchRα7 mRNA, consistent with a corticosteroid mediated effect of CRS. While the mechanism behind these observations is as yet unclear, they may be neuroprotective against the damaging effects of CRS or an example of adaptation to the allostatic load produced by CRS. PMID:20153739

  20. Early chronic blockade of NR2B subunits and transient activation of NMDA receptors modulate LTP in mouse auditory cortex.

    PubMed

    Mao, Yuting; Zang, Shaoyun; Zhang, Jiping; Sun, Xinde

    2006-02-16

    In the auditory cortex, the properties of NMDA receptors depend primarily on the ratio of NR2A and NR2B subunits. NR2B subunit expression is high at the beginning of critical period and lower in adulthood. Because NMDA receptors are crucial in triggering long-term potentiation (LTP) and long-term depression, developmental or experience-dependent modification of NMDAR subunit composition is likely to influence synaptic plasticity. To examine how NMDA subunit change during postnatal development affect the adult synaptic plasticity, we employed chronic ifenprodil blockade of NR2B subunits and analyzed evoked field potentials in adult C57BL/6 mice auditory cortex (AC). We found that chronic loss of NR2B activity led to a decline in LTP magnitude in the AC of adult mice. Adding NMDA to the artificial cerebrospinal fluid (ACSF) in blocked mice had the opposite effect, producing LTP magnitudes at or exceeding those found in treated or untreated animals. These results suggest that, even in adulthood when NR2B expression is downregulated, these receptor subunits play an important role in experience-dependent plasticity of mouse auditory cortex. Blockade from P60 did not result in any decrease of LTP amplitude, suggesting that chronic block in postnatal period may permanently affect cortical circuits so that they cannot produce significant LTP in adulthood.

  1. Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR.

    PubMed

    Kanno, Yuichiro; Tanuma, Nobuaki; Yazawa, Saki; Zhao, Shuai; Inaba, Miki; Nakamura, Satoshi; Nemoto, Kiyomitsu; Inouye, Yoshio

    2016-08-01

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

  2. Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors

    PubMed Central

    Gibson, Scott K.; Gilman, Alfred G.

    2006-01-01

    Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between α2-adrenergic receptors (α2R) and various combinations of Giα and Gβ subunit isoforms by measuring changes in FRET between Giα–yellow fluorescent protein and cyan fluorescent protein–Gβ chimeras in HeLa cells. All combinations of Giα1, -2, or -3 with Gβ1, -2, or -4 were activated to some degree by endogenous α2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Giα and Gβ subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of α2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express α2a- and α2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the α2aR subtype minimized the differences among coupling preferences for Giα and Gβ isoforms. The molecular properties of each Giα, Gβ, and α2-adrenergic receptor subtype influence signaling efficiency for the α2-adrenergic receptor-mediated signaling pathway. PMID:16371464

  3. Kainate receptor pore‐forming and auxiliary subunits regulate channel block by a novel mechanism

    PubMed Central

    Brown, Patricia M. G. E.; Aurousseau, Mark R. P.; Musgaard, Maria; Biggin, Philip C.

    2016-01-01

    Key points Kainate receptor heteromerization and auxiliary subunits, Neto1 and Neto2, attenuate polyamine ion‐channel block by facilitating blocker permeation.Relief of polyamine block in GluK2/GluK5 heteromers results from a key proline residue that produces architectural changes in the channel pore α‐helical region.Auxiliary subunits exert an additive effect to heteromerization, and thus relief of polyamine block is due to a different mechanism.Our findings have broad implications for work on polyamine block of other cation‐selective ion channels. Abstract Channel block and permeation by cytoplasmic polyamines is a common feature of many cation‐selective ion channels. Although the channel block mechanism has been studied extensively, polyamine permeation has been considered less significant as it occurs at extreme positive membrane potentials. Here, we show that kainate receptor (KAR) heteromerization and association with auxiliary proteins, Neto1 and Neto2, attenuate polyamine block by enhancing blocker permeation. Consequently, polyamine permeation and unblock occur at more negative and physiologically relevant membrane potentials. In GluK2/GluK5 heteromers, enhanced permeation is due to a single proline residue in GluK5 that alters the dynamics of the α‐helical region of the selectivity filter. The effect of auxiliary proteins is additive, and therefore the structural basis of polyamine permeation and unblock is through a different mechanism. As native receptors are thought to assemble as heteromers in complex with auxiliary proteins, our data identify an unappreciated impact of polyamine permeation in shaping the signalling properties of neuronal KARs and point to a structural mechanism that may be shared amongst other cation‐selective ion channels. PMID:26682513

  4. The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor.

    PubMed Central

    Zhu, Y; Pless, M; Inhorn, R; Mathey-Prevot, B; D'Andrea, A D

    1996-01-01

    Cytokines regulate cell growth and differentiation by inducing the expression of specific target genes. We have recently isolated a cytokine-inducible, immediate-early cDNA, DUB-1, that encodes a deubiquitinating enzyme. The DUB-1 mRNA was specifically induced by the receptors for interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5, suggesting a role for the beta common (betac subunit known to be shared by these receptors. In order to identify the mechanism of cytokine induction, we isolated a murine genomic clone for DUB-1 containing a functional promoter region. The DUB-1 gene contains two exons, and the nucleotide sequence of its coding region is identical to the sequence of DUB-1 cDNA. Various regions of the 5' flanking region of the DUB-1 gene were assayed for cytokine-inducible activity. An enhancer region that retains the beta c-specific inducible activity of the DUB-1 gene was identified. Enhancer activity was localized to a 112-bp fragment located 1.4 kb upstream from the ATG start codon. Gel mobility shift assays revealed two specific protein complexes that bound to this minimal enhancer region. One complex was induced by betac signaling, while the other was noninducible. Finally, the membrane-proximal region of human betac was required for DUB-1 induction. In conclusion, DUB-1 is the first example of an immediate-early gene that is induced by a specific subunit of a cytokine receptor. Further analysis of the DUB-1 enhancer element may reveal specific determinants of a betac-specific signaling pathway. PMID:8756639

  5. Immunohistochemical Localization of AMPA Type Glutamate Receptor Subunits in the Striatum of Rhesus Monkey

    PubMed Central

    Deng, Yun-Ping; Shelby, Evan; Reiner, Anton J.

    2010-01-01

    Corticostriatal and thalamostriatal projections utilize glutamate as their neurotransmitter. Their influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1-4 subunits. While the cellular localization of AMPA-type subunits in the basal ganglia has been well characterized in rodents, the cellular localization of AMPA subunits in primate basal ganglia is not. We thus carried out immunohistochemical studies of GluR1-4 distribution in rhesus monkey basal ganglia in conjunction with characterization of each major neuron type. In striatum, about 65% of striatal neurons immunolabeled for GluR1, 75%-79% immunolabeled for GluR2 or GluR2/3, and only 2.5% possessed GluR4. All neurons the large size of cholinergic interneurons (mean diameter 26.1μm) were moderately labeled for GluR1, while all neurons in the size range of parvalbuminergic interneurons (mean diameter 13.8μm) were intensely rich in GluR1. Additionally, somewhat more than half of neurons in the size range of projection neurons (mean diameter 11.6μm) immunolabeled for GluR1, and about one third of these were very rich in GluR1. About half of neurons the size of cholinergic interneurons were immunolabeled for GluR2, and the remainder of the neurons that were immunolabeled for GluR2 coincided with projection neurons in size and shape (GluR2 diameter=10.7μm), indicating that the vast majority of striatal projection neurons possess immunodectible GluR2. Similar results were observed with GluR2/3 immunolabeling. Half of the neurons the size of cholinergic interneurons immunolabeled for GluR4 and seemingly all neurons in the size range of parvalbuminergic interneurons possessed GluR4. These results indicate that AMPA receptor subunit combinations for striatal projection neurons in rhesus monkey are similar to those for the corresponding neuron types in rodents, and thus their AMPA responses to glutamate likely to be similar to those demonstrated

  6. Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating.

    PubMed

    De Rosa, María José; Corradi, Jeremías; Bouzat, Cecilia

    2008-02-01

    The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.

  7. Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons

    PubMed Central

    2014-01-01

    Background Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity. Results Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), β3 (4-fold) and β4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and β4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and β4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund’s adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice. Conclusions

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

    PubMed Central

    Wright, Amanda; Vissel, Bryce

    2012-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are comprised of different combinations of GluA1–GluA4 (also known asGluR1–GluR4 and GluR-A to GluR-D) subunits. The GluA2 subunit is subject to RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Gln; Q), present in the GluA2 gene, to a codon for arginine (Arg; R) found in the mRNA. AMPA receptors are calcium (Ca2+)-permeable if they contain the unedited GluA2(Q) subunit or if they lack the GluA2 subunit. While most AMPA receptors in the brain contain the edited GluA2(R) subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable AMPA receptors are important in synaptic plasticity, learning, and disease. Strong evidence supports the notion that Ca2+-permeable AMPA receptors are usually GluA2-lacking AMPA receptors, with little evidence to date for a significant role of unedited GluA2 in normal brain function. However, recent detailed studies suggest that Ca2+-permeable AMPA receptors containing unedited GluA2 do in fact occur in neurons and can contribute to excitotoxic cell loss, even where it was previously thought that there was no unedited GluA2.This review provides an update on the role of GluA2 RNA editing in the healthy and diseased brain and summarizes recent insights into the mechanisms that control this process. We suggest that further studies of the role of unedited GluA2 in normal brain function and disease are warranted, and that GluA2 editing should be considered as a possible contributing factor when Ca2+-permeable AMPA receptors are observed. PMID:22514516

  9. The diversity of GABA(A) receptor subunit distribution in the normal and Huntington's disease human brain.

    PubMed

    Waldvogel, H J; Faull, R L M

    2015-01-01

    GABA(A) receptors are assembled into pentameric receptor complexes from a total of 19 different subunits derived from a variety of different subunit classes (α1-6, β1-3, γ1-3, δ, ɛ, θ, and π) which surround a central chloride ion channel. GABA(A) receptor complexes are distributed heterogeneously throughout the brain and spinal cord and are activated by the extensive GABAergic inhibitory system. In this chapter, we describe the heterogeneous distribution of six of the most widely distributed subunits (α1, α2, α3, β2,3, and γ2) throughout the human basal ganglia. This review describes the studies we have carried out on the normal and Huntington's disease human basal ganglia using autoradiographic labeling and immunohistochemistry in the human basal ganglia. GABA(A) receptors are known to react to changing conditions in the brain in neurological disorders, especially in Huntington's disease and display a high degree of plasticity which is thought to compensate for loss of function caused by disease. In Huntington's disease, the variable loss of GABAergic medium spiny striatopallidal projection neurons is associated with a loss of GABA(A) receptor subunits in the striosome and/or the matrix compartments of the striatum. By contrast in the globus pallidus, a loss of the GABAergic striatal projection neurons results in a dramatic upregulation of subunits on the large postsynaptic pallidal neurons; this is thought to be a compensatory plastic mechanism resulting from the loss of striatal GABAergic input. Most interestingly, our studies have revealed that the subventricular zone overlying the caudate nucleus contains a variety of proliferating progenitor stem cells that possess a heterogeneity of GABA(A) receptor subunits which may play a role in human brain repair mechanisms.

  10. 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.

    PubMed

    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.

  11. Localisation of GABA(A) receptor epsilon-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the theta-subunit in rat brain.

    PubMed

    Moragues, N; Ciofi, P; Tramu, G; Garret, M

    2002-01-01

    In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA(A) receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA(A) receptor epsilon-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to epsilon with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, epsilon-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, epsilon-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all epsilon-immunoreactive. Also, epsilon-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that epsilon mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of epsilon with that of the recently cloned theta-subunit of the GABA(A) receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No theta-mRNA signals were detected in cholinergic cell-groups. Taken together with previously published evidence of the presence of the alpha3-subunit in monoamine- or acetylcholine-containing systems, our data suggest

  12. cDNA cloning and expression of a gamma-aminobutyric acid A receptor epsilon-subunit in rat brain.

    PubMed

    Moragues, N; Ciofi, P; Lafon, P; Odessa, M F; Tramu, G; Garret, M

    2000-12-01

    A cDNA encoding a GABA(A) receptor subunit was isolated from rat brain. The predicted protein is 70% identical to the human epsilon-subunit. It was recently reported [Sinkkonen et al. (2000), J. Neurosci., 20, 3588-3595] that the rodent epsilon-subunit mRNA encoded an additional sequence ( approximately 400 residues). We provide evidence that human and rat epsilon-subunit are similar in size. The distribution of cells expressing the GABA(A) epsilon-subunit was examined in the rat brain. In situ hybridization histochemistry revealed that epsilon-subunit mRNA is expressed by neurons located in septal and preoptic areas, as well as in various hypothalamic nuclei, including paraventricular, arcuate, dorsomedial and medial tuberal nuclei. The mRNA was also detected in major neuronal groups with broad-range influence, such as the cholinergic (basal nucleus), dopaminergic (substantia nigra compacta), serotonergic (raphe nuclei), and noradrenergic (locus coeruleus) systems. Immunohistochemistry using an affinity-purified antiserum directed towards the N-terminal sequence unique to the rat epsilon-subunit revealed the presence of epsilon-subunit immunoreactivity over the somatodendritic domain of neurons with a distribution closely matching that of mRNA-expressing cells. Moreover, using in situ hybridization, alpha3, theta and epsilon GABA(A) subunit mRNAs were all detected with an overlapping distribution in neurons of the dorsal raphe and the locus coeruleus. Our results suggest that novel GABA(A) receptors may regulate, neuroendocrine and modulatory systems in the brain.

  13. Role of the large cytoplasmic loop of the alpha 7 neuronal nicotinic acetylcholine receptor subunit in receptor expression and function.

    PubMed

    Valor, Luis M; Mulet, José; Sala, Francisco; Sala, Salvador; Ballesta, Juan J; Criado, Manuel

    2002-06-25

    The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus oocyte membrane was tested. Surface nAChR expression was abolished upon removal of sequences at two regions, a 29-amino acid segment close to the N-terminus of the loop (amino acids 297-325) and adjacent to the third transmembrane region and an 11-amino acid segment near the fourth transmembrane region. Some residues (amino acids 317-322) within the 29 amino acids N-terminal segment could be substituted by others but not deleted without loss of expression, suggesting that a certain structure, determined by the number of amino acids rather than by their identity, has to be maintained in this region. The contiguous sequence M323 K324 R325 did not tolerate deletions and substitutions. Removal of the rest of the cytoplasmic loop was not deleterious; even higher expression levels (2-4-fold) were obtained upon large deletions of the loop (Delta399-432 and Delta339-370). High expression levels were observed provided that a minimal sequence of three amino acids (E371, G372, and M373) was present. In addition, some electrophysiological properties of mutant nAChRs were modified. Substitution of the EGM sequence by other protein segments produced a variety of effects, but, in general, insertions were not well tolerated, suggesting the existence of tight structural restrictions in the large cytoplasmic region of the rat alpha7 subunit.

  14. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

    PubMed

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

    2017-03-01

    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  15. The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling.

    PubMed

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas

    2016-05-01

    Antigen-receptor-mediated activation of lymphocytes relies on a signalosome comprising CARMA1 (also known as CARD11), BCL10 and MALT1 (the CBM complex). The CBM activates nuclear factor κB (NF-κB) transcription factors by recruiting the 'linear ubiquitin assembly complex' (LUBAC), and unleashes MALT1 paracaspase activity. Although MALT1 enzyme shapes NF-κB signaling, lymphocyte activation and contributes to lymphoma growth, the identity of its substrates continues to be elucidated. Here, we report that the LUBAC subunit HOIL1 (also known as RBCK1) is cleaved by MALT1 following antigen receptor engagement. HOIL1 is also constitutively processed in the 'activated B-cell-like' (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), which exhibits aberrant MALT1 activity. We further show that the overexpression of MALT1-insensitive HOIL1 mitigates T-cell-receptor-mediated NF-κB activation and subsequent cytokine production in lymphocytes. Thus, our results unveil HOIL1 as a negative regulator of lymphocyte activation cleaved by MALT1. This cleavage could therefore constitute an appealing therapeutic target for modulating immune responses.

  16. Amino-terminal domains of kainate receptors determine the differential dependence on Neto auxiliary subunits for trafficking.

    PubMed

    Sheng, Nengyin; Shi, Yun Stone; Nicoll, Roger A

    2017-01-31

    The kainate receptor (KAR), a subtype of glutamate receptor, mediates excitatory synaptic responses at a subset of glutamatergic synapses. However, the molecular mechanisms underlying the trafficking of its different subunits are poorly understood. Here we use the CA1 hippocampal pyramidal cell, which lacks KAR-mediated synaptic currents, as a null background to determine the minimal requirements for the extrasynaptic and synaptic expression of the GluK2 subunit. We find that the GluK2 receptor itself, in contrast to GluK1, traffics to the neuronal surface and synapse efficiently and the auxiliary subunits Neto1 and Neto2 caused no further enhancement of these two trafficking processes. However, the regulation of GluK2 biophysical properties by Neto proteins is the same as that of GluK1. We further determine that it is the amino-terminal domains (ATDs) of GluK1 and GluK2 that control the strikingly different trafficking properties between these two receptors. Moreover, the ATDs are critical for synaptic expression of heteromeric receptors at mossy fiber-CA3 synapses and also mediate the differential dependence on Neto proteins for surface and synaptic trafficking of GluK1 and GluK2. These results highlight the fundamental differences between the two major KAR subunits and their interplay with Neto auxiliary proteins.

  17. The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.

    PubMed

    Calver, A R; Medhurst, A D; Robbins, M J; Charles, K J; Evans, M L; Harrison, D C; Stammers, M; Hughes, S A; Hervieu, G; Couve, A; Moss, S J; Middlemiss, D N; Pangalos, M N

    2000-01-01

    GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.

  18. Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

    PubMed Central

    Kornstein, M. J.; Asher, O.; Fuchs, S.

    1995-01-01

    Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis. Images Figure 2 Figure 3 PMID:7778671

  19. Amyloid-β effects on synapses and memory require AMPA receptor subunit GluA3

    PubMed Central

    Reinders, Niels R.; Pao, Yvonne; Renner, Maria C.; da Silva-Matos, Carla M.; Lodder, Tessa R.; Malinow, Roberto; Kessels, Helmut W.

    2016-01-01

    Amyloid-β (Aβ) is a prime suspect for causing cognitive deficits during the early phases of Alzheimer’s disease (AD). Experiments in AD mouse models have shown that soluble oligomeric clusters of Aβ degrade synapses and impair memory formation. We show that all Aβ-driven effects measured in these mice depend on AMPA receptor (AMPAR) subunit GluA3. Hippocampal neurons that lack GluA3 were resistant against Aβ-mediated synaptic depression and spine loss. In addition, Aβ oligomers blocked long-term synaptic potentiation only in neurons that expressed GluA3. Furthermore, although Aβ-overproducing mice showed significant memory impairment, memories in GluA3-deficient congenics remained unaffected. These experiments indicate that the presence of GluA3-containing AMPARs is critical for Aβ-mediated synaptic and cognitive deficits. PMID:27708157

  20. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.; Propping, P.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.

  1. Differential agonist sensitivity of glycine receptor alpha2 subunit splice variants.

    PubMed

    Miller, Paul S; Harvey, Robert J; Smart, Trevor G

    2004-09-01

    1. The glycine receptor (GlyR) alpha2A and alpha2B splice variants differ by a dual, adjacent amino acid substitution from alpha2A(V58,T59) to alpha2B(I58,A59) in the N-terminal extracellular domain. 2. Comparing the effects of the GlyR agonists, glycine, beta-alanine and taurine, on the GlyR alpha2 isoforms, revealed a significant increase in potency for all three agonists at the alpha2B variant. 3. The sensitivities of the splice variants to the competitive antagonist, strychnine, and to the biphasic modulator Zn(2+), were comparable. In contrast, the allosteric inhibitor picrotoxin was more potent on GlyR alpha2A compared to GlyR alpha2B receptors. 4. Coexpression of alpha2A or alpha2B subunits with the GlyR beta subunit revealed that the higher agonist potencies observed with the alpha2B homomer were retained for the alpha2Bbeta heteromer. 5. The identical sensitivity to strychnine combined with a reduction in the maximum current induced by the partial agonist taurine at the GlyR alpha2A homomer, suggested that the changed sensitivity to agonists is in accordance with a modulation of agonist efficacy rather than agonist affinity. 6. An effect on agonist efficacy was also supported by using a structural model of the GlyR, localising the region of splice variation to the proposed docking region between GlyR loop 2 and the TM2-3 loop, an area associated with channel activation. 7. The existence of a spasmodic mouse phenotype linked to a GlyR alpha1(A52S) mutation, the equivalent position to the source of the alpha2 splice variation, raises the possibility that the GlyR alpha2 splice variants may be responsible for distinct roles in neuronal function.

  2. Subunit rotation models activation of serotonin 5-HT3AB receptors by agonists

    NASA Astrophysics Data System (ADS)

    Maksay, Gábor; Simonyi, Miklós; Bikádi, Zsolt

    2004-10-01

    The N-terminal extracellular regions of heterooligomeric 3AB-type human 5-hydroxytryptamine receptors (5-HT 3ABR) were modelled based on the crystal structure of snail acetylcholine binding protein AChBP. Stepwise rotation of subunit A by 5° was performed between -10° and 15° to mimic agonist binding and receptor activation. Anticlockwise rotation reduced the size of the binding cavity in interface AB and reorganised the network of hydrogen bonds along the interface. AB subunit dimers with different rotations were applied for docking of ligands with different efficacies: 5-HT, m-chlorophenylbiguanide, SR 57227, quinolinyl piperazine and lerisetron derivatives. All ligands were docked into the dimer with -10° rotation representing ligand-free, open binding cavities similarly, without pharmacological discrimination. Their ammonium ions were in hydrogen bonding distance to the backbone carbonyl of W183. Anticlockwise rotation and contraction of the binding cavity led to distinctive docking interactions of agonists with E129 and cation-π interactions of their ammonium ions. Side chains of several further amino acids participating in docking (Y143, Y153, Y234 and E236) are in agreement with the effects of point mutations in the binding loops. Our model postulates that 5-HT binds to W183 in a hydrophobic cleft as well as to E236 in a hydrophilic vestibule. Then it elicits anticlockwise rotation to draw in loop C via π-cation-π interactions of␣its ammonium ion with W183 and Y234. Finally, closure of the binding cavity might end in rebinding of 5-HT to E129 in the hydrophilic vestibule.

  3. Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors.

    PubMed

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2008-02-12

    Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Galpha-subunits (Galpha16 or Galphai2) with the kappa-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S3107.33, F3147.37, and I3167.39 to Y3207.43) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S3117.34 and N3267.49 were found to be sensitive in Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.

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

    PubMed Central

    Kachel, Hamid S.; Patel, Rohit N.; Franzyk, Henrik; Mellor, Ian R.

    2016-01-01

    Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-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 in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity. PMID:27901080

  5. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2008-11-10

    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  6. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  7. Open G2 strings

    NASA Astrophysics Data System (ADS)

    de Boer, Jan; de Medeiros, Paul; El-Showk, Sheer; Sinkovics, Annamaria

    2008-02-01

    We consider an open string version of the topological twist previously proposed for sigma-models with G2 target spaces. We determine the cohomology of open strings states and relate these to geometric deformations of calibrated submanifolds and to flat or anti-self-dual connections on such submanifolds. On associative three-cycles we show that the worldvolume theory is a gauge-fixed Chern-Simons theory coupled to normal deformations of the cycle. For coassociative four-cycles we find a functional that extremizes on anti-self-dual gauge fields. A brane wrapping the whole G2 induces a seven-dimensional associative Chern-Simons theory on the manifold. This theory has already been proposed by Donaldson and Thomas as the higher-dimensional generalization of real Chern-Simons theory. When the G2 manifold has the structure of a Calabi-Yau times a circle, these theories reduce to a combination of the open A-model on special Lagrangians and the open B + B-bar-model on holomorphic submanifolds. We also comment on possible applications of our results.

  8. UPSS and G2

    NASA Technical Reports Server (NTRS)

    Dito, Scott J.

    2014-01-01

    The Universal Propellant Servicing System (UPSS) is a dedicated mobile launcher propellant delivery method that will minimize danger and complexity in order to allow vehicles to be serviced and ultimately launched from a variety of locations previously not seen fit for space launch. The UPPS/G2 project is the development of a model, simulation, and ultimately a working application that will control and monitor the cryogenic fluid delivery to the rocket for testing purposes. To accomplish this, the project is using the programming language/environment Gensym G2. The environment is an all-inclusive application that allows development, testing, modeling, and finally operation of the unique application through graphical and programmatic methods. We have learned G2 through classes and trial-and-error, and are now in the process of building the application that will soon be able to be tested on apparatuses here at Kennedy Space Center, and eventually on the actual unit. The UPSS will bring near-autonomous control of launches to those that need it, as well it will be a great addition to NASA and KSC's operational viability and the opportunity to bring space launches to parts of the world, and in time constraints, once not thought possible.

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

    ERIC Educational Resources Information Center

    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.…

  10. Loop-F of the α-subunit determines the pharmacologic profile of novel competitive inhibitors of GABAA receptors.

    PubMed

    Mihalik, Balázs; Pálvölgyi, Adrienn; Bogár, Ferenc; Megyeri, Katalin; Ling, István; Barkóczy, József; Bartha, Ferenc; Martinek, Tamás A; Gacsályi, István; Antoni, Ferenc A

    2017-03-05

    The neurotransmitter γ-amino butyric acid (GABA) has a fundamental role in CNS function and ionotropic (GABAA) receptors that mediate many of the actions of GABA are important therapeutic targets. This study reports the mechanism of action of novel GABAA antagonists based on a tricyclic oxazolo-2,3-benzodiazepine scaffold. These compounds are orthosteric antagonists of GABA on heteropentameric GABAA receptors of αxβ2γ2 configuration expressed in HEK293 cells. In silico modelling predicted that the test compounds docked in the GABA binding-pocket and would interact with amino-acid residues in the α- and β-subunit interface that are known to be important for the binding of GABA. Intriguingly, optimal docking also required an interaction with the non-conserved amino-terminal segment of Loop-F of the α-subunit. Testing of a compound with altered regiochemistry of the oxazolone moiety supported the model with respect to the conserved GABA-interacting residues in vitro as well as in vivo. The prediction regarding loop-F was examined by replacing the amino-terminal variable segment of loop-F of the α5-subunit with the corresponding residues in the α1- and α2-subunits. When tested with the novel inhibitors, the receptors formed by the modified α5-subunits displayed the pharmacologic phenotype of the source of loop-F. In summary, these data show that the variable amino-terminal segment of loop-F of the α-subunit determines the pharmacologic selectivity of the novel tricyclic inhibitors of GABAA receptors.

  11. Diminution of the NMDA receptor NR2B subunit in cortical and subcortical areas of WAG/Rij rats.

    PubMed

    Karimzadeh, Fariba; Soleimani, Mansoureh; Mehdizadeh, Mehdi; Jafarian, Maryam; Mohamadpour, Maliheh; Kazemi, Hadi; Joghataei, Mohammad-Taghi; Gorji, Ali

    2013-12-01

    Modulation of glutamatergic NMDA receptors affects the synchronization of spike discharges in in WAG/Rij rats, a valid genetic animal model of absence epilepsy. In this study, we describe the alteration of NR2B subunit of NMDA receptors expression in WAG/Rij rats in different somatosensory cortical layers and in hippocampal CA1 area. Experimental groups were divided into four groups of six rats of both WAG/Rij and Wistar strains with 2 and 6 months of age. The distribution of NR2B receptors was assessed by immunohistochemical staining in WAG/Rij and compared with age-matched Wistar rats. The expression of NR2B subunit was significantly decreased in different somatosensory cortical layers in 2- and 6-month-old WAG/Rij rats. In addition, the distribution of NR2B in hippocampal CA1 area was lower in 6-month-old WAG/Rij compared with age-matched Wistar rats. The reduction of NR2B receptors in different brain areas points to disturbance of glutamate receptors expression in cortical and subcortical areas in WAG/Rij rats. An altered subunit assembly of NMDA receptors may underlie cortical hyperexcitability in absence epilepsy.

  12. GluN2A Subunit-Containing NMDA Receptors Are the Preferential Neuronal Targets of Homocysteine

    PubMed Central

    Sibarov, Dmitry A.; Abushik, Polina A.; Giniatullin, Rashid; Antonov, Sergei M.

    2016-01-01

    Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated

  13. PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization.

    PubMed

    Lin, Da-Ting; Huganir, Richard L

    2007-12-12

    Changes in surface trafficking of AMPA receptors play an important role in synaptic plasticity. Phosphorylation of the C terminus of the AMPA receptor (AMPAR) subunit glutamate receptor 2 (GluR2) and the binding of GluR2 to the PDZ [postsynaptic density-95/Discs large/zona occludens-1]-domain containing protein, protein interacting with protein kinase C (PICK1), have been proposed to play an important role in NMDA receptor dependent internalization of GluR2. However, the fate of internalized GluR2 after NMDA receptor (NMDAR) activation is still unclear. Both recycling and degradation of GluR2 after the activation of NMDAR have been reported. Here, we used a pH-sensitive green fluorescent protein variant, pHluorin, tagged to the N terminus of GluR2 (pH-GluR2) to study the dynamic internalization and recycling of GluR2 after NMDAR activation. Using fluorescence recovery after photobleach (FRAP), we directly demonstrate that internalized pH-GluR2 subunits recycle back to the cell surface after NMDAR activation. We further demonstrate that changing the phosphorylation state of the S880 residue at the C terminus of GluR2 does not affect NMDAR-dependent GluR2 internalization, but alters the recycling of GluR2 after NMDAR activation. In addition, mutation of the N-ethylmaleimide-sensitive fusion protein (NSF) binding site in the pH-GluR2 slows receptor recycling. Finally, neurons lacking PICK1 display normal NMDAR dependent GluR2 internalization compared with wild-type neurons, but demonstrate accelerated GluR2 recycling after NMDAR activation. These results indicate that phosphorylation of GluR2 S880 and NSF and PICK1 binding to GluR2 dynamically regulate GluR2 recycling.

  14. Expression and characterization of a glycine-binding fragment of the N-methyl-D-aspartate receptor subunit NR1.

    PubMed Central

    Miyazaki, J; Nakanishi, S; Jingami, H

    1999-01-01

    N-Methyl-D-aspartate receptor channels are composed of an NR1 subunit and at least one of the NR2 subunits (NR2A-D). Activation of the N-methyl-d-aspartate receptor requires the co-agonists glycine and glutamate. It has been proposed that the NR1 subunit possesses a glycine-binding site. We have expressed a soluble form of the NR1 subunit, which was produced by connecting the N-terminal extracellular region with the extracellular loop between the third and fourth membrane segments, by a baculovirus system along with full-length and truncated membrane-bound forms. The soluble NR1 receptor was efficiently secreted into the culture medium and showed a high affinity for ligands. The Kd of a glycine-site antagonist, [3H]MDL 105,519 [(E)-3-(2-phenyl-2-carboxyethenyl)-4, 6-dichloro-1H-indole-2-carboxylic acid], for the soluble receptor was 3.89+/-0.97 nM, which was comparable to the Kd of 4.47+/-1.39 nM for the membrane-bound full-length form. These values were close to the values reported previously with the use of rat brain membranes and Chinese hamster ovary cells expressing the full-length form of the NR1 subunit. The Ki values of other glycine-site antagonists, L-689,560 (trans-2-carboxy-5,7-dichloro - 4 - phenylaminocarbonylamino - 1,2,3,4 - tetrahydroquinoline), 5, 7-dichlorokynurenate and 5,7-dinitroquinoxaline-2,3-dione, for the soluble receptor were also similar to those for the full-length form of NR1. [3H]MDL 105,519 binding was also inhibited by the agonists glycine and d-serine. Thus the affinity and selectivity of ligand-binding characteristics of the NR1 subunit is conferred on the soluble form of the NR1 subunit. This soluble receptor provides a good experimental tool for initiating a biophysical analysis of the N-methyl-d-aspartate receptor channel protein. PMID:10359652

  15. Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

    PubMed

    Martin, Jessica A; Garczynski, Stephen F

    2016-04-01

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

  16. Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor.

    PubMed

    Lorberboum-Galski, H; Kozak, R W; Waldmann, T A; Bailon, P; FitzGerald, D J; Pastan, I

    1988-12-15

    IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation.

  17. Nicotine normalizes intracellular subunit stoichiometry of nicotinic receptors carrying mutations linked to autosomal dominant nocturnal frontal lobe epilepsy.

    PubMed

    Son, Cagdas D; Moss, Fraser J; Cohen, Bruce N; Lester, Henry A

    2009-05-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (alpha4)(3)(beta2)(2) versus (alpha4)(2)(beta2)(3) subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in alpha4, V287L, and V287M in beta2. alpha4 and beta2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Förster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the alpha4 and beta2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (alpha4)(3)(beta2)(2) carrying fluorescent alpha4 subunits were significantly higher than for (alpha4)(2)(beta2)(3); the converse was found for fluorescent beta2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (alpha4)(3)(beta2)(2) receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 muM) exposure increased the proportion of (alpha4)(2)(beta2)(3) in WT receptors and also returned subunit stoichiometry to WT levels for alpha4S248F and beta2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.

  18. Complex Control of GABA(A) Receptor Subunit mRNA Expression: Variation, Covariation, and Genetic Regulation

    PubMed Central

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

    2012-01-01

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

  19. Developmental origin dictates interneuron AMPA and NMDA receptor subunit composition and plasticity.

    PubMed

    Matta, Jose A; Pelkey, Kenneth A; Craig, Michael T; Chittajallu, Ramesh; Jeffries, Brian W; McBain, Chris J

    2013-08-01

    Disrupted excitatory synapse maturation in GABAergic interneurons may promote neuropsychiatric disorders such as schizophrenia. However, establishing developmental programs for nascent synapses in GABAergic cells is confounded by their sparsity, heterogeneity and late acquisition of subtype-defining characteristics. We investigated synaptic development in mouse interneurons targeting cells by lineage from medial ganglionic eminence (MGE) or caudal ganglionic eminence (CGE) progenitors. MGE-derived interneuron synapses were dominated by GluA2-lacking AMPA-type glutamate receptors (AMPARs), with little contribution from NMDA-type receptors (NMDARs) throughout development. In contrast, CGE-derived cell synapses had large NMDAR components and used GluA2-containing AMPARs. In neonates, both MGE- and CGE-derived interneurons expressed primarily GluN2B subunit-containing NMDARs, which most CGE-derived interneurons retained into adulthood. However, MGE-derived interneuron NMDARs underwent a GluN2B-to-GluN2A switch that could be triggered acutely with repetitive synaptic activity. Our findings establish ganglionic eminence-dependent rules for early synaptic integration programs of distinct interneuron cohorts, including parvalbumin- and cholecystokinin-expressing basket cells.

  20. GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia.

    PubMed

    Lu, Junjie; Zhang, Qian; Tan, Dongmei; Luo, Wenping; Zhao, Hai; Ma, Jing; Liang, Hao; Tan, Yi

    2016-07-01

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE.

  1. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    PubMed

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  2. Mutations in the main cytoplasmic loop of the GABA(A) receptor α4 and δ subunits have opposite effects on surface expression.

    PubMed

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

    2014-07-01

    We examined the role of putative trafficking sequences in two GABA(A) receptor subunits: α4 and δ. These subunits assemble with a β subunit to form a subtype of GABA(A) receptor involved in generating the "tonic" outward current. Both α4 and δ subunits contain dibasic retention motifs in homologous positions. When basic residues are mutated to alanine in the α4 subunit, surface expression of epitope-tagged δ subunits is increased. When basic residues in homologous regions of the δ subunit are mutated, however, surface expression is reduced. We focused on the mutants that had the maximal effects to increase (in α4) or reduce (in δ) surface expression. The total expression of δ subunits is significantly decreased by the δ mutation, suggesting an effect on subunit maturation. We also examined surface expression of the β2 subunit. Expression of the mutated α4 subunit resulted in increased surface expression of β2 compared with wild-type α4, indicating enhanced forward trafficking. In contrast, mutated δ resulted in decreased surface expression of β2 compared with wild-type δ and to α4 and β2 in the absence of any δ. This observation suggests that the mutated δ incorporates into multimeric receptors and reduces the overall forward trafficking of receptors. These observations indicate that the roles of trafficking motifs are complex, even when located in homologous positions in related subunits. The physiologic properties of receptors containing mutated subunits were not significantly affected, indicating that the mutations in the α4 subunit will be useful to enhance surface expression.

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

    PubMed Central

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

    2008-01-01

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

  4. Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus☆

    PubMed Central

    Lees, Kristin; Jones, Andrew K.; Matsuda, Kazuhiko; Akamatsu, Miki; Sattelle, David B.; Woods, Debra J.; Bowman, Alan S.

    2014-01-01

    Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides. PMID:24291321

  5. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  6. NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus

    PubMed Central

    Swanger, Sharon A.; Vance, Katie M.; Pare, Jean-François; Sotty, Florence; Fog, Karina; Smith, Yoland

    2015-01-01

    The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)–P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease. SIGNIFICANCE STATEMENT The subthalamic nucleus (STN) is a key component of the basal ganglia, a group of subcortical nuclei that control movement and are dysregulated in movement disorders such as Parkinson's disease. Subthalamic neurons receive direct excitatory input, but the pharmacology of excitatory

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

    PubMed

    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

  8. Med1 Subunit of the Mediator Complex in Nuclear Receptor-Regulated Energy Metabolism, Liver Regeneration, and Hepatocarcinogenesis

    PubMed Central

    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

  9. Null mutation of the β2 nicotinic acetylcholine receptor subunit attenuates nicotine withdrawal-induced anhedonia in mice.

    PubMed

    Stoker, Astrid K; Marks, Michael J; Markou, Athina

    2015-04-15

    The anhedonic signs of nicotine withdrawal are predictive of smoking relapse rates in humans. Identification of the neurobiological substrates that mediate anhedonia will provide insights into the genetic variations that underlie individual responses to smoking cessation and relapse. The present study assessed the role of β2 nicotinic acetylcholine receptor (nACh receptor) subunits in nicotine withdrawal-induced anhedonia using β2 nACh receptor subunit knockout (β2(-/-)) and wildtype (β2(+/+)) mice. Anhedonia was assessed with brain reward thresholds, defined as the current intensity that supports operant behavior in the discrete-trial current-intensity intracranial self-stimulation procedure. Nicotine was delivered chronically through osmotic minipumps for 28 days (40 mg/kg/day, base), and withdrawal was induced by either administering the broad-spectrum nicotinic receptor antagonist mecamylamine (i.e., antagonist-precipitated withdrawal) in mice chronically treated with nicotine or terminating chronic nicotine administration (i.e., spontaneous withdrawal). Mecamylamine (6 mg/kg, salt) significantly elevated brain reward thresholds in nicotine-treated β2(+/+) mice compared with saline-treated β2(+/+) mice and nicotine-treated β2(-/-) mice. Spontaneous nicotine withdrawal similarly resulted in significant elevations in thresholds in nicotine-withdrawing β2(+/+) mice compared with saline-treated β2(+/+) and nicotine-treated β2(-/-) mice, which remained at baseline levels. These results showed that precipitated and spontaneous nicotine withdrawal-induced anhedonia was attenuated in β2(-/-) mice. The reduced expression of anhedonic signs during nicotine withdrawal in β2(-/-) mice may have resulted from the lack of neuroadaptations in β2 nACh receptor subunit expression and function that may have occurred during either nicotine exposure or nicotine withdrawal in wildtype mice. In conclusion, individuals with genetic variations that result in diminished

  10. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

    PubMed

    Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

    2015-06-01

    Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment.

  11. Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons.

    PubMed

    Qiang, Mei; Denny, Ashley D; Ticku, Maharaj K

    2007-07-01

    A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.

  12. Nicotine-induced up-regulation and desensitization of alpha4beta2 neuronal nicotinic receptors depend on subunit ratio.

    PubMed

    López-Hernández, Gretchen Y; Sánchez-Padilla, Javier; Ortiz-Acevedo, Alejandro; Lizardi-Ortiz, José; Salas-Vincenty, Janice; Rojas, Legier V; Lasalde-Dominicci, José A

    2004-09-03

    Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.

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

    PubMed

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

    1998-11-01

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

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

    ERIC Educational Resources Information Center

    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…

  15. Cloning of the mouse GABA-benzodiazepine receptor. alpha. 1 subunit in a study of alcohol neurosensitivity

    SciTech Connect

    Keir, W.J.; Deitrich, R.A.; Sikela, J.M. )

    1989-02-09

    The inhibitory action of gamma amino butyric acid (GABA) is mediated by its binding to the benzodiazepine (BDZ) receptor and opening of a chloride channel. This receptor contains a variety of binding sites for several behavorially active drugs. Recent studies with SS and LS mice which were selected for differential neurosensitivity to ethanol, suggest that the GABAergic system plays a role in this differential sensitivity. Thus genes controlling the GABAergic system may also influence the acute hypnotic actions of ethanol. As a fist step towards verifying this hypothesis we have cloned and partially sequenced the mouse GABA-BDZ {alpha}1 subunit cDNA using a 40 bp oligonucleotide derived from the N terminus of a published bovine {alpha} subunit cDNA. A positive clone from a mouse brain cDNA library was identified and contains an insert of approximately 2.5 Kb. Partial sequence analysis indicates that this clone corresponds to the mouse homolog of the {alpha}1 subunit of the GABA-BDZ receptor. This clone is being used as a probe to identify restriction fragment length polymorphisms in several mouse genotypes which differ in their neurosensitivity to ethanol in an attempt to identify molecular genetic changes in the GABA-BDZ receptor that are related to differential ethanol neurosensitivity.

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

    SciTech Connect

    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.

  17. Methanandamide allosterically inhibits in vivo the function of peripheral nicotinic acetylcholine receptors containing the alpha 7-subunit.

    PubMed

    Baranowska, Urszula; Göthert, Manfred; Rudz, Radoslaw; Malinowska, Barbara

    2008-09-01

    Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.

  18. Constitutive Effects of Lead on Aryl Hydrocarbon Receptor Gene Battery and Protection by β-carotene and Ascorbic Acid in Human HepG2 Cells.

    PubMed

    Darwish, Wageh S; Ikenaka, Yoshinori; Nakayama, Shouta M M; Mizukawa, Hazuki; Ishizuka, Mayumi

    2016-01-01

    Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb-induced adverse effects were done using some phytochemicals like β-carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase-1 mRNA expression in a concentration-dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like β-carotene (10 μM) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.

  19. Orphan Glutamate Receptor δ1 Subunit Required for High-Frequency Hearing▿

    PubMed Central

    Gao, Jiangang; Maison, Stéphane F.; Wu, Xudong; Hirose, Keiko; Jones, Sherri M.; Bayazitov, Ildar; Tian, Yong; Mittleman, Guy; Matthews, Douglas B.; Zakharenko, Stanislav S.; Liberman, M. Charles; Zuo, Jian

    2007-01-01

    The function of the orphan glutamate receptor delta subunits (GluRδ1 and GluRδ2) remains unclear. GluRδ2 is expressed exclusively in the Purkinje cells of the cerebellum, and GluRδ1 is prominently expressed in inner ear hair cells and neurons of the hippocampus. We found that mice lacking the GluRδ1 protein displayed significant cochlear threshold shifts for frequencies of >16 kHz. These deficits correlated with a substantial loss of type IV spiral ligament fibrocytes and a significant reduction of endolymphatic potential in high-frequency cochlear regions. Vulnerability to acoustic injury was significantly enhanced; however, the efferent innervation of hair cells and the classic efferent inhibition of outer hair cells were unaffected. Hippocampal and vestibular morphology and function were normal. Our findings show that the orphan GluRδ1 plays an essential role in high-frequency hearing and ionic homeostasis in the basal cochlea, and the locus encoding GluRδ1 represents a candidate gene for congenital or acquired high-frequency hearing loss in humans. PMID:17438141

  20. Retinal waves in mice lacking the beta2 subunit of the nicotinic acetylcholine receptor.

    PubMed

    Sun, Chao; Warland, David K; Ballesteros, Jose M; van der List, Deborah; Chalupa, Leo M

    2008-09-09

    The structural and functional properties of the visual system are disrupted in mutant animals lacking the beta2 subunit of the nicotinic acetylcholine receptor. In particular, eye-specific retinogeniculate projections do not develop normally in these mutants. It is widely thought that the developing retinas of beta2(-/-) mutants do not manifest correlated activity, leading to the notion that retinal waves play an instructional role in the formation of eye-specific retinogeniculate projections. By multielectrode array recordings, we show here that the beta2(-/-) mutants have robust retinal waves during the formation of eye-specific projections. Unlike in WT animals, however, the mutant retinal waves are propagated by gap junctions rather than cholinergic circuitry. These results indicate that lack of retinal waves cannot account for the abnormalities that have been documented in the retinogeniculate pathway of the beta2(-/-) mutants and suggest that other factors must contribute to the deficits in the visual system that have been noted in these animals.

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

    PubMed Central

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

    2009-01-01

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

  2. Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues

    PubMed Central

    Schaik, R H N van; Wierikx, C D J; Timmerman, M A; Oomen, M H; Weerden, W M van; Kwast, T H van der; Steenbrugge, G J van; Jong, F H de

    1999-01-01

    The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels. © 2000 Cancer Research Campaign PMID:10638976

  3. Antineoplastic effects of α-santalol on estrogen receptor-positive and estrogen receptor-negative breast cancer cells through cell cycle arrest at G2/M phase and induction of apoptosis.

    PubMed

    Santha, Sreevidya; Bommareddy, Ajay; Rule, Brittny; Guillermo, Ruth; Kaushik, Radhey S; Young, Alan; Dwivedi, Chandradhar

    2013-01-01

    Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor (ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.

  4. Pharmacological characterisation of a cell line expressing GABA B1b and GABA B2 receptor subunits.

    PubMed

    Hirst, Warren D; Babbs, Adam J; Green, Andrew; Minton, Jayne A L; Shaw, Tracy E; Wise, Alan; Rice, Simon Q; Pangalos, Menelas N; Price, Gary W

    2003-04-01

    The gamma-aminobutyric acid (GABA(B)) receptor has been shown to be a heterodimer consisting of two receptor subunits, GABA(B1) and GABA(B2). We have stably co-expressed these two subunits in a CHO cell line, characterised its pharmacology and compared it to the native receptor in rat brain membranes. Radioligand binding using [3H]CGP54626A demonstrated a similar rank order of potency between recombinant and native receptors: CGP62349>CGP54626A>SCH 50911>3-aminopropylphosphinicacid(3-APPA)>GABA>baclofen>saclofen>phaclofen. However, differences were observed in the affinity of agonists, which were higher at the native receptor, suggesting that in the recombinant system a large number of the receptors were in the low agonist affinity state. In contrast, [35S]GTPgammaS binding studies did not show any differences between recombinant and native receptors with the full agonists GABA and 3-APPA. Measurement of cAMP accumulation in the cells revealed a degree of endogenous coupling of the receptors to G-proteins. This is most likely to be due to the high expression levels of receptors (B(max)=22.5+/-2.5pmol/mg protein) in this experimental system. There was no evidence of GABA(B2) receptors, when expressed alone, binding [3H]CGP54626A, [3H]GABA, [3H]3-APPA nor of GABA having any effect on basal [35S]GTPgammaS binding or cAMP levels.

  5. NMDA receptor subunit expression in the supraoptic nucleus of adult rats: Dominance of NR2B and NR2D

    PubMed Central

    Doherty, Faye C; Sladek, Celia D

    2011-01-01

    The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory neurons (MNC) which synthesize and release the peptide hormones vasopressin and oxytocin. Glutamate is a prominent excitatory neurotransmitter in the SON and regulates MNC excitability. NMDA receptors (NMDAR), a type of ionotropic glutamate receptor, mediate synaptic plasticity of MNCs and are necessary for characteristic burst firing patterns which serve to maximize hormone release. NMDARs are di- or tri-heteromeric complexes of NR1 and NR2 subunits. Receptor properties depend on NR2 subunit composition and variable splicing of NR1. We investigated the expression profile of NR1 and NR2 subunits in the SON at the mRNA and protein levels, plus protein expression of NR1 splice variants in control and salt-loaded adult rats. There was robust mRNA expression of all subunits, with NR2D levels being the highest. At the protein level, NR1, NR2B and NR2D were robustly expressed, while NR2A was weakly expressed. NR2C protein was not detected with either of two antibodies. All four NR1 splice variant cassettes (N1, C1, C2, C2’) were detected in the SON, though NR1 N1 expression was too low for accurate analysis. Three days of salt-loading did not alter mRNA, protein or splice variant expression of NMDAR subunits in the SON. Robust NR2D protein expression has not been previously shown in MNCs, and is uncommon in the adult brain. Though the functional significance of this unusual expression profile is unknown, it may contribute to important physiological characteristics of SON neurons, such as burst firing and resistance to excitotoxicity. PMID:21397592

  6. Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels.

    PubMed

    Boué-Grabot, Eric; Toulmé, Estelle; Emerit, Michel B; Garret, Maurice

    2004-12-10

    ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.

  7. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  8. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Sumikawa, K; Houghton, M; Smith, J C; Bell, L; Richards, B M; Barnard, E A

    1982-01-01

    A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle. Images PMID:6183641

  9. Localization of a gene for a glutamate binding subunit of a NMDA receptor (GRINA) to 8q24

    SciTech Connect

    Lewis, T.B.; DuPont, B.R.; Leach, R.

    1996-02-15

    This article reports on the localization of a gene for a glutamate binding subunit of an N-methyl-D-aspartate (NMDA) receptor, called GRINA, to human chromosome 8q24 using fluorescence in situ hybridization and radiation hybridization mapping. This gene mapped outside the critical region for benign familial neonatal convulsions (BFNC), a rare form of epilepsy; however, GRINA could be the causative genetic factor inducing idiopathic generalized epilepsy. Further studies need to be conducted. 15 refs., 2 figs.

  10. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  11. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  12. Parafascicular thalamic nucleus deep brain stimulation decreases NMDA receptor GluN1 subunit gene expression in the prefrontal cortex.

    PubMed

    Fernández-Cabrera, Mónica R; Selvas, Abraham; Miguéns, Miguel; Higuera-Matas, Alejandro; Vale-Martínez, Anna; Ambrosio, Emilio; Martí-Nicolovius, Margarita; Guillazo-Blanch, Gemma

    2017-04-21

    The rodent parafascicular nucleus (PFn) or the centromedian-parafascicular complex of primates is a posterior intralaminar nucleus of the thalamus related to cortical activation and maintenance of states of consciousness underlying attention, learning and memory. Deep brain stimulation (DBS) of the PFn has been proved to restore arousal and consciousness in humans and to enhance performance in learning and memory tasks in rats. The primary expected effect of PFn DBS is to induce plastic changes in target neurons of brain areas associated with cognitive function. In this study, Wistar rats were stimulated for 20mins in the PFn following a DBS protocol that had previously facilitated memory in rats. NMDA and GABAB receptor binding, and gene expression of the GluN1subunit of the NMDA receptor (NMDAR) were assessed in regions related to cognitive functions, such as the prefrontal cortex and hippocampus. The results showed that PFn DBS induced a decrease in NMDAR GluN1 subunit gene expression in the cingulate and prelimbic cortices, but no significant statistical differences were found in the density of NMDA or GABAB receptors in any of the analyzed regions. Taken together, our findings suggest a possible role for the NMDAR GluN1 subunit in the prefrontal cortex in the procognitive actions of the PFn DBS.

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

    ERIC Educational Resources Information Center

    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…

  14. Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

    PubMed

    Mehta, Ashok K; Marutha Ravindran, C R; Ticku, Maharaj K

    2007-08-24

    In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

  15. The Optimization of TaqMan Real-Time RT-PCR Assay for Transcriptional Profiling of GABA-A Receptor Subunit Plasticity

    PubMed Central

    Gangisetty, Omkaram; Reddy, Doodipala Samba

    2009-01-01

    The GABA-A receptor plays a critical role in inhibitory neurotransmission in the brain. Quantitation of GABA-A receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. However, conventional RNA assays are tedious and less sensitive for use in studies of subunit plasticity. Here we describe optimization of a sensitive assay of GABA-A receptor subunit gene expression by TaqMan real-time PCR. For each subunit gene, a set of primers and TaqMan fluorogenic probe were designed to specifically amplify the target template. The TaqMan methodology was optimized for quantification of mouse GABA-A receptor subunits (α1–6, β1–3, γ2, and δ) and GAPDH. The TaqMan reaction detected very low levels of gene expression (~100 template copies of cDNA). A standard curve for GAPDH and one of the target genes, constructed using the cDNA, revealed slopes around −3.4 (r2=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor α4 subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the α4-subunit expression increased threefold in the hippocampus following neurosteroid withdrawal in mice. The TaqMan PCR assay allows sensitive, high-throughput transcriptional profiling of complete GABA-A receptor subunit family, and thus provides specific tool for studies of GABA-A receptor subunit plasticity in neurological and psychiatric animal models. PMID:19406150

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

    PubMed

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

    2005-10-14

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

  17. Distinct Contributions of T1R2 and T1R3 Taste Receptor Subunits to the Detection of Sweet Stimuli

    SciTech Connect

    Nie,Y.; Vigues, S.; Hobbs, J.; Conn, G.; Munger, S.

    2005-01-01

    The molecular mechanisms by which G protein-coupled receptor (GPCR)-type chemosensory receptors of animals selectively interact with their cognate ligands remain poorly understood. There is growing evidence that many chemosensory receptors exist in multimeric complexes, though little is known about the relative contributions of individual subunits to receptor functions. This study showed that each of the two subunits in the mammalian heteromeric T1R2:T1R3 sweet taste receptor binds sweet stimuli, though with distinct affinities and conformational changes. Furthermore, ligand affinities for T1R3 are drastically reduced by the introduction of a single amino acid change associated with decreased sweet taste sensitivity in mice. Thus, individual T1R subunits increase the receptive range of the sweet taste receptor, offering a functional mechanism for phenotypic variations in sweet taste.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    Cox, David J; Racca, Claudia

    2013-06-15

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

  1. Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor α1 and α2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides.

    PubMed

    Dederer, Helene; Werr, Margaret; Ilg, Thomas

    2011-01-01

    Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR α subunits Cfα1, Cfα2, and Cfα3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) β2 (Ggβ2) subunit, these cat flea α subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cfα2/Ggβ2 were resistant to α-bungarotoxin, while those of Cfα1/Ggβ2 were sensitive to this snake toxin. The pharmacological profiles of Cfα1/Ggβ2, Cfα2/Ggβ2 and the chicken neuronal receptor Ggα4/Ggβ2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cfα1/Ggβ2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cfα2/Ggβ2 or Ggα4/Ggβ2: for the anti flea neonicotinoid market compound imidacloprid the respective EC₅₀s were 0.02 μM, 1.31 μM and 10 μM. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dmα1 and Dmα2 subunits as hybrid receptors with Ggβ2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Ggβ2 hybrid receptor setting, insect α1 subunits confer higher sensitivity to neonicotinoids than α2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class.

  2. γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type.

    PubMed

    Eaton, Megan M; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry; Akk, Gustav

    2014-12-01

    Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1-3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes.

  3. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Huff, Murray W

    2003-10-01

    The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

  4. Assemblies of glutamate receptor subunits with post-synaptic density proteins and their alterations in Parkinson's disease.

    PubMed

    Gardoni, Fabrizio; Ghiglieri, Veronica; Di Luca, Monica; Calabresi, Paolo

    2010-01-01

    N-methyl-D-aspartate (NMDA) receptors have been implicated as a mediator of neuronal injury associated with many neurological disorders including ischemia, epilepsy, brain trauma, dementia and neurodegenerative disorders such as Parkinson's disease (PD) and Alzheimer's disease. To this, non-selective NMDA receptor antagonists have been tried and have been shown to be effective in many experimental animal models of disease, and some of these compounds have moved into clinical trials. However, the initial enthusiasm for this approach has waned, because the therapeutic index for most NMDA antagonists is quite poor, with significant adverse effects at clinically effective doses, thus limiting their utility. More recently, the concept that the exact pathways downstream NMDA receptor activation could represent a key variable element among neurological disorders has been put forward. In particular, variations in NMDA receptor subunit composition could be important in different disorders, both in the pathophysiological mechanisms of cell death and in the application of specific symptomatic therapies. As to PD, NMDA receptor complex has been shown to be altered in experimental models of parkinsonism and in PD in humans. Further, it has become increasingly evident that the NMDA receptor complex is intimately involved in the regulation of corticostriatal long-term potentiation, which is altered in experimental parkinsonism. The following sections will examine the modifications of specific NMDA receptor subunits as well as post-synaptic associated signalling complex including kinases and scaffolding proteins in experimental parkinsonism. These findings may allow the identification of specific molecular targets whose pharmacological or genetic manipulation might lead to innovative therapies for PD.

  5. Structurally dissimilar antimanic agents modulate synaptic plasticity by regulating AMPA glutamate receptor subunit GluR1 synaptic expression.

    PubMed

    Du, Jing; Gray, Neil A; Falke, Cynthia; Yuan, Peixiong; Szabo, Steven; Manji, Husseini K

    2003-11-01

    A growing body of data from clinical and preclinical studies suggests that the glutamatergic system may represent a novel therapeutic target for severe recurrent mood disorders. Since synapse-specific glutamate receptor expression/localization is known to play critical roles in synaptic plasticity, we investigated the effects of mood stabilizers on AMPA receptor expression. Rats were treated chronically with lithium or valproate, hippocampal synaptosomes were isolated, and GluR1 levels were determined. Additionally, hippocampal neurons were prepared from E18 rat embryos and treated with lithium or valproate. Surface expression of GluR1 was determined using a biotinylation assay, and double-immunostaining with anti-GluR1 and anti-synaptotagmin antibodies was used to determine synaptic GluR1 levels. The AMPA receptor subunit GluR1 expression in hippocampal synaptosomes was significantly reduced by both chronic lithium and valproate. Overall, these studies show that AMPA receptor subunit GluR1 is a common target for two structurally highly dissimilar, but highly efficacious, mood stabilizers, lithium and valproate. These studies suggest that regulation of glutamatergically mediated synaptic plasticity may play a role in the treatment of mood disorders, and raise the possibility that agents more directly affecting synaptic GluR1 may represent novel therapies for this devastating illness.

  6. T Cell Receptor Engagement Triggers Its CD3ε and CD3ζ Subunits to Adopt a Compact, Locked Conformation

    PubMed Central

    Risueño, Ruth M.; Schamel, Wolfgang W. A.; Alarcón, Balbino

    2008-01-01

    How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3ε subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3ε and CD3ζ subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3ε and CD3ζ, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails. PMID:18320063

  7. The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits α1, β2, and δ along their extrasynaptic cell membrane.

    PubMed

    Milenkovic, I; Vasiljevic, M; Maurer, D; Höger, H; Klausberger, T; Sieghart, W

    2013-12-19

    Neuronal circuitries in the hippocampus are involved in navigation and memory and are controlled by major networks of GABAergic interneurons. Parvalbumin (PV)-expressing interneurons in the dentate gyrus (DG) are identified as fast-spiking cells, playing a crucial role in network oscillation and synchrony. The inhibitory modulation of these interneurons is thought to be mediated mainly through GABAA receptors, the major inhibitory neurotransmitter receptors in the brain. Here we show that all PV-positive interneurons in the granular/subgranular layer (GL/SGL) of the mouse DG express high levels of the GABAA receptor δ subunit. PV-containing interneurons in the hilus and the molecular layer, however, express the δ subunit to a lower extent. Only 8% of the somatostatin-containing interneurons express the δ subunit, whereas calbindin- or calretinin-containing interneurons in the DG seem not to express the GABAA receptor δ subunit at all. Hence, these cells receive a GABAergic control different from that of PV-containing interneurons in the GL/SGL. Experiments investigating a possible co-expression of GABAA receptor α1, α2, α3, α4, α5, β1, β2, β3, or γ2 subunits with PV and δ subunits indicated that α1 and β2 subunits are co-expressed with δ subunits along the extrasynaptic membranes of PV-interneurons. These results suggest a robust tonic GABAergic control of PV-containing interneurons in the GL/SGL of the DG via δ subunit-containing receptors. Our data are important for better understanding of the neuronal circuitries in the DG and the role of specific cell types under pathological conditions.

  8. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  9. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  10. Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum

    PubMed Central

    Leppä, Elli; Linden, Anni-Maija; Aller, Maria I.; Wulff, Peer; Vekovischeva, Olga; Luscher, Bernhard; Lüddens, Hartmut; Wisden, William; Korpi, Esa R.

    2016-01-01

    Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABAA receptors. In recombinant receptors and genetic mouse models their actions depend on the α, β, and δ subunits of the receptor, especially on those that form extrasynaptic GABAA receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched γ2 subunit-containing receptors and even on αβ binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5β-pregnan-3α-ol-20-one is altered in genetically engineered mouse models that have deficient GABAA receptor-mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABAA receptor γ2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5β-pregnan-3α-ol-20-one. Motor-impairing effects of 5β-pregnan-3α-ol-20-one were strongly increased in all three mouse models in which γ2 subunit-dependent synaptic GABAA responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABAA receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell γ2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of γ2 subunit has removed synaptic GABAA receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic αβ or αβδ receptors lead to enhanced changes in the cerebellum-generated behavior. PMID:27833556

  11. SETDB1 HISTONE METHYLTRANSFERASE REGULATES MOOD-RELATED BEHAVIORS AND EXPRESSION OF THE NMDA RECEPTOR SUBUNIT NR2B

    PubMed Central

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

    2010-01-01

    Histone methyltransferases specific for the histone H3-lysine 9 (H3K9) residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to less than 1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture (“3C”) and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30Kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wildtype mice, systemic treatment with the NR2B antagonist, Ro-256981, and hippocampal siRNA-mediated NR2B/Grin2b knockdown, resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations. PMID:20505083

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

    PubMed

    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.

  13. Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Gershoni, J M; Fridkin, M; Fuchs, S

    1985-01-01

    Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site. Images PMID:2582416

  14. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.

    PubMed Central

    Protti, M P; Manfredi, A A; Straub, C; Howard, J F; Conti-Tronconi, B M

    1990-01-01

    In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation. Images PMID:2145582

  15. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

    PubMed Central

    1996-01-01

    The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck. PMID:8642247

  16. Location- and Subunit-Specific NMDA Receptors Determine the Developmental Sevoflurane Neurotoxicity Through ERK1/2 Signaling.

    PubMed

    Wang, Wen-Yuan; Jia, Li-Jie; Luo, Yan; Zhang, Hong-Hai; Cai, Fang; Mao, Hui; Xu, Wei-Cai; Fang, Jun-Biao; Peng, Zhi-You; Ma, Zheng-Wen; Chen, Yan-Hong; Zhang, Juan; Wei, Zhen; Yu, Bu-Wei; Hu, Shuang-Fei

    2016-01-01

    It is well established that developmental exposure of sevoflurane (an inhalational anesthetic) is capable of inducing neuronal apoptosis and subsequent learning and memory disorders. Synaptic NMDA receptors activity plays an essential role in cell survival, while the extra-synaptic NMDA receptors activation is usually associated with cell death. However, whether synaptic or extra-synaptic NMDA receptors mediate developmental sevoflurane neurotoxicity is largely unknown. Here, we show that developmental sevoflurane treatment decreased NR2A, but increased NR2B subunit expression both in vitro and in vivo. Sevoflurane-induced neuronal apoptosis was attenuated by synaptic NMDA receptors activation or low dose of exogenous NMDA in vitro. Interestingly, these effects could be abolished by NR2A inhibitor PEAQX, but not NR2B inhibitor Ifenprodil in vitro. In contrast, activation of extra-synaptic NMDA receptors alone had no effects on sevoflurane neurotoxicity. In the scenario of extra-synaptic NMDA receptors stimulation, however, sevoflurane-induced neuronal apoptosis could be prevented by addition of Ifenprodil, but not by PEAQX in vitro. In addition, sevoflurane neurotoxicity could also be rescued by memantine, an uncompetitive antagonist for preferential blockade of extra-synaptic NMDA receptors both in vitro and in vivo. Furthermore, we found that developmental sevoflurane-induced phospho-ERK1/2 inhibition was restored by synaptic NMDA receptor activation (in vitro), low dose of NMDA (in vitro) or memantine (in vivo). And the neuroprotective role of synaptic NMDA activity was able to be reversed by MEK1/2 inhibitor U0126 in vitro. Finally, administration of memantine or NMDA significantly improved spatial learning and memory dysfunctions induced by developmental sevoflurane exposure without influence on locomotor activity. These results indicated that activation of synaptic NR2A-containing NMDA receptors, or inhibition of extra-synaptic NR2B-containing NMDA receptors

  17. Effect of unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway on GABA(A) receptor subunit gene expression in the rodent basal ganglia and thalamus.

    PubMed

    Chadha, A; Dawson, L G; Jenner, P G; Duty, S

    2000-01-01

    In Parkinson's disease, changes in GABAergic activity occurring downstream of the striatal dopamine loss are accompanied by reciprocal changes in GABA(A) receptor binding, the underlying molecular mechanisms for which are unknown. This study examined whether changes in expression of the genes encoding known GABA(A) receptor subunits (alpha(1-4), beta(1-3), gamma(1-3) and delta) could account for this receptor plasticity using a rodent model of Parkinson's disease with a 6-hydroxydopamine-induced nigrostriatal lesion. Analysis of autoradiograms of the basal ganglia and thalamus revealed changes in expression of only four of the 11 subunits studied. Expression of alpha1 and beta2 subunit genes was altered in a parallel manner following a 6-hydroxydopamine lesion; messenger RNA levels for both were significantly increased in the substantia nigra pars reticulata (11 +/- 4% and 17 +/- 1%, respectively), and significantly reduced in the globus pallidus (18 +/- 3% and 16 +/- 3%, respectively) and parafascicular nucleus (19 +/- 3% and 16 +/- 5%, respectively). Smaller changes in the messenger RNA levels encoding the alpha1 subunit in the lateral amygdala (8 +/- 1% decrease) and the alpha4 and gamma2 subunits in the striatum (10 +/- 2% and 6 +/- 1% increase, respectively) were also observed. No changes in expression were noted for any other subunits in any region studied. Clearly, both region- and subunit-specific regulation of GABA(A) receptor subunit gene expression occurs following a nigrostriatal tract lesion. The changes in expression of the alpha1 and beta2 subunit genes probably contribute to the documented changes in GABA(A) receptor binding following striatal dopamine depletion. Moreover, they provide a molecular basis by which the pathological changes in GABAergic activity in Parkinson's disease may be partially compensated.

  18. A single gene codes for the nicotinic acetylcholine receptor alpha-subunit in Torpedo marmorata: structural and developmental implications.

    PubMed Central

    Klarsfeld, A; Devillers-Thiéry, A; Giraudat, J; Changeux, J P

    1984-01-01

    We have used Southern blot hybridization to analyze the genomic structure encoding the alpha-subunit of the acetylcholine receptor (AChR) in Torpedo marmorata, with cDNA probes isolated from the electric organ. Four different radiolabelled probes, corresponding to various parts of the alpha-subunit mRNA, hybridized to several genomic fragments of T. marmorata DNA generated by digestion with the restriction enzymes SstI, PvuII and PstI. The same hybridization pattern was observed after washing the blots under low- or high-stringency conditions. As a check for detection sensitivity of heterologous sequences, the same probes were hybridized to PvuII-digested chicken DNA, revealing bands at low stringency which disappeared at higher stringencies. Unambiguously, two of our probes (one of them entirely within the coding region) hybridized to a single genomic fragment from T. marmorata DNA. This feature, as well as the results of an extensive study of the whole hybridization pattern, points towards the uniqueness of alpha-subunit-specific sequences in the genome of T. marmorata. Since overall more bands were found than expected from the cDNA sequence, this alpha-subunit gene must be split by several introns (at least four, possibly more). The length of this gene is at least 20 kb. The existence of a single alpha-subunit gene is consistent with the absence of chemical heterogeneity in the NH2-terminal sequence of the purified alpha-chain, and supports the view that the two alpha-chains belonging to one AChR oligomer have an identical primary structure. It also suggests that localization and stabilization of the AChR in well-defined post-synaptic areas of T. marmorata electric organ basically relies, during development, on 'epigenetic' mechanisms. Images Fig. 2. Fig. 3. Fig. 4. PMID:6323168

  19. Tumor necrosis factor receptor-associated protein 1 improves hypoxia-impaired energy production in cardiomyocytes through increasing activity of cytochrome c oxidase subunit II.

    PubMed

    Xiang, Fei; Ma, Si-Yuan; Zhang, Dong-Xia; Zhang, Qiong; Huang, Yue-Sheng

    2016-10-01

    Tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes against hypoxia, but the underlying mechanisms are not completely understood. In the present study, we used gain- and loss-of-function approaches to explore the effects of tumor necrosis factor receptor-associated protein 1 and cytochrome c oxidase subunit II on energy production in hypoxic cardiomyocytes. Hypoxia repressed ATP production in cultured cardiomyocytes, whereas overexpression of tumor necrosis factor receptor-associated protein 1 significantly improved ATP production. Conversely, knockdown of tumor necrosis factor receptor-associated protein 1 facilitated the hypoxia-induced decrease in ATP synthesis. Further investigation revealed that tumor necrosis factor receptor-associated protein 1 induced the expression and activity of cytochrome c oxidase subunit II, a component of cytochrome c oxidase that is important in mitochondrial respiratory chain function. Moreover, lentiviral-mediated overexpression of cytochrome c oxidase subunit II antagonized the decrease in ATP synthesis caused by knockdown of tumor necrosis factor receptor-associated protein 1, whereas knockdown of cytochrome c oxidase subunit II attenuated the increase in ATP synthesis caused by overexpression of tumor necrosis factor receptor-associated protein 1. In addition, inhibition of cytochrome c oxidase subunit II by a specific inhibitor sodium azide suppressed the ATP sy nthesis induced by overexpressed tumor necrosis factor receptor-associated protein 1. Hence, tumor necrosis factor receptor-associated protein 1 protects cardiomyocytes from hypoxia at least partly via potentiation of energy generation, and cytochrome c oxidase subunit II is one of the downstream effectors that mediates the tumor necrosis factor receptor-associated protein 1-mediated energy generation program.

  20. A lasting effect of postnatal sevoflurane anesthesia on the composition of NMDA receptor subunits in rat prefrontal cortex.

    PubMed

    Zhang, Xiaoyu; Shen, Fengyan; Xu, Daojie; Zhao, Xuan

    2016-11-01

    Sevoflurane is widely used in pediatric anesthesia and studies have shown that it is capable of inducing neurodegeneration and subsequent cognitive disorders in the developing brain. However, the evidence that anesthetics are toxic to the human brain is insufficient. N-Methyl-d-aspartate (NMDA) receptors, critical for learning and memory, display expression changes with age and can be modulated by inhalation anesthetics. Generally, NMDA receptor (NR) type 1 is expressed at birth, peaks around the third postnatal week, and then declines slightly to adult levels. NR2Bs slowly decrease and NR2As gradually increase during postnatal development. These developmental switches of NMDA receptor subunits composition mark the transition from immature to adult neural processing and allow for the final maturation of associative learning abilities. In this study, we aimed to evaluate the effect of repeated sevoflurane anesthesia on NMDA receptor subunits composition in the developing rat brain and related behavioral disorders. Six-day-old male Sprague Dawley rats were randomly allocated into either a control group (group con) or a sevoflurane group (group sevo). Group sevo inhaled 2.1% sevoflurane carried by 70% oxygen for 2h each day from postnatal day (PND) 6 to PND 8. The same procedure, without applying the sevoflurane, was executed in group con. The membrane protein expression of NR1, NR2A and NR2B in the prefrontal cortex (PFC) and hippocampus was assessed at the end of the three days of anesthesia and at PND 21. An open field test was carried out to assess spontaneous locomotion on PNDs 21, 28 and 35. Y maze performance was used to assess attention and working memory on PND 28. Sevoflurane induced upregulation of NR1 and NR2B in the PFC at the end of anesthesia. On PND 21, NR1 and NR2B receptors were significantly increased whereas NR2A receptors were significantly decreased in the PFC in group sevo. Sevoflurane-treated rats showed hyper-locomotion and impairment of

  1. The glutamate receptor subunit delta1 is highly expressed in hair cells of the auditory and vestibular systems.

    PubMed

    Safieddine, S; Wenthold, R J

    1997-10-01

    In the inner ear, fast excitatory synaptic transmission is mediated by ionotropic glutamate receptors, including AMPA, kainate, and NMDA receptors. The recently identified delta1 and delta2 glutamate receptors share low homology with the other three types, and no clear response or ligand binding has been obtained from cells transfected with delta alone or in combination with other ionotropic receptors. Studies of mice lacking expression of delta2 show that this subunit plays a crucial role in plasticity of cerebellar glutamatergic synapses. In addition, these mice show a deficit in vestibular compensation. These findings and the nature of glutamatergic synapses between vestibulocochlear hair cells and primary afferent dendrites suggest that delta receptors may be functionally important in the inner ear and prompted us to investigate the expression of delta receptors in the cochlea and peripheral vestibular system. Reverse transcription and DNA amplification by PCR combined with immunocytochemistry and in situ hybridization were used. Our results show that the expression of delta1 in the organ of Corti is intense and restricted to the inner hair cells, whereas delta1 is expressed in all spiral ganglion neurons as well as in their satellite glial cells. In the vestibular end organ, delta1 was highly expressed in both hair cell types and also was expressed in the vestibular ganglion neurons. The prominent expression of delta1 in inner hair cells and in type I and type II vestibular hair cells suggests a functional role in hair cell neurotransmission.

  2. Agonist-specific conformational changes in the α1-γ2 subunit interface of the GABA A receptor.

    PubMed

    Eaton, Megan M; Lim, You Bin; Bracamontes, John; Steinbach, Joe Henry; Akk, Gustav

    2012-08-01

    The GABA(A) receptor undergoes conformational changes upon the binding of agonist that lead to the opening of the channel gate and a flow of small anions across the cell membrane. Besides the transmitter GABA, allosteric ligands such as the general anesthetics pentobarbital and etomidate can activate the receptor. Here, we have investigated the agonist specificity of structural changes in the extracellular domain of the receptor. We used the substituted cysteine accessibility method and focused on the γ2(S195C) site (loop F). We show that modification of the site with (2-sulfonatoethyl)methanethiosulfonate (MTSES) results in an enhanced response to GABA, indicating accessibility of the resting receptor to the modifying agent. Coapplication of GABA or muscimol, but not of gabazine, with MTSES prevented the effect, suggesting that GABA and muscimol elicit a conformational change that reduces access to the γ2(S195C) site. Exposure of the receptors to MTSES in the presence of the allosteric activators pentobarbital and etomidate resulted in an enhanced current response indicating accessibility and labeling of the γ2(S195C) site. However, comparison of the rates of modification indicated that labeling in the presence of etomidate was significantly faster than that in the presence of pentobarbital or gabazine or in resting receptors. We infer from the data that the structure of the α1-γ2 subunit interface undergoes agonist-specific conformational changes.

  3. Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

    PubMed Central

    Klarsfeld, A; Changeux, J P

    1985-01-01

    In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases approximately equal to 2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor alpha subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of alpha-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (approximately equal to 17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation of the corresponding mRNAs. Images PMID:2989833

  4. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

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

    SciTech Connect

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

    2011-09-01

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

  6. Antigen-specific modulation of experimental myasthenia gravis: nasal tolerization with recombinant fragments of the human acetylcholine receptor alpha-subunit.

    PubMed

    Barchan, D; Souroujon, M C; Im, S H; Antozzi, C; Fuchs, S

    1999-07-06

    Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AcChoR) is the major autoantigen. The immune response in these diseases is heterogeneous and is directed to a wide variety of T and B cell epitopes of AcChoR. Candidate molecules for specific immunotherapy of MG should, therefore, have a broad specificity. We used recombinant fragments of the human AcChoR, encompassing the extracellular domain of the alpha-subunit, or shorter fragments derived from it, in experiments to modulate EAMG. We have demonstrated that intranasal administration of these recombinant fragments, which represent a major portion of epitopes involved in MG, prevents the induction of EAMG in rats and immunosuppresses an ongoing disease, as assessed by clinical symptoms, weight loss, and muscle AcChoR content. These effects on EAMG were accompanied by a marked reduction in the proliferative T-cell response and IL-2 production in response to AcChoR, in reduced anti-self AcChoR antibody titers and in an isotype switch of AcChoR-specific antibodies, from IgG2 to IgG1. We conclude that nasal tolerance induced by appropriate recombinant fragments of human AcChoR is effective in suppressing EAMG and might possibly be considered as a therapeutic modality for MG.

  7. Antigen-specific modulation of experimental myasthenia gravis: Nasal tolerization with recombinant fragments of the human acetylcholine receptor α-subunit

    PubMed Central

    Barchan, Dora; Souroujon, Miriam C.; Im, Sin-Hyeog; Antozzi, Carlo; Fuchs, Sara

    1999-01-01

    Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AcChoR) is the major autoantigen. The immune response in these diseases is heterogeneous and is directed to a wide variety of T and B cell epitopes of AcChoR. Candidate molecules for specific immunotherapy of MG should, therefore, have a broad specificity. We used recombinant fragments of the human AcChoR, encompassing the extracellular domain of the α-subunit, or shorter fragments derived from it, in experiments to modulate EAMG. We have demonstrated that intranasal administration of these recombinant fragments, which represent a major portion of epitopes involved in MG, prevents the induction of EAMG in rats and immunosuppresses an ongoing disease, as assessed by clinical symptoms, weight loss, and muscle AcChoR content. These effects on EAMG were accompanied by a marked reduction in the proliferative T-cell response and IL-2 production in response to AcChoR, in reduced anti-self AcChoR antibody titers and in an isotype switch of AcChoR-specific antibodies, from IgG2 to IgG1. We conclude that nasal tolerance induced by appropriate recombinant fragments of human AcChoR is effective in suppressing EAMG and might possibly be considered as a therapeutic modality for MG. PMID:10393952

  8. Evidence for the participation of peripheral α5 subunit-containing GABAA receptors in GABAA agonists-induced nociception in rats.

    PubMed

    Bravo-Hernández, Mariana; Feria-Morales, Luis Alberto; Torres-López, Jorge Elías; Cervantes-Durán, Claudia; Delgado-Lezama, Rodolfo; Granados-Soto, Vinicio; Rocha-González, Héctor Isaac

    2014-07-05

    The activation of GABAA receptor by γ-amino butyric acid (GABA) in primary afferent fibers produces depolarization. In normal conditions this depolarization causes a reduction in the release of neurotransmitters. Therefore, this depolarization remains inhibitory. However, previous studies have suggested that in inflammatory pain, GABA shifts its signaling from inhibition to excitation by an increased GABA-induced depolarization. The contribution of peripheral α5 subunit-containing GABAA receptors to the inflammatory pain is unknown. The purpose of this study was to investigate the possible pronociceptive role of peripheral α5 subunit-containing GABAA receptors in the formalin test. Formalin (0.5%) injection into the dorsum of the right hind paw produced flinching behavior in rats. Ipsilateral local peripheral pre-treatment (-10min) with exogenous GABA (0.003-0.03µg/paw) or common GABAA receptor agonists muscimol (0.003-0.03µg/paw), diazepam (0.017-0.056µg/paw) or phenobarbital (1-100µg/paw) significantly increased 0.5% formalin-induced nociceptive behavior. The pronociceptive effects of GABA (0.03µg/paw), muscimol (0.03µg/paw), diazepam (0.056µg/paw) and phenobarbital (100µg/paw) were prevented by either the GABAA receptor antagonist bicuculline (0.01-0.1µg/paw) or selective α5 subunit-containing GABAA receptor inverse agonist L-655,708 (0.017-0.17µg/paw). The α5 subunit-containing GABAA receptor protein was expressed in dorsal root ganglion (DRG) and dorsal spinal cord of naïve rats. The formalin injection did not modify α5 subunit-containing GABAA receptor expression. Overall, these results suggest that peripheral α5 subunit-containing GABAA receptors play a pronociceptive role in the rat formalin test.

  9. Synthesis of GABAA Receptor Agonists and Evaluation of their α-Subunit Selectivity and Orientation in the GABA Binding Site

    PubMed Central

    Jansen, Michaela; Rabe, Holger; Strehle, Axelle; Dieler, Sandra; Debus, Fabian; Dannhardt, Gerd; Akabas, Myles H.; Lüddens, Hartmut

    2008-01-01

    Drugs used to treat various disorders target GABAA receptors. To develop α subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [3H]muscimol binding and in patch-clamp experiments with heterologously expressed GABAA αiβ3γ2 receptors (i = 1–6). The effects of 5-aminomethyl-3H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all α subunit isoforms. 5-piperidin-4-yl-3H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3H- [1,3,4]oxadiazol-2-thione 6a were weak agonists at α3–, α3–, and α5–containing receptors. When coapplied with GABA they were antagonistic inα2–, α4–, and α6–containing receptors and potentiated α3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants α1F64C and α1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the α1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing α subtype selective GABA mimetic drugs. PMID:18651727

  10. Expression of GABA(A) receptor alpha3-, theta-, and epsilon-subunit mRNAs during rat CNS development and immunolocalization of the epsilon subunit in developing postnatal spinal cord.

    PubMed

    Pape, J-R; Bertrand, S S; Lafon, P; Odessa, M-F; Chaigniau, M; Stiles, J K; Garret, M

    2009-04-21

    Ionotropic GABA(A) receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABA(A) receptors consist of alpha1-, beta2-, and gamma2-subunits that are co-expressed in numerous brain areas. alpha3-, theta-, And epsilon-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of epsilon and theta transcripts before birth, to a theta and alpha3 expression at birth, and finally to a grouped epsilon, theta and alpha3 expression postpartum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), epsilon and theta appeared to have a greater spatial distribution before the dominance of the alpha3 subunit transcript around birth. We also revealed expression of alpha3, theta, and epsilon in the developing spinal cord and identified neurons that express epsilon in the postnatal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of alpha3-, theta- and epsilon-subunits may be assembled at a regional and developmental level in the brain.

  11. EXPRESSION OF GABAA RECEPTOR α3-, θ-, AND ε-SUBUNIT mRNAs DURING RAT CNS DEVELOPMENT AND IMMUNOLOCALIZATION OF THE ε SUBUNIT IN DEVELOPING POSTNATAL SPINAL CORD

    PubMed Central

    PAPE, J.-R.; BERTRAND, S. S.; LAFON, P.; ODESSA, M.-F.; CHAIGNIAU, M.; STILES, J. K.; GARRET, M.

    2010-01-01

    Ionotropic GABAA receptors are heteromeric structures composed of a combination of five from at least 16 different subunits. Subunit genes are expressed in distinct cell types at specific times during development. The most abundant native GABAA receptors consist of α1-, β2-, and γ2-subunits that are co-expressed in numerous brain areas. α3-, θ-, And ε-subunits are clustered on the X chromosome and show striking overlapping expression patterns throughout the adult rat brain. To establish whether these subunits are temporally and spatially co-expressed, we used in situ hybridization to analyze their expression throughout rat development from embryonic stage E14 to postnatal stage P12. Each transcript exhibited a unique or a shared regional and temporal developmental expression profile. The thalamic expression pattern evolved from a restricted expression of ε and θ transcripts before birth, to a θ and α3 expression at birth, and finally to a grouped ε, θ and α3 expression post-partum. However, strong similarities occurred, such as a grouped expression of the three subunits within the hypothalamus, tegmentum and pontine nuclei throughout the developmental process. At early stages of development (E17), ε and θ appeared to have a greater spatial distribution before the dominance of the α3 subunit transcript around birth. We also revealed expression of α3, θ, and ε in the developing spinal cord and identified neurons that express ε in the post-natal dorsal horn, intermediolateral column and motoneurons. Our findings suggest that various combinations of α3-, θ- and ε-subunits may be assembled at a regional and developmental level in the brain. PMID:19249336

  12. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  13. Modulation of recombinant, α2*, α3* or α4*-nicotinic acetylcholine receptor (nAChR) function by nAChR β3 subunits.

    PubMed

    Dash, Bhagirathi; Bhakta, Minoti; Chang, Yongchang; Lukas, Ronald J

    2012-05-01

    The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3(V9'S) subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.

  14. HIV-1 gp120Bal down-regulates phosphorylated NMDA receptor subunit 1 in cortical neurons via activation of glutamate and chemokine receptors

    PubMed Central

    Ru, Wenjuan; Tang, Shao-Jun

    2015-01-01

    HIV-1 envelope glycoprotein gp120 (gp120) is a major virulence protein implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Although gp120 has been suggested to cause synaptic and neuronal injuries by disrupting NMDA receptor (NMDAR) function, the underlying mechanism is unclear. Here, we show that gp120Bal down-regulates the phosphorylation of the NMDAR subunit 1 NR1 (at Ser896 and Ser897), which is essential for NMDAR function. This effect of gp120Bal is blocked by specific antagonists of both NMDA and AMPA receptors, indicating a critical role of synaptic activation. Furthermore, AMD3100 and maraviroc, antagonists of CCR5 and CXCR4 chemokine receptors, respectively, inhibit the effect of gp120Bal on NR1, suggesting that CXCR4 and CCR5 activation are involved. These findings may provide mechanistic insights into the synaptopathogenesis caused by HIV-1 infection. PMID:26582091

  15. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  16. Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α-ε subunit interface.

    PubMed

    Webster, Richard; Liu, Wei-Wei; Chaouch, Amina; Lochmüller, Hanns; Beeson, David

    2014-02-01

    Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management.

  17. [New approaches to the immunotherapy of Alzheimer's disease with the synthetic fragments of alpha7 subunit of the acetylcholine receptor].

    PubMed

    Vol'pina, O M; Volkova, T D; Titova, M A; Gershovich, Iu G; medvinskaia, N I; Samokhin, A N; Kamynina, A V; Shalgunov, V S; Koroev, D O; Filatova, M P; Oboznaia, M B; Bobkova, N V

    2008-01-01

    The effect of immunization with the synthetic fragments of the alpha7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neuro-degenetrative disease of Alzheimer's type, was studied. Mice of the NMRI line were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the alpha7 subunit extraxcellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenetrative disease of Alzheimer's type, and then the state of the spartial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with the N-terminal 1-23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159-167)-(179-188) consisting of two hydrophilic exposed regions of alpha7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of the animals were educated. Thus, the development of immunotherapy with peptide (159-167)-(179-188) seems to be a promising approach to prophylaxis and treatment of Alzheimer's disease.

  18. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    PubMed

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

  19. The NMDA receptor GluN2C subunit controls cortical excitatory-inhibitory balance, neuronal oscillations and cognitive function

    PubMed Central

    Gupta, Subhash C.; Ravikrishnan, Aparna; Liu, Jinxu; Mao, Zhihao; Pavuluri, Ratnamala; Hillman, Brandon G.; Gandhi, Pauravi J.; Stairs, Dustin J.; Li, Ming; Ugale, Rajesh R.; Monaghan, Daniel T.; Dravid, Shashank M.

    2016-01-01

    Despite strong evidence for NMDA receptor (NMDAR) hypofunction as an underlying factor for cognitive disorders, the precise roles of various NMDAR subtypes remains unknown. The GluN2C-containing NMDARs exhibit unique biophysical properties and expression pattern, and lower expression of GluN2C subunit has been reported in postmortem brains from schizophrenia patients. We found that loss of GluN2C subunit leads to a shift in cortical excitatory-inhibitory balance towards greater inhibition. Specifically, pyramidal neurons in the medial prefrontal cortex (mPFC) of GluN2C knockout mice have reduced mEPSC frequency and dendritic spine density and a contrasting higher frequency of mIPSCs. In addition a greater number of perisomatic GAD67 puncta was observed suggesting a potential increase in parvalbumin interneuron inputs. At a network level the GluN2C knockout mice were found to have a more robust increase in power of oscillations in response to NMDAR blocker MK-801. Furthermore, GluN2C heterozygous and knockout mice exhibited abnormalities in cognition and sensorimotor gating. Our results demonstrate that loss of GluN2C subunit leads to cortical excitatory-inhibitory imbalance and abnormal neuronal oscillations associated with neurodevelopmental disorders. PMID:27922130

  20. Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement

    PubMed Central

    Baur, Roland; Sigel, Erwin

    2017-01-01

    The major subunit isoform of GABAA receptors is α1β2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2β2α1β2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. β2-α1-γ2/β2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected μM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent. PMID:28114407

  1. Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.

    PubMed

    Morgan, Neil V; Brueton, Louise A; Cox, Phillip; Greally, Marie T; Tolmie, John; Pasha, Shanaz; Aligianis, Irene A; van Bokhoven, Hans; Marton, Tamas; Al-Gazali, Lihadh; Morton, Jenny E V; Oley, Christine; Johnson, Colin A; Trembath, Richard C; Brunner, Han G; Maher, Eamonn R

    2006-08-01

    Multiple pterygium syndromes (MPSs) comprise a group of multiple-congenital-anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). In addition, a variety of developmental defects (e.g., vertebral anomalies) may occur. MPSs are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. To elucidate the pathogenesis of MPS, we undertook a genomewide linkage scan of a large consanguineous family and mapped a locus to 2q36-37. We then identified germline-inactivating mutations in the embryonal acetylcholine receptor gamma subunit (CHRNG) in families with both lethal and nonlethal MPSs. These findings extend the role of acetylcholine receptor dysfunction in human disease and provide new insights into the pathogenesis and management of fetal akinesia syndromes.

  2. Kalirin binds the NR2B subunit of the NMDA receptor, altering its synaptic localization and function

    PubMed Central

    Kiraly, Drew D.; Lemtiri-Chlieh, Fouad; Levine, Eric S.; Mains, Richard E.; Eipper, Betty 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 (GEF) 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 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. PMID:21880917

  3. Kalirin binds the NR2B subunit of the NMDA receptor, altering its synaptic localization and function.

    PubMed

    Kiraly, Drew D; Lemtiri-Chlieh, Fouad; Levine, Eric S; Mains, Richard E; Eipper, Betty A

    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 (Kal7(KO)) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7(KO) mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7(KO) 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 Kal7(KO) 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.

  4. In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons.

    PubMed

    Ferando, Isabella; Mody, Istvan

    2015-01-01

    Perisynaptic and extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) mediate tonic conductances in many neurons. On principal cells of the neocortex and hippocampus they comprise α4 subunits, whereas they usually contain α1 on various interneurons. Specific characteristics of δ-GABAARs are their pharmacology and high plasticity. In particular δ-GABAARs are sensitive to low concentrations of neurosteroids (NS) and during times of altered NS production (stress, puberty, ovarian cycle and pregnancy) δ-GABAARs expression varies in many neurons regardless of the α subunits they contain, with direct consequences for neuronal excitability and network synchrony. For example δ-GABAARs plasticity on INs underlies modifications in hippocampal γ oscillations during pregnancy or over the ovarian cycle. Most δ-GABAAR-expressing INs in CA3 stratum pyramidale (SP) are parvalbumin (PV) + INs, whose fundamental role in γ oscillations generation and control has been extensively investigated. In this study we reduced or deleted δ-subunits in PV + INs, with the use of a PV/Cre-Gabrd/floxed genetic system. We find that in vitro CA3 γ oscillations of both PV-Gabrd(+/-)and PV-Gabrd(-/-) mice are characterized by higher frequencies than WT controls. The increased frequencies could be lowered to control levels in PV-Gabrd(+/-) by the NS allopregnanolone (3α,5α-tetrahydroprogesterone, 100 nM) but not the synthetic δ-GABAAR positive allosteric modulator 4-Chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl] benzamide (DS-2, 10 μM). This is consistent with the idea that DS-2, in contrast to ALLO, selectively targets α4/δ-GABAARs but not the α1/δ-GABAARs found on INs. Therefore, development of drugs selective for IN-specific α1/δ-GABAARs may be useful in neurological and psychiatric conditions correlated with altered PV + IN function and aberrant γ oscillations.

  5. Exonic Sp1 sites are required for neural-specific expression of the glycine receptor beta subunit gene.

    PubMed Central

    Tintrup, H; Fischer, M; Betz, H; Kuhse, J

    2001-01-01

    The gene encoding the beta subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5' non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5' region of the beta subunit gene and the corresponding regions of the homologous GlyR alpha subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5'-untranslated portion of the GlyR beta mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58 kb of upstream flanking region and 180 bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5' flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR beta subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites. PMID:11256962

  6. Diastereomers of the Brominated Flame Retardant 1,2-Dibromo-4-(1,2 dibromoethyl)cyclohexane Induce Androgen Receptor Activation in the HepG2 Hepatocellular Carcinoma Cell Line and the LNCaP Prostate Cancer Cell Line

    PubMed Central

    Khalaf, Hazem; Larsson, Anders; Berg, Håkan; McCrindle, Robert; Arsenault, Gilles; Olsson, Per-Erik

    2009-01-01

    Background Reported incidences of prostate cancer and masculinization of animals indicate a release of compounds with androgenic properties into the environment. Large numbers of environmental pollutants have been screened to identify such compounds; however, not until recently was 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) identified as the first potent activator of the human androgen receptor (hAR). TBECH has been found in beluga whales and bird eggs and has also been found to be maternally transferred in zebrafish. Objectives In the present study we investigated interaction energies between TBECH diastereomers (α, β, γ, and δ) and the hAR, and their ability to activate the receptor and induce prostate-specific antigen (PSA) expression in vitro. Methods We performed computational modeling to determine interaction energies between the ligand and the AR ligand-binding site, and measured in vitro competitive binding assays for AR by polarization fluorometry analysis. We used enzyme-linked immunosorbent assays to determine PSA activity in LNCaP and HepG2 cells. Results We found the γ and δ diastereomers to be more potent activators of hAR than the α and β diastereomers, which was confirmed in receptor binding studies. All TBECH diastereomers induced PSA expression in LNCaP cells even though the AR present in these cells is mutated (T877A). Modeling studies of LNCaP AR revealed that TBECH diastereomers bound to the receptor with a closer distance to the key amino acids in the ligand-binding domain, indicating stronger binding to the mutated receptor. Conclusions The present study demonstrates the ability of TBECH to activate the hAR, indicating that it is a potential endocrine disruptor. PMID:20049203

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

    SciTech Connect

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

    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.

  8. Deficiency of the beta 3 subunit of the type A gamma-aminobutyric acid receptor causes cleft palate in mice.

    PubMed

    Culiat, C T; Stubbs, L J; Woychik, R P; Russell, L B; Johnson, D K; Rinchik, E M

    1995-11-01

    In addition to its function in the nervous system, gamma-aminobutyric acid (GABA) has been implicated in mouse craniofacial development by the results of both teratological, and genetic studies. We previously reported that disruption of the cleft palate 1 (cp1) locus, closely linked to the pink-eyed dilution (p) locus on mouse chromosome 7, causes a 95% penetrant, recessive, neonatally-lethal cleft palate (CP) in mice homozygous for the p(4THO-II) deletion. We proposed that the beta 3 subunit gene (Gabrb3) of the GABAA receptor might be a candidate for cp1 (ref. 4); our earlier studies had localized cp1 to an interval beginning distal to the gene for the GABAA receptor alpha 5 subunit (Gabra5) and ending within the Gabrb3 coding region. To test the hypothesis that deletion of Gabrb3, and not another gene in the interval, causes CP, we performed an experiment to rescue the CP phenotype by introducing a Gabrb3 transgene into p(4THO-II) homozygotes. We now show that such transgenic mice are phenotypically normal, indicating that Gabrb3 is indeed the cp1 locus.

  9. Hypoplasia of spiral and Scarpa's ganglion cells in GABA(A) receptor beta(3) subunit knockout mice.

    PubMed

    Koo, Ja-Won; Homanics, Gregg E; Balaban, Carey D

    2002-05-01

    This study documents morphologic alterations in the spiral ganglion and Scarpa's ganglion from gamma-aminobutyric acid A (GABA(A)) receptor beta(3) subunit null mutant mice. The ganglion cells of the mutant mice were hypoplastic in hematoylin&eosin-stained sections. Hypoplasia was observed at every location of the spiral ganglion and Scarpa's ganglion except the apical cochlear turn. Calretinin immunostaining demonstrated a selective hypoplasia of calretinin-negative cells at every location of spiral and Scarpa's ganglion cells, while the soma area of calretinin-positive cells was not affected by the gene deletion. Meanwhile, in the spiral ganglion of both wild type and knockout mice, there were apical to basal gradients in the soma size and the proportion of calretinin-positive cells. The absence of statistically significant hypoplasia in hematoylin&eosin sections through the apical turn of the cochlea can be explained by the relatively higher proportion of calretinin-positive ganglion cells, which were unaffected by the gene deletion. These findings suggest that GABA(A) receptor isoforms containing the beta(3) subunit may play an important role in the development and differentiation of non-calyceal terminals of Scarpa's ganglion cells and type II and smaller type I spiral ganglion cells.

  10. Unique domain anchoring of Src to synaptic NMDA receptors via the mitochondrial protein NADH dehydrogenase subunit 2

    PubMed Central

    Gingrich, Jeffrey R.; Pelkey, Kenneth A.; Fam, Sami R.; Huang, Yueqiao; Petralia, Ronald S.; Wenthold, Robert J.; Salter, Michael W.

    2004-01-01

    Src is the prototypic protein tyrosine kinase and is critical for controlling diverse cellular functions. Regions in Src define structural and functional domains conserved in many cell signaling proteins. Src also contains a region of low sequence conservation termed the unique domain, the function of which has until now remained enigmatic. Here, we show that the unique domain of Src is a protein–protein interaction region and we identify NADH dehydrogenase subunit 2 (ND2) as a Src unique domain-interacting protein. ND2 is a subunit of complex I in mitochondria, but we find that ND2 interacts with Src outside this organelle at excitatory synapses in the brain. ND2 acts as an adapter protein anchoring Src to the N-methyl-d-aspartate (NMDA) receptor complex, and is crucial for Src regulation of synaptic NMDA receptor activity. By showing an extramitochondrial action for a protein encoded in the mitochondrial genome, we identify a previously unsuspected means by which mitochondria regulate cellular function, suggesting a new paradigm that may be of general relevance for control of Src signaling. PMID:15069201

  11. Cloning and characterization of glutamate receptor subunit 4 (GLUA4) and its alternatively spliced isoforms in turtle brain.

    PubMed

    Sabirzhanov, Boris; Keifer, Joyce

    2011-07-01

    Ionotropic glutamate receptors sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GluAs, play an important role in neural development, synaptic plasticity, and neurodegeneration. Previous studies using an in vitro model of eyeblink classical conditioning in pond turtles suggested that acquisition of conditioning is associated with synaptic delivery of AMPA receptors containing GluA4 subunits. However, sequences of the GluA4 subunit, expression profile, and its alternatively spliced isoforms in turtle brain have not been previously determined. The sequence and domain structure of turtle GluA4 (tGluA4) and its splice variants was characterized. We found ten isoforms of tGluA4 including several previously unidentified truncated variants. Analysis of the nucleotide sequences of tGluA4 flip/flop, tGluA4c flip/flop, and tGluA4s showed they are highly similar to known isoforms of the GluA4 subunit identified in chick. Examination of the relative abundance of mRNA expression for the tGluA4 variants showed that the flip and flop versions of tGluA4 and tGluA4c, and a novel truncated variant, tGluA4trc1, which is also expressed as protein, are major forms in the adult turtle brain. Identification of these alternatively spliced isoforms of tGluA4 will provide a unique opportunity to assess their role in synaptic plasticity through the application of short interfering RNAs.

  12. A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface.

    PubMed

    Frugier, Guillaume; Coussen, Françoise; Giraud, Marie-France; Odessa, Marie-Françoise; Emerit, Michel B; Boué-Grabot, Eric; Garret, Maurice

    2007-02-09

    Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

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

    SciTech Connect

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

    2010-12-01

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

  14. Further evidence for clustering of human GABA[sub A] receptor subunit genes: Localization of the [alpha][sub 6]-subunit gene (GABRA6) to distal chromosome 5q by linkage analysis

    SciTech Connect

    Hicks, A.A.; Kamphuis, W.; Darlison, M.G. ); Bailey, M.E.S.; Johnson, K.J. ); Riley, B.P. ); Siciliano, M.J. )

    1994-03-15

    GABA[sub A] receptors are hetero-oligomeric ion-channel complexes that are composed of combinations of [alpha], [beta], [gamma], and [delta] subunits and play a major role in inhibitory neurotransmission in the mammalian brain. The authors report here a microsatellite polymorphism within the human [alpha][sub 6]-subunit gene (GABRA6). Mapping of this marker in a human-hamster hybrid cell-line panel and typing of the repeat in the Centre d'Etude du Polymorphisme Humain (CEPH) reference families enabled the localization of this gene to chromosome 5q and established its linkage to the GABA[sub A] receptor [alpha][sub 1]-subunit gene (GA-BRA1) with a maximum lod score (Z[sub max]) of 39.87 at a [theta] of 0.069 (males) and 0.100 (females). These results reveal the clustering of GABRA6, GABRA1, and the GABA[sub A] receptor [gamma][sub 2]-subunit gene (GABRG2) on distal chromosome 5q. 17 refs., 1 fig., 1 tab.

  15. Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors.

    PubMed

    Gehring, U; Mugele, K; Arndt, H; Busch, W

    1987-09-01

    Apparent molecular weights of wild-type and nti ('increased nuclear transfer') mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of Mr 328,000 and 298,000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of Mr 130,000 and 63,000 of the wild-type and mutant, respectively, were obtained at 300 mM KCl and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nti receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nti receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nti receptors. We conclude that the large forms of wild-type and nti receptors are heteromeric and contain only one hormone-building polypeptide per complex.

  16. Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus.

    PubMed

    Cardona-Gómez, Gloria Patricia; Arango-Davila, Cesar; Gallego-Gómez, Juan Carlos; Barrera-Ocampo, Alvaro; Pimienta, Hernan; Garcia-Segura, Luis Miguel

    2006-08-21

    During cerebral ischemia, part of the damage associated with the hyperactivation of glutamate receptors results from the hyperphosphorylation of the microtubule-associated protein Tau. Previous studies have shown that estradiol treatment reduces neural damage after cerebral ischemia. Here, we show that transient occlusion of the middle cerebral artery results in the hyperphosphorylation of Tau and in a significant increase in the association of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type glutamate receptor subunits 2/3 in the hippocampus. Estradiol treatment decreased hippocampal injury, inhibited glycogen synthase kinase-3beta and decreased the hyperphosphorylation of Tau and the interaction of Tau with glycogen synthase kinase-3beta and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor. These findings suggest that ischemia produces a strong association between Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor, and estradiol can exert at least part of its neuroprotective activity through inhibition of glycogen synthase kinase-3beta.

  17. Ethyl 2,4,6-trihydroxybenzoate is an agonistic ligand for liver X receptor that induces cholesterol efflux from macrophages without affecting lipid accumulation in HepG2 cells.

    PubMed

    Hoang, Minh-Hien; Jia, Yaoyao; Jun, Hee-jin; Lee, Ji-Hae; Lee, Dong-Ho; Hwang, Bang-Yeon; Kim, Woo-Jin; Lee, Hak-Ju; Lee, Sung-Joon

    2012-06-15

    The present study reports a novel liver X receptor (LXR) activator, ethyl 2,4,6-trihydroxybenzoate (ETB), isolated from Celtis biondii. Using a reporter gene assay, time-resolved fluorescence resonance energy transfer (TR-FRET), and surface plasmon resonance (SPR) analysis, we showed that ETB directly bound to and stimulated the transcriptional activity of LXR-α and LXR-β. In macrophages, hepatocytes, and intestinal cells, ETB suppressed cellular cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes. Notably, ETB did not induce lipogenic gene expression or cellular triglyceride accumulation in hepatocytes. These results suggest that ETB is a dual-LXR modulator that regulates the expression of key genes in cholesterol homeostasis in multiple cells without inducing lipid accumulation in HepG2 cells.

  18. Phytol directly activates peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) and regulates gene expression involved in lipid metabolism in PPAR{alpha}-expressing HepG2 hepatocytes

    SciTech Connect

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kato, Sota; Egawa, Kahori; Ebisu, Shogo; Moriyama, Tatsuya; Fushiki, Tohru; Kawada, Teruo . E-mail: fat@kais.kyoto-u.ac.jp

    2005-11-18

    The peroxisome proliferator-activated receptor (PPAR) is one of the indispensable transcription factors for regulating lipid metabolism in various tissues. In our screening for natural compounds that activate PPAR using luciferase assays, a branched-carbon-chain alcohol (a component of chlorophylls), phytol, has been identified as a PPAR{alpha}-specific activator. Phytol induced the increase in PPAR{alpha}-dependent luciferase activity and the degree of in vitro binding of a coactivator, SRC-1, to GST-PPAR{alpha}. Moreover, the addition of phytol upregulated the expression of PPAR{alpha}-target genes at both mRNA and protein levels in PPAR{alpha}-expressing HepG2 hepatocytes. These findings indicate that phytol is functional as a PPAR{alpha} ligand and that it stimulates the expression of PPAR{alpha}-target genes in intact cells. Because PPAR{alpha} activation enhances circulating lipid clearance, phytol may be important in managing abnormalities in lipid metabolism.

  19. Reporter mutation studies show that nicotinic acetylcholine receptor (nAChR) α5 Subunits and/or variants modulate function of α6*-nAChR.

    PubMed

    Dash, Bhagirathi; Chang, Yongchang; Lukas, Ronald J

    2011-11-04

    To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3(V9)'(S) subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.

  20. Expression of striatal adenosine and dopamine receptors in mice deficient in the p50 subunit of NF-κB

    PubMed Central

    Xie, Xiaobin; Jhaveri, Krishna A.; Ding, Ming; Hughes, Larry F.; Toth, Linda A.; Ramkumar, Vickram

    2007-01-01

    The striatal dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AAR) exhibit mutually antagonistic effects through physical interactions and by differential modulation of post-receptor signaling pathways. The expression of the A2AAR and the D2R are differentially regulated by nuclear factor-κB (NFkB). In this report, we determined the role of NFkB in regulation of these receptors by comparing mice deficient in the NFκB p50 subunit (p50 KO) with genetically intact B6129PF2/J (F2) mice. Quantification of adenosine receptor (AR) subtypes in mouse striatum by real time PCR, immunocytochemistry and radioligand binding assays showed more A2AAR but less A1AR in p50 KO mice as compared with F2 mice. Striata from p50 KO mice also had less D2R mRNA and [3H]-methylspiperone binding than did striata from F2 mice. Gαolf and Gαs proteins, which are transducers of A2AAR signals, were also present at a higher level in striata from the p50 KO versus F2 mice. In contrast, the Gαi1 protein, which transduces signals from the A1AR and D2R, was significantly reduced in striata from p50-/ mice. Behaviorally, p50 KO mice exhibited increased locomotor activity relative to that of F2 mice after caffeine ingestion. These data are consistent with a role for the NFkB in the regulation of A1AR, A2AAR, D2R and possibly their coupling G proteins in the striatum. Dysregulation of these receptors in the striata of p50 KO mice might sensitize these animals to locomotor stimulatory action of caffeine. PMID:17869311

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

    PubMed

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

    1996-02-01

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

  2. G2 Gas Cloud Simulation

    NASA Video Gallery

    This simulation shows the future behavior of the G2 gas cloud now approaching Sgr A*, the supermassive black hole at the center of the Milky Way. X-ray emission from the cloud's tidal interaction w...

  3. Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

    PubMed

    McClure-Begley, T D; Papke, R L; Stone, K L; Stokes, C; Levy, A D; Gelernter, J; Xie, P; Lindstrom, J; Picciotto, M R

    2014-03-01

    Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4β2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4β2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4β2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4β2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

  4. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

    PubMed Central

    Thomsen, Morten Skøtt; Zwart, Ruud; Ursu, Daniel; Jensen, Majbrit Myrup; Pinborg, Lars Hageman; Gilmour, Gary; Wu, Jie; Sher, Emanuele; Mikkelsen, Jens Damsgaard

    2015-01-01

    The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands. PMID:26086615

  5. Two steps of insulin receptor internalization depend on different domains of the beta-subunit [published erratum appears in J Cell Biol 1993 Nov;123(4):1047

    PubMed Central

    1993-01-01

    The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin- coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit. PMID:8376461

  6. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  7. Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

    PubMed

    Christensen, Mark H; Kohlmeier, Kristi A

    2016-03-01

    The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry.

  8. Interferon-α Induces Neurotoxicity Through Activation of the Type I Receptor and the GluN2A Subunit of the NMDA Receptor

    PubMed Central

    Kessing, Cari F.

    2015-01-01

    Elevated levels of interferon-alpha (IFNα) in the central nervous system (CNS) are linked to cognitive dysfunction in patients with inflammatory CNS diseases such as HIV-associated neurocognitive disorders (HAND). Increased CNS IFNα has also been found to be associated with cognitive dysfunction in a HAND mouse model. Here, we corroborate previous studies showing a dose-dependent decrease in dendritic branching and length caused by IFNα treatment and extend those studies. Because both direct and indirect mechanisms of IFNα-induced neurotoxicity are likely involved, the cell signaling pathway involving the IFNα receptor (IFNAR) was initially evaluated. Rat neuronal cultures exposed to IFNα demonstrate increased phosphorylation of STAT1 and increased interferon stimulating gene 15 (ISG15) expression, indicators of IFNAR engagement. However, specific blocking antibodies to the IFNAR were found to only partially protect neurons from IFNα-induced neurotoxicity. Additionally, inhibiting the GluN2A subunit of N-methyl-D-asparate receptor (NMDAR) was also found to be partially protective against IFNα-induced neurotoxicity compared with the GluN2B subunit. Neurotoxicity is evident in neurons extracted from IFNAR KO mice treated with IFNα as well, further indicating that IFNAR signaling is not required for IFNα neurotoxicity. The neurotoxic actions of IFNα are mediated through both the IFNAR as well as the GluN2A subunit of the NMDAR to reduce dendritic arborization in neurons. Complete protection from IFNα-induced neurotoxicity was demonstrated when both pathways were blocked. Blocking these pathways could lead to potential therapies for cognitive dysfunction during neuroinflammation and specifically lead to better treatments for HAND. PMID:25517826

  9. Delta-subunit-containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

    PubMed Central

    Marowsky, Anne; Vogt, Kaspar E.

    2014-01-01

    The intercalated paracapsular cells (pcs) are small GABAergic interneurons that form densely populated clusters surrounding the basolateral (BLA) complex of the amygdala. Their main task in the amygdala circuitry appears to be the control of information flow, as they act as an inhibitory interface between input and output nuclei. Modulation of their activity is thus thought to affect amygdala output and the generation of fear and anxiety. Recent evidence indicates that pcs express benzodiazepine (BZ)-sensitive GABAA receptor (GABAAR) variants containing the α2- and α3-subunit for transmission of post-synaptic currents, yet little is known about the expression of extrasynaptic GABAARs, mediating tonic inhibition and regulating neuronal excitability. Here, we show that pcs from the lateral and medial intercalated cell cluster (l- and mITC, respectively) express a tonic GABAergic conductance that could be significantly increased in a concentration-dependent manner by the δ-preferring GABAAR agonist THIP (0.5–10 μM), but not by the BZ diazepam (1 μM). The neurosteroid THDOC (300 nM) also increased tonic currents in pcs significantly, but only in the presence of additional GABA (5 μM). Immunohistochemical stainings revealed that both the δ-GABAAR and the α4-GABAAR subunit are expressed throughout all ITCs, while no staining for the α5-GABAAR subunit could be detected. Moreover, 1 μM THIP dampened excitability in pcs most likely by increasing shunting inhibition. In line with this, THIP significantly decreased lITC-generated inhibition in target cells residing in the BLA nucleus by 30%. Taken together these results demonstrate for the first time that pcs express a tonic inhibitory conductance mediated most likely by α4/δ-containing GABAARs. This data also suggest that δ-GABAAR targeting compounds might possibly interfere with pcs-related neuronal processes such as fear extinction. PMID:24723854

  10. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype

    PubMed Central

    Paloneva, Juha; Manninen, Tuula; Christman, Grant; Hovanes, Karine; Mandelin, Jami; Adolfsson, Rolf; Bianchin, Marino; Bird, Thomas; Miranda, Roxana; Salmaggi, Andrea; Tranebjærg, Lisbeth; Konttinen, Yrjö; Peltonen, Leena

    2002-01-01

    Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype. PMID:12080485

  11. Masking of an endoplasmic reticulum retention signal by its presence in the two subunits of the asialoglycoprotein receptor.

    PubMed

    Shenkman, M; Ehrlich, M; Lederkremer, G Z

    2000-01-28

    Human asialoglycoprotein receptor H1 and H2b subunits assemble into a hetero-oligomer that travels to the cell surface. The H2a variant on the other hand is a precursor of a cleaved soluble form that is secreted. Uncleaved H2a precursor molecules cannot exit the endoplasmic reticulum (ER), a lumenal juxtamembrane pentapeptide being responsible for their retention. Insertion of this pentapeptide into H1 (H1i5) causes its complete ER retention but not fast degradation as happens to H2a. Cotransfection of H2a elicited, by heterodimerization, the Golgi processing of H1i5 and its surface expression. This occurred to a much lesser extent by cotransfection of H2b. Likewise, coexpression of H1i5 and not H1 stabilized H2a and caused its export to the cell surface. Homodimerization of molecules containing the pentapeptide did not cancel the retention. Thus, only when the pentapeptide is present in both subunits is the ER retention efficiently abrogated. The results show the unexpected finding that identical ER retention signals present in two associated chains can mask and cancel each other's effect. This could have important implications as similar abrogation of ER retention of other proteins could eventually be obtained by engineering and coexpressing an associated protein containing the same retention signal.

  12. The N-methyl-D-aspartate receptor's neglected subunit - GluN1 matters under normal and hyperbaric conditions.

    PubMed

    Bliznyuk, Alice; Aviner, Ben; Golan, Hava; Hollmann, Michael; Grossman, Yoram

    2015-10-01

    Professional deep-water divers exposed to hyperbaric pressure (HP) above 1.1 MPa develop high-pressure neurological syndrome, which is associated with central nervous system hyperexcitability. It was previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. In addition, we have reported that HP (10.1 MPa) differentially affects ionic currents, measured by the two-electrode voltage-clamp technique, of eight specific NMDAR subtypes generated by the co-expression of GluN1-1a or GluN1-1b with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. We now report that eight GluN1 splice variants, when co-expressed with GluN2A, mediate different ionic currents at normal and HP (5.1 MPa). These data, in conjunction with our previous results, indicate that both GluN1 and GluN2 subunits play a critical role in determining NMDAR currents under normal and HP conditions. These data, given the differential spatial distribution of the different NMDAR subtypes in the central nervous system, may offer a partial explanation for the mechanism governing the complex signs and symptoms of high-pressure neurological syndrome, and an explanation for the suspected long-term HP health decrement due to repetitive deep dives by professional divers.

  13. Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

    PubMed Central

    Standley, Steve; Petralia, Ronald S.; Hamilton, Rebecca; Wang, Ya-Xian; Schubert, Manfred

    2012-01-01

    NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets. PMID:22761831

  14. Localization of two major GABA(A) receptor subunits in the dentate gyrus of the rat and cell type-specific up-regulation following entorhinal cortex lesion.

    PubMed

    Simbürger, E; Plaschke, M; Fritschy, J M; Nitsch, R

    2001-01-01

    GABA(A) receptor subunits show a specific regional distribution in the CNS during development and in the adult animal. In the hippocampal formation, individual subsets of GABAergic interneurons are highly immunoreactive for the alpha1-subunit, whereas granule and pyramidal cells show a strong expression of the alpha2-subunit. Using confocal microscopy and digital image analysis, we demonstrate that in the dentate gyrus the alpha1-subunit immunolabeling appears in differently sized clusters. The large clusters, which are confined to dendrites of interneurons, show no alpha2 labeling, whereas the smaller ones coincide with alpha2-subunit-positive clusters. In the molecular layer, the clusters of both alpha-subunits co-localize with the anchoring protein gephyrin. In the granule cell layer and hilus, we found alpha1- and alpha2-subunit-positive clusters which were devoid of gephyrin labeling. Lesions of the medial entorhinal cortex led to the deafferentation of dendrites in the middle molecular layer of the dentate gyrus. This resulted in a significantly increased concentration of alpha2-subunit-positive clusters. We also observed an increase of alpha1-subunit immunolabeling in the deafferented area. We found no change in the co-localization between alpha1 and alpha2, and no significant change in the number of large alpha1-positive clusters along individual dendritic segments of interneurons. In a previous study, we demonstrated that calbindin-immunoreactive dendrites of granule cells revealed a significant increase in gephyrin immunoreactivity following lesion, whereas parvalbumin-positive dendrites showed no such alterations. The predominant localization of small gephyrin clusters in dendrites of granule cells, which was also described in this study, leads to the conclusion that the increase of the alpha2-subunit-positive clusters, demonstrated in the present study, indicates that, following entorhinal cortex lesion, new GABAergic synapses may be formed and that

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

    PubMed

    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.

  16. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25.

    PubMed

    Hung, Rayjean J; McKay, James D; Gaborieau, Valerie; Boffetta, Paolo; Hashibe, Mia; Zaridze, David; Mukeria, Anush; Szeszenia-Dabrowska, Neonilia; Lissowska, Jolanta; Rudnai, Peter; Fabianova, Eleonora; Mates, Dana; Bencko, Vladimir; Foretova, Lenka; Janout, Vladimir; Chen, Chu; Goodman, Gary; Field, John K; Liloglou, Triantafillos; Xinarianos, George; Cassidy, Adrian; McLaughlin, John; Liu, Geoffrey; Narod, Steven; Krokan, Hans E; Skorpen, Frank; Elvestad, Maiken Bratt; Hveem, Kristian; Vatten, Lars; Linseisen, Jakob; Clavel-Chapelon, Françoise; Vineis, Paolo; Bueno-de-Mesquita, H Bas; Lund, Eiliv; Martinez, Carmen; Bingham, Sheila; Rasmuson, Torgny; Hainaut, Pierre; Riboli, Elio; Ahrens, Wolfgang; Benhamou, Simone; Lagiou, Pagona; Trichopoulos, Dimitrios; Holcátová, Ivana; Merletti, Franco; Kjaerheim, Kristina; Agudo, Antonio; Macfarlane, Gary; Talamini, Renato; Simonato, Lorenzo; Lowry, Ray; Conway, David I; Znaor, Ariana; Healy, Claire; Zelenika, Diana; Boland, Anne; Delepine, Marc; Foglio, Mario; Lechner, Doris; Matsuda, Fumihiko; Blanche, Helene; Gut, Ivo; Heath, Simon; Lathrop, Mark; Brennan, Paul

    2008-04-03

    Lung cancer is the most common cause of cancer death worldwide, with over one million cases annually. To identify genetic factors that modify disease risk, we conducted a genome-wide association study by analysing 317,139 single-nucleotide polymorphisms in 1,989 lung cancer cases and 2,625 controls from six central European countries. We identified a locus in chromosome region 15q25 that was strongly associated with lung cancer (P = 9 x 10(-10)). This locus was replicated in five separate lung cancer studies comprising an additional 2,513 lung cancer cases and 4,752 controls (P = 5 x 10(-20) overall), and it was found to account for 14% (attributable risk) of lung cancer cases. Statistically similar risks were observed irrespective of smoking status or propensity to smoke tobacco. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3 and CHRNB4). Such subunits are expressed in neurons and other tissues, in particular alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to N'-nitrosonornicotine and potential lung carcinogens. A non-synonymous variant of CHRNA5 that induces an amino acid substitution (D398N) at a highly conserved site in the second intracellular loop of the protein is among the markers with the strongest disease associations. Our results provide compelling evidence of a locus at 15q25 predisposing to lung cancer, and reinforce interest in nicotinic acetylcholine receptors as potential disease candidates and chemopreventative targets.

  17. Sex-Dependent Anti-Stress Effect of an α5 Subunit Containing GABAA Receptor Positive Allosteric Modulator

    PubMed Central

    Piantadosi, Sean C.; French, Beverly J.; Poe, Michael M.; Timić, Tamara; Marković, Bojan D.; Pabba, Mohan; Seney, Marianne L.; Oh, Hyunjung; Orser, Beverley A.; Savić, Miroslav M.; Cook, James M.; Sibille, Etienne

    2016-01-01

    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 min 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 the hippocampus after chronic treatment with α5-PAM in females only, and these expression changes correlated with behavioral emotionality. Conclusion: 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

  18. Short-term steroid treatment increases δ GABAA receptor subunit expression in rat CA1 hippocampus: Pharmacological and behavioral effects

    PubMed Central

    Shen, Hui; Gong, Qi Hua; Yuan, Maoli; Smith, Sheryl S.

    2010-01-01

    In this study, 48 h administration of 3α-OH-5β-pregnan-20-one (3α,5β-THP) or 17β-estradiol (E2)+progesterone (P) to female rats increased expression of the δ subunit of the GABAA receptor (GABAR) in CA1 hippocampus. Coexpression of α4 and δ subunits was suggested by an increased response of isolated pyramidal cells to the GABA agonist 4,5,6,7- tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), following 48 h steroid treatment, and nearly complete blockade by 300 μM lanthanum (La3+). Because α4βδ GABAR are extrasynaptic, we also recorded pharmacologically isolated GABAergic holding current from CA1 hippocampal pyramidal cells in the slice. The La3+-sensitive THIP current, representative of current gated by α4βδ GABAR, was measurable only following 48 h steroid treatment. In contrast, the bicuculline-sensitive current was not altered by steroid treatment, assessed with or without 200 nM gabazine to block synaptic current. However, 48 h steroid treatment resulted in a tonic current insensitive to the benzodiazepine agonists lorazepam (10 μM) and zolpidem (100 nM). These results suggest that 48 h steroid treatment increases expression of α4βδ GABAR which replace the ambient receptor population. Increased anxiolytic effects of THIP were also observed following 48 h steroid treatment. The findings from the present study may be relevant for alterations in mood and benzodiazepine sensitivity reported across the menstrual cycle. PMID:15950994

  19. Interaction of p72syk with the gamma and beta subunits of the high-affinity receptor for immunoglobulin E, Fc epsilon RI.

    PubMed Central

    Shiue, L; Green, J; Green, O M; Karas, J L; Morgenstern, J P; Ram, M K; Taylor, M K; Zoller, M J; Zydowsky, L D; Bolen, J B

    1995-01-01

    Activation of protein tyrosine kinases is one of the initial events following aggregation of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on RBL-2H3 cells, a model mast cell line. The protein tyrosine kinase p72syk (Syk), which contains two Src homology 2 (SH2) domains, is activated and associates with phosphorylated Fc epsilon RI subunits after receptor aggregation. In this report, we used Syk SH2 domains, expressed in tandem or individually, as fusion proteins to identify Syk-binding proteins in RBL-2H3 lysates. We show that the tandem Syk SH2 domains selectively associate with tyrosine-phosphorylated forms of the gamma and beta subunits of Fc epsilon RI. The isolated carboxy-proximal SH2 domain exhibited a significantly higher affinity for the Fc epsilon RI subunits than did the amino-proximal domain. When in tandem, the Syk SH2 domains showed enhanced binding to phosphorylated gamma and beta subunits. The conserved tyrosine-based activation motifs contained in the cytoplasmic domains of the gamma and beta subunits, characterized by two YXXL/I sequences in tandem, represent potential high-affinity binding sites for the dual SH2 domains of Syk. Peptide competition studies indicated that Syk exhibits a higher affinity for the phosphorylated tyrosine activation motif of the gamma subunit than for that of the beta subunit. In addition, we show that Syk is the major protein in RBL-2H3 cells that is affinity isolated with phosphorylated peptides corresponding to the phosphorylated gamma subunit motif. These data suggest that Syk associates with the gamma subunit of the high-affinity receptor for immunoglobulin E through an interaction between the tandem SH2 domains of SH2 domains of Syk and the phosphorylated tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Fc epsilon RI tyrosine activation motif of the gamma subunit and that Syk may be the major signaling protein that binds to Dc epsilon

  20. Tyrosine phosphorylation of the NR2B subunit of the NMDA receptor in the spinal cord contributes to chronic visceral pain in rats.

    PubMed

    Luo, Xiao-Qing; Cai, Qin-Yan; Chen, Yu; Guo, Li-Xia; Chen, Ai-Qin; Wu, Zhen-Quan; Lin, Chun

    2014-01-13

    The roles of spinal N-methyl-d-aspartic acid receptor 2B (NR2B) subunit in central sensitization of chronic visceral pain were investigated. A rat model with irritable bowel syndrome (IBS) was established by colorectal distention (CRD) on post-natal days 8-14. Responses of the external oblique muscle of the abdomen to CRD were measured to evaluate the sensitivity of visceral pain in rats. The sensitivity of visceral pain significantly increased in IBS-like rats. Expressions of spinal NR2B subunit and phosphorylated NR2B subunit significantly increased by 50-55% in IBS-like rats when compared with those in control rats. Ro 25-6981, a selective antagonist of NR2B subunit, has a dose-dependent anti-allodynic and anti-hyperalgesic effect without causing motor dysfunction in IBS-like rats. Furthermore, the activation mechanism of the spinal NR2B subunit in chronic visceral pain was also investigated. Spinal administration of genistein, a specific inhibitor of tyrosine kinases, also decreased the visceral pain hypersensitivity of IBS-like rats in a dose-dependent manner. In addition, the expression of phosphorylated NR2B subunit was decreased after spinal administration of Ro 25-6981 or genistein in IBS-like rats. In conclusion, tyrosine kinase activation-induced phosphorylation of NR2B subunit may play a crucial role in central sensitization of chronic visceral pain.

  1. G2 Flywheel Module Design

    NASA Technical Reports Server (NTRS)

    Jensen, Ralph H.; Dever, Timothy P.

    2006-01-01

    Design of a flywheel module, designated the G2 module, is described. The G2 flywheel is a 60,000 RPM, 525 W-hr, 1 kW system designed for a laboratory environment; it will be used for component testing and system demonstrations, with the goal of applying flywheels to aerospace energy storage and integrated power and attitude control (IPACS) applications. G2 has a modular design, which allows for new motors, magnetic bearings, touchdown bearings, and rotors to be installed without a complete redesign of the system. This design process involves several engineering disciplines, and requirements are developed for the speed, energy storage, power level, and operating environment. The G2 rotor system consists of a multilayer carbon fiber rim with a titanium hub on which the other components mount, and rotordynamics analysis is conducted to ensure rigid and flexible rotor modes are controllable or outside of the operating speed range. Magnetic bearings are sized using 1-D magnetic circuit analysis and refined using 3-D finite element analysis. The G2 magnetic bearing system was designed by Texas A&M and has redundancy which allows derated operation after the loss of some components, and an existing liquid cooled two pole permanent magnet motor/generator is used. The touchdown bearing system is designed with a squeeze film damper system allowing spin down from full operating speed in case of a magnetic bearing failure. The G2 flywheel will enable module level demonstrations of component technology, and will be a key building block in system level attitude control and IPACS demonstrations.

  2. Differential dependence of the D1 and D5 dopamine receptors on the G protein gamma 7 subunit for activation of adenylylcyclase.

    PubMed

    Wang, Q; Jolly, J P; Surmeier, J D; Mullah, B M; Lidow, M S; Bergson, C M; Robishaw, J D

    2001-10-19

    The D(1) dopamine receptor, G protein gamma(7) subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein gamma(7) subunit in HEK 293 cells stably expressing the human D(1) dopamine receptor. Prior in vitro analysis revealed that the gamma(7) ribozyme possessed cleavage activity directed exclusively toward the gamma(7) RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997) J. Biol. Chem. 272, 26040-26048). In vivo analysis of cells transfected with the gamma(7) ribozyme showed a specific reduction in the expression of the gamma(7) protein. Coincident with the loss of the gamma(7) protein, there was a noticeable reduction in the expression of the beta(1) protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the beta(1) and gamma(7) proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D(1) dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the beta(1) and gamma(7) proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D(5) dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D(1) and D(5) dopamine receptor subtypes utilize G proteins composed of distinct betagamma subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D(1) dopamine receptor and the G protein gamma(7) subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein gamma(7) subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

  3. The g2 Structure Function

    SciTech Connect

    Matthias Burkardt

    2009-07-01

    Polarized structure functions at low $Q^2$ have the physical interpretation of (generalized) spin polarizabilities. At high $Q^2$, the polarized parton distribution $g_2(x)$ provides access to quark-gluon correlations in the nucleon. We discuss the interpretation of the $x^2$ moment of $\\bar{g}_2(x)$ as an average transverse force on quarks in deep-inelastic scattering from a transversely polarized target. Qualitative connections with generalized parton distributions are emphasized. The $x^2$ moment of the chirally-odd twist-3 parton distribution $e(x)$ provides information on the dependence of the average transverse force on the transversity of the quark.

  4. Cellular protein and mRNA expression of β1 nicotinic acetylcholine receptor (nAChR) subunit in brain, skeletal muscle and placenta.

    PubMed

    Aishah, Atqiya; Hinton, Tina; Machaalani, Rita

    2017-01-30

    The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.

  5. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer.

    PubMed Central

    Karunagaran, D; Tzahar, E; Beerli, R R; Chen, X; Graus-Porta, D; Ratzkin, B J; Seger, R; Hynes, N E; Yarden, Y

    1996-01-01

    Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling. Images PMID:8617201

  6. Stimulation of transactivation of the largemouth bass estrogen receptors alpha, beta-a, and beta-b by methoxychlor and its mono- and bis-demethylated metabolites in HepG2 cells

    PubMed Central

    Blum, Jason L.; James, Margaret O.; Stuchal, Leah D.; Denslow, Nancy D.

    2008-01-01

    The purpose of this study was to determine the mechanisms by which the pesticide, methoxychlor (MXC), acts as an environmental endocrine disruptor through interaction with the three largemouth bass (Micropterus salmoides) estrogen receptors (ERs) α, βa, and βb. MXC is a less-environmentally persistent analog of DDT that behaves as a weak estrogen. Using transient transfection assays in HepG2 cells, we have previously shown that each receptor is responsive to the endogenous ligand 17β-estradiol (E2) in a dose-dependent manner. The parent compound, MXC, showed dose-dependent stimulation of transcriptional activation through all three ERs. In addition to the parent molecule, each of the metabolites was also estrogenic with all three ERs. The order of potency for ERα and ERβb was HPTE > OH-MXC > MXC, while the opposite order was seen for ERβa. HepG2 cells did not substantially metabolize MXC to the active metabolites, thus the activity of MXC was not due to metabolism. When examining the effects of increasing concentrations of MXC at a fixed concentration of E2, all three ERs show increased activity compared to that with E2 alone, showing that the effects of MXC and E2 are additive. However, when this experiment was repeated with increasing concentrations of HPTE at a fixed concentration of E2, the activity of ERα was decreased, that of ERβb was increased, while that of ERβa was unaffected compared to E2 alone. These experiments suggest that HPTE functions as an E2 antagonist with Erα, an E2 agonist with ERβb and does not perturb E2 stimulation of ERβa. While it is clear the ERβ subtypes are the products of different genes (due to a gene duplication in teleosts) the differences in their responses to MXC and its metabolites indicate that their functions diverge, both in their in vivo molecular response to E2, as well as to their interaction with endocrine disrupting compounds found in the wild. PMID:17949972

  7. Quinazolin-4-one derivatives: A novel class of non-competitive NR2C/D subunit-selective N-methyl-D-aspartate receptor antagonists

    PubMed Central

    Mosley, Cara A.; Acker, Timothy M.; Hansen, Kasper B.; Mullasseril, Praseeda; Andersen, Karen T.; Le, Phuong; Vellano, Kimberly M.; Bräuner-Osborne, Hans; Liotta, Dennis C.; Traynelis, Stephen F.

    2010-01-01

    We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists. PMID:20684595

  8. Cloning of the. gamma. -aminobutyric acid (GABA). rho. sub 1 cDNA: A GABA receptor subunit highly expressed in the retina

    SciTech Connect

    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. Johns Hopkins Univ. School of Medicine, Baltimore, MD )

    1991-04-01

    Type A {gamma}-aminobutyric acid (GABA{sub 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{sub 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 {rho}{sub 1}, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.

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

    PubMed

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

    2015-02-19

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

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

    PubMed Central

    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

  11. 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.

    PubMed

    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.

  12. Muon g-2 Experiment Shimming

    SciTech Connect

    Kiburg, Brendan

    2016-06-28

    The Muon g-2 experiment at Fermilab will use as its primary instrument a 52-foot-wide electromagnet that creates a precise magnetic field. In this video, Fermilab's Brendan Kiburg explains the lengthy process of finely "shimming" that magnetic field into shape.

  13. Muon g-2 Experiment Shimming

    ScienceCinema

    Kiburg, Brendan

    2016-07-12

    The Muon g-2 experiment at Fermilab will use as its primary instrument a 52-foot-wide electromagnet that creates a precise magnetic field. In this video, Fermilab's Brendan Kiburg explains the lengthy process of finely "shimming" that magnetic field into shape.

  14. 17β-Estradiol Regulation of the mRNA Expression of T-type Calcium Channel subunits: Role of Estrogen Receptor α and Estrogen Receptor β

    PubMed Central

    Bosch, Martha A.; Hou, Jingwen; Fang, Yuan; Kelly, Martin J.; Rønnekleiv., Oline K.

    2009-01-01

    Low voltage-activated (T-type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T-type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, 3.2 and 3.3, and each subtype has distinct kinetic characteristics. Although 17β-estradiol modulates T-type calcium channel expression and function, little is known about the molecular mechanisms involved. Presently, we used real-time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2-treated C57BL/6 mice to elucidate E2-mediated regulation of Cav3.1, 3.2 and 3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and 3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2-treatment and Cav3.2 and 3.3 were decreased. In order to examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα- and ERβ-deficient C57BL/6 mice and explored the effects of E2 on T-type channel subtypes. Indeed, we found that the E2-induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2-induced effects in the pituitary were dependent on only the expression of ERα. The robust E2-regulation of the T-type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion. PMID:19003958

  15. Expression of G protein-coupled receptor 19 in human lung cancer cells is triggered by entry into S-phase and supports G(2)-M cell-cycle progression.

    PubMed

    Kastner, Stefan; Voss, Tilman; Keuerleber, Simon; Glöckel, Christina; Freissmuth, Michael; Sommergruber, Wolfgang

    2012-10-01

    It has long been known that G protein-coupled receptors (GPCR) are subject to illegitimate expression in tumor cells. Presumably, hijacking the normal physiologic functions of GPCRs contributes to all biologic capabilities acquired during tumorigenesis. Here, we searched for GPCRs that were expressed in lung cancer: the mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found frequently overexpressed in tissue samples obtained from patients with small cell lung cancer. Several observations indicate that overexpression of Gpr19 confers a specific advantage to lung cancer cells by accelerating transition through the cell-cycle. (i) Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines. (ii) Cell-cycle progression through G(2)-M-phase was impaired in cells transfected with siRNAs directed against Gpr19 and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. (iii) The expression levels of Gpr19 mRNA varied along the cell-cycle with a peak observed in S-phase. (iv) The putative control of Gpr19 expression by E2F transcription factors was verified by chromatin immunoprecipitation: antibodies directed against E2F-1 to -4 allowed for the recovery of the Gpr19 promoter. (v) Removal of E2F binding sites in the Gpr19 promoter diminished the expression of a luciferase reporter. (vi) E2f and Gpr19 expression correlated in lung cancer patient samples. To the best of knowledge, this is the first example of a GPCR showing cell-cycle-specific mRNA expression. Our data also validate GPR19 as a candidate target when overexpressed in lung cancer.

  16. Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

    PubMed

    Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

    1998-07-24

    The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

  17. Superagonist, Full Agonist, Partial Agonist, and Antagonist Actions of Arylguanidines at 5-Hydroxytryptamine-3 (5-HT3) Subunit A Receptors.

    PubMed

    Alix, Katie; Khatri, Shailesh; Mosier, Philip D; Casterlow, Samantha; Yan, Dong; Nyce, Heather L; White, Michael M; Schulte, Marvin K; Dukat, Małgorzata

    2016-11-16

    Introduction of minor variations to the substitution pattern of arylguanidine 5-hydroxytryptamine-3 (5-HT3) receptor ligands resulted in a broad spectrum of functionally-active ligands from antagonist to superagonist. For example, (i) introduction of an additional Cl-substituent(s) to our lead full agonist N-(3-chlorophenyl)guanidine (mCPG, 2; efficacy % = 106) yielded superagonists 7-9 (efficacy % = 186, 139, and 129, respectively), (ii) a positional isomer of 2, p-Cl analog 11, displayed partial agonist actions (efficacy % = 12), and (iii) replacing the halogen atom at the meta or para position with an electron donating OCH3 group or a stronger electron withdrawing (i.e., CF3) group resulted in antagonists 13-16. We posit based on combined mutagenesis, crystallographic, and computational analyses that for the 5-HT3 receptor, the arylguanidines that are better able to simultaneously engage the primary and complementary subunits, thus keeping them in close proximity, have greater agonist character while those that are deficient in this ability are antagonists.

  18. Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

    SciTech Connect

    Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung; Kim, Jae-Sung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2005-12-30

    {alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, a kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.

  19. Human chorionic gonadotropin and its free β-subunit stimulate trophoblast invasion independent of LH/hCG receptor.

    PubMed

    Lee, Cheuk-Lun; Chiu, Philip C N; Hautala, Laura; Salo, Tuula; Yeung, William S B; Stenman, Ulf-Håkan; Koistinen, Hannu

    2013-08-15

    Both paracrine and autocrine factors are involved in the regulation of trophoblast invasion. One of these factors is human chorionic gonadotropin (hCG), which stimulates trophoblast invasion. The stimulatory activity has especially been ascribed to a hyperglycosylated form of hCG (hCG-h) that is expressed in early pregnancy. We compared the stimulatory activities of different forms of hCG and its free β-subunit (hCGβ) on trophoblast invasion. hCG, hCG-h, hCGβ, and its hyperglycosylated form (hCGβ-h) stimulated the invasion of JEG-3 choriocarcinoma cells. The stimulatory effect of hCGβ was also confirmed with primary human trophoblasts. Down-regulation of the LH/hCG receptor by RNA-interference did not significantly reduce the effect of hCGβ and hCG on cell invasion. Increased invasion was associated with increased levels of MMP-2, MMP-9 and activity of uPA. Our findings suggest that hCG, hCGβ and their hyperglycosylated forms stimulate the invasion of trophoblast cells independent of the classical LH/hCG-receptor.

  20. Lynx1 Shifts α4β2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum*

    PubMed Central

    Nichols, Weston A.; Henderson, Brandon J.; Yu, Caroline; Parker, Rell L.; Richards, Christopher I.; Lester, Henry A.; Miwa, Julie M.

    2014-01-01

    Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)3(β2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein. PMID:25193667

  1. NMDA Receptor Subunit Composition Controls Dendritogenesis of Hippocampal Neurons through CAMKII, CREB-P and H3K27ac.

    PubMed

    Bustos, Fernando J; Jury, Nur; Martinez, Pablo; Ampuero, Estibaliz; Campos, Matias; Abarzúa, Sebastian; Jaramillo, Karen; Ibing, Susanne; Mardones, Muriel D; Haensgen, Henny; Kzhyshkowska, Julia; Tevy, Maria Florencia; Neve, Rachael; Sanhueza, Magdalena; Varela-Nallar, Lorena; Montecino, Martín; van Zundert, Brigitte

    2017-02-04

    Dendrite arbor growth, or dendritogenesis, is choreographed by a diverse set of cues, including the NMDA receptor (NMDAR) subunits NR2A and NR2B. While NR1NR2B receptors are predominantly expressed in immature neurons and promote plasticity, NR1NR2A receptors are mainly expressed in mature neurons and induce circuit stability. How the different subunits regulate these processes is unclear, but this is likely related to the presence of their distinct C-terminal sequences that couple different signaling proteins. Calcium-calmodulin-dependent protein kinase II (CaMKII) is an interesting candidate as this protein can be activated by calcium influx through NMDARs. CaMKII triggers a series of biochemical signaling cascades, involving the phosphorylation of diverse targets. Among them, the activation of cAMP response element-binding protein (CREB-P) pathway triggers a plasticity-specific transcriptional program through unknown epigenetic mechanisms. Here we found that dendritogenesis in hippocampal neurons is impaired by several well-characterized constructs (i.e. NR2B-RS/QD) and peptides (i.e. tatCN21) that specifically interfere with the recruitment and interaction of CaMKII with the NR2B C-terminal domain. Interestingly, we found that transduction of NR2AΔIN, a mutant NR2A construct with increased interaction to CaMKII, reactivates dendritogenesis in mature hippocampal neurons in vitro and in vivo. To gain insights into the signaling and epigenetic mechanisms underlying NMDAR-mediated dendritogenesis, we used immunofluorescence staining to detect CREB-P and acetylated lysine 27 of histone H3 (H3K27ac), an activation-associated histone tail mark. In contrast to control mature neurons, our data shows that activation of the NMDAR/CaMKII/ERK-P/CREB-P signaling axis in neurons expressing NR2AΔIN is not correlated with increased nuclear H3K27ac levels. This article is protected by copyright. All rights reserved.

  2. Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy

    PubMed Central

    Mott, David D; Erreger, Kevin; Banke, Tue G; Traynelis, Stephen F

    2001-01-01

    The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. Serotonin activated whole cell currents with an EC50 value for the peak response of 2 μm and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean ±s.e.m. 10-90 % rise time 12.5 ± 1.6 ms; n = 9 patches) for 100 μm serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s−1. The 5-HT3A receptor response to 100 μm serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches τslow 1006 ± 139 ms, amplitude 31 % τfast 176 ± 25 ms, amplitude 69 %). Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches τslow 838 ± 217 ms, 55 % amplitude; τfast 213 ± 44 ms, 45 % amplitude). In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of

  3. A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes*

    PubMed Central

    Woll, Kellie A.; Murlidaran, Sruthi; Pinch, Benika J.; Hénin, Jérôme; Wang, Xiaoshi; Salari, Reza; Covarrubias, Manuel; Dailey, William P.; Brannigan, Grace; Garcia, Benjamin A.; Eckenhoff, Roderic G.

    2016-01-01

    Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. PMID:27462076

  4. Human GABAA receptor alpha 1 and alpha 3 subunits genes and alcoholism.

    PubMed

    Parsian, A; Cloninger, C R

    1997-05-01

    gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABAA receptor, causing the opening of an integral chloride-ion channel. The GABAA antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABAA agonists. To determine the role of the GABAA receptor (GABR) genes in the development of alcoholism, we have used alpha 1 and alpha 3 simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABR alpha 1 gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABR alpha 3, the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABR alpha 1 and GABR alpha 3, were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample that will allow haplotype relative risk or affected sib-pair analysis.

  5. NMDA receptor mediated phosphorylation of GluR1 subunits contributes to the appearance of calcium-permeable AMPA receptors after mechanical stretch injury

    PubMed Central

    Spaethling, Jennifer; Le, Linda; Meaney, David F

    2016-01-01

    Alterations in neuronal cytosolic calcium is a key mediator of the traumatic brain injury (TBI) pathobiology, but less is known of the role and source of calcium in shaping early changes in synaptic receptors and neural circuits after TBI. In this study, we examined the calcium source and potential phosphorylation events leading to insertion of calcium-permeable AMPARs (CP-AMPARs) after in vitro traumatic brain injury, a receptor subtype that influences neural circuit dynamics for hours to days following injury. We found that both synaptic and NR2B-containing NMDARs contribute significantly to the calcium influx following stretch injury. Moreover, an early and sustained phosphorylation of the S-831 site of the GluR1 subunit appeared after mechanical injury, and this phosphorylation was blocked with the inhibition of either synaptic NMDARs or NR2B-containing NMDARs. In comparison, mechanical injury led to no significant change in the S-845 phosphorylation of the GluR1 subunit. Although no change in S-845 phosphorylation appeared in injured cultures, we observed that inhibition of NR2B-containing NMDARs significantly increased S-845 phosphorylation one hour after injury while blockade of synaptic NMDARs did not change S-845 phosphorylation at any time point following injury. These findings show that a broad class of NMDARs are activated in parallel and that targeting either subpopulation will reverse some of the consequences of mechanical injury, providing distinct paths to treat the effects of mechanical injury on neural circuits after TBI. PMID:22426393

  6. Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

    PubMed

    Collins, L; Tsien, W H; Seals, C; Hakimi, J; Weber, D; Bailon, P; Hoskings, J; Greene, W C; Toome, V; Ju, G

    1988-10-01

    Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.

  7. Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

    PubMed Central

    Collins, L; Tsien, W H; Seals, C; Hakimi, J; Weber, D; Bailon, P; Hoskings, J; Greene, W C; Toome, V; Ju, G

    1988-01-01

    Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity. PMID:3051003

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

    PubMed

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

    2011-05-25

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

  9. Cellular distribution of AMPA receptor subunits and mGlu5 following acute and repeated administration of morphine or methamphetamine.

    PubMed

    Herrold, Amy A; Persons, Amanda L; Napier, T Celeste

    2013-08-01

    Ionotropic AMPA receptors (AMPAR) and metabotropic glutamate group I subtype 5 receptors (mGlu5) mediate neuronal and behavioral effects of abused drugs. mGlu5 stimulation increases expression of striatal-enriched tyrosine phosphatase isoform 61 (STEP61 ) which internalizes AMPARs. We determined the rat brain profile of these proteins using two different classes of abused drugs, opiates, and stimulants. STEP61 levels, and cellular distribution/expression of AMPAR subunits (GluA1, GluA2) and mGlu5, were evaluated via a protein cross-linking assay in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP) harvested 1 day after acute, or fourteen days after repeated morphine (8 mg/kg) or methamphetamine (1 mg/kg) (treatments producing behavioral sensitization). Acute morphine decreased GluA1 and GluA2 surface expression in mPFC and GluA1 in NAc. Fourteen days after repeated morphine or methamphetamine, mGlu5 surface expression increased in VP. In mPFC, mGlu5 were unaltered; however, after methamphetamine, STEP61 levels decreased and GluA2 surface expression increased. Pre-treatment with a mGlu5-selective negative allosteric modulator, blocked methamphetamine-induced behavioral sensitization and changes in mPFC GluA2 and STEP61 . These data reveal (i) region-specific distinctions in glutamate receptor trafficking between acute and repeated treatments of morphine and methamphetamine, and (ii) that mGlu5 is necessary for methamphetamine-induced alterations in mPFC GluA2 and STEP61 .

  10. Matter from G(2) Manifolds

    NASA Astrophysics Data System (ADS)

    Berglund, Per; Brandhuber, Andreas

    2003-04-01

    We describe how chiral matter charged under SU(N) and SO(2N) gauge groups arises from codimension seven singularities in compactifications of M-theory on manifolds with G(2) holonomy. The geometry of these spaces is that of a cone over a six-dimensional Einstein space which can be constructed by (multiple) unfolding of hyper-Kahler quotient spaces. In type IIA the corresponding picture is given by stacks of intersecting D6-branes and chiral matter arises from open strings stretching between them. Usually one obtains (bi)fundamental representations but by including orientifold six-planes in the type IIA picture we find more exotic representations like the anti-symmetric, which is important for the study of SU(5) grand unification, and trifundamental representations. We also exhibit many cases where the G(2) metrics can be described explicitly, although in general the metrics on the spaces constructed via unfolding are not known.

  11. Methionine choline reverses lead-induced cognitive and N-methyl-d-aspartate receptor subunit 1 deficits.

    PubMed

    Fan, Guangqin; Feng, Chang; Wu, Fengyun; Ye, Weiwei; Lin, Fen; Wang, Chunhong; Yan, Ji; Zhu, Gaochun; Xiao, Yuanmei; Bi, Yongyi

    2010-06-04

    The principal effects of Pb(2+) exposure in children are attention, memory and learning deficits that persist into adulthood. The application of the conventional chelators in children is somewhat prohibited by adverse health effects and is not effective in reversing learning deficits once they have occurred. In this study, we applied the nutrients, methionine and choline, to prevent Pb(2+)-induced cognitive impairment. Male weanling Sprague-Dawley rats were divided into five groups. Three groups of rats were exposed to Pb(2+) in drinking water containing 400mg/L Pb(2+) acetate, of which two groups were concurrently administered by oral gavage once a day, 6 days per week, with low or high doses of methionine and choline for 60 days. The normal control group received distilled water alone, and the reagent control received methionine choline chloride alone. Methionine choline treatment reversed long-term deficits in spatial learning and memory caused by Pb(2+) exposure in rats. Enhanced learning performance of Pb(2+)-exposed rats was associated with recovery of deficits in N-methyl-d-aspartate receptor (NMDAR) subunit 1 (NR1) mRNA and protein expression in the hippocampus. The effect of methionine choline on NR1 gene and protein expression was somewhat specific to Pb(2+)-exposed rats and did not affect the NR2A and NR2B subunits of the NMDAR measured in the same animals. Moreover, methionine choline treatment did not lower brain Pb(2+) content in Pb(2+)-exposed rats, although it reduced blood and bone Pb(2+) content. Methionine and choline reversed cognitive and NR1 deficits induced by Pb(2+) exposure, a beneficial effect that has significant clinical implications for the treatment of childhood Pb(2+) intoxication.

  12. Mice devoid of γ-aminobutyrate type A receptor β3 subunit have epilepsy, cleft palate, and hypersensitive behavior

    PubMed Central

    Homanics, Gregg E.; DeLorey, Timothy M.; Firestone, Leonard L.; Quinlan, Joseph J.; Handforth, Adrian; Harrison, Neil L.; Krasowski, Matthew D.; Rick, Caroline E. M.; Korpi, Esa R.; Mäkelä, Riikka; Brilliant, Murray H.; Hagiwara, Nobuko; Ferguson, Carolyn; Snyder, Kimberly; Olsen, Richard W.

    1997-01-01

    γ-Aminobutyric acid type A receptors (GABAA-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The β3 subunit is an essential component of the GABAA-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a β3 gene inactivated by gene targeting. GABAA-R density is approximately halved in brain of β3-deficient mice, and GABAA-R function is severely impaired. Most β3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. β3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the β3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. β3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects. PMID:9108119

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

    PubMed

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

    2016-07-07

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

  14. The residence time of GABAARs at inhibitory synapses is determined by direct binding of the receptor α1 subunit to gephyrin

    PubMed Central

    Mukherjee, Jayanta; Kretschmannova, Karla; Gouzer, Geraldine; Maric, Hans-Michael; Ramsden, Sarah; Tretter, Verena; Harvey, Kirsten; Davies, Paul A; Triller, Antoine; Schindelin, Hermann

    2011-01-01

    Summary The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive, α1 subunit-containing GABAARs; however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat hippocampal neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ~20 μM, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of miniature inhibitory postsynaptic currents (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 phospho-mimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, receptor accumulation at synapses and the amplitude of mIPSCs. Finally, single particle tracking reveals that gephyrin reduces the diffusion of α1 subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition. PMID:21994384

  15. Downregulation of the spinal NMDA receptor NR2B subunit during electro-acupuncture relief of chronic visceral hyperalgesia.

    PubMed

    Liu, Hongping; Zhang, Yuhua; Qi, Debo; Li, Weimin

    2017-01-01

    The involvement of spinal NR2B, a N-methyl-D-aspartate (NMDA) receptor subunit, in the therapeutic effect of electro-acupuncture (EA) on chronic visceral hyperalgesia was investigated. Chronic visceral hyperalgesia was induced using an irritable bowel syndrome (IBS) model in rats. Graded colorectal distention (CRD) stimuli at strengths of 20, 40, 60 and 80 mmHg were applied, and behavioral tests were performed to measure the abdominal withdrawal reflex (AWR) in response to the CRD stimuli and assess the severity of the visceral hyperalgesia. Rats were randomly divided into four groups: normal intact (control) group, IBS model (model) group, EA-treated IBS rats (EA) group and sham EA-treated IBS rats (sham EA) group. For the EA treatment, electric stimuli were applied through needles inserted into two acupoints [Zu-san-li (ST-36) and Shang-ju-xu (ST-37)] in both hind limbs, while the sham EA treatment consisted of only the insertion of needles into these same acupoints without an application of electric stimuli. Our results showed that AWR scores of the model group responding to CRD stimuli of 20, 40, 60 and 80 mmHg were significantly increased. These increased scores subsequently decreased following EA treatment (P < 0.05) compared with those for the other groups. The expression of NR2B in the superficial laminae (SDH, laminae I and II), nucleus proprius (NP, laminae III and IV), neck of the dorsal horn (NECK, laminae V and VI) and central canal region (lamina X) at thoracolumbar (T13-L2) and lumbosacral (L6-S2) segmental level significantly increased in the model group versus the control group (P < 0.05) and significantly decreased after EA treatment (P < 0.05). There were no significant changes in neither AWR scores nor expression of the NR2B subunit in these spinal regions after the sham EA treatment. These results confirm that EA can relieve chronic visceral hyperalgesia in IBS model rats and suggest that such an effect is possibly mediated through the

  16. Phenoxy herbicides and fibrates potently inhibit the human chemosensory receptor subunit T1R3

    PubMed Central

    Maillet, Emeline L.; Margolskee, Robert F.; Mosinger, Bedrich

    2009-01-01

    We show that phenoxy-auxin herbicides and lipid-lowering fibrates inhibit human but not rodent T1R3. T1R3 as a co-receptor in taste cells responds to sweet compounds and amino-acids; in endocrine cells of gut and pancreas T1R3 contributes to glucose sensing. Thus, certain effects of fibrates in treating hyperlipidemia and type II diabetes may be via actions on T1R3. Likewise, phenoxy-herbicides may have adverse metabolic effects in humans that would have gone undetected in studies on rodents. PMID:19817384

  17. Phenotypic consequences of deletion of the gamma 3, alpha 5, or beta 3 subunit of the type A gamma-aminobutyric acid receptor in mice.

    PubMed

    Culiat, C T; Stubbs, L J; Montgomery, C S; Russell, L B; Rinchik, E M

    1994-03-29

    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the gamma 3, alpha 5, and beta 3 subunits of the type A gamma-aminobutyric acid receptor, respectively, are known to map near the pink-eyed dilution (p) locus in mouse chromosome 7. This region shares homology with a segment of human chromosome 15 that is implicated in Angelman syndrome, an inherited neurobehavioral disorder. By mapping Gabrg3 on a panel of p-locus deletions, we have determined that the order of genes within this cluster is centromere-p(D15S12h)-Gabrg3-Gabra5-Gabrb3-telom ere. Like Gabrb3, neither the Gabra5 nor Gabrg3 gene is functionally imprinted in adult mouse brain. Mice deleted for all three subunits die at birth with a cleft palate, although there are rare survivors (approximately 5%) that do not have a cleft palate but do exhibit a neurological abnormality characterized by tremor, jerky gait, and runtiness. We have previously suggested that deficiency of the beta 3 subunit may be responsible for the clefting defect. Most notably, however, in this report we describe mice carrying two overlapping, complementing p deletions that fail to express the gamma 3 transcript, as well as mice from another line that express neither the gamma 3 nor alpha 5 transcripts. Surprisingly, mice from both of these lines are phenotypically normal and do not exhibit any of the neurological symptoms characteristic of the rare survivors that are deleted for all three (gamma 3, alpha 5, and beta 3) subunits. These mice therefore provide a whole-organism type A gamma-aminobutyric-acid receptor background that is devoid of any receptor subtypes that normally contain the gamma 3 and/or alpha 5 subunits. The absence of an overt neurological phenotype in mice lacking the gamma 3 and/or alpha 5 subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  18. Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

    PubMed Central

    Schwimmbeck, P L; Dyrberg, T; Drachman, D B; Oldstone, M B

    1989-01-01

    The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetylcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetylcholine receptor (HuAChR) alpha-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR alpha-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of alpha-bungarotoxin to the receptor. The HuAChR alpha-subunit 160-167 peptide demonstrated specific immunological cross-reactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR alpha-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia. Images PMID:2551924

  19. Enhancement of Inhibitory Neurotransmission by GABAA Receptors Having α2,3-Subunits Ameliorates Behavioral Deficits in a Mouse Model of Autism

    PubMed Central

    Han, Sung; Tai, Chao; Jones, Christina J.; Scheuer, Todd; Catterall, William A.

    2014-01-01

    SUMMARY Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T+ Itpr3tf/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low non-sedating/non-anxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABAA receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABAA receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit-specific—the α2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the α1-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and α2,3-subunit-selective positive GABAA receptor modulation may be an effective treatment. PMID:24656250

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

    PubMed

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

    2016-04-01

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

  1. Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model.

    PubMed

    Benjamin, Sailas; Flotho, Silke; Börchers, Torsten; Spener, Friedrich

    2013-02-01

    The purpose of this study was to examine the induction profiles (as judged by quantitative reverse transcription polymerase chain reaction (qRT-PCR)) of peroxisome proliferator-activated receptor (PPAR) α, β, γ subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element (PPRE) in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid (9-CLA) or trans-10,cis-12-octadecadienoic acid (10-CLA) or their precursor fatty acids (FAs). HepG2 cells were treated with 100 μmol/L 9-CLA or 10-CLA or their precursor FAs, viz., oleic, linoleic, and trans-11-vaccenic acids against bezafibrate control to evaluate the induction/expression profiles of PPAR α, β, γ subtypes and major PPAR-target genes bearing a functional PPRE, i.e., fatty acid transporter (FAT), glucose transporter-2 (GLUT-2), liver-type FA binding protein (L-FABP), acyl CoA oxidase-1 (ACOX-1), and peroxisomal bifunctional enzyme (PBE) with reference to β-actin as house keeping gene. Of the three housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, and ubiquitin), β-actin was found to be stable. Dimethyl sulfoxide (DMSO), the common solubilizer of agonists, showed a significantly higher induction of genes analyzed. qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT, GLUT-2, and L-FABP (~0.5-2.0-fold). Compared to 10-CLA, 9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE, while 10-CLA decreased the induction of PBE less than did ACOX-1. Both CLAs and precursor FAs upregulated PPRE-bearing genes, but with comparatively less or marginal activation of PPAR subtypes. This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation, thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE. To sum up, the expression profiles of these candidate genes showed that

  2. GABAA receptor ɛ-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat

    PubMed Central

    Kasparov, Sergey; Davies, Katy A; Patel, Umesh A; Boscan, Pedro; Garret, Maurice; Paton, Julian F R

    2001-01-01

    Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABAA receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex. In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 μm) did not Significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 μm) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50–100 μm) was effective in enhancing GABAA-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM; 1 or 10 μm), failed to depress GABA-induced currents in the cNTS. Microinjections of midazolam (10 and 100 μm solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 μm) significantly and reversibly depressed it (gain decrease to 53 ± 11 % of control, P < 0.01). Reverse transcriptase polymerase chain reaction revealed the presence of α1, α2, β2, β3 and γ2 GABAA receptor subunit mRNA in the cNTS. No alternatively spliced variants of the α1- and γ2-subunits were revealed. Moreover, GABAAɛ-subunit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts. Immunocytochemical analysis revealed numerous GABAAɛ-subunit-positive neurones within the cNTS with significantly fewer ɛ subunit-positive cells in the rNTS. As incorporation of the ɛ-subunit in recombinant GABAA receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of ɛ-subunit expression. This is the first demonstration of a possible physiological impact of the ɛ -subunit on native GABAA

  3. Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the alpha4 subunit of GABA(A) receptors in an animal model of epilepsy.

    PubMed

    Sun, Chengsan; Mtchedlishvili, Zakaria; Erisir, Alev; Kapur, Jaideep

    2007-11-14

    In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABA(A) receptors on dentate granule cells (DGCs) is diminished; the molecular mechanism underlying this phenomenon remains unclear. The current study investigated a mechanism for loss of neurosteroid sensitivity of synaptic GABA(A) receptors in TLE. Synaptic currents recorded from DGCs of epileptic animals (epileptic DGCs) were less frequent, larger in amplitude, and less sensitive to allopregnanolone modulation than those recorded from DGCs of control animals (control DGCs). Synaptic currents recorded from epileptic DGCs were less sensitive to diazepam and had altered sensitivity to benzodiazepine inverse agonist RO 15-4513 (ethyl-8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) and furosemide than those recorded from control DGCs. Properties of synaptic currents recorded from epileptic DGCs appeared similar to those of recombinant receptors containing the alpha4 subunit. Expression of the alpha4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the alpha4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The alpha4 immunogold labeling was present more commonly within the synapse in epileptic DGCs compared with control DGCs, in which the subunit was extrasynaptic. These studies demonstrate that, in epileptic DGCs, the neurosteroid modulation of synaptic currents is diminished and alpha4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

  4. The Ca2+ channel β subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

    PubMed Central

    Heneghan, John F.; Mitra-Ganguli, Tora; Stanish, Lee F.; Liu, Liwang; Zhao, Rubing

    2009-01-01

    In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AA's concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and

  5. Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit.

    PubMed

    Jubinsky, P T; Laurie, A S; Nathan, D G; Yetz-Aldepe, J; Sieff, C A

    1994-12-15

    To determine the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha chain (GMR alpha) during hematopoiesis and on leukemic cells, monoclonal antibodies were raised by immunizing mice with cells expressing high levels of human GMR alpha. A pool of five antibodies isolated from three different mice was used to characterize GMR alpha. This antibody pool (anti-GMR alpha) immunoprecipitated a protein with the expected molecular weight of GMR alpha from COS cells transiently transfected with the GMR alpha gene. In factor-dependent cells, GMR alpha existed as a phosphoprotein. However, its phosphorylation was not stimulated by the presence of GM-CSF. Anti-GMR alpha inhibited the GM-CSF-dependent growth of cell lines and normal bone marrow cells and inhibited the binding of iodinated GM-CSF to its receptor. Cell surface expression of GMR alpha was examined using anti-GMR alpha and flow cytometry. GMR alpha was readily detectable on both blood monocytes and neutrophils. In adherence-depleted normal bone marrow, two separate populations expressed GMR alpha. The most positive cells were predominantly macrophages, whereas the cells that expressed less GMR alpha were largely myelocytes and metamyelocytes. A small population of lin-CD34+ or CD34+CD38- cells also expressed GMR alpha, but they were not capable of significant growth in colony-forming assays. In contrast, the majority of lin-CD34+ and CD34+CD38- cells were GMR alpha-, yet they produced large numbers of myeloid and erythroid colonies in the same assay. Malignant cells from patients with leukemia were also tested for GMR alpha expression. All of the myeloid leukemias and only rare lymphoid leukemias surveyed tested positive for GMR alpha. These results show that anti-GMR alpha is useful for the functional characterization of the GMR alpha and for the detection of myeloid leukemia and that GMR alpha is expressed on certain lineages throughout hematopoietic

  6. Voltage-dependent regulation of CaV2.2 channels by Gq-coupled receptor is facilitated by membrane-localized β subunit.

    PubMed

    Keum, Dongil; Baek, Christina; Kim, Dong-Il; Kweon, Hae-Jin; Suh, Byung-Chang

    2014-10-01

    G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca(2+), and phosphatidylinositol 4,5-bisphosphate (PIP2), to modulate N-type voltage-gated Ca(2+) (CaV2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which GqPCRs inhibit CaV2.2 channel current are not completely understood. Here, we report that the location of CaV β subunits is key to determining the voltage dependence of CaV2.2 channel modulation by GqPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M1 receptors, together with CaV N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the current-voltage relationship for CaV2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of CaV2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of CaV2.2 current after M1 receptor activation was markedly reduced and delayed, whereas the delay between PIP2 hydrolysis and inhibition of CaV2.2 current was decreased. When the Gβγ-insensitive CaV2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M1 receptors act through Gβγ to inhibit CaV2.2 channels bearing membrane-localized CaV β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylation-negative mutant β2a(C3,4S), reduced the voltage dependence of M1 muscarinic inhibition of CaV2.2 channels, whereas it increased inhibition mediated by PIP2 depletion. Together, our results indicate that, with membrane-localized CaV β subunits, CaV2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of

  7. The β2 nicotinic acetylcholine receptor subunit differentially influences ethanol behavioral effects in the mouse.

    PubMed

    Dawson, Anton; Miles, Micheal F; Damaj, M Imad

    2013-03-01

    The high co-morbidity between alcohol (ethanol) and nicotine abuse suggests that nicotinic acetylcholine receptors (nAChRs), thought to underlie nicotine dependence, may also be involved in alcohol dependence. The β2* nAChR subtype serves as a potential interface for these interactions since they are the principle mediators of nicotine dependence and have recently been shown to modulate some acute responses to ethanol. Therefore, the aim of this study was to more fully characterize the role of β2* nAChRs in ethanol-responsive behaviors in mice after acute exposure to the drug. We conducted a battery of tests in mice lacking the β2* coding gene (Chrnb2) or pretreated with a selective β2* nAChR antagonist for a range of ethanol-induced behaviors including locomotor depression, hypothermia, hypnosis, and anxiolysis. We also tested the effect of deletion on voluntary escalated ethanol consumption in an intermittent access two-bottle choice paradigm to determine the extent of these effects on drinking behavior. Our results showed that antagonism of β2* nAChRs modulated some acute behaviors, namely by reducing recovery time from hypnosis and enhancing the anxiolytic-like response produced by acute ethanol in mice. Chrnb2 deletion had no effect on ethanol drinking behavior, however. We provide further evidence that β2* nAChRs have a measurable role in mediating specific behavioral effects induced by acute ethanol exposure without affecting drinking behavior directly. We conclude that these receptors, along with being key components in nicotine dependence, may also present viable candidates in the discovery of the molecular underpinnings of alcohol dependence.

  8. Transformation of postingestive glucose responses after deletion of sweet taste receptor subunits or gastric bypass surgery

    PubMed Central

    Geraedts, Maartje C. P.; Takahashi, Tatsuyuki; Vigues, Stephan; Markwardt, Michele L.; Nkobena, Andongfac; Cockerham, Renee E.; Hajnal, Andras; Dotson, Cedrick D.; Rizzo, Mark A.

    2012-01-01

    The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K+ (KATP) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3−/− mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3+/+, but not T1R3−/−, ileum explants; this secretion was not mimicked by the KATP channel blocker glibenclamide. T1R2−/− mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was KATP channel-dependent and glucose-specific emerged in the large intestine of T1R3−/− mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB. PMID:22669246

  9. Gerbes on G2 manifolds

    NASA Astrophysics Data System (ADS)

    Oliveira, Goncalo

    2017-04-01

    On a projective complex manifold, the Abelian group of divisors maps surjectively onto that of holomorphic line bundles (the Picard group). On a G2-manifold we use coassociative submanifolds to define an analogue of the divisors, and a gauge theoretical equation for a connection on a gerbe to define an analogue of the Picard group. Then, we construct a map from the former to the later. We also prove that the canonical map from our analogue of the Picard group to the third cohomology group with integer coefficients is surjective. As a side remark we make an observation relating the topological type of coassociative submanifolds and the cohomology classes they represent.

  10. Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

    PubMed

    Harris, R A; Osterndorff-Kahanek, E; Ponomarev, I; Homanics, G E; Blednov, Y A

    2011-01-13

    Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in α1 knock-in mice and three in the α2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the α2 mutants, and about 50% in the α1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between α2 mutants and wild-type, but they were enhanced for α1 knock-in mice. These results indicate that introduction of these mutations in the α2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the α1 subunit produce more pronounced changes, especially in GABAergic function.

  11. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    SciTech Connect

    Sigalov, Alexander B.; Hendricks, Gregory M.

    2009-11-13

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including {zeta}{sub cyt} and CD3{epsilon}{sub cyt} all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  12. Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.

    PubMed

    Nakamura, Yasuko; Morrow, Danielle H; Modgil, Amit; Huyghe, Deborah; Deeb, Tarek Z; Lumb, Michael J; Davies, Paul A; Moss, Stephen J

    2016-06-03

    The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, β, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.

  13. The nicotinic acetylcholine receptor α7 subunit is an essential negative regulator of bone mass.

    PubMed

    Mito, Kazuaki; Sato, Yuiko; Kobayashi, Tami; Miyamoto, Kana; Nitta, Eriko; Iwama, Atsushi; Matsumoto, Morio; Nakamura, Masaya; Sato, Kazuki; Miyamoto, Takeshi

    2017-03-28

    The nicotinic receptor α7nAchR reportedly regulates vagal nerve targets in brain and cardiac tissue. Here we show that nAchR7(-/-) mice exhibit increased bone mass due to decreased osteoclast formation, accompanied by elevated osteoprotegerin/RANKL ratios in serum. Vagotomy in wild-type mice also significantly increased the serum osteoprotegerin/RANKL ratio, and elevated bone mass seen in nAchR7(-/-) mice was reversed in α7nAchR/osteoprotegerin-doubly-deficient mice. α7nAchR loss significantly increased TNFα expression in Mac1-positive macrophages, and TNFα increased the osteoprotegerin/RANKL ratio in osteoblasts. Targeting TNFα in nAchR7(-/-) mice normalized both serum osteoprotegerin/RANKL ratios and bone mass. Administration of nicotine, an α7nAchR ligand, to wild-type mice increased serum RANKL levels. Thus, vagal nerve stimulation of macrophages via α7nAchR regulates bone mass by modulating osteoclast formation.

  14. The nicotinic acetylcholine receptor α7 subunit is an essential negative regulator of bone mass

    PubMed Central

    Mito, Kazuaki; Sato, Yuiko; Kobayashi, Tami; Miyamoto, Kana; Nitta, Eriko; Iwama, Atsushi; Matsumoto, Morio; Nakamura, Masaya; Sato, Kazuki; Miyamoto, Takeshi

    2017-01-01

    The nicotinic receptor α7nAchR reportedly regulates vagal nerve targets in brain and cardiac tissue. Here we show that nAchR7−/− mice exhibit increased bone mass due to decreased osteoclast formation, accompanied by elevated osteoprotegerin/RANKL ratios in serum. Vagotomy in wild-type mice also significantly increased the serum osteoprotegerin/RANKL ratio, and elevated bone mass seen in nAchR7−/− mice was reversed in α7nAchR/osteoprotegerin-doubly-deficient mice. α7nAchR loss significantly increased TNFα expression in Mac1-positive macrophages, and TNFα increased the osteoprotegerin/RANKL ratio in osteoblasts. Targeting TNFα in nAchR7−/− mice normalized both serum osteoprotegerin/RANKL ratios and bone mass. Administration of nicotine, an α7nAchR ligand, to wild-type mice increased serum RANKL levels. Thus, vagal nerve stimulation of macrophages via α7nAchR regulates bone mass by modulating osteoclast formation. PMID:28349965

  15. Deficit in acoustic signal-in-noise detection in glycine receptor α3 subunit knockout mice.

    PubMed

    Tziridis, Konstantin; Buerbank, Stefanie; Eulenburg, Volker; Dlugaiczyk, Julia; Schulze, Holger

    2017-02-01

    Hearing is an essential sense for communication in animals and humans. Normal function of the cochlea of higher vertebrates relies on a fine-tuned interplay of afferent and efferent innervation of both inner and outer hair cells. Efferent inhibition is controlled via olivocochlear feedback loops, mediated mainly by acetylcholine, γ-aminobutyric acid (GABA) and glycine, and is one of the first sites affected by synapto- and neuropathy in the development of hearing loss. While the functions of acetylcholine, GABA and other inhibitory transmitters within these feedback loops are at least partially understood, especially the function of glycine still remains elusive. To address this question, we investigated hearing in glycine receptor (GlyR) α3 knockout (KO) and wildtype (WT) mice. We found no differences in pure tone hearing thresholds at 11.3 and 16 kHz between the two groups as assessed by auditory brainstem response (ABR) measurements. Detailed analysis of the ABR waves at 11.3 kHz, however, revealed a latency decrease of wave III and an amplitude increase of wave IV in KO compared to WT animals. GlyRα3 KO animals showed significantly impaired prepulse inhibition of the auditory startle response in a noisy environment, indicating that GlyRα3-mediated glycinergic inhibition is important for signal-in-noise detection.

  16. Reduction in focal ictal activity following transplantation of MGE interneurons requires expression of the GABAA receptor α4 subunit

    PubMed Central

    Jaiswal, Manoj K.; Keros, Sotirios; Zhao, Mingrui; Inan, Melis; Schwartz, Theodore H.; Anderson, Stewart A.; Homanics, Gregg E.; Goldstein, Peter A.

    2015-01-01

    Despite numerous advances, treatment-resistant seizures remain an important problem. Loss of neuronal inhibition is present in a variety of epilepsy models and is suggested as a mechanism for increased excitability, leading to the proposal that grafting inhibitory interneurons into seizure foci might relieve refractory seizures. Indeed, transplanted medial ganglionic eminence interneuron progenitors (MGE-IPs) mature into GABAergic interneurons that increase GABA release onto cortical pyramidal neurons, and this inhibition is associated with reduced seizure activity. An obvious conclusion is that inhibitory coupling between the new interneurons and pyramidal cells underlies this effect. We hypothesized that the primary mechanism for the seizure-limiting effects following MGE-IP transplantation is the tonic conductance that results from activation of extrasynaptic GABAA receptors (GABAA-Rs) expressed on cortical pyramidal cells. Using in vitro and in vivo recording techniques, we demonstrate that GABAA-R α4 subunit deletion abolishes tonic currents (Itonic) in cortical pyramidal cells and leads to a failure of MGE-IP transplantation to attenuate cortical seizure propagation. These observations should influence how the field proceeds with respect to the further development of therapeutic neuronal transplants (and possibly pharmacological treatments). PMID:25914623

  17. Association of the Nicotinic Receptor α7 Subunit Gene (CHRNA7) with Schizophrenia and Visual Backward Masking.

    PubMed

    Bakanidze, George; Roinishvili, Maya; Chkonia, Eka; Kitzrow, Werner; Richter, Sarina; Neumann, Konrad; Herzog, Michael H; Brand, Andreas; Puls, Imke

    2013-01-01

    The nicotinic system is involved in the pathophysiology of schizophrenia. However, very little is known about its genetic basis and how it relates to clinical symptoms and potentially pharmacological intervention. Here, we investigated five single nucleotide polymorphisms (SNPs) [rs3826029] [rs2337506] [rs982574] [rs904952] [rs2337980] of the cholinergic nicotinic receptor gene, alpha 7 subunit (CHRNA7) and their association to schizophrenia. We found an association with rs904952 (p = 0.009) in a German sample of 224 schizophrenic patients and 224 healthy control subjects. The same trend was shown in an independent Georgian sample of 50 schizophrenic patients, 57 first order unaffected relatives, and 51 healthy controls. In addition, visual backward masking (VBM), a sensitive test for early visual information processing, was assessed in the Georgian sample. In line with prior studies, VBM performance deficits were much more pronounced in schizophrenic patients and their unaffected relatives compared to healthy controls (schizophrenic patients: 156 ms; unaffected relatives: 60 ms; healthy controls: 33 ms). VBM was strongly correlated with SNP rs904952 (H[2] = 7.3, p = 0.026). Our results further support the notion that changes in the nicotinic system are involved in schizophrenia and open the avenue for pharmacological intervention.

  18. Association of the Nicotinic Receptor α7 Subunit Gene (CHRNA7) with Schizophrenia and Visual Backward Masking

    PubMed Central

    Bakanidze, George; Roinishvili, Maya; Chkonia, Eka; Kitzrow, Werner; Richter, Sarina; Neumann, Konrad; Herzog, Michael H.; Brand, Andreas; Puls, Imke

    2013-01-01

    The nicotinic system is involved in the pathophysiology of schizophrenia. However, very little is known about its genetic basis and how it relates to clinical symptoms and potentially pharmacological intervention. Here, we investigated five single nucleotide polymorphisms (SNPs) [rs3826029] [rs2337506] [rs982574] [rs904952] [rs2337980] of the cholinergic nicotinic receptor gene, alpha 7 subunit (CHRNA7) and their association to schizophrenia. We found an association with rs904952 (p = 0.009) in a German sample of 224 schizophrenic patients and 224 healthy control subjects. The same trend was shown in an independent Georgian sample of 50 schizophrenic patients, 57 first order unaffected relatives, and 51 healthy controls. In addition, visual backward masking (VBM), a sensitive test for early visual information processing, was assessed in the Georgian sample. In line with prior studies, VBM performance deficits were much more pronounced in schizophrenic patients and their unaffected relatives compared to healthy controls (schizophrenic patients: 156 ms; unaffected relatives: 60 ms; healthy controls: 33 ms). VBM was strongly correlated with SNP rs904952 (H[2] = 7.3, p = 0.026). Our results further support the notion that changes in the nicotinic system are involved in schizophrenia and open the avenue for pharmacological intervention. PMID:24155726

  19. Mouse mutants for the nicotinic acetylcholine receptor ß2 subunit display changes in cell adhesion and neurodegeneration response genes.

    PubMed

    Rubin, Carol M; van der List, Deborah A; Ballesteros, Jose M; Goloshchapov, Andrey V; Chalupa, Leo M; Chapman, Barbara

    2011-04-25

    Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2) display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC) axons to their dorsal lateral geniculate nuclei (dLGNs). Transcriptomes of LGN tissue from two independently generated Chrnb2-/- mutants and from wildtype mice were obtained at postnatal day 4 (P4), during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1), a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1) and chemokine (C-C motif) ligand 21 (Ccl21) mRNAs in Chrnb2-/- mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2-/- mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2-/- mutant strains reveals the effects of genetic background upon gene expression.

  20. Negative regulatory elements upstream of a novel exon of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene.

    PubMed Central

    Bessis, A; Savatier, N; Devillers-Thiéry, A; Bejanin, S; Changeux, J P

    1993-01-01

    The expression of the nicotinic acetylcholine receptor alpha 2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mechanism underlying this regulation, we have investigated the structural and regulatory properties of the 5' sequence of this gene. A strategy based on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was found approximately 3kb upstream from the first coding exon, and multiple transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nuclear proteins, suggesting that the 1 kb flanking region contains at least a portion of the promoter of the gene. We have analysed the negative regulatory elements present within this region and found that a silencer region located between nucleotide -144 and +76 is active in fibroblasts as well as in neurons. This silencer is composed of six tandem repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member of the Oct family. Moreover these motifs were found to act as a silencer only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested. Images PMID:8502560

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

    PubMed

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

    1995-12-01

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

  2. The Brassinosteroid-Activated BRI1 Receptor Kinase Is Switched off by Dephosphorylation Mediated by Cytoplasm-Localized PP2A B' Subunits.

    PubMed

    Wang, Ruiju; Liu, Mengmeng; Yuan, Min; Oses-Prieto, Juan A; Cai, Xingbo; Sun, Ying; Burlingame, Alma L; Wang, Zhi-Yong; Tang, Wenqiang

    2016-01-04

    Brassinosteroid (BR) binding activates the receptor kinase BRI1 by inducing heterodimerization with its co-receptor kinase BAK1; however, the mechanisms that reversibly inactivate BRI1 remain unclear. Here we show that cytoplasm-localized protein phosphatase 2A (PP2A) B' regulatory subunits interact with BRI1 to mediate its dephosphorylation and inactivation. Loss-of-function and overexpression experiments showed that a group of PP2A B' regulatory subunits, represented by B'η, negatively regulate BR signaling by decreasing BRI1 phosphorylation. BR increases the expression levels of these B' subunits, and B'η interacts preferentially with phosphorylated BRI1, suggesting that the dynamics of BR signaling are modulated by the PP2A-mediated feedback inactivation of BRI1. Compared with PP2A B'α and B'β, which promote BR responses by dephosphorylating the downstream transcription factor BZR1, the BRI1-inactivating B' subunits showed similar binding to BRI1 and BZR1 but distinct subcellular localization. Alteration of the nuclear/cytoplasmic localization of the B' subunits revealed that cytoplasmic PP2A dephosphorylates BRI1 and inhibits the BR response, whereas nuclear PP2A dephosphorylates BZR1 and activates the BR response. Our findings not only identify the PP2A regulatory B subunits that mediate the binding and dephosphorylation of BRI1, but also demonstrate that the subcellular localization of PP2A specifies its substrate selection and distinct effects on BR signaling.

  3. GABA(A) receptor epsilon-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat.

    PubMed

    Kasparov, S; Davies, K A; Patel, U A; Boscan, P; Garret, M; Paton, J F

    2001-11-01

    1. Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABA(A) receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex. 2. In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 microM) did not significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 microM) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50-100 microM) was effective in enhancing GABA(A)-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; 1 or 10 microM), failed to depress GABA-induced currents in the cNTS. 3. Microinjections of midazolam (10 and 100 microM solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 microM) significantly and reversibly depressed it (gain decrease to 53 +/- 11 % of control, P < 0.01). 4. Reverse transcriptase polymerase chain reaction revealed the presence of alpha(1), alpha(2), beta(2), beta(3) and gamma(2) GABA(A) receptor subunit mRNA in the cNTS. No alternatively spliced variants of the alpha(1)- and gamma(2)-subunits were revealed. Moreover, GABA(A) epsilon-unit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts. 5. Immunocytochemical analysis revealed numerous GABA(A) epsilon-subunit-positive neurones within the cNTS with significantly fewer epsilon-subunit-positive cells in the rNTS. 6. As incorporation of the epsilon-subunit in recombinant GABA(A) receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of epsilon-subunit

  4. ZD0947, a sulphonylurea receptor modulator, detects functional sulphonylurea receptor subunits in murine vascular smooth muscle ATP-sensitive K(+) channels.

    PubMed

    Yamamoto, Tadashi; Takahara, Kohei; Uchida, Keiichiro; Teramoto, Noriyoshi

    2017-02-16

    In order to identify functional sulphonylurea receptor (SUR.x) subunits of native ATP-sensitive K(+) channels (KATP channels) in mouse portal vein, the effects of ZD0947, a SUR.x modulator, were investigated on spontaneous portal vein contractions, macroscopic membrane currents and unitary currents recorded (using patch-clamp techniques) in freshly dispersed mouse portal vein myocytes. Spontaneous contractions in mouse portal vein were reversibly reduced by ZD0947 in a concentration-dependent manner (Ki =293nM). The relaxation elicited by 3µM ZD0947 was antagonized by the additional application of glibenclamide (300nM), but not gliclazide (100-300nM). In the conventional whole-cell configuration, 100µM ZD0947 elicited inward glibenclamide-sensitive currents at a holding potential of -60mV that demonstrated selectivity for K(+)(i.e. KATP currents). The peak amplitude of the membrane current elicited by 30µM or 100µM ZD0947 was smaller than that elicited by 100µM pinacidil at -60mV. In the cell-attached mode, 100µM ZD0947 activated glibenclamide-sensitive K(+) channels with a conductance (35 pS) similar to that of recombinant Kir6.1/SUR2B channels that were expressed in HEK293 cells and activated by 100µM ZD0947. These results demonstrate that ZD0947 caused a significant vascular relaxation through the activation of KATP channels and that SUR2B may be the major functional subunit of SUR.x in mouse portal vein KATP channels, based on its pharmacological selectivity.

  5. Identification of the binding subunit of the D/sub 1/-dopamine receptor by photoaffinity crosslinking

    SciTech Connect

    Amlaiky, N.; Berger, J.G.; Chang, W.; McQuade, R.D.; Caron, M.G.

    1986-03-01

    A derivative of the potent D/sub 1/ antagonist SCH-23390 has been synthesized. This compound, (R,S)-1-(m-aminophenyl)-7-chloro-8-hydroxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-38548), has been radioiodinated by a chloramine T procedure yielding 3 radioiodinated products. One of these separated isomers (R/sub f/ = 0.35; CH/sub 2/Cl/sub 2/:MEOH:TEA; 82.5:17.5:0.1 on TLC) binds reversibly to rat striatal membranes with high affinity (K/sub D/ approx. 80 pM) appropriate stereoselectivity and D/sub 1/-dopaminergic specificity. (/sup 125/I)SCH-38548 can be covalently incorporated into a peptide of M/sub r/ approx. 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino) hexanoate. Covalent incorporation of (/sup 125/I) SCH-38548 into the M/sub r/ approx. 72,000 peptide can be blocked by dopaminergic agents with D/sub 1/-dopaminergic specificity (for agonists: SKF 38393 > apomorphine > dopamine; for antagonists: SCH-23390 > cis-flupentixol >>> trans-flupentixol). The D/sub 1/-dopaminergic selectivity and specificity of the labeling was further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine and alprenolol did not compete for the covalent labeling of the M/sub r/ approx. 72,000 peptide. This new radioligand should be useful in the molecular characterization of the D/sub 1/-dopaminergic receptor from various sources.

  6. Cannabinoid receptor type 1 activation by arachidonylcyclopropylamide in rat aortic rings causes vasorelaxation involving calcium-activated potassium channel subunit alpha-1 and calcium channel, voltage-dependent, L type, alpha 1C subunit.

    PubMed

    Sánchez-Pastor, E; Andrade, F; Sánchez-Pastor, J M; Elizalde, A; Huerta, M; Virgen-Ortiz, A; Trujillo, X; Rodríguez-Hernández, A

    2014-04-15

    Cannabinoids are key regulators of vascular tone, some of the mechanisms involved include the activation of cannabinoid receptor types 1 and 2 (CB); the transient receptor potential cation channel, subfamily V, member 1 (TRPV1); and non-(CB(1))/non-CB2 receptors. Here, we used the potent, selective CB(1) agonist arachidonylcyclopropylamide (ACPA) to elucidate the mechanism underlying vascular tone regulation. Immunohistochemistry and confocal microscopy revealed that CB(1) was expressed in smooth muscle and endothelial cells in rat aorta. We performed isometric tension recordings in aortic rings that had been pre-contracted with phenylephrine. In these conditions, ACPA caused vasorelaxation in an endothelium-independent manner. To confirm that the effect of ACPA was mediated by CB(1) receptor, we repeated the experiment after blocking these receptors with a selective antagonist, AM281. In these conditions, ACPA did not cause vasorelaxation. We explored the role of K(+) channels in the effect of ACPA by applying high-K(+) solution to induce contraction in aortic rings. In these conditions, the ACPA-induced vasorelaxation was about half that observed with phenylephrine-induced contraction. Thus, K(+) channels were involved in the ACPA effect. Furthermore, the vasorelaxation effect was similarly reduced when we specifically blocked calcium-activated potassium channel subunit alpha-1 (KCa1.1) (MaxiK; BKCa) prior to adding ACPA. Finally, ACPA-induced vasorelaxation was also diminished when we specifically blocked the calcium channel, voltage-dependent, L type, alpha 1C subunit (Ca(v)1.2). These results showed that ACPA activation of CB(1) in smooth muscle caused vasorelaxation of aortic rings through a mechanism involving the activation of K(Ca)1.1 and the inhibition of Ca(v)1.2.

  7. Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K/sup +/ channel subunits and target size analysis

    SciTech Connect

    Seagar, M.J.; Labbe-Jullie, C.; Granier, C.; Goll, A.; Glossmann, H.; Rietschoten, J.V.; Couraud, F.

    1986-07-01

    Two photoreactive apamin derivatives were prepared with an aryl azide group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca/sup 2 +/-activated K/sup +/ channel. /sup 125/I-(..cap alpha..-ANPAA-Cys/sub 1/)apamin labeled a single M/sub r/ 86,000 chain in cultured neurons whereas two bands corresponding to M/sub r/ 86,000 and 59,000 were detected in synaptic membrane preparations, suggesting that the M/sub r/ 59,000 polypeptide may be a degradation product. Randomly modified /sup 125/I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K/sup +/ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in /sup 125/I-apamin receptor capacity, yielding a target size for the functional binding unit of M/sub r/ 84,000-115,000, which could be constituted by the M/sub r/ 86,000 subunit alone or by the M/sub r/ 86,000 subunit in conjunction with one of the two smaller subunits.

  8. A partial genomic DNA clone for the alpha subunit of the mouse complement receptor type 3 and cellular adhesion molecule Mac-1.

    PubMed Central

    Sastre, L; Roman, J M; Teplow, D B; Dreyer, W J; Gee, C E; Larson, R S; Roberts, T M; Springer, T A

    1986-01-01

    A genomic clone coding for the alpha subunit of the mouse complement receptor type 3 and the cellular adhesion molecule Mac-1 has been isolated directly from a genomic library using synthetic oligonucleotide probes based on the amino-terminal amino acid sequence of the protein. The identity of the clone has been established by DNA sequencing and in vitro translation of hybrid-selected mRNA. The gene is present in a single copy in the murine genome. The region containing the amino-terminal exon has been sequenced. RNA gel blotting shows that the Mac-1 alpha-subunit mRNA is 6 kilobases in length. Mac-1 alpha-subunit mRNA is present in macrophages but not T lymphoma or L cells. During gamma interferon-stimulated maturation of the mouse premyelocytic cell line M1, Mac-1 alpha-subunit mRNA is induced. This corresponds with the tissue distribution of the Mac-1 alpha subunit, showing expression is regulated at least partially at the message level. Images PMID:2942940

  9. Sex differences in anxiety, sensorimotor gating and expression of the α4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal

    PubMed Central

    Gulinello, M.; Orman, R.; Smith, S. S.

    2010-01-01

    In a progesterone withdrawal (PWD) model of premenstrual anxiety, we have previously demonstrated that increased hippocampal expression of the α4 subunit of the GABAA receptor (GABAA-R) is closely associated with higher anxiety levels in the elevated plus maze. However, several studies indicate that sex differences in regulation of the GABAA-R in specific brain regions may be an important factor in the observed gender differences in mood disorders. Thus, we investigated possible sex differences in GABAA-R subunit expression and anxiety during PWD. To this end, we utilized the acoustic startle response (ASR) to assess anxiety levels in male and female rats undergoing PWD as the ASR is also applicable to the assessment of human anxiety responses. We also investigated GABAA-R α4 subunit expression in the amygdala, as the amygdala directly regulates the primary startle circuit. Female rats exhibited a greater ASR during PWD than controls, indicating higher levels of anxiety and arousal. In contrast, male rats undergoing PWD did not demonstrate an increased ASR. The sex differences in the ASR were paralleled by sex differences in the expression of the GABAA-R α4 subunit in the amygdala such that α4 subunit expression was up-regulated in females during PWD whereas α4 levels in males undergoing PWD were not altered relative to controls. These findings might have implications regarding gender differences in human mood disorders and the aetiology of premenstrual anxiety. PMID:12581182

  10. Expression of functional human α6β2β3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers.

    PubMed

    Kuryatov, Alexandre; Lindstrom, Jon

    2011-01-01

    α6β2β3* acetylcholine receptors (AChRs) on dopaminergic neurons are important targets for drugs to treat nicotine addiction and Parkinson's disease. However, it has not been possible to efficiently express functional α6β2β3* AChRs in oocytes or transfected cells. α6/α3 subunit chimeras permit expression of functional AChRs and reveal that parts of the α6 M1 transmembrane domain and large cytoplasmic domain impair assembly. Concatameric subunits permit assembly of functional α6β2β3* AChRs with defined subunit compositions and subunit orders. Assembly of accessory subunits is limiting in formation of mature AChRs. A single linker between the β3 accessory subunit and an α4 or α6 subunit is sufficient to permit assembly of complex β3-(α4β2)(α6β2) or β3-(α6β2)(α4β2) AChRs. Concatameric pentamers such as β3-α6-β2-α4-β2 have been functionally characterized. α6β2β3* AChRs are sensitive to activation by drugs used for smoking cessation therapy (nicotine, varenicline, and cytisine) and by sazetidine. All these are partial agonists. (α6β2)(α4β2)β3 AChRs are most sensitive to agonists. (α6β2)₂β3 AChRs have the greatest Ca²+ permeability. (α4β2)(α6β2)β3 AChRs are most efficiently transported to the cell surface, whereas (α6β2)₂β3 AChRs are the least efficiently transported. Dopaminergic neurons may have special chaperones for assembling accessory subunits with α6 subunits and for transporting (α6β2)₂β3 AChRs to the cell surface. Concatameric pentamers and pentamers formed from combinations of trimers, dimers, and monomers exhibit similar properties, indicating that the linkers between subunits do not alter their functional properties. For the first time, these concatamers allow analysis of functional properties of α6β2β3* AChRs. These concatamers should enable selection of drugs specific for α6β2β3* AChRs.

  11. Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices.

    PubMed

    Small, D L; Poulter, M O; Buchan, A M; Morley, P

    1997-08-29

    Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

  12. Deletion of GIRK2 Subunit of GIRK Channels Alters the 5-HT1A Receptor-Mediated Signaling and Results in a Depression-Resistant Behavior

    PubMed Central

    Llamosas, Nerea; Bruzos-Cidón, Cristina; Rodríguez, José Julio; Ugedo, Luisa

    2015-01-01

    Background: Targeting dorsal raphe 5-HT1A receptors, which are coupled to G-protein inwardly rectifying potassium (GIRK) channels, has revealed their contribution not only to behavioral and functional aspects of depression but also to the clinical response to its treatment. Although GIRK channels containing GIRK2 subunits play an important role controlling excitability of several brain areas, their impact on the dorsal raphe activity is still unknown. Thus, the goal of the present study was to investigate the involvement of GIRK2 subunit-containing GIRK channels in depression-related behaviors and physiology of serotonergic neurotransmission. Methods: Behavioral, functional, including in vivo extracellular recordings of dorsal raphe neurons, and neurogenesis studies were carried out in wild-type and GIRK2 mutant mice. Results: Deletion of the GIRK2 subunit promoted a depression-resistant phenotype and determined the behavioral response to the antidepressant citalopram without altering hippocampal neurogenesis. In dorsal raphe neurons of GIRK2 knockout mice, and also using GIRK channel blocker tertiapin-Q, the basal firing rate was higher than that obtained in wild-type animals, although no differences were observed in other firing parameters. 5-HT1A receptors were desensitized in GIRK2 knockout mice, as demonstrated by a lower sensitivity of dorsal raphe neurons to the inhibitory effect of the 5-HT1A receptor agonist, 8-OH-DPAT, and the antidepressant citalopram. Conclusions: Our results indicate that GIRK channels formed by GIRK2 subunits determine depression-related behaviors as well as basal and 5-HT1A receptor-mediated dorsal raphe neuronal activity, becoming alternative therapeutic targets for psychiatric diseases underlying dysfunctional serotonin transmission. PMID:25956878

  13. Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation.

    PubMed

    Jones, K L; Bagley, C J; Butcher, C; Barry, S C; Vadas, M A; D'Andrea, R J

    2001-06-21

    A mutant form of the common beta-subunit of the GM-CSF, interleukin-3 (IL3) and IL5 receptors is activated by a 37 residue duplicated segment which includes the WSXWS motif and an adjacent, highly conserved, aliphatic/basic element. Haemopoietic expression of this mutant, hbeta(c)FIDelta, in mice leads to myeloproliferative disease. To examine the mechanism of activation of this mutant we targetted the two conserved motifs in each repeat for mutagenesis. Here we show that this mutant exhibits constitutive activity in BaF-B03 cells in the presence of mouse or human GM-CSF receptor alpha-subunit (GMRalpha) and this activity is disrupted by mutations of the conserved motifs in the first repeat. In the presence of these mutations the receptor reverts to an alternative conformation which retains responsiveness to human IL3 in a CTLL cell line co-expressing the human IL3 receptor alpha-subunit (hIL3Ralpha). Remarkably, the activated conformation is maintained in the presence of substitutions, deletions or replacement of the second repeat. This suggests that activation occurs due to insertion of extra sequence after the WSXWS motif and is not dependent on the length or specific sequence of the insertion. Thus hbeta(c) displays an ability to fold into functional receptor conformations given insertion of up to 37 residues in the membrane-proximal region. Constitutive activation most likely results from a specific conformational change which alters a dormant, inactive receptor complex, permitting functional association with GMRalpha and ligand-independent mitogenic signalling.

  14. Roles of the AMPA receptor subunit GluA1 but not GluA2 in synaptic potentiation and activation of ERK in the anterior cingulate cortex.

    PubMed

    Toyoda, Hiroki; Zhao, Ming-Gao; Ulzhöfer, Bettina; Wu, Long-Jun; Xu, Hui; Seeburg, Peter H; Sprengel, Rolf; Kuner, Rohini; Zhuo, Min

    2009-08-10

    Cortical areas including the anterior cingulate cortex (ACC) are important for pain and pleasure. Recent studies using genetic and physiological approaches have demonstrated that the investigation of basic mechanism for long-term potentiation (LTP) in the ACC may reveal key cellular and molecular mechanisms for chronic pain in the cortex. Glutamate N-methyl D-aspartate (NMDA) receptors in the ACC are critical for the induction of LTP, including both NR2A and NR2B subunits. However, cellular and molecular mechanisms for the expression of ACC LTP have been less investigated. Here, we report that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, GluA1 but not GluA2 contributes to LTP in the ACC using genetic manipulated mice lacking GluA1 or GluA2 gene. Furthermore, GluA1 knockout mice showed decreased extracellular signal-regulated kinase (ERK) phosphorylation in the ACC in inflammatory pain models in vivo. Our results demonstrate that AMPA receptor subunit GluA1 is a key mechanism for the expression of ACC LTP and inflammation-induced long-term plastic changes in the ACC.

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

    PubMed

    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

  16. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABA(A) receptors on parvalbumin interneurons.

    PubMed

    Ferando, Isabella; Mody, Istvan

    2013-01-01

    Gamma (γ) oscillations (30-120 Hz), an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs) and the principal (pyramidal) cells. PV+BCs express δ-subunit-containing GABA(A)Rs (δ-GABA(A)Rs) and NMDA receptors (NMDA-Rs) that balance the frequency of γ oscillations. Neuroactive steroids (NS), such as the progesterone-derived (3α,5α)-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO), modulate the expression of δ-GABA(A)Rs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABA(A)Rs expression. Here we show that in CA3, where most PV+ interneurons (INs) express δ-GABA(A)Rs, expression of δ-GABA(A)Rs on INs diminishes during pregnancy, but reverts to control levels within 48 h postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy) may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABA(A)Rs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABA(A)R expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

  17. Etomidate Impairs Long-Term Potentiation In Vitro by Targeting α5-Subunit Containing GABAA Receptors on Nonpyramidal Cells

    PubMed Central

    Rodgers, F. Clifford; Zarnowska, Ewa D.; Laha, Kurt T.; Engin, Elif; Zeller, Anja; Keist, Ruth; Rudolph, Uwe

    2015-01-01

    Previous experiments using genetic and pharmacological manipulations have provided strong evidence that etomidate impairs synaptic plasticity and memory by modulating α5-subunit containing GABAA receptors (α5-GABAARs). Because α5-GABAARs mediate tonic inhibition (TI) in hippocampal CA1 pyramidal cells and etomidate enhances TI, etomidate enhancement of TI in pyramidal cells has been proposed as the underlying mechanism (Martin et al., 2009). Here we tested this hypothesis by selectively removing α5-GABAARs from pyramidal neurons (CA1–pyr–α5–KO) and comparing the ability of etomidate to enhance TI and block LTP in fl–α5 (WT), global–α5–KO (gl–α5–KO), and CA1–pyr–α5–KO mice. Etomidate suppressed LTP in slices from WT and CA1–pyr–α5–KO but not gl–α5–KO mice. There was a trend toward reduced TI in both gl–α5–KO and CA1–pyr–α5–KO mice, but etomidate enhanced TI to similar levels in all genotypes. The dissociation between effects of etomidate on TI and LTP in gl–α5–KO mice indicates that increased TI in pyramidal neurons is not the mechanism by which etomidate impairs LTP and memory. Rather, the ability of etomidate to block LTP in WT and CA1–pyr–α5–KO mice, but not in gl–α5–KO mice, points toward α5-GABAARs on nonpyramidal cells as the essential effectors controlling plasticity in this in vitro model of learning and memory. PMID:26134653

  18. Anxious phenotypes plus environmental stressors are related to brain DNA damage and changes in NMDA receptor subunits and glutamate uptake.

    PubMed

    Réus, Gislaine Z; Abaleira, Helena M; Michels, Monique; Tomaz, Débora B; dos Santos, Maria Augusta B; Carlessi, Anelise S; Matias, Beatriz I; Leffa, Daniela D; Damiani, Adriani P; Gomes, Vitor de C; Andrade, Vanessa M; Dal-Pizzol, Felipe; Landeira-Fernadez, Jesus; Quevedo, João

    2015-02-01

    This study aimed at investigating the effects of chronic mild stress on DNA damage, NMDA receptor subunits and glutamate transport levels in the brains of rats with an anxious phenotype, which were selected to represent both the high-freezing (CHF) and low-freezing (CLF) lines. The anxious phenotype induced DNA damage in the hippocampus, amygdala and nucleus accumbens (NAc). CHF rats subjected to chronic stress presented a more pronounced DNA damage in the hippocampus and NAc. NMDAR1 were increased in the prefrontal cortex (PC), hippocampus and amygdala of CHF, and decreased in the hippocampus, amygdala and NAc of CHF stressed. NMDAR2A were decreased in the amygdala of the CHF and stressed; and increased in CHF stressed. NMDRA2A in the NAc was increased after stress, and decreased in the CLF. NMDAR2B were increased in the hippocampus of CLF and CHF. In the amygdala, there was a decrease in the NMDAR2B for stress in the CLF and CHF. NMDAR2B in the NAc were decreased for stress and increased in the CHF; in the PC NMDAR2B increased in the CHF. EAAT1 increased in the PC of CLF+stress. In the hippocampus, EAAT1 decreased in all groups. In the amygdala, EAAT1 decreased in the CLF+stress and CHF. EAAT2 were decreased in the PC for stress, and increased in CHF+control. In the hippocampus, the EAAT2 were increased for the CLF and decreased in the CLF+stress. In the amygdala, there was a decrease in the EATT2 in the CLF+stress and CHF. These findings suggest that an anxious phenotype plus stress may induce a more pronounced DNA damage, and promote more alterations in the glutamatergic system. These findings may help to explain, at least in part, the common point of the mechanisms involved with the pathophysiology of depression and anxiety.

  19. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABAA receptors on parvalbumin interneurons

    PubMed Central

    Ferando, Isabella; Mody, Istvan

    2013-01-01

    Gamma (γ) oscillations (30–120 Hz), an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs) and the principal (pyramidal) cells. PV+BCs express δ-subunit-containing GABAARs (δ-GABAARs) and NMDA receptors (NMDA-Rs) that balance the frequency of γ oscillations. Neuroactive steroids (NS), such as the progesterone-derived (3α,5α)-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO), modulate the expression of δ-GABAARs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABAARs expression. Here we show that in CA3, where most PV+ interneurons (INs) express δ-GABAARs, expression of δ-GABAARs on INs diminishes during pregnancy, but reverts to control levels within 48 h postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy) may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABAARs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABAAR expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments. PMID:24062647

  20. Association of GABAA receptor α2 subunit gene (GABRA2) with alcohol dependence-related aggressive behavior.

    PubMed

    Strac, Dubravka Svob; Erjavec, Gordana Nedic; Perkovic, Matea Nikolac; Sviglin, Korona Nenadic; Borovecki, Fran; Pivac, Nela

    2015-12-03

    Alcohol dependence is a common chronic disorder precipitated by the complex interaction between biological, genetic and environmental risk factors. Recent studies have demonstrated that polymorphisms of the gene encoding the GABAA receptor α2 subunit (GABRA2) are associated with alcohol dependence in different populations of European ancestry. As aggression often occurs in the context of alcohol dependence, the aim of this study was to examine the allelic and haplotypic association of GABRA2 gene with alcohol dependence and related aggressive behavior in subjects of Eastern European (Croatian) origin. Genotyping of the 3 single nucleotide polymorphisms (SNPs) across the GABRA2 gene (rs567926, rs279858 and rs9291283) was performed in patients with alcohol dependence (N=654) and healthy control subjects (N=574). Alcohol-dependent participants were additionally subdivided according to the presence/absence of aggressive behavior and type of alcohol dependence according to the Cloninger's classification. The association of rs279858 with alcohol dependence yielded nominal significance level. Haplotype analysis revealed a high degree of linkage disequilibrium (LD) for rs567926 and rs279858, but not for rs9291283 polymorphism in the GABRA2 gene. In patients with alcohol dependence, the A-C (rs567926 and rs279858) haplotype carriers were more likely to demonstrate aggressive behavior. The same haplotype (present only in 1.6% of all subjects) was significantly more often present in patients with a combination of early onset alcohol abuse and aggression, corresponding to the Cloninger's type II alcoholism subgroup. These findings support the involvement of GABRA2 gene in alcohol dependence-related aggressive behavior.

  1. Mouse Mutants for the Nicotinic Acetylcholine Receptor ß2 Subunit Display Changes in Cell Adhesion and Neurodegeneration Response Genes

    PubMed Central

    Rubin, Carol M.; van der List, Deborah A.; Ballesteros, Jose M.; Goloshchapov, Andrey V.; Chalupa, Leo M.; Chapman, Barbara

    2011-01-01

    Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2) display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC) axons to their dorsal lateral geniculate nuclei (dLGNs). Transcriptomes of LGN tissue from two independently generated Chrnb2−/− mutants and from wildtype mice were obtained at postnatal day 4 (P4), during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1), a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1) and chemokine (C-C motif) ligand 21 (Ccl21) mRNAs in Chrnb2−/− mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2−/− mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2−/− mutant strains reveals the effects of genetic background upon gene expression. PMID:21547082

  2. Altered expression of GABAA receptors (α4, γ2 subunit), potassium chloride cotransporter 2 and astrogliosis in tremor rat hippocampus.

    PubMed

    Mao, Xiaoyuan; Ma, Ping; Cao, Danfeng; Sun, Chunyan; Ji, Zhong; Min, Dongyu; Sun, Hongli; Xie, Ni; Cai, Jiqun; Cao, Yonggang

    2011-11-25

    Impaired GABAergic inhibitory neurotransmission plays an essential role in the pathogenesis of epilepsy. GABA(A) receptor (GABA(A)R), potassium chloride cotransporter 2 (KCC2) and astrocytes are of particular importance to GABAergic transmission and thus involved in the development of increased seizure susceptibility. The tremor rat (TRM: tm/tm), a genetic mutant discovered in a Kyoto-Wistar colony, can manifest both absence-like seizures and tonic convulsions without any external stimuli. So far, there are no reports that can elucidate the effects of GABA(A)R (α4, γ2 subunit), KCC2 and astrocytes on TRMs. The present study was undertaken to detect the expressions of GABA(A)R α4, GABA(A)R γ2 and KCC2 in TRMs hippocampus at mRNA and protein levels. In this work, mRNA and protein expressions of GABA(A)R α4 were significantly elevated while GABA(A)R γ2 and KCC2 were both evidently decreased in TRMs hippocampus by real-time RT-PCR and western blot, respectively. Furthermore, a dramatic elevation of KCC2 protein level was found after cerebroventricular injection with K252a to TRMs than that in the DMSO-treated TRMs. Besides, our present study also demonstrated that GFAP (a major component of astrocyte) immunoreactivity was much more intense in TRMs hippocampal CA1, CA3 and DG regions than that in control group with immnohistochemistry and confocal microscopic analyses. The protein expression of GFAP was also markedly elevated in TRMs hippocampus, suggesting that astrogliosis appeared in the TRM model. These data demonstrate that altered expressions of GABA(A)R (α4, γ2) and KCC2 and astrogliosis observed in TRMs hippocampus may provide us good therapeutic targets for the treatment of genetic epilepsy.

  3. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

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

    PubMed

    Baez, Maria Veronica; Oberholzer, Maria Victoria; Cercato, Magali Cecilia; Snitcofsky, Marina; Aguirre, Alejandra Ines; Jerusalinsky, Diana Alicia

    2013-01-01

    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.

  5. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    PubMed

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

    2014-01-01

    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  6. Regional mRNA expression of GABAergic receptor subunits in brains of C57BL/6J and 129P3/J mice: Strain and heroin effects

    PubMed Central

    Brownstein, A.J.; Zhang, Y.; Ho, A.; Kreek, M.J.

    2014-01-01

    C57BL/6J and 129 substrains of mice are known to differ in their basal levels of anxiety and behavioral response to drugs of abuse. We have previously shown strain differences in heroin-induced conditioned place preference (CPP) between C57BL/6J (C57) and 129P3/J (129) mice, and in the regional expression of several receptor and peptide mRNAs. In this study, we examined the contribution of the GABAergic system in the cortex, nucleus accumbens (NAc), caudate putamen (CPu) and the region containing the substantia nigra and ventral tegmental area (SN/VTA) to heroin reward by measuring mRNA levels of 7 of the most commonly expressed GABA-A receptor subunits, and both GABA-B receptor subunits, in these same mice following saline (control) or heroin administration in a CPP design. Using real-time PCR, we studied the effects of strain and heroin administration on GABA-A α1, α2, α3, β2, and γ2 subunits, which typically constitute synaptic GABA-A receptors, GABA-A α4 and δ subunits, which typically constitute extrasynaptic GABA-A receptors, and GABA-B R1 and R2 subunits. In saline-treated animals, we found an experiment-wise significant strain difference in GABA-Aα2 mRNA expression in the SN/VTA. Point-wise significant strain differences were also observed in GABA-Aα2, GABA-Aα3, and GABA-Aα4 mRNA expression in the NAc, as well as GABA-BR2 mRNA expression in the NAc and CPu, and GABA-BR1 mRNA expression in the cortex. For all differences, 129 mice had higher mRNA expression compared to C57 animals, with the exception of GABA-BR1 mRNA in the cortex where we observed lower levels in 129 mice. Therefore, it may be possible that known behavioral differences between these two strains are, in part, due to differences in their GABAergic systems. While we did not find heroin dose-related changes in mRNA expression levels in C57 mice, we did observe dose-related differences in 129 mice. These results may relate to our earlier behavioral finding that 129 mice are

  7. Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit*

    PubMed Central

    Jin, Hongbing; Chiou, Tzu-Ting; Serwanski, David R.; Miralles, Celia P.; Pinal, Noelia; De Blas, Angel L.

    2014-01-01

    We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation. PMID:25193658

  8. First and second transmembrane segments of alpha3, alpha4, beta2, and beta4 nicotinic acetylcholine receptor subunits influence the efficacy and potency of nicotine.

    PubMed

    Rush, Ray; Kuryatov, Alexander; Nelson, Mark E; Lindstrom, Jon

    2002-06-01

    The first three transmembrane segments (M1-M3) of human nicotinic acetylcholine receptors (nAChRs) have been implicated in determining the efficacy of nicotine by studies of alpha3/alpha4 subunit chimeras. Nicotine has full efficacy on the alpha4beta2 nAChR and partial efficacy on the alpha3beta2 nAChR. Now, we have exchanged individually three amino acids between the alpha4 and the alpha3 subunits at positions 226(M1), 258(M2), and 262(M2). Also, similar exchanges were made in the beta2 and beta4 subunits at positions 224(M1), 226(M1), and 254(M2) (using alpha subunit numbering). Expression of these mutated nAChRs in Xenopus laevis oocytes showed that the mutated M1 amino acids were important in influencing the potency of ACh and nicotine. It is hypothesized that these M1 amino acids affect the stability between the resting and activated states of the nAChR. M2 amino acids altered the efficacy of nicotine, usually without altering its potency. When the residue located at position 258 in the M2 region of the alpha subunit was valine (as in the alpha3 subunit), the resulting nAChR exhibited partial efficacy for nicotine that was voltage-dependent. Therefore, we believe that these M2 amino acids contribute to the formation of a binding site for nicotine in the alpha3beta2 nAChR channel, which results in a low-affinity channel block, causing the lower efficacy of nicotine on this nAChR.

  9. N-methyl-D-aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model.

    PubMed

    Peng, Wei-Feng; Ding, Jing; Li, Xin; Fan, Fan; Zhang, Qian-Qian; Wang, Xin

    2016-01-01

    Depression is a common comorbidity in patients with epilepsy with unclear mechanisms. This study is to explore the role of glutamate N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. Lithium chloride (Licl)-pilocarpine chronic rat epilepsy model was established and rats were divided into epilepsy with depression (EWD) and epilepsy without depression (EWND) subgroups based on forced swim test. Expression of NMDA receptor NR1, NR2A and NR2B subunits was measured by western blot and immunofluorescence methods. The immobility time (IMT) was significantly greater in Licl-pilocarpine model group than in Control group, which was also greater in EWD group than in EWND group. No differences of spontaneous recurrent seizure