Sample records for receptor beta subunit

  1. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity. (United States)

    Ratra, G S; Kamita, S G; Casida, J E


    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  2. 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. (United States)

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


    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.

  3. Slow-dissociation effect of common signaling subunit beta c on IL5 and GM-CSF receptor assembly. (United States)

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


    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.

  4. GABA{sub A} receptor beta 3 subunit gene is possibly paternally imprinted in humans

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    As the gene for GABA{sub A} receptor beta 3 subunit (GABRB3) is encompassed by a small molecular deletion in chromosome 15q11-q13, which is the critical region for Angelman syndrome(AS), the GABRB3 gene could be a candidate gene for AS. The abnormal phenotype of AS is manifested only when the deletion is inherited from the mother, not from the father. Therefore, a candidate gene for AS should be paternally imprinted. Although it was reported that the GABRB3 gene was expressed equally from either the maternal or paternal chromosome in mouse brain (i.e., not imprinted), it is well known that imprinting shows tissue specificity, and it remains to be determined if all genes imprinted in the mouse are also imprinted in humans. 4 refs., 1 fig.

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

    Wagoner, Kelly R; Czajkowski, Cynthia


    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.

  6. Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

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    King, K.; Caron, M.G.; Lefkowitz, R.J. (Duke Univ. Medical Center, Durham, NC (USA)); Dohlman, H.G.; Thorner, J. (Univ. of California, Berkeley (USA))


    To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR and a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

  7. NMDA receptor subunit composition determines beta-amyloid-induced neurodegeneration and synaptic loss


    Tackenberg, C; Grinschgl, S; Trutzel, A; Santuccione, A C; Frey, M C; Konietzko, U; Grimm, J.; Brandt, R.; Nitsch, R M


    Aggregates of amyloid-beta (Aβ) and tau are hallmarks of Alzheimer's disease (AD) leading to neurodegeneration and synaptic loss. While increasing evidence suggests that inhibition of N-methyl--aspartate receptors (NMDARs) may mitigate certain aspects of AD neuropathology, the precise role of different NMDAR subtypes for Aβ- and tau-mediated toxicity remains to be elucidated. Using mouse organotypic hippocampal slice cultures from arcAβ transgenic mice combined with Sindbis virus-mediated ex...

  8. Further studies on the covalent crosslinking of thyrotropin to its receptor: evidence that both the alpha and beta subunits of thyrotropin are crosslinked to the receptor. (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N


    Highly purified alpha- and beta-subunits of thyrotropin were individually radioiodinated and, subsequently, recombined with their unlabeled complementary subunits. This procedure resulted in the formation of [125I]thyrotropin(TSH) hybrid molecules which were labeled on only one hormone subunit. Characterization of the binding properties of these two hybrid molecules demonstrated that both yielded nonlinear Scatchard plots with Kd and Bmax values similar to those obtained with radioiodinated native TSH and that both were capable of interaction with the high- and low-affinity binding components of the TSH receptor. The recombined [125I]TSH molecules were then crosslinked to the TSH receptor using disuccinimidyl suberate. Following electrophoresis and autoradiography, two labeled TSH-receptor complexes with Mr of 68,000 and 80,000 were observed. These two complexes exhibited hormone specificity and electrophoretic mobility identical to those previously observed using native [125I]TSH. Crosslinking with increasing concentrations of disuccinimidyl suberate suggested that the formation of the 68,000 and 80,000 complexes was sequential with the 68,000 appearing before the 80,000. Furthermore, the two bands were labeled regardless of which TSH subunit of the hybrid TSH was radioiodinated. These data strongly suggest that the 68,000 and 80,000 TSH-receptor complexes are the result of crosslinking to the TSH alpha-beta dimer and not to one subunit in the case of the 68,000 complex and to the TSH alpha-beta dimer in the case of the 80,000 complex, as had been hypothesized previously.

  9. Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor. (United States)

    Fabbi, M; Acuto, O; Bensussan, A; Poole, C B; Reinherz, E L


    To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

  10. PPP1R16A, the membrane subunit of protein phosphatase 1beta, signals nuclear translocation of the nuclear receptor constitutive active/androstane receptor. (United States)

    Sueyoshi, Tatsuya; Moore, Rick; Sugatani, Junko; Matsumura, Yonehiro; Negishi, Masahiko


    Constitutive active/androstane receptor (CAR), a member of the nuclear steroid/thyroid hormone receptor family, activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. We have now identified a membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A) as a novel CAR-binding protein. When CAR and R16A are coexpressed in mouse liver, CAR translocates into the nucleus. Close association of R16A and CAR molecule on liver membrane was shown by fluorescence resonance energy transfer (FRET) analysis using expressed yellow fluorescent protein (YFP)-CAR and CFP-R16A fusion proteins. R16A can form dimer through its middle region, where protein kinase A phosphorylation sites are recently identified. Translocation of CAR by R16A correlates with the ability of R16A to form an intermolecular interaction via the middle region. Moreover, this interaction is enhanced by PB treatment in mouse liver. R16A specifically interacted with PP1beta in HepG2 cells despite the highly conserved structure of PP1 family molecules. PP1beta activity was inhibited by R16A in vitro and coexpression of PP1beta in liver can prevent YFP-CAR translocation into mouse liver. Taken together, R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1beta activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial.

  11. Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes. (United States)

    Scibek, Jeffery J; Evergren, Emma; Zahn, Stefan; Canziani, Gabriela A; Van Ryk, Donald; Chaiken, Irwin M


    To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

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

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


    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

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

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    Cunyou Zhao

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

  14. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I;


    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  15. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

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    Machaalani, Rita, E-mail: [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Say, Meichien [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); Waters, Karen A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia)


    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black

  16. Prefrontal beta2 subunit-containing and alpha7 nicotinic acetylcholine receptors differentially control glutamatergic and cholinergic signaling. (United States)

    Parikh, Vinay; Ji, Jinzhao; Decker, Michael W; Sarter, Martin


    One-second-long increases in prefrontal cholinergic activity ("transients") were demonstrated previously to be necessary for the incorporation of cues into ongoing cognitive processes ("cue detection"). Nicotine and, more robustly, selective agonists at alpha4beta2* nicotinic acetylcholine receptors (nAChRs) enhance cue detection and attentional performance by augmenting prefrontal cholinergic activity. The present experiments determined the role of beta2-containing and alpha7 nAChRs in the generation of prefrontal cholinergic and glutamatergic transients in vivo. Transients were evoked by nicotine, the alpha4beta2* nAChR agonist ABT-089 [2-methyl-3-(2-(S)-pyrrolindinylmethoxy) pyridine dihydrochloride], or the alpha7 nAChR agonist A-582941 [2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole]. Transients were recorded in mice lacking beta2 or alpha7 nAChRs and in rats after removal of thalamic glutamatergic or midbrain dopaminergic inputs to prefrontal cortex. The main results indicate that stimulation of alpha4beta2* nAChRs evokes glutamate release and that the presence of thalamic afferents is necessary for the generation of cholinergic transients. ABT-089-evoked transients were completely abolished in mice lacking beta2* nAChRs. The amplitude, but not the decay rate, of nicotine-evoked transients was reduced by beta2* knock-out. Conversely, in mice lacking the alpha7 nAChR, the decay rate, but not the amplitude, of nicotine-evoked cholinergic and glutamatergic transients was attenuated. Substantiating the role of alpha7 nAChR in controlling the duration of release events, stimulation of alpha7 nAChR produced cholinergic transients that lasted 10- to 15-fold longer than those evoked by nicotine. alpha7 nAChR-evoked cholinergic transients are mediated in part by dopaminergic activity. Prefrontal alpha4beta2* nAChRs play a key role in evoking and facilitating the transient glutamatergic-cholinergic interactions that are necessary for cue detection

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

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


    2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1......)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced...

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

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


    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…

  19. Co-expression of alpha7 and beta2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons. (United States)

    Azam, L; Winzer-Serhan, U; Leslie, F M


    Nicotine enhances cognitive and attentional processes through stimulation of the basal forebrain cholinergic system. Although muscarinic cholinergic autoreceptors have been well characterized, pharmacological characterization of nicotinic autoreceptors has proven more difficult. The present study used double-labeling in situ hybridization to determine expression of nicotinic acetylcholine receptor (nAChR) subunit mRNAs within basal forebrain cholinergic neurons in order to gain information about possible nAChR autoreceptor properties. Cholinergic cells of the mesopontine tegmentum and striatal interneurons were also examined, as were septohippocampal GABAergic neurons that interact with cholinergic neurons to regulate hippocampal activity. alpha7 and beta2 nAChR mRNAs were found to be co-expressed in almost all cholinergic cells and in the majority of GABAergic neurons examined. alpha4 nAChR mRNA expression was restricted to cholinergic cells of the nucleus basalis magnocellularis, and to non-cholinergic cells of the medial septum and mesopontine tegmentum. These data suggest possible regional differences in the pharmacological properties of nicotinic autoreceptors on cholinergic cells. Whereas most cholinergic cells express rapidly desensitizing alpha7 homomers or alpha7beta2 heteromers, cortical projection neurons may also express a pharmacologically distinct alpha4beta2 nAChR subtype. There may also be differential nAChR regulation of cholinergic and non-cholinergic cells within the mesopontine tegmentum that are implicated in acquisition of nicotine self-administration.

  20. GABA receptor subunit composition relative to insecticide potency and selectivity. (United States)

    Ratra, G S; Casida, J E


    Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

  1. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA. (United States)

    Vela, J; Vitorica, J; Ruano, D


    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  2. Phenotypic consequences of deletion of the {gamma}{sub 3}, {alpha}{sub 5}, or {beta}{sub 3} subunit of the type A {gamma}-aminobutyric acid receptor in mice

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    Culia, C.T.; Stubbs, L.J.; Montgomery, C.S.; Russell, L.B.; Rinchik, E.M. [Oak Ridge National Lab., TN (United States)


    Three genes (Gabrg3, Gabra5, and Gabrb3) encoding the {gamma}{sub 3}, {alpha}{sub 5}, and {beta}{sub 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-Gabra5-Gabrb3-telomere. 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. The authors have previously suggested that deficiency of the {beta}{sub 3} subunit may be responsible for the clefting defect. Most notably, however, in this report they describe mice carrying two overlapping, complementing p deletions that fail to express the {gamma}{sub 3} transcript, as well as mice from another line that express neither the {gamma}{sub 3} nor {alpha}{sub 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}{sub 3}, {alpha}{sub 5}, and {beta}{sub 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}{sub 3} and/or {alpha}{sub 5} subunits. The absence of an overt neurological phenotype in mice lacking the {gamma}{sub 3} and/or {alpha}{sub 5} subunits also suggests that mutations in these genes are unlikely to provide useful animal models for Angelman syndrome in humans.

  3. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo. (United States)

    Jackson, K J; Walters, C L; Damaj, M I


    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  4. Stoichiometry of δ subunit containing GABAA receptors (United States)

    Patel, B; Mortensen, M; Smart, T G


    Background and Purpose Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Experimental Approach Using site-directed mutagenesis, we inserted a highly characterized 9′ serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Key Results Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose–response curves of cells co-expressing WT subunits with their respective L9′S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Conclusions and Implications Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. PMID:24206220

  5. Effects of hippocampal injections of a novel ligand selective for the alpha 5 beta 2 gamma 2 subunits of the GABA/benzodiazepine receptor on Pavlovian conditioning. (United States)

    Bailey, David J; Tetzlaff, Julie E; Cook, James M; He, Xiaohui; Helmstetter, Fred J


    Benzodiazepine pharmacology has led to greater insight into the neural mechanisms underlying learning and anxiety. The synthesis of new compounds capable of modulating responses produced by these receptors has been made possible by the development of an isoform model of the GABA(A)/benzodiazepine receptor complex. In the current experiment, rats were pretreated with several concentrations of the novel ligand RY024 (an alpha 5 beta 2 gamma 2 -selective benzodiazepine receptor inverse agonist) in the hippocampus and were trained in a Pavlovian fear conditioning paradigm. RY024 independently produced fear-related behavior prior to training and, at the highest concentration, decreased the strength of conditioning observed 24 h after training. These data provide further evidence for the involvement of hippocampal GABA(A)/benzodiazepine receptors in learning and anxiety.

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

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


    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.

  7. GABA B receptor subunit expression in glia. (United States)

    Charles, K J; Deuchars, J; Davies, C H; Pangalos, M N


    GABA(B) receptor subunits are widely expressed on neurons throughout the CNS, at both pre- and postsynaptic sites, where they mediate the late, slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. The existence of functional GABA(B) receptors on nonneuronal cells has been reported previously, although the molecular composition of these receptors has not yet been described. Here we demonstrate for the first time, using immunohistochemistry the expression of GABA(B1a), GABA(B1b), and GABA(B2) on nonneuronal cells of the rat CNS. All three principle GABA(B) receptor subunits were expressed on these cells irrespective of whether they had been cultured or found within brain tissue sections. At the ultrastructural level GABA(B) receptor subunits were expressed on astrocytic processes surrounding both symmetrical and assymetrical synapses in the CA1 subregion of the hippocampus. In addition, GABA(B1a), GABA(B1b), and GABA(B2) receptor subunits were expressed on activated microglia in culture but were not found on myelin forming oligodendrocytes in the white matter of rat spinal cord. Together these data demonstrate that the obligate subunits of functional GABA(B) receptors are expressed in astrocytes and microglia in the rat CNS.

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

    Directory of Open Access Journals (Sweden)

    Brandon H. Cline


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

  9. Threonine 788 in integrin subunit beta1 regulates integrin activation

    DEFF Research Database (Denmark)

    Nilsson, Stina; Kaniowska, Dorota; Brakebusch, Cord


    was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate......In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1...... integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing...

  10. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.


    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J.; Rogers, N C; J. M. Smith; Stark, G R; Gardiner, K.; Mogensen, K E


    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two ...

  11. Expression of BK Ca channels and the modulatory beta-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander


    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  12. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W


    changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements......Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational...... from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  13. The human [gamma]-aminobutyric acid receptor subunit [beta]3 and [alpha]5 gene cluster in chromosome 15q11-q13 is rich in highly polymorphic (CA)[sub n] repeats

    Energy Technology Data Exchange (ETDEWEB)

    Glatt, K.; Lalande, M. (Howard Hughes Medical Institute, Boston, MA (United States)); Sinnett, D. (Harvard Medical School, Boston, MA (United States))


    The [gamma]-aminobutyric acid (GABA[sub A]) receptor [beta]33 (GABRB3) and [alpha]5 (GABRA5) subunit genes have been localized to the Angelman and Prader-Willi syndrome region of chromosome 15q11-q13. GABRB3, which encompasses 250 kb, is located 100 kb proximal of GABRA5, with the two genes arranged in head-to-head transcriptional orientation. In screening 135 kb of cloned DNA within a 260-kb interval extending from within GABRB3 to the 5[prime] end of GABRA5, 10 new (CA), repeats have been identified. Five of these have been analyzed in detail and found to be highly polymorphic, with the polymorphism information content (PIC) ranging from 0.7 to 0.85 and with heterozygosities of 67 to 94%. In the clones from GABRB3/GABRA5 region, therefore, the frequency of (CA)[sub n] with PICs [ge] 0.7 is 1 per 27 kb. Previous estimates of the density of (CA)[sub n] with PICs [ge] 0.7 in the human genome have been approximately 10-fold lower. The GABRB3/GABRA5 region appears, therefore, to be enriched for highly informative (CA)[sub n]. This set of closely spaced, short tandem repeat polymorphisms will be useful in the molecular analyses of Prader-Willi and Angelman syndromes and in high-resolution studies of genetic recombination within this region. 21 refs., 2 figs., 1 tab.

  14. Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster. (United States)

    Lutfalla, G; Holland, S J; Cinato, E; Monneron, D; Reboul, J; Rogers, N C; Smith, J M; Stark, G R; Gardiner, K; Mogensen, K E


    The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.

  15. Substitution of isoleucine for methionine at position 1153 in the beta-subunit of the human insulin receptor. A mutation that impairs receptor tyrosine kinase activity, receptor endocytosis, and insulin action. (United States)

    Cama, A; Quon, M J; de la Luz Sierra, M; Taylor, S I


    The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase. The receptor tyrosine kinase is stimulated by insulin binding to the extracellular domain of the receptor. Previously, we have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Ile for Met1153 in the tyrosine kinase domain of the receptor near the cluster of the three major autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163). In this investigation, the Ile1153 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. The mutation impairs receptor tyrosine kinase activity and also inhibits the ability of insulin to stimulate 2-deoxyglucose uptake and thymidine incorporation. These data support the hypothesis that the receptor tyrosine activity plays a necessary role in the ability of the receptor to mediate insulin action in vivo. Furthermore, expression of the Ile1153 mutant receptor exerted a dominant negative effect to inhibit the ability of endogenous murine receptors for insulin and insulin-like growth factor I to mediate their actions upon the cell. This observation is consistent with previous suggestions that mutant receptors dimerize with wild type receptors, thereby creating hybrid molecules which lack biological activity. The dominant negative effect of the mutant receptor may explain the dominant mode of inheritance of insulin resistance caused by the Ile1153 mutation. Finally, the mutation inhibits the ability of insulin to stimulate receptor endocytosis. This may explain the normal number of insulin receptors on the surface of the patient's cells in vivo. Despite the presence of markedly elevated levels of insulin in the patient's plasma, the receptors were resistant to down-regulation.

  16. Mapping of the {alpha}{sub 4} subunit gene (GABRA4) to human chromosome 4 defines an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 1} gene cluster: Further evidence that modern GABA{sub a} receptor gene clusters are derived from an ancestral cluster

    Energy Technology Data Exchange (ETDEWEB)

    McLean, P.J.; Farb, D.H.; Russek, S.J. [Boston Univ. School of Medicine, MA (United States)] [and others


    We demonstrated previously that an {alpha}{sub 1}-{beta}{sub 2}-{gamma}{sub 2} gene cluster of the {gamma}-aminobutyric acid (GABA{sub A}) receptor is located on human chromosome 5q34-q35 and that an ancestral {alpha}-{beta}-{gamma} gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the {alpha}{sub 4} gene (GABRA4) maps to human chromosome 4p14-q12, defining a cluster comprising the {alpha}{sub 2}, {alpha}{sub 4}, {beta}{sub 1}, and {gamma}{sub 1} genes. The existence of an {alpha}{sub 2}-{alpha}{sub 4}-{beta}{sub 1}-{gamma}{sub 2} cluster on chromosome 4 and an {alpha}{sub 1}-{alpha}{sub 6}-{beta}{sub 2}-{gamma}{sub 2} cluster on chromosome 5 provides further evidence that the number of ancestral GABA{sub A} receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the {alpha} gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of a subunit should be located on human chromosome 15q11-q13 within an {alpha}{sub 5}-{alpha}{sub x}-{beta}{sub 3}-{gamma}{sub 3} gene cluster at the locus for Angelman and Prader-Willi syndromes. 34 refs., 6 figs., 1 tab.

  17. Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus. (United States)

    Gullick, W J; Lindstrom, J M


    The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

  18. Stoichiometry of δ subunit containing GABA(A) receptors. (United States)

    Patel, B; Mortensen, M; Smart, T G


    Although the stoichiometry of the major synaptic αβγ subunit-containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach. Using site-directed mutagenesis, we inserted a highly characterized 9' serine to leucine mutation into the second transmembrane (M2) region of α4, β3 and δ subunits that increases receptor sensitivity to GABA. Whole-cell, GABA-activated currents were recorded from HEK-293 cells co-expressing different combinations of wild-type (WT) and/or mutant α4(L297S), β3(L284S) and δ(L288S) subunits. Recombinant receptors containing one or more mutant subunits showed increased GABA sensitivity relative to WT receptors by approximately fourfold, independent of the subunit class (α, β or δ) carrying the mutation. GABA dose-response curves of cells co-expressing WT subunits with their respective L9'S mutants exhibited multiple components, with the number of discernible components enabling a subunit stoichiometry of 2α, 2β and 1δ to be deduced for α4β3δ receptors. Varying the cDNA transfection ratio by 10-fold had no significant effect on the number of incorporated δ subunits. Subunit stoichiometry is an important determinant of GABAA receptor function and pharmacology, and δ subunit-containing receptors are important mediators of tonic inhibition in several brain regions. Here we demonstrate a preferred subunit stoichiometry for α4β3δ receptors of 2α, 2β and 1δ. © 2013 The British Pharmacological Society.

  19. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis. (United States)

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G


    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  20. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits. (United States)

    Wong, Kelly A; Lodish, Harvey F


    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  1. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls (United States)

    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)


    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

  2. Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells. (United States)

    Zhang, X L; Danto, S I; Borok, Z; Eber, J T; Martín-Vasallo, P; Lubman, R L


    The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be

  3. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G


    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses...

  4. Functional characterization of Kv channel beta-subunits from rat brain. (United States)

    Heinemann, S H; Rettig, J; Graack, H R; Pongs, O


    1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

  5. Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels. (United States)

    Aman, Teresa K; Grieco-Calub, Tina M; Chen, Chunling; Rusconi, Raffaella; Slat, Emily A; Isom, Lori L; Raman, Indira M


    The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.

  6. Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist

    DEFF Research Database (Denmark)

    Elster, L; Hansen, Gert Helge; Belhage, B;


    or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization...... composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist....

  7. A-Raf kinase is a new interacting partner of protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G


    In a search for protein kinase CK2 beta subunit binding proteins using the two-hybrid system, more than 1000 positive clones were isolated. Beside clones for the alpha' and beta subunit of CK2, there were clones coding for a so far unknown protein, whose partial cDNA sequence was already deposite...

  8. Inhibin subunits, follistatin and activin receptors in the human teratocarcinoma cell line Tera-2. (United States)

    de Jong, F H; de Winter, J P; Wesseling, J G; Timmerman, M A; van Genesen, S; van den Eijnden-van Raaij, A J; van Zoelen, E J


    The expression of mRNAs for inhibin subunits was studied in the human teratocarcinoma cell line Tera-2 clone 13 before and after differentiation with retinoic acid (RA). Both alpha- and beta B-subunits of inhibin were expressed. Subsequently, inhibin bio- and immunoactivity in the conditioned media of the Tera-2 cells were determined by studying the release of follicle-stimulating hormone from rat pituitary cells, by immunoassay and by immunoprecipitation using inhibin alpha- and beta B-subunit specific antibodies. Strikingly dissimilar high bio- and low immuno-activities were found. The ensuing hypothesis that the high bioactivity might be due to the presence of the activin-binding protein follistatin was confirmed by immunoprecipitation of 34 and 37 kDa labelled proteins, using a polyclonal anti-follistatin antiserum after culture of the Tera-2 cells with [35S]-methionine. Furthermore, expression of activin receptor types II and IIB mRNA was found in the cells. Addition of 5 microM RA to monolayer cultures of Tera-2 cells resulted in differentiation to flat endoderm-like cells and caused a slight suppression of the expression of the mRNA encoding the inhibin alpha- and beta B-subunits. The expression of follistatin and activin receptor type IIB was strongly suppressed, whereas the expression of the activin receptor type II was not affected. We conclude that Tera-2 cells secrete follistatin and express inhibin subunits and activin receptors differentially during RA-induced differentiation. The role of the decreased expression of follistatin and activin receptor IIB mRNA after addition of RA in the mechanism of RA-induced differentiation remains to be elucidated.

  9. The nicotinic receptor in the rat pineal gland is an alpha3beta4 subtype. (United States)

    Hernandez, Susan C; Vicini, Stefano; Xiao, Yingxian; Dávila-García, Martha I; Yasuda, Robert P; Wolfe, Barry B; Kellar, Kenneth J


    The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of approximately 100 pM to nAChRs in the pineal gland, and the density of these sites is approximately 5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at alpha3beta4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous alpha3beta4 nAChRs. Measurements of mRNA indicated the presence of transcripts for alpha3, beta2, and beta4 nAChR subunits but not those for alpha2, alpha4, alpha5, alpha6, alpha7, or beta3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained alpha3 and beta4 subunits associated in one complex. The beta2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the alpha3beta4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.

  10. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    Full Text Available BACKGROUND: The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  11. Shared receptor components but distinct complexes for alpha and beta interferons. (United States)

    Lewerenz, M; Mogensen, K E; Uzé, G


    The type I interferon family includes 13 alpha, one omega and one beta subtypes recognized by a complex containing the receptor subunits ifnar1 and ifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1. To investigate the reported differences in the way that alpha and beta interferons signal through the receptor, we introduced alanine-substitutions in the ifnar2 extracellular domain, and expressed the mutants in U5A cells, lacking endogenous ifnar2. A selection, designed to recover mutants that responded preferentially to alpha or beta interferon yielded three groups: I, neutral; II, sensitive to alpha interferon, partially resistant to beta interferon; III, resistant to alpha interferon, partially sensitive to beta interferon. A mutant clone, TMK, fully resistant to alpha interferon with good sensitivity to beta interferon, was characterized in detail and compared with U5A cells complemented with wild-type ifnar2 and also with Tyk2-deficient 11.1 cells, which exhibit a similar alpha-unresponsive phenotype with a partial beta interferon response. Using anti-receptor antibodies and mutant forms of beta interferon, three distinct modes of ligand interaction could be discerned: (i) alpha interferon with ifnar1 and ifnar2; (ii) beta interferon with ifnar1 and ifnar2; (iii) beta interferon with ifnar2 alone. We conclude that alpha and beta interferons signal differently through their receptors because the two ligand subtypes interact with the receptor subunits ifnar 1 and ifnar2 in entirely different ways.

  12. Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively



    The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony- stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice...

  13. Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures. (United States)

    Zheng, T M; Caruncho, H J; Zhu, W J; Vicini, S; Ikonomovic, S; Grayson, D R; Costa, E


    Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

  14. GABAB(1) receptor subunit isoforms differentially regulate stress resilience (United States)

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


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

  15. Early diagnosis of sepsis using serum hemoglobin subunit Beta. (United States)

    Yoo, Hayoung; Ku, Sae-Kwang; Kim, Shin-Woo; Bae, Jong-Sup


    The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBβ) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBβ in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBβ in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBβ were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBβ levels when compared to patients with severe sepsis. Furthermore, the HBβ levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBβ blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBβ can be used as a biomarker to determine the severity of sepsis.

  16. The alpha3 laminin subunit, alpha6beta4 and alpha3beta1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

    DEFF Research Database (Denmark)

    Goldfinger, L E; Hopkinson, S B; deHart, G W


    Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithe......-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results....... in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix...... the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function...

  17. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Johansson, Helle Wulf; Hay-Schmidt, Anders


    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expresse...

  18. Beta adrenergic receptors in human cavernous tissue

    Energy Technology Data Exchange (ETDEWEB)

    Dhabuwala, C.B.; Ramakrishna, C.V.; Anderson, G.F.


    Beta adrenergic receptor binding was performed with /sup 125/I iodocyanopindolol on human cavernous tissue membrane fractions from normal tissue and transsexual procedures obtained postoperatively, as well as from postmortem sources. Isotherm binding studies on normal fresh tissues indicated that the receptor density was 9.1 fmoles/mg. with a KD of 23 pM. Tissue stored at room temperature for 4 to 6 hours, then at 4C in saline solution for 19 to 20 hours before freezing showed no significant changes in receptor density or affinity, and provided evidence for the stability of postmortem tissue obtained within the same time period. Beta receptor density of 2 cavernous preparations from transsexual procedures was not significantly different from normal control tissues, and showed that high concentrations of estrogen received by these patients had no effect on beta adrenergic receptor density. Displacement of /sup 125/iodocyanopindolol by 5 beta adrenergic agents demonstrated that 1-propranolol had the greatest affinity followed by ICI 118,551, zinterol, metoprolol and practolol. When the results of these displacement studies were subjected to Scatfit, non- linear regression line analysis, a single binding site was described. Based on the relative potency of the selective beta adrenergic agents it appears that these receptors were of the beta 2 subtype.

  19. Subunit structure of the acetylcholine receptor from Electrophorus electricus. (United States)

    Conti-Tronconi, B M; Hunkapiller, M W; Lindstrom, J M; Raftery, M A


    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.

  20. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O;


    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...

  1. Misfolded amyloid ion channels present mobile beta-sheet subunits in contrast to conventional ion channels. (United States)

    Jang, Hyunbum; Arce, Fernando Teran; Capone, Ricardo; Ramachandran, Srinivasan; Lal, Ratnesh; Nussinov, Ruth


    In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Abeta channels consist of the solid-state NMR-based U-shaped beta-strand-turn-beta-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that beta-sheet channels break into loosely associated mobile beta-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for beta-sheet channels formed by cytolytic PG-1 beta-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by beta-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting alpha-helices that robustly prevent ion leakage, rather than hydrogen-bonded beta-strands. The simulations suggest why conventional gated channels evolved to consist of interacting alpha-helices rather than hydrogen-bonded beta-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.

  2. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons. (United States)

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


    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.

  3. Association of single nucleotide polymorphisms of nicotinic acetylcholine receptor subunits with cervical neoplasia (United States)

    Calleja-Macias, Itzel; Osann, Kathryn; Remedios-Chan, Mariana; Barrera-Saldana, Hugo A.; Illades-Aguiar, Berenice; Anton-Culver, Hoda; Chikova, Anna K.; Grando, Sergei A.; Bernard, Hans-Ulrich


    Aims Cholinergic signaling, particularly in response to non-physiological ligands like nicotine, stimulates carcinogenesis of a variety of tissue types including epithelia of the cervix uteri. Cholinergic signaling is mediated by nicotinic acetylcholine receptors (nAChRs), which are pentamers formed by subsets of 16 nAChR subunits. Recent literature suggests that single nucleotide polymorphisms (SNPs) of some of these subunits, notably alpha5, are risk factors for developing lung cancer in smokers as well as in non-smokers. Main methods We have studied the prevalence of four SNPs in the alpha5, alpha9, and beta1 subunits, which are expressed in cervical cells, in 456 patients with cervical cancers, precursor lesions, and healthy controls from two cohorts in Mexico. Key findings A SNP in the alpha9 subunit, the G allele of rs10009228 (alpha9, A>G) shows a significant trend in the combined cohort, indicating that this allele constitutes a risk factor for neoplastic progression. The A allele of the SNP rs16969968 (alpha5, G>A), which correlates with the development of lung cancer, shows a non-significant trend to be associated with cervical lesions. Two other SNPs, rs55633891 (alpha9, C>T) and rs17856697 (beta1, A>G), did not exhibit a significant trend. Significance Our study points to a potential risk factor of cervical carcinogenesis with importance for DNA diagnosis and as a target for intervention. PMID:22406075

  4. Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies. (United States)

    Ikeda, M; Kadowaki, H; Ikeda, H; Moritani, C; Kanazawa, H


    The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

  5. Molecular investigations of BK(Ca) channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander


    arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BK(Ca) channel alpha-subunit and beta-subunit proteins. The BK(Ca) channel alpha-subunit RNA and protein distribution patterns were......-PCR in porcine basilar and middle cerebral arteries. However, at the protein level, only, the beta1-subunit protein was found by western blotting....

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (UWA); (St. Vincent); (Queensland)


    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.

  7. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A


    an altered sedimentation coefficient. The holoenzymes reconstituted with substituted mutants beta A 55,57, beta A55-57, and, to a lesser extent, beta A 59-61, beta A63,64, and beta A5,6 display a basal activity which is higher (up to 4-fold) than that of the wild type holoenzyme.(ABSTRACT TRUNCATED AT 250......Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C......-terminal part (mutants beta delta 209-215, beta delta 194-215, beta delta 181-215, beta delta 171-215, beta delta 150-215) or have undergone Ala substitutions for the acidic and basic residues which are concentrated in the sequences 55-70 and 171-180, respectively. All these mutants have been examined...

  8. Subunit structure of the acetylcholine receptor from Electrophorus electricus.


    Conti-Tronconi, B M; Hunkapiller, M. W.; Lindstrom,J.M.; Raftery, M A


    The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Compa...

  9. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A


    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  10. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes. (United States)

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G


    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  11. Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin. (United States)

    Acevedo, H F; Kellen, J A


    A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.

  12. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4. (United States)

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung


    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  13. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes. (United States)

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A


    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  14. Characterization of the subunit structure of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. (United States)

    Gennick, S E; Thomas, C G; Nayfeh, S N


    incremental cross-linking of TSH alpha-beta dimer to receptor subunits; and 2) the TSH receptor may be an oligomer composed of three different subunits, 31,000, 17,000, and 63,000, of which only the 31,000 subunit binds TSH.

  15. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G


    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  16. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G


    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  17. cDNA cloning of the beta subunit of teleost thyrotropin.


    Ito, M.; Koide, Y.; Takamatsu, N; Kawauchi, H.; Shiba, T.


    cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human fo...

  18. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B;


    Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition......, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin...

  19. E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit. (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E


    Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

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

    Directory of Open Access Journals (Sweden)

    Simon C. Haering


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

  1. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors


    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.


    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors co...

  2. Suggestive association between the C825T polymorphism of the G-protein beta3 subunit gene (GNB3) and clinical improvement with antipsychotics in schizophrenia. (United States)

    Müller, Daniel J; De Luca, Vincenzo; Sicard, Tricia; King, Nicole; Hwang, Rudi; Volavka, Jan; Czobor, Pal; Sheitman, Brian B; Lindenmayer, Jean-Pierre; Citrome, Leslie; McEvoy, Joseph P; Lieberman, Jeffrey A; Meltzer, Herbert Y; Kennedy, James L


    G-proteins are composed of alpha, beta and gamma subunits. Once activated, these subunits play a major role in the conversion of external receptor activation into intracellular signals. The functional C825T polymorphism of the beta3 subunit gene (GNB3) has recently been shown to modulate antidepressant response, with the T-allele conferring an increased signaling and being associated with favorable antidepressant response. We hypothesized that this polymorphism may be associated with response to antipsychotics in a population of 145 chronic schizophrenic patients deriving from two study-samples and being mainly treated with clozapine for up to 6 months. Overall, the C/C genotype was significantly associated with relative clinical improvement as measured by Brief Psychiatric Rating Scale (BPRS) change scores after 6 and 12 weeks (ppoint to the role of intracellular mechanisms in antipsychotic response.

  3. Activin receptor subunits in normal and dysfunctional adult human testis

    DEFF Research Database (Denmark)

    Dias, V.; Meachem, S.; Rajpert-De, Meyts E.


    , carcinoma in situ (CIS), seminoma, non-seminoma and gonadotropin-deprived human testis. ActRIIA mRNA was localized by in situ hybridization. RESULTS: ALK2, ALK4 and ActRIIB proteins were observed in Sertoli cells, spermatogonia and some spermatocytes within normal and gonadotropin-suppressed adult human...... testis; all three receptor subunits were also detected in CIS, seminoma and non-seminoma cells. ActRIIA immunoreactivity was faint to absent in the normal testis and in CIS and non-seminoma cells, whereas some seminoma cells displayed a strong signal. Also in contrast to the normal testis, a majority...

  4. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Directory of Open Access Journals (Sweden)

    Ariel Avila


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

  5. Cortisone Dissociates the Shaker Family K Channels from their Beta Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Y.; Weng, J; Kabaleeswaran, V; Li, H; Cao, Y; Bholse, R; Zhou, M


    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with {Beta} subunits (Kv{Beta}s), and certain Kv{Beta}s, for example Kv{Beta}1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kv{Beta}1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kv{Beta}1. A crystal structure of the K{Beta}v-cortisone complex was solved to 1.82-{angstrom}resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kv{Beta}. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.

  6. Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear. (United States)

    Aridon, Paolo; Marini, Carla; Di Resta, Chiara; Brilli, Elisa; De Fusco, Maurizio; Politi, Fausta; Parrini, Elena; Manfredi, Irene; Pisano, Tiziana; Pruna, Dario; Curia, Giulia; Cianchetti, Carlo; Pasqualetti, Massimo; Becchetti, Andrea; Guerrini, Renzo; Casari, Giorgio


    Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.

  7. Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry. (United States)

    Kultas-Ilinsky, K; Leontiev, V; Whiting, P J


    In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and

  8. Cloning and gene expression of a cDNA for the chicken follicle-stimulating hormone (FSH)-beta-subunit. (United States)

    Shen, San-Tai; Yu, John Yuh-Lin


    Follicle-stimulating hormone (FSH) is a member of pituitary glycoprotein hormones that are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSH-beta in avian species. For better understanding of the phylogenic diversity and evolution of FSH molecule, we have isolated and sequenced the complete complementary DNA (cDNA) encoding chicken FSH-beta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned chicken FSH-beta cDNA consists of 2457-bp nucleotides, including 44-bp nucleotides of the 5'-untranslated region (UTR), 396 bp of the open reading frame, and an extraordinarily long 3'-UTR of 2001-bp nucleotides followed by a poly(A)((16)) tail. It encodes a 131-amino-acid precursor molecule of FSH-beta-subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the chicken FSH-beta-subunit. Four proline residues, presumably responsible for changing the backbone direction of protein structure, are conserved in chicken FSH-beta-subunit as well. The nucleotide sequence of chicken FSH-beta cDNA shows high homology with quail FSH-beta cDNA, 97% homology in the open reading frame, and 85% homology in the 3'-UTR. The deduced amino acid sequence of chicken FSH-beta-subunit shows a remarkable similarity to other avian FSH-beta-subunits, 98% homology with quail, and 93% homology with ostrich, whereas a lower similarity (66 to 70%) is noted when compared with mammalian FSH-beta-subunits. By contrast, when comparing with the beta-subunits of chicken luteinizing hormone and thyroid-stimulating hormone, the homologies are as low as 37 and 40%, respectively. FSH-beta mRNA was only expressed in pituitary gland out of various

  9. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;


    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  10. Distinct Subunit Domains Govern Synaptic Stability and Specificity of the Kainate Receptor

    Directory of Open Access Journals (Sweden)

    Christoph Straub


    Full Text Available Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.

  11. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P


    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  12. Estrogen dissociates Tau and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit in postischemic hippocampus. (United States)

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


    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.

  13. Inhibition of voltage-gated calcium channels by sequestration of beta subunits. (United States)

    Cuchillo-Ibañez, Inmaculada; Aldea, Marcos; Brocard, Jacques; Albillos, Almudena; Weiss, Norbert; Garcia, Antonio G; De Waard, Michel


    The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.

  14. Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting (United States)

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


    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

  15. Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit. (United States)

    Senior, A E; al-Shawi, M K


    Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.

  16. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil. (United States)

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan


    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

  17. Monitoring of beta-receptor sensitivity in cardiac surgery

    DEFF Research Database (Denmark)

    Yndgaard, S; Lippert, F K; Bigler, Dennis Richard


    To determine the repeatability of the hemodynamic response to repeated isoproterenol challenge doses to validate the standardized isoproterenol sensitivity test as an index of cardiovascular beta-receptor function.......To determine the repeatability of the hemodynamic response to repeated isoproterenol challenge doses to validate the standardized isoproterenol sensitivity test as an index of cardiovascular beta-receptor function....

  18. Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey. (United States)

    Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L


    The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

  19. Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors. (United States)

    Mercik, Katarzyna; Pytel, Maria; Cherubini, Enrico; Mozrzymas, Jerzy W


    Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

  20. Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor. (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N


    The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains

  1. Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia


    Glausier, Jill R; Lewis, David A.


    Levels of messenger RNA (mRNA) for the α1 subunit of the GABAA receptor, which is present in 60% of cortical GABAA receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABAA α1 subunit mRNA expression...

  2. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B


    , comparison of the far-UV CD spectrum of reconstituted CK-2 with the spectra of the subunits indicates that conformational changes occur in the backbone region upon association. Such changes may explain the increased enzyme activity of the holoenzyme relative to that of the alpha subunit itself. In contrast......, presumably by its binding to the polylysine stretch at amino acid positions 74-77. Heat denaturation experiments (25-90 degrees C) support the notion that heparin may provide a local protective function. A similar but much larger effect was also observed in the presence of the beta subunit only, which...... supports previous suggestions of a protective function for this subunit. These results indicate that the protection provided by the beta subunit and the increased enzyme activity of the holoenzyme may arise, in part, from a stabilization of the conformation of the enzyme complex and an increase in alpha...

  3. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)


    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  4. Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4

    DEFF Research Database (Denmark)

    Richter, Wito; Day, Peter; Agrawal, Rani


    Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing...

  5. Directed mutagenesis of the dicyclohexylcarbodiimide-reactive carboxyl residues in beta-subunit of F1-ATPase of Escherichia coli. (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E


    Previous studies in which dicyclohexylcarbodiimide (DCCD) was used to inactivate F1-ATPase enzymes have suggested that two glutamate residues in the beta-subunit are essential for catalysis. In the Escherichia coli F1-ATPase, these are residues beta-Glu-181 and beta-Glu-192. Oligonucleotide-directed mutagenesis was used to change these residues to beta-Gln-181 and beta-Gln-192. The beta-Gln-181 mutation produced strong impairment of oxidative phosphorylation in vivo and also of ATPase and ATP-driven proton-pumping activities in membranes assayed in vitro. A low level of each activity was detected and an F1-ATPase appeared to be assembled normally on the membranes. Therefore, it is suggested that the carboxyl side chain at residue beta-181 is important, although not absolutely required, for catalysis in both directions on E. coli F1-ATPase. The beta-Gln-192 mutation produced partial inhibition of oxidative phosphorylation in vivo and membrane ATPase activity was reduced by 78%. These results contrast with the complete or near-complete inactivation seen when E. coli F1-ATPase is reacted with DCCD and imply that DCCD-inactivation is attributable more to the attachment of the bulky DCCD molecule than to the derivatization of the carboxyl side chain of residue beta-Glu-192. M. Ohtsubo and colleagues (Biochem. Biophys. Res. Commun. (1987) 146, 705-710) described mutagenesis of the F1-beta-subunit of thermophilic bacterium PS3. Mutations (Glu----Gln) of the residues homologous to Glu-181 and Glu-192 of E. coli F1-beta-subunit both caused total inhibition of ATPase activity. Therefore, there was a marked difference in results obtained when the same residues were modified in the PS3 and E. coli F1-beta-subunits.

  6. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus. (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M


    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  7. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

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


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

  8. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores. (United States)

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A


    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  9. Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis. (United States)

    van Nierop, Pim; Bertrand, Sonia; Munno, David W; Gouwenberg, Yvonne; van Minnen, Jan; Spafford, J David; Syed, Naweed I; Bertrand, Daniel; Smit, August B


    We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

  10. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli. (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y


    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  11. Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits. (United States)

    Isailovic, Dragan; Sultana, Ishrat; Phillips, Gregory J; Yeung, Edward S


    Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

  12. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis;


    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep...

  13. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo


    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

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

    Directory of Open Access Journals (Sweden)

    Morgane Riou

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

  15. Expression of transforming growth factor-beta (TGF-beta) receptors, TGF-beta 1 and TGF-beta 2 production and autocrine growth control in osteosarcoma cells. (United States)

    Kloen, P; Jennings, C L; Gebhardt, M C; Springfield, D S; Mankin, H J


    Transforming growth factor-beta (TGF-beta) is a polypeptide with multiple physiological functions. Isoforms of this growth factor have important roles in control of the cell cycle, in regulation of cell-cell interactions and in growth and development. Malignant transformation has been shown to be associated with increased expression of TGF-beta. Since bone is the largest storage site and producer of TGF-beta, we speculated on the existence of an autocrine mechanism in osteosarcoma, a malignant bone tumor. Expression of TGF-beta cell surface receptors, effects on growth of TGF-beta and TGF-beta antibodies and production of 2 TGF-beta isoforms were studied in a panel of 7 osteosarcoma cell lines. In contrast to most previous reports on the effects of TGF-beta on osteosarcoma cell growth, we found a mitogenic effect of TGF-beta 1 in 4 of 7 osteosarcoma cell lines. Receptor profiles for TGF-beta were aberrant in 5 of the 7 cell lines tested, and production of TGF-beta 1 and TGF-beta 2 varied among cell lines. Addition of anti-TGF-beta antagonized the effects of endogenous TGF-beta. Our results suggest a potential role of TGF-beta in autocrine growth control of osteosarcoma cells.

  16. Phenylthiophenecarboxamide antagonists of the olfactory receptor co-receptor subunit from a mosquito.

    Directory of Open Access Journals (Sweden)

    Sisi Chen

    Full Text Available Insects detect environmental chemicals using chemosensory receptors, such as the ORs, a family of odorant-gated ion channels. Insect ORs are multimeric complexes of unknown stoichiometry, formed by a common subunit (the odorant receptor co-receptor subunit, Orco and one of many variable subunits that confer odorant specificity. The recent discovery of Orco directed ligands, including both agonists and antagonists, suggests Orco as a promising target for chemical control of insects. In addition to competitively inhibiting OR activation by Orco agonists, several Orco antagonists have been shown to act through a non-competitive mechanism to inhibit OR activation by odorants. We previously identified a series of Orco antagonists, including N-(4-ethylphenyl-2-thiophenecarboxamide (OX1a, previously referred to as OLC20. Here, we explore the chemical space around the OX1a structure to identify more potent Orco antagonists. Cqui\\Orco+Cqui\\Or21, an OR from Culex quinquefasciatus (the Southern House Mosquito that responds to 3-methylindole (skatole and is thought to mediate oviposition behavior, was expressed in Xenopus oocytes and receptor function assayed by two-electrode voltage clamp electrophysiology. 22 structural analogs of OX1a were screened for antagonism of OR activation by an Orco agonist. By varying the moieties decorating the phenyl and thiophene rings, and altering the distance between the rings, we were able to identify antagonists with improved potency. Detailed examination of three of these compounds (N-mesityl-2-thiophenecarboxamide, N-(4-methylbenzyl-2-thiophenecarboxamide and N-(2-ethylphenyl-3-(2-thienyl-2-propenamide demonstrated competitive inhibition of receptor activation by an Orco agonist and non-competitive inhibition of receptor activation by an odorant. The ability to inhibit OR activation by odorants may be a general property of this class of Orco antagonist, suggesting that odorant mediated behaviors can be manipulated

  17. Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines beta100 and beta110 in porcine luteinizing hormone: metastability and hypothetical isomerization of the two disulfide bridges of its beta-subunit seatbelt. (United States)

    Belghazi, Maya; Klett, Danièle; Cahoreau, Claire; Combarnous, Yves


    Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, alpha and beta, that are non-covalently associated. The heterodimer is stabilized by a region of the beta-subunit called the "seatbelt" because it wraps around the alpha-subunit and it is fastened by a disulfide bridge between cysteines beta26 and beta110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines beta26 and beta110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines beta100 and beta110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysbeta93 and beta26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges beta26-beta110 and beta93-beta100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.

  18. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O


    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  19. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast (United States)

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


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

  20. Interaction of Plasminogen Activator Inhibitor-2 and Proteasome Subunit, Beta Type 1

    Institute of Scientific and Technical Information of China (English)

    JingFAN; Yu-QingZHANG; PingLI; MinHOU; LiTAN; XiaWANG; Yun-SongZHU


    The apoptosis protection by plasminogen activator inhibitor-2(PAI-2) is dependent on a 33 amino acid fragment between helix C and D of PAI-2 which is probably due to the interaction of PAI-2 with unknown intracellular proteins. In this study, we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cell cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone encoding 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMβ1) which plays important roles in NF-κB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMβ1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interation with PSMβ1.

  1. Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta

    DEFF Research Database (Denmark)

    Boldyreff, B; James, P; Staudenmann, W;


    Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced...

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

    DEFF Research Database (Denmark)

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


    -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...

  3. Function of the integrin alpha 6 beta 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor

    DEFF Research Database (Denmark)

    Shaw, L M; Chao, C; Wewer, U M;


    The involvement of the alpha 6 beta a integrin, a laminin receptor, in breast carcinoma progression needs to be addressed rigorously. We report that a human breast carcinoma cell line, MDA-MB-435, known to be highly invasive and metastatic, expresses three potential integrin laminin receptors...... function that involved expression of a cytoplasmic domain deletion mutant of the beta 4 integrin subunit by cDNA transfection. Stable transfectants of MDA-MB-435 cells that expressed this mutant beta 4 subunit were inhibited dramatically in their ability to adhere and migrate on laminin matrices......, and their capacity to invade Matrigel was reduced significantly. These findings support the hypothesis that alpha 6 beta 1 is important for breast cancer progression. Moreover, this approach is a powerful method that should be useful in assessing the role of alpha 6 beta 1 in other cells....

  4. Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene. (United States)

    Medeiros-Neto, G; de Lacerda, L; Wondisford, F E


    Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

  5. The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme. (United States)

    Tomer, G; Reuven, N B; Livneh, Z


    The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

  6. Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits. (United States)

    Takagi, J; Beglova, N; Yalamanchili, P; Blacklow, S C; Springer, T A


    Integrin beta subunits contain four cysteine-rich repeats in a long extracellular stalk that connects the headpiece to the membrane. Most mAbs to integrin activation epitopes map to these repeats, and they are important in propagating conformational signals from the membrane/cytosol to the ligand-binding headpiece. Sequence analysis of a protein containing only 10 integrin-like, cysteine-rich repeats suggests that these repeats start one cysteine earlier than previously reported. By using the new repeat boundaries, statistically significant sequence homology to epidermal growth factor-like domains is found, and a disulfide bond connectivity of the eight cysteines is predicted that differs in three of four disulfides from a previous prediction of epidermal growth factor-like modules [Berg, R. W., Leung, E., Gough, S., Morris, C., Yao, W.-P., Wang, S.-x., Ni, J. & Krissansen, G. W. (1999) Genomics 56, 169-178]. N-terminally truncated beta2 integrin stalk fragments were well expressed and secreted from 293 T cells when they began at repeat boundaries but not when they began one cysteine earlier or later. Furthermore, peptides that correspond to module 3 or modules 2 + 3 were expressed in bacteria and refolded. The module 2 + 3 fragment was as reactive with three mAbs to activation epitopes as a beta2 fragment expressed in eukaryotic cells, indicating a native fold. Only one residue intervenes between the last cysteine of one module and the first cysteine of the next. This arrangement is consistent with a tight intermodule connection, a prerequisite for signal propagation from the membrane to the ligand binding headpiece.

  7. [Beta 3 adrenergic receptor polymorphism and obesity]. (United States)

    Yoshida, T; Umekawa, T


    The beta 3-adrenoceptor plays a significant role in the control of lipolysis and thermogenesis in the brown adipose tissue of rodents and humans. In human beta 3-adrenoceptor, a Trp to Arg replacement has recently been discovered. This change which occurs at position 64, in the first coding exon, has been correlated with increased weight gain, difficulty in losing weight, insulin resistance syndrome, and worsened diabetic situation. Higher percentages of this mutation are observed in Pima Indians (over 30%) and Japanese (20%). The possible functional mechanism of Trp54Arg is reported using human HEK293 cell line stably expressing the wild type and the [Arg64] beta 3-adrenoceptor type. Beta 3-adrenoceptor agonists available for humans are been also developing. In this paper we describe these points up-to-date.

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

    DEFF Research Database (Denmark)

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


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

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

    DEFF Research Database (Denmark)

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


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

  10. Α2 GABAA receptor sub-units in the ventral hippocampus and α5 GABAA receptor sub-units in the dorsal hippocampus mediate anxiety and fear memory. (United States)

    McEown, K; Treit, D


    Temporary neuronal inactivation of the ventral hippocampus with the GABAA agonist muscimol suppresses unconditioned fear behavior (anxiety) but inactivation of the dorsal hippocampus does not. On the other hand, inactivating the dorsal hippocampus disrupts fear memory, while inactivating the ventral hippocampus does not. Here we investigate the roles of hippocampal GABAA receptor sub-units in mediating these anxiolytic and amnesic effects of GABAA receptor agonists. We microinfused TPA023 (α2 agonist) or TB-21007 (inverse α5 agonist) into the dorsal or ventral hippocampus prior to testing rats in two animal models of anxiety: the elevated plus-maze and shock-probe burying test. Twenty-four hours later rats were re-tested in the shock-probe chamber with a non-electrified probe to assess their memory of the initial shock-probe experience (i.e., fear memory). We found that TPA023 was anxiolytic in the plus-maze and shock-probe burying tests when microinfused into the ventral hippocampus. However, TPA023 did not affect anxiety-related behavior when infused into the dorsal hippocampus. Conversely, we found that the α5 sub-unit inverse agonist TB-21007 impaired rats' memory of the initial shock-probe experience when infused into the dorsal hippocampus, but not when infused into the ventral hippocampus. This double dissociation suggests that α2 GABAA receptor sub-units in the ventral hippocampus mediate unconditioned fear or anxiety, while α5 GABAA receptor sub-units in the dorsal hippocampus mediate conditioned fear memory.

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

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

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

  12. GABAA receptor β3 subunit expression regulates tonic current in developing striatopallidal medium spiny neurons

    Directory of Open Access Journals (Sweden)

    Megan eJanssen


    Full Text Available The striatum is a key structure for movement control, but the mechanisms that dictate the output of distinct subpopulations of medium spiny projection neurons (MSNs, striatonigral projecting and dopamine D1 receptor- (D1+ or striatopallidal projecting and dopamine D2 receptor- (D2+ expressing neurons, remains poorly understood. GABA-mediated tonic inhibition largely controls neuronal excitability and action potential firing rates, and we previously suggested with pharmacological analysis that the GABAA receptor β3 subunit plays a large role in the basal tonic current seen in D2+ MSNs from young mice (Ade et al, 2008; Janssen et al, 2009. In this study, we demonstrated the essential role of the β3 GABAA receptor subunit in mediating MSN tonic currents using conditional β3 subunit knock-out (β3f/fDrd2 mice. Cre-lox genetics were used to generate conditional knock-out animals where Cre recombinase was expressed under the D2 receptor (Drd2 promoter. We show that while the wild-type MSN tonic current pattern demonstrates a high degree of variability, tonic current patterns from β3f/fDrd2 mice are narrow, suggesting that the β3 subunit is essential to striatal MSN GABA-mediated tonic current. Our data also suggest that a distinct population of synaptic receptors upregulate due to β3 subunit removal. Further, deletion of this subunit significantly decreases the D2+ MSN excitability. These results offer insight for target mechanisms in Parkinson’s disease, where symptoms arise due to the imbalance in striatal D1+ and D2+ MSN excitability and output.

  13. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    Energy Technology Data Exchange (ETDEWEB)

    Khatoon, S.; Slevin, J.T.; Haley, B.E.


    A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeled proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.

  14. Disulfide bond formation between the COOH-terminal domain of the beta subunits and the gamma and epsilon subunits of the Escherichia coli F1-ATPase. Structural implications and functional consequences. (United States)

    Aggeler, R; Haughton, M A; Capaldi, R A


    A set of mutants of the Escherichia coli F1F0-type ATPase has been generated by site-directed mutagenesis as follows: beta E381C, beta S383C, beta E381C/epsilon S108C, and beta S383C/epsilon S108C. Treatment of ECF1 isolated from any of these mutants with CuCl2 induces disulfide bond formation. For the single mutants, beta E381C and beta S383C, a disulfide bond is formed in essentially 100% yield between a beta subunit and the gamma subunit, probably at Cys87 based on the recent structure determination of F1 (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). In the double mutants, two disulfide bonds are formed, again in essentially full yield, one between beta and gamma, the other between a beta and the epsilon subunit via Cys108. The same two cross-links are produced with CuCl2 treatment of ECF1F0 isolated from either of the double mutants. These results show that the parts of gamma around residue 87 (a short alpha-helix) and the epsilon subunit interact with different beta subunits. The yield of covalent linkage of beta to gamma is nucleotide dependent and highest in ATP and much lower with ADP in catalytic sites. The yield of covalent linkage of beta to epsilon is also nucleotide dependent but in this case is highest in ADP and much lower in ATP. Disulfide bond formation between either beta and gamma, or beta and epsilon inhibits the ATPase activity of the enzyme in proportion to the yield of the cross-linked product. Chemical modification of the Cys at either position 381 or 383 of the beta subunit inhibits ATPase activity in a manner that appears to be dependent on the size of the modifying reagent. These results are as expected if movements of the catalytic site-containing beta subunits relative to the gamma and epsilon subunits are an essential part of the cooperativity of the enzyme.

  15. Mouse Leydig cells express multiple P2X receptor subunits



    ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X1–P2X7) and seven heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6, P2X4/6, P2X4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca2+]i and testosterone secretion, supporting the hypothesis that Ca2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Ley...

  16. Flexible subunit stoichiometry of functional human P2X2/3 heteromeric receptors. (United States)

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


    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.

  17. Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa). (United States)

    Concas, A; Imperatore, R; Santoru, F; Locci, A; Porcu, P; Cristino, L; Pierobon, P


    γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABAA receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABAA receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABAA receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

  18. Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii. (United States)

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


    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.

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

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


    Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABA(A) receptor alpha subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABA(A) receptor alpha-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABA(A) receptor alpha subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6weeks to 49years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABA(A) receptor alpha1 and alpha5 subunits during the first few years of postnatal development, with a 60% decrease in alpha5 mRNA expression and a doubling of alpha1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p=0.0012) and decreased alpha5 mRNA expression (p=0.038) in the DLPFC with no significant change of other alpha subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical alpha1 or alpha2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of alpha5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts. Copyright 2009 Elsevier Ltd. All rights reserved.

  20. Functions of nucleotide binding subunits in the tonoplast ATPase from Beta vulgaris L

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M.F.; Poole, R.J.


    Partial purification of NO/sub 3/ sensitive H/sup +/-ATPases from the vacuolar membranes of high plants reveal two prominent polypeptides of approximately 60 and 70 kDa. Both polypeptides appear to contain nucleotide binding sites. The photoactive affinity analog of ATP, BzATP, cannot be hydrolyzed by the tonoplast ATPase but is a potential inhibitor (apparent K/sub I/ = 11 /sup 32/P-BzATP was shown to specifically photolabel the 60 kDa polypeptide. In contrast, Mandala and Taiz have shown the photoincorporation of /sup 32/P-azidoATP to the 70 kDa polypeptide. This sterically different photoaffinity probe can be hydrolyzed although with a low affinity. Azido and benzophenone derivatives of the product, ADP, are currently being examined with respect to their inhibition kinetics of, and their photoincorporation into, the tonoplast ATPase from Beta vulgaris L. Kinetic data will be integrated with patterns of photoincorporation using analogs of both substrate and product, in order to illuminate the functions of the two nucleotide binding subunits.

  1. Cortisone dissociates voltage-dependent K+ channel from its beta subunit (United States)

    Pan, Yaping; Weng, Jun; Kabaleeswaran, Venkataraman; Li, Huiguang; Cao, Yu; Bhosle, Rahul C.; Zhou, Ming


    The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with beta subunits (Kvβ) and certain Kvβs, for example Kvβ1, have an N-terminal segment that closes a channel by the N-type inactivation mechanism. In principle dissociation of Kvβ1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases mammalian (rat) Kv1 channel activity by binding to Kvβ1. A crystal structure of the Kvβ-cortisone complex was solved to 1.82 Å resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvβ. The new mode of channel modulation may be explored by native or synthetic ligands to fine tune cellular excitability. PMID:18806782

  2. Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases (United States)

    Printz, Bruno; Gutsch, Annelie; Behr, Marc; Renaut, Jenny; Hausman, Jean-Francois


    The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given. PMID:28207764

  3. Expression of a novel beta adaptin subunit mRNA splice variant in human testes

    Institute of Scientific and Technical Information of China (English)

    Xin-Dong Zhang; Lan-Lan Yin; Ying Zheng; Li Lu; Zuo-Min Zhou; Jia-Hao Sha


    Aim: To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis. Methods: Using a human testis cDNA microarray, a clone (Ap2β-NY),which was strongly expressed in adult testes but weakly expressed in embryo testes, was sequenced and analyzed.Using polymerase chain reaction (PCR), the tissue distribution and expression time pattern of Ap2β-NY were determined.Results: Ap2β-NY was identified and has been deposited in the GenBank (AY341427). The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis. PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis, spleen, thymus, prostate, ovary, blood leukocyte and brain, but not in the heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. In addition, Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome, which implied that, in the testis, Ap2β-NY was restrictively expressed in germ cells. Conclusion: Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.

  4. Interactions of protein kinase CK2beta subunit within the holoenzyme and with other proteins

    DEFF Research Database (Denmark)

    Kusk, M; Ahmed, R; Thomsen, B;


    in alpha-beta and beta-beta interactions. We also detected an intramolecular beta interaction within the amino acid stretch 132-165. Using CK2beta as a bait in a two-hybrid library screening several new putative cellular partners have been identified, among them the S6 kinase p90rsk, the putative tumor...

  5. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice. (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji


    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  6. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E


    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides...

  7. Ligand-guided homology modelling of the GABAB2 subunit of the GABAB receptor. (United States)

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


    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.

  8. Structure of the T cell receptor in a Ti alpha V beta 2, alpha V beta 8-positive T cell line

    DEFF Research Database (Denmark)

    Hou, X; Dietrich, J; Kuhlmann, J


    The T cell receptor (TcR) is composed of at least six different polypeptide chains consisting of the clonotypic Ti heterodimer (Ti alpha beta or Ti gamma delta) and the noncovalently associated CD3 chains (CD3 gamma delta epsilon zeta). The exact number of subunits constituting the TcR is still...... not known; however, it has been suggested that each TcR contains two Ti dimers. To gain insight into the structure of the TcR we constructed a Ti alpha V beta 2, alpha V beta 8-positive T cell line which expressed the endogenous human TiV beta 8 and the transfected mouse TiV beta 2 both in association...... with the endogenous Ti alpha and CD3 chains at the cell surface. Preclearing experiments with radioiodinated cell lysate prepared with digitonin lysis buffer demonstrated that depleting the lysate of Ti alpha V beta 8 by immunoprecipitation with anti V beta 8 monoclonal antibody (mAb) did not reduce the amount of Ti...

  9. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons (United States)

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


    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…

  10. d Subunit-Containing GABA[subscript A] Receptor Prevents Overgeneralization of Fear in Adult Mice (United States)

    Zhang, Wen-Hua; Zhou, Jin; Pan, Han-Qing; Wang, Xiao-Yang; Liu, Wei-Zhu; Zhang, Jun-Yu; Yin, Xiao-Ping; Pan, Bing-Xing


    The role of d subunit-containing GABA[subscript A] receptor (GABA[subscript A](d)R) in fear generalization is uncertain. Here, by using mice with or without genetic deletion of GABA[subscript A](d)R and using protocols in which the conditioned tone stimuli were cross presented with different nonconditioned stimuli, we observed that when the two…

  11. LTP requires a reserve pool of glutamate receptors independent of subunit type. (United States)

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


    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.

  12. Differences in AMPA and kainate receptor interactomes facilitate identification of AMPA receptor auxiliary subunit GSG1L

    National Research Council Canada - National Science Library

    Shanks, Natalie F; Savas, Jeffrey N; Maruo, Tomohiko; Cais, Ondrej; Hirao, Atsushi; Oe, Souichi; Ghosh, Anirvan; Noda, Yasuko; Greger, Ingo H; Yates, 3rd, John R; Nakagawa, Terunaga


    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...

  13. Estrogen receptor beta treats Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    Zhu Tian; Jia Fan; Yang Zhao; Sheng Bi; Lihui Si; Qun Liu


    In vitro studies have shown that estrogen receptor β can attenuate the cytotoxic effect of amyloid β protein on PC12 cells through the Akt pathway without estrogen stimulation. In this study, we aimed to observe the effect of estrogen receptor β in Alzheimer's disease rat models established by intraventricular injection of amyloid β protein. Estrogen receptor β lentiviral particles delivered via intraventricular injection increased Akt content in the hippocampus, decreased interleukin-1β mRNA, tumor necrosis factor α mRNA and amyloid β protein levels in the hippocampus, and improved the learning and memory capacities in Alzheimer's disease rats. Estrogen receptor β short hairpin RNA lentiviral particles delivered via intraventricular injection had none of the above impacts on Alzheimer's disease rats. These experimental findings indicate that estrogen receptor β, independent from estrogen, can reduce inflammatory reactions and amyloid β deposition in the hippocampus of Alzheimer's disease rats, and improve learning and memory capacities. This effect may be mediated through activation of the Akt pathway.

  14. SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits


    Heidelberg, Laura S.; Warren, James W.; Fisher, Janet L.


    Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete popu...

  15. Spectral and Temporal Properties of the Alpha and Beta Subunits and (alpha Beta) Monomer Isolated from Nostoc SP. Using Picosecond Laser Spectroscopy. (United States)

    Dagen, Aaron J.


    The fluorescence decay profiles, relative quantum yield and transmission of the (alpha), (beta) and ((alpha)(beta)) complexes from phycoerythrin isolated from the photosynthetic antenna system of Nostoc sp. and measured by single picosecond laser spectroscopic techniques is studied. The fluorescence decay profiles of all three complexes are found to be intensity independent for the intensity range investigated ((TURN)4 x 10('13) to (TURN)4 x 10('15) photons-cm('-2) per pulse). The apparent decrease in the relative quantum yield of all three complexes as intensity increases is offset by a corresponding increase in the relative transmission. This evidence, along with the intensity independent fluorescence kinetics, suggests that exciton annihilation is absent in these complexes. The decay profiles are fit to models assuming energy transfer amongst fluorescing chromophores. The intraprotein transfer rate is found to be 100 ps in the (alpha) subunit, 666 ps in the (beta) subunit. Constraining these rates to be identical in the monomer results in explaining the monomer kinetics by an increase in the nonradiative rate of the f(,(beta)) chromophore, an apparent result of aggregation effects.

  16. Altered GABAA Receptor Subunit Expression and Pharmacology in Human Angelman Syndrome Cortex (United States)

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


    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 β3-α5-γ3 GABAA 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 GABAA 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 β3/β2 and α5/α1 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 GABAA receptor currents was then performed. Studies of GABAA receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the α1-selective benzodiazepine site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABAA receptor affinity and modulation by neurosteroids were unchanged. This shift in GABAA 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. PMID:20692323

  17. Expression of nicotinic acetylcholine receptor subunits from parasitic nematodes in Caenorhabditis elegans. (United States)

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


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

  18. Analysis of NR3A receptor subunits in human native NMDA receptors

    DEFF Research Database (Denmark)

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


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

  19. An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars. (United States)

    Foltz, David W


    A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

  20. Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

    DEFF Research Database (Denmark)

    Sarno, S; Vaglio, P; Marin, O


    Protein kinase CK2 is a ubiquitous pleiotropic serine/threonine protein kinase whose holoenzyme is comprised of two catalytic (alpha and/or alpha') and two non-catalytic, beta-subunits. The beta-subunit possesses antagonist functions that can be physically dissected by generating synthetic...... fragments encompassing its N-terminal and C-terminal domains. Here we show that by mutating basic residues in the 74-77 and in the 191-198 regions of the alpha-subunit, the negative regulation by the beta-subunit and by its N-terminal synthetic fragment CK2beta-(1-77), which is observable using calmodulin...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

  1. Parathyroid hormone receptor recycling: role of receptor dephosphorylation and beta-arrestin. (United States)

    Chauvin, Stephanie; Bencsik, Margaret; Bambino, Tom; Nissenson, Robert A


    The recovery of PTH receptor (PTHR) function after acute homologous receptor desensitization and down-regulation in bone and kidney cells has been attributed to receptor recycling. To determine the role of receptor dephosphorylation in PTHR recycling, we performed morphological and functional assays on human embryonic kidney 293 cells stably expressing wild-type (wt) or mutant PTHRs. Confocal microscopy and ligand binding assays revealed that the wt PTHR is rapidly recycled back to the plasma membrane after removal of the agonist. Receptors that were engineered to either lack the sites of phosphorylation or to resemble constitutively phosphorylated receptors were able to recycle back to the plasma membrane with the same kinetics as the wt PTHR. The PTHR was found to be dephosphorylated by an enzyme apparently distinct from protein phosphatases 1 or 2A. The PTHR and beta-arrestin-2-green fluorescent protein (GFP) were found to stably colocalize during PTHR internalization, whereas after agonist removal and during receptor recycling, the colocalization slowly disappeared. Experiments using phosphorylation-deficient PTHRs and a dominant-negative form of beta-arrestin showed that beta-arrestin does not regulate the efficiency of PTHR recycling. These studies indicate that, unlike many G protein-coupled receptors, PTHR recycling does not require receptor dephosphorylation or its dissociation from beta-arrestin.

  2. The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. (United States)

    Aydar, Ebru; Palmer, Christopher P; Klyachko, Vitaly A; Jackson, Meyer B


    The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

  3. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B;


    -subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

  4. [Molecular cloning of the DNA sequence of activin beta A subunit gene mature peptides from panda and related species and its application in the research of phylogeny and taxonomy]. (United States)

    Wang, Xiao-Jing; Wang, Xiao-Xing; Wang, Ya-Jun; Wang, Xi-Zhong; He, Guang-Xin; Chen, Hong-Wei; Fei, Li-Song


    Activin, which is included in the transforming growth factor-beta (TGF beta) superfamily of proteins and receptors, is known to have broad-ranging effects in the creatures. The mature peptide of beta A subunit of this gene, one of the most highly conserved sequence, can elevate the basal secretion of follicle-stimulating hormone (FSH) in the pituitary and FSH is pivotal to organism's reproduction. Reproduction block is one of the main reasons which cause giant panda to extinct. The sequence of Activin beta A subunit gene mature peptides has been successfully amplified from giant panda, red panda and malayan sun bear's genomic DNA by using polymerase chain reaction (PCR) with a pair of degenerate primers. The PCR products were cloned into the vector pBlueScript+ of Esherichia coli. Sequence analysis of Activin beta A subunit gene mature peptides shows that the length of this gene segment is the same (359 bp) and there is no intron in all three species. The sequence encodes a peptide of 119 amino acid residues. The homology comparison demonstrates 93.9% DNA homology and 99% homology in amino acid among these three species. Both GenBank blast search result and restriction enzyme map reveal that the sequences of Activin beta A subunit gene mature peptides of different species are highly conserved during the evolution process. Phylogeny analysis is performed with PHYLIP software package. A consistent phylogeny tree has been drawn with three different methods. The software analysis outcome accords with the academic view that giant panda has a closer relationship to the malayan sun bear than the red panda. Giant panda should be grouped into the bear family (Uersidae) with the malayan sun bear. As to the red panda, it would be better that this animal be grouped into the unique family (red panda family) because of great difference between the red panda and the bears (Uersidae).

  5. Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist. (United States)

    Rossjohn, J; McKinstry, W J; Woodcock, J M; McClure, B J; Hercus, T R; Parker, M W; Lopez, A F; Bagley, C J


    Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)

  6. A novel biological activity of praziquantel requiring voltage-operated Ca2+ channel beta subunits: subversion of flatworm regenerative polarity.

    Directory of Open Access Journals (Sweden)

    Taisaku Nogi

    Full Text Available BACKGROUND: Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug-praziquantel (PZQ-has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s of praziquantel remain undefined. METHODS AND FINDINGS: Here, we provide fresh perspective on the molecular basis of praziquantel efficacy in vivo consequent to the discovery of a remarkable action of PZQ on regeneration in a species of free-living flatworm (Dugesia japonica. Specifically, PZQ caused a robust (100% penetrance and complete duplication of the entire anterior-posterior axis during flatworm regeneration to yield two-headed organisms with duplicated, integrated central nervous and organ systems. Exploiting this phenotype as a readout for proteins impacting praziquantel efficacy, we demonstrate that PZQ-evoked bipolarity was selectively ablated by in vivo RNAi of voltage-operated calcium channel (VOCC beta subunits, but not by knockdown of a VOCC alpha subunit. At higher doses of PZQ, knockdown of VOCC beta subunits also conferred resistance to PZQ in lethality assays. CONCLUSIONS: This study identifies a new biological activity of the antischistosomal drug praziquantel on regenerative polarity in a species of free-living flatworm. Ablation of the bipolar regenerative phenotype evoked by PZQ via in vivo RNAi of VOCC beta subunits provides the first genetic evidence implicating a molecular target crucial for in vivo PZQ activity and supports the 'VOCC hypothesis' of PZQ efficacy. Further, in terms of regenerative biology and Ca(2+ signaling, these data highlight a novel role for voltage-operated Ca(2+ entry in regulating in vivo stem cell differentiation and regenerative patterning.

  7. Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

    DEFF Research Database (Denmark)

    Buchou, Thierry; Vernet, Muriel; Blond, Olivier


    Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in m....... Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.......Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene...

  8. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    Energy Technology Data Exchange (ETDEWEB)

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J. (National Heart and Lung Institute, Brompton Hospital, London (England))


    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand (125I)pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed.

  9. Amiloride interacts with renal. cap alpha. - and. beta. -adrenergic receptors

    Energy Technology Data Exchange (ETDEWEB)

    Howard, M.J.; Mullen, M.D.; Insel, P.A.


    The authors have used radioligand binding techniques to assess whether amiloride and certain analogues of amiloride (ethylisopropyl amiloride and benzamil) can bind to adrenergic receptors in the kidney. They found that amiloride could compete for (/sup 3/H)rauwolscine (..cap alpha../sub 2/-adrenergic receptors), (/sup 3/H)prazosin (..cap alpha../sub 1/-adrenergic receptors), and (/sup 125/I)iodocyanopindolol (..beta..-adrenergic receptors) binding in rat renal cortical membranes with inhibitor constants of 13.6 /plus minus/ 5.7, 24.4 /plus minus/ 7.4, and 8.36 /plus minus/ 13.5, respectively. Ethylisopropyl amiloride and benzamil were from 2- to 25-fold more potent than amiloride in competing for radioligand binding sites in studies with these membranes. In addition, amiloride and the two analogues competed for (/sup 3/H)prazosin sites on intact Madin-Darby canine kidney cells and amiloride blocked epinephrine-stimulated prostaglandin E/sub 2/ production in these cells. They conclude that amiloride competes for binding to several classes of renal adrenergic receptors with a rank order of potency of ..cap alpha../sub 2/ > ..cap alpha../sub 1/ > ..beta... Binding to, and antagonism of, adrenergic receptors occurs at concentrations of amiloride that are lower than previously observed nonspecific interactions of this agent.

  10. Biological significance of glucocorticoid receptor beta

    Institute of Scientific and Technical Information of China (English)


    Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, termed hGRα and hGRβ. hGRα is a ligand-activated transcription factor which, in the hormone-bound state, modulates the expression of glucocorticoid-responsive genes by binding to specific glucocorticoid response element (GRE) DNA sequences. In contrast, hGRβ dose not bind glucocorticoids and is transcriptionally inactive. We demonstrate here that hGRβ inhibits the hormone-induced, hGRα-mediated stimulations of gene expression, including glucocorticoid-responsive reporter gene (cat) and endogenous p21 gene. We also demonstrate that hGRβ can inhibit hGRα-mediated regulation of proliferation and differentiation of a human osteosarcoma cell line (HOS-8603). Our studies on the expression of hGR mRNA in nephrotic syndrome patients indicate that the hGRα/hGRβ mRNA ratio in peripheral white blood cell of hormone-resistant patients is lower than that of hormone-sensitive patients and health volunteers. These results indicate that hGRβ may be a physiologically and pathophysiologically relevant endogenous inhibitor of hGRα

  11. Directed mutagenesis of the strongly conserved aspartate 242 in the beta-subunit of Escherichia coli proton-ATPase. (United States)

    Al-Shawi, M K; Parsonage, D; Senior, A E


    Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the beta-subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in beta-subunits of F1-ATPase enzymes, in a region of sequence which shows homology with numerous nucleotide-binding proteins. By analogy with adenylate kinase (Fry, D.C., Kuby, S.A., and Mildvan, A.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 907-911), beta-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+ or through hydrogen bonding to the substrate(s); an acid-base catalytic role is also plausible. The substitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue beta-Asp-242. Both mutations significantly impaired oxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental than Val-242. Purified soluble mutant F1-ATPases had normal molecular size and subunit composition, and displayed 7% (beta-Asn-242) and 17% (beta-Val-242) of normal specific Mg-ATPase activity. The relative MgATPase activities of both mutant enzymes showed similar pH dependence to normal. Relative MgATPase and CaATPase activities of normal and mutant enzymes were compared at widely varied pMg and pCa. The mutations had little effect on KM MgATP, but KM CaATP was reduced. The data showed that the carboxyl side-chain of beta-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linked mechanism, nor is it likely to be directly involved in liganding to substrate Mg2+ during the reaction. Specificity constants (kcat/KM) for MgATP and CaATP were reduced in both mutant enzymes, showing that the mutations destabilized interactions between the catalytic nucleotide-binding domain and the transition state.

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

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    Natalie F. Shanks


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

  13. Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex

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    Robichon Alain


    Full Text Available Abstract Background We investigated how the synthesis of cAMP, stimulated by isoproterenol acting through β-adrenoreceptors and Gs, is strongly amplified by simultaneous incubation with baclofen. Baclofen is an agonist of δ-aminobutyric acid type B receptors [GABAB], known to inhibit adenylyl cyclase via Gi. Because these agents have opposite effects on cAMP levels, the unexpected increase in cAMP synthesis when they are applied simultaneously has been intensively investigated. From previous reports, it appears that cyclase type II contributes most significantly to this phenomenon. Results We found that simultaneous application of isoproterenol and baclofen specifically influences the association/dissociation of molecules involved in the induction and termination of cyclase activity. Beta/gamma from [GABA]B receptor-coupled Gi has a higher affinity for adenylyl cyclase isoform(s when these isoforms are co-associated with Gs. Our data also suggest that, when beta/gamma and Gαs are associated with adenylyl cyclase isoform(s, beta/gamma from [GABA]B receptor-coupled Gi retards the GTPase activity of Gαs from adrenergic receptor. These reciprocal regulations of subunits of the adenylyl cyclase complex might be responsible for the drastic increase of cAMP synthesis in response to the simultaneous signals. Conclusions Simultaneous signals arriving at a particular synapse converge on molecular detectors of coincidence and trigger specific biochemical events. We hypothesize that this phenomenon comes from the complex molecular architectures involved, including scaffolding proteins that make reciprocal interactions between associated molecules possible. The biochemistry of simultaneous signaling is addressed as a key to synaptic function.

  14. Estrogen receptor beta agonists in neurobehavioral investigations. (United States)

    Choleris, Elena; Clipperton, Amy E; Phan, Anna; Kavaliers, Martin


    Neurobehavioral investigations into the functions of estrogen receptor (ER)alpha and ERbeta have utilized 'knockout' mice, phytoestrogens and, more recently, ER-specific agonists. Feeding, sexual, aggressive and social behavior, anxiety, depression, drug abuse, pain perception, and learning (and associated synaptic plasticity) are affected by ERalpha and ERbeta in a manner that is dependent upon the specific behavior studied, gender and developmental stage. Overall, ERalpha and ERbeta appear to function together to foster sociosexual behavior while inhibiting behaviors that, if occurring at the time of behavioral estrous, may compete with reproduction (eg, feeding). Recently developed pharmacological tools have limited selectivity and availability to the research community at large, as they are not commercially available. The development of highly selective, commercially available ERbeta-specific antagonists would greatly benefit preclinical and applied research.

  15. Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus. (United States)

    Ekuni, A; Watanabe, H; Kuroda, N; Sawada, K; Murakami, H; Kanazawa, H


    An engineered gamma subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag gamma), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F1 containing alpha, beta, and His-tagged gamma exhibited sixty percent of the wild-type ATPase activity. The reconstituted alphabeta His-tag gamma complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. ATPase activity was eluted specifically with imidazole. These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity. The reconstituted alphabeta His-tag gamma complex, even after its binding to the resin, exhibited ATPase activity suggesting that the gamma subunit, when fixed to a solid phase, may rotate the alphabeta complex. This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.

  16. Identification, expression and serological evaluation of the recombinant ATP synthase beta subunit of Mycoplasma pneumoniae

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    Nuyttens Hélène


    Full Text Available Abstract Background Mycoplasma pneumoniae is responsible for acute respiratory tract infections (RTIs common in children and young adults. As M. pneumoniae is innately resistant to β-lactams antibiotics usually given as the first-line treatment for RTIs, specific and early diagnosis is important in order to select the right treatment. Serology is the most used diagnostic method for M. pneumoniae infections. Results In this study, we identified the M. pneumoniae ATP synthase beta subunit (AtpD by serologic proteome analysis and evaluated its usefulness in the development of a serological assay. We successfully expressed and purified recombinant AtpD (rAtpD protein, which was recognised by serum samples from M. pneumoniae-infected patient in immunoblots. The performance of the recombinant protein rAtpD was studied using a panel of serum samples from 103 infected patients and 86 healthy blood donors in an in-house IgM, IgA and IgG enzyme-linked immunosorbent assay (ELISA. The results of this assay were then compared with those of an in-house ELISA with a recombinant C-terminal fragment of the P1 adhesin (rP1-C and of the commercial Ani Labsystems ELISA kit using an adhesin P1-enriched whole-cell extract. Performances of the rAtpD and rP1-C antigen combination were further assessed by binary logistic regression analysis. We showed that combination of rAtpD and rP1-C discriminated maximally between the patients infected with M. pneumoniae (children and adults and the healthy subjects for the IgM class, performing better than the single recombinant antigens or the commercial whole-cell extract. Conclusion These results suggest that AtpD can be used as an antigen for the immunodiagnosis of early and acute M. pneumoniae infection in association with adhesin P1, providing an excellent starting point for the development of point-of-care diagnostic assays.

  17. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes. (United States)

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


    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.

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

    Institute of Scientific and Technical Information of China (English)

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS


    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  19. Mechanism of functional interaction between potassium channel Kv1.3 and sodium channel NavBeta1 subunit (United States)

    Kubota, Tomoya; Correa, Ana M.; Bezanilla, Francisco


    The voltage-gated potassium channel subfamily A member 3 (Kv1.3) dominantly expresses on T cells and neurons. Recently, the interaction between Kv1.3 and NavBeta1 subunits has been explored through ionic current measurements, but the molecular mechanism has not been elucidated yet. We explored the functional interaction between Kv1.3 and NavBeta1 through gating current measurements using the Cut-open Oocyte Voltage Clamp (COVC) technique. We showed that the N-terminal 1–52 sequence of hKv1.3 disrupts the channel expression on the Xenopus oocyte membrane, suggesting a potential role as regulator of hKv1.3 expression in neurons and lymphocytes. Our gating currents measurements showed that NavBeta1 interacts with the voltage sensing domain (VSD) of Kv1.3 through W172 in the transmembrane segment and modifies the gating operation. The comparison between G-V and Q-V with/without NavBeta1 indicates that NavBeta1 may strengthen the coupling between hKv1.3-VSD movement and pore opening, inducing the modification of kinetics in ionic activation and deactivation. PMID:28349975

  20. Steady-state levels of G-protein beta-subunit expression are regulated by treatment of cells with bacterial toxins

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.


    Cultures of 3T3-L1 cells were incubated with either 10 ng/ml cholera toxin or 10 ng/ml pertussis toxin from 4 days prior to the initiation of differentiation and throughout the subsequent incubation. Toxin concentrations were sufficient to completely prevent the labelling of alpha-subunits with (/sup 32/P)NAD/sup +/ and pertussis toxin and to prevent by more than 90% the labelling with (/sup 32/P)NAD/sup +/ and cholera toxin in membranes prepared from these cells. Neither toxin prevented the differentiation to the adipocyte phenotype. Neither toxin prevented the increases in the relative amounts of G-proteins which occur upon differentiation. Both toxins dramatically decreased the amount of beta-subunits. As measured by quantitative immunoblotting with antisera specific for both the 35 kDa and 36 kDa beta-subunits, levels of beta-subunit were decreased by more than 50% of steady-state level of control cells. Thus, bacterial toxins which modifies G-protein alpha-subunits are capable of modulating the levels of beta-subunits in vivo. The basis for the regulation of G-protein subunit expression by bacterial toxins is under study.

  1. Pathological reorganization of NMDA receptors subunits and postsynaptic protein PSD-95 distribution in Alzheimer's disease. (United States)

    Leuba, Genevieve; Vernay, Andre; Kraftsik, Rudolf; Tardif, Eric; Riederer, Beat Michel; Savioz, Armand


    In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

  2. [Functional analysis of transforming growth factor-beta type II dominant negative receptor]. (United States)

    Takarada, M


    The transforming growth factor-beta (TGF-beta) is a multifunctional homodimeric protein with an apparent molecular weight of 25 KDa. TGF-beta transduces signals by forming heteromeric complexes of their type-I (T beta R-I) and type-II (T beta R-II) serin/threonine kinase receptors. TGF-beta binds first to T beta R-II receptor, and then the ligand in this complex is recognized by T beta R-I, resulting in formation of a heteromeric receptor complex composed of T beta R-I and T beta R-II. Once received, T beta R-I becomes phosphorylated in the GS domain by the associated constitutively active T beta R-II and transmits the downstream signal. It has been reported that formation of the heteromeric complex is indispensible at least in epithelial cells for growth inhibition and extracellular matrix production induced by TGF-beta. In this study, the functional role of T beta R-II for the TGF-beta-induced signals in osteoblastic cells was investigated by using a dominant negative type of T beta R-II mutant receptors (T beta RIIDNR). ROS 17/2.8 and MG 63 cells were found to express T beta R-I, T beta R-II, and T beta R-III, and their cell growth was inhibited by TGF-beta, whereas alkaline phosphatase activity was stimulated. Cells that were stably transfected with the T beta RIIDNR plasmid showed decreased response to TGF-beta during growth and alkaline phosphatase activity. These results indicate that the intracellular serine/threonine kinase domain of T beta R-II is essential for signal transduction of the TGF-beta-induced alkaline phosphatase activity as well as growth inhibition.

  3. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies. (United States)

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


    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.

  4. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse (United States)

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


    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

  5. The Nicotinic Acetylcholine Receptor α5 Subunit Plays a Key Role in Attention Circuitry and Accuracy (United States)

    Bailey, Craig D. C.; De Biasi, Mariella; Fletcher, Paul J.; Lambe, Evelyn K.


    Stimulation of the prefrontal cortex by acetylcholine is critical for attention; however, the cellular mechanisms underlying its influence on attention pathways within the brain are not well understood. Pyramidal neurons in layer VI of the prefrontal cortex are believed to play an important role in this process because they are excited by acetylcholine and provide a major source of feedback projections to the thalamus. Here, we show using whole-cell electrophysiology that the relatively rare α5 subunit of the nicotinic acetylcholine receptor powerfully enhances nicotinic currents in layer VI pyramidal neurons in prefrontal cortical brain slices from adult mice. In addition, behavioral experiments using the five-choice serial reaction time test show that the presence of the nicotinic receptor α5 subunit also increases the accuracy of adult mice on this visual attention task under highly demanding conditions. Together, these findings demonstrate a novel and important role for the nicotinic receptor α5 subunit in adult brain circuitry required for attentional performance. PMID:20610759

  6. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜


    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  7. The crystal structure of the complex of Zea mays alpha subunit with a fragment of human beta subunit provides the clue to the architecture of protein kinase CK2 holoenzyme

    DEFF Research Database (Denmark)

    Battistutta, R; Sarno, S; De Moliner, E


    The crystal structure of a complex between the catalytic alpha subunit of Zea mays CK2 and a 23-mer peptide corresponding the C-terminal sequence 181-203 of the human CK2 regulatory beta subunit has been determined at 3.16-A resolution. The complex, composed of two alpha chains and two peptides......, presents a molecular twofold axis, with each peptide interacting with both alpha chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The beta...... subunit can influence the catalytic activity both directly and by promoting the formation of the alpha2 dimer, in which each alpha chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two...

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

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    Dian-Shi Wang


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

  9. NO-sensitive guanylyl cyclase beta1 subunit is peripherally associated to chromosomes during mitosis. Novel role in chromatin condensation and cell cycle progression. (United States)

    Pifarré, Paula; Baltrons, María Antonia; Földi, Istvan; García, Agustina


    NO-sensitive guanylyl cyclase (GC(NO)), the major NO target, is involved in important regulatory functions in the cardiovascular, gastrointestinal and central nervous systems. GC(NO) exists as heterodimers of alpha(1/2) and beta1 subunits. Deletion of the obligate beta1 dimerizing partner abrogates NO/cGMP signaling and shortens the life span of KO mice. Localization studies in the CNS have shown that beta1 is more widespread than alpha subunits and in some areas is the only GC(NO) subunit expressed, suggesting that beta1 may have GC(NO)-independent functions. GC(NO) is predominantly cytosolic, but association to membranes and other intracellular structures has been described. Here, we show localization of beta1 in cytoplasm and nucleus of cells expressing alpha subunits and GC(NO) activity (astrocytes, C6 cells), as well as in cells devoid of alpha subunits and GC(NO) activity (microglia). In both cell types beta1 associates peripherally to chromosomes in all phases of mitosis. Immunodepletion of beta1 in C6 cells enhances chromatin condensation in an in vitro assay. Moreover, silencing beta1 by siRNA induces cell cycle re-entry as determined by flow cytometry, and increases proliferation rate in a MTT-assay, whereas infection with beta1-containing adenovirus has the opposite effect. These actions are independent of cGMP formation. We postulate that beta1 is a multifunctional protein that regulates chromatin condensation and cell cycle progression, in addition to being an obligate monomer in functional GC(NO) heterodimers.

  10. Differential modulation of alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors expressed in Xenopus oocytes by flufenamic acid and niflumic acid. (United States)

    Zwart, R; Oortgiesen, M; Vijverberg, H P


    Effects of flufenamic acid (FFA) and niflumic acid (NFA), which are often used to block Ca(2+)-activated Cl- current, have been investigated in voltage-clamped Xenopus oocytes expressing alpha 3 beta 2 and alpha 3 beta 4 nicotinic ACh receptors (nAChRs). NFA and FFA inhibit alpha 3 beta 2 nAChR-mediated inward currents and potentiate alpha 3 beta 4 nAChR-mediated inward currents in normal, Cl(-)-free and Ca(2+)-free solutions to a similar extent. The concentration-dependence of the inhibition of alpha 3 beta 2 nAChR-mediated ion current yields IC50 values of 90 microM for FFA and of 260 microM for NFA. The potentiation of alpha 3 beta 4 nAChR-mediated ion current by NFA yields an EC50 value of 30 microM, whereas the effect of FFA does not saturate for concentrations of up to 1 mM. At 100 microM, FFA reduces the maximum of the concentration-effect curve of ACh for alpha 3 beta 2 nAChRs, but leaves the EC50 of ACh unaffected. The same concentration of FFA potentiates alpha 3 beta 4 nAChR-mediated ion currents for all ACh concentrations and causes a small shift of the concentration-effect curve of ACh to lower agonist concentrations. The potentiation, like the inhibition, is most likely due to a noncompetitive effect of FFA. Increasing ACh-induced inward current either by raising the agonist concentration from 10 microM to 200 microM or by coapplication of 10 microM ACh and 200 microM FFA causes a similar enhancement of block of the alpha 3 beta 4 nAChR-mediated ion current by Mg2+. This suggests that the effects of FFA and of an increased agonist concentration result in a similar functional modification of the alpha 3 beta 4 nAChR-operated ion channel. It is concluded that alpha 3 beta 4 and alpha 3 beta 2 nAChRs are oppositely modulated by FFA and NFA through a direct beta-subunit-dependent effect.

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

    Directory of Open Access Journals (Sweden)

    Sayyed Alireza Talaei


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

  12. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Directory of Open Access Journals (Sweden)

    Caudle Robert M


    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  13. β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+ pathway in xenopus gastrulation.

    Directory of Open Access Journals (Sweden)

    Katharina Seitz

    Full Text Available β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+ cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+ signaling cascade upstream of Protein Kinase C (PKC and Ca(2+/Calmodulin-dependent Protein Kinase II (CamKII. We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

  14. Peptide insertions in domain 4 of hbeta(c), the shared signalling receptor subunit for GM-CSF, IL3 and IL5, induce ligand-independent activation. (United States)

    Jones, K L; Bagley, C J; Butcher, C; Barry, S C; Vadas, M A; D'Andrea, R J


    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.

  15. A linkage study between the GABAA beta2 and GABAA gamma2 subunit genes and major psychoses. (United States)

    Ambrósio, Alda M; Kennedy, James L; Macciardi, Fabio; King, Nicole; Azevedo, Maria H; Oliveira, Catarina R; Pato, Carlos N


    Alterations of the gamma-aminobutyric acid (GABA) system have been implicated in the pathophysiology of major psychoses. Restriction fragment length polymorphisms associated with the human gamma-aminobutyric acid type A (GABAA) beta2 and GABAA gamma2 subunit genes on chromosome 5q32-q35 were tested to determine whether they confer susceptibility to major psychoses. Thirty-two schizophrenic families and 25 bipolar families were tested for linkage. Nonparametric linkage (NPL) analysis performed by GENEHUNTER showed no significant NPL scores for both genes in schizophrenia (GABAA beta2: NPL narrow= -0.450; NPL broad= -0.808; GABAA gamma2: NPL narrow=0.177; NPL broad= -0.051) or bipolar disorder (GABAA beta2: NPL narrow=0.834; NPL broad=0.783; GABAA gamma2: NPL narrow= -0.159; NPL broad=0.070). Linkage analysis does not support the hypothesis that variants within the GABAA beta2 and GABAA gamma2 genes are significantly linked to major psychoses in a Portuguese population.

  16. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;


    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis...... of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...

  17. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons (United States)

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


    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.

  18. Auxiliary Subunit GSG1L Acts to Suppress Calcium-Permeable AMPA Receptor Function (United States)

    McGee, Thomas P.; Bats, Cécile


    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

  19. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost


    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...... correlates with its functional effect, suggesting that the relative orientation of β modulates ion binding at the α subunit. β2 is primarily expressed in granule neurons and glomeruli in the cerebellum, and we propose that its unique functional characteristics are important to respond appropriately...

  20. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms. (United States)

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


    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 V as 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 of

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

    Directory of Open Access Journals (Sweden)

    Ben-Wen Li


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

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

    Directory of Open Access Journals (Sweden)

    Torrão A.S.


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

  3. The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression


    Sonal Shruti; Joanna Urban-Ciecko; Fitzpatrick, James A.; Robert Brenner; Bruchez, Marcel P.; Alison L Barth


    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we fi...

  4. Discs-large (DLG is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila

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    Featherstone David E


    Full Text Available Abstract Background Drosophila discs-large (DLG is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors. Results There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1 receptors that contain the subunit GluRIIA, and 2 receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors. Conclusions Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the

  5. NR2 subunits and NMDA receptors on lamina II inhibitory and excitatory interneurons of the mouse dorsal horn

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    MacDermott Amy B


    Full Text Available Abstract Background NMDA receptors expressed by spinal cord neurons in the superficial dorsal horn are involved in the development of chronic pain associated with inflammation and nerve injury. The superficial dorsal horn has a complex and still poorly understood circuitry that is mainly populated by inhibitory and excitatory interneurons. Little is known about how NMDA receptor subunit composition, and therefore pharmacology and voltage dependence, varies with neuronal cell type. NMDA receptors are typically composed of two NR1 subunits and two of four NR2 subunits, NR2A-2D. We took advantage of the differences in Mg2+ sensitivity of the NMDA receptor subtypes together with subtype preferring antagonists to identify the NR2 subunit composition of NMDA receptors expressed on lamina II inhibitory and excitatory interneurons. To distinguish between excitatory and inhibitory interneurons, we used transgenic mice expressing enhanced green fluorescent protein driven by the GAD67 promoter. Results Analysis of conductance ratio and selective antagonists showed that lamina II GABAergic interneurons express both the NR2A/B containing Mg2+ sensitive receptors and the NR2C/D containing NMDA receptors with less Mg2+ sensitivity. In contrast, excitatory lamina II interneurons express primarily NR2A/B containing receptors. Despite this clear difference in NMDA receptor subunit expression in the two neuronal populations, focally stimulated synaptic input is mediated exclusively by NR2A and 2B containing receptors in both neuronal populations. Conclusions Stronger expression of NMDA receptors with NR2C/D subunits by inhibitory interneurons compared to excitatory interneurons may provide a mechanism to selectively increase activity of inhibitory neurons during intense excitatory drive that can provide inhibitory feedback.

  6. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block. (United States)

    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


    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.


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


    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. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase. (United States)

    Dallmann, H G; Flynn, T G; Dunn, S D


    The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1

  9. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport. (United States)

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


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

  10. BAT3 interacts with transforming growth factor-beta (TGF-beta) receptors and enhances TGF-beta1-induced type I collagen expression in mesangial cells. (United States)

    Kwak, Joon Hyeok; Kim, Sung Il; Kim, Jin Kuk; Choi, Mary E


    Transforming growth factor-beta1 (TGF-beta1) plays essential roles in a wide array of cellular processes, such as in development and the pathogenesis of tissue fibrosis, including that associated with progressive kidney diseases. Tight regulation of its signaling pathways is critical, and proteins that associate with the TGF-beta receptors may exert positive or negative regulatory effects on TGF-beta signaling. In the present study we employed a yeast-based two-hybrid screening system to identify BAT3 (HLA-B-associated transcript 3) as a TGF-beta receptor-interacting protein. Analysis of endogenously expressed BAT3 in various tissues including the kidney reveals the existence of approximately 140-kDa full-length protein as well as truncated forms of BAT3 whose expression is developmentally regulated. Endogenous BAT3 protein interacts with TGF-beta receptors type I and type II in renal mesangial cells. Functional assays show that expression of full-length BAT3 results in enhancement of TGF-beta1-stimulated transcriptional activation of p3TP-Lux reporter, and these effects require the presence of functional TGF-beta signaling receptors as demonstrated in R-1B and DR-26 mutant cells. Moreover, expression of full-length BAT3, but not C-terminal truncated mutant of BAT3, enhanced TGF-beta1-induced type I collagen expression in mesangial cells, whereas knock down of BAT3 protein expression by small interfering RNA suppressed the expression of type I collagen induced by TGF-beta1. Our findings suggest that BAT3, a TGF-beta receptor-interacting protein, is capable of modulating TGF-beta signaling and acts as a positive regulator of TGF-beta1 stimulation of type I collagen expression in mesangial cells.

  11. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.


    Pellegrini-Giampietro, D E; Zukin, R.S.; Bennett, M V; Cho, S; Pulsinelli, W. A.


    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeab...

  12. Evaluation of native GABA(A) receptors containing an alpha 5 subunit. (United States)

    Li, M; Szabo, A; Rosenberg, H C


    The type A receptor for gamma-aminobutyric acid (GABA), or GABA(A) receptor, is a pentamer of highly variable quaternary structure. It includes two alpha subunits, drawn from a pool of six genes, which largely determine benzodiazepine pharmacology of the receptor. In brain sections, both [(3)H]RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4]benzodiazepine-3-carboxylate) and [(3)H]L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), which are selective for the benzodiazepine site of alpha 5 subunit-containing receptors, showed high-affinity, specific binding, but to fewer regions than did the nonselective benzodiazepine, [(3)H]flunitrazepam. The pattern mirrored alpha 5 mRNA distribution, and was similar to that previously reported for [(3)H]L-655,708 binding. Displacement of [(3)H]RY-80 bound to hippocampal homogenates, and of [(3)H]flunitrazepam bound to cerebellar and hippocampal homogenates showed comparable displacement by flumazenil (K(i)'s 5--7 nM). However, the K(i)'s for diazepam and for clobazam to displace [(3)H]RY-80 binding in hippocampus were about fourfold higher than for [(3)H]flunitrazepam, and the K(i) for clonazepam was sixfold larger, suggesting that these benzodiazepine receptor agonists bind with relatively lower affinity at hippocampal alpha 5-containing receptors.

  13. New disguises for an old channel: MaxiK channel beta-subunits. (United States)

    Orio, Patricio; Rojas, Patricio; Ferreira, Gonzalo; Latorre, Ramón


    Ca(2+)-activated K(+) channels of large conductance (MaxiK or BK channels) control a large variety of physiological processes, including smooth muscle tone, neurosecretion, and hearing. Despite being coded by a single gene (Slowpoke), the diversity of MaxiK channels is great. Regulatory b-subunits, splicing, and metabolic regulation create this diversity fundamental to the adequate function of many tissues.

  14. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A


    , and protection against thermal denaturation. Deletions delta 171-215 and delta 150-215, however, give rise to truncated molecules which are unable to associate with the alpha-subunit. The intermediate deletion delta 181-215 is still compatible with association, albeit the reconstituted holoenzyme exhibits...

  15. GABAA receptors containing ρ1 subunits contribute to in vivo effects of ethanol in mice.

    Directory of Open Access Journals (Sweden)

    Yuri A Blednov

    Full Text Available GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR, and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ1" antagonist, when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests, but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R-4-amino-cyclopent-1-enyl butylphosphinic acid ("ρ2" antagonist did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.

  16. Species differences in the localization and number of CNS beta adrenergic receptors: Rat versus guinea pig

    Energy Technology Data Exchange (ETDEWEB)

    Booze, R.M.; Crisostomo, E.A.; Davis, J.N.


    The localization and number of beta adrenergic receptors were directly compared in the brains of rats and guinea pigs. The time course of association and saturability of (125I)cyanopindolol (CYP) binding to slide-mounted tissue sections was similar in rats (Kd = 17 pM) and guinea pigs (Kd = 20 pM). The beta-1 and beta-2 receptor subtypes were examined through the use of highly selective unlabeled receptor antagonists, ICI 118,551 (50 nM) and ICI 89,406 (70 nM). Dramatic species differences between rats and guinea pigs were observed in the neuroanatomical regional localization of the beta adrenergic receptor subtypes. For example, in the thalamus prominent beta-1 and beta-2 receptor populations were identified in the rat; however, the entire thalamus of the guinea pig had few, if any, beta adrenergic receptors of either subtype. Hippocampal area CA1 had high levels of beta-2 adrenergic receptors in both rats and guinea pigs but was accompanied by a widespread distribution of beta-2 adrenergic receptors only in rats. Quantitative autoradiographic analyses of 25 selected neuroanatomical regions (1) confirmed the qualitative differences in CNS beta adrenergic receptor localization, (2) determined that guinea pigs had significantly lower levels of beta adrenergic receptors than rats and (3) indicated a differential pattern of receptor subtypes between the two species. Knowledge of species differences in receptor patterns may be useful in designing effective experiments as well as in exploring the relationships between receptor and innervation patterns. Collectively, these data suggest caution be used in extrapolation of the relationships of neurotransmitters and receptors from studies of a single species.

  17. The GluK4 kainate receptor subunit regulates memory, mood, and excitotoxic neurodegeneration. (United States)

    Lowry, E R; Kruyer, A; Norris, E H; Cederroth, C R; Strickland, S


    Though the GluK4 kainate receptor subunit shows limited homology and a restricted expression pattern relative to other kainate receptor subunits, its ablation results in distinct behavioral and molecular phenotypes. GluK4 knockout mice demonstrated impairments in memory acquisition and recall in a Morris water maze test, suggesting a previously unreported role for kainate receptors in spatial memory. GluK4 knockout mice also showed marked hyperactivity and impaired pre-pulse inhibition, thereby mirroring two of the hallmark endophenotypes of patients with schizophrenia and bipolar disorder. Furthermore, we found that GluK4 is a key mediator of excitotoxic neurodegeneration: GluK4 knockout mice showed robust neuroprotection in the CA3 region of the hippocampus following intrahippocampal injection of kainate and widespread neuroprotection throughout the hippocampus following hypoxia-ischemia. Biochemical analysis of kainate- or sham-treated wild-type and GluK4 knockout hippocampal tissue suggests that GluK4 may act through the JNK pathway to regulate the molecular cascades that lead to excitotoxicity. Together, our findings suggest that GluK4 may be relevant to the understanding and treatment of human neuropsychiatric and neurodegenerative disorders.

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

    Directory of Open Access Journals (Sweden)

    Christine Laura Dixon


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

  19. The α5 subunit containing GABAA receptors contribute to chronic pain. (United States)

    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


    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.

  20. Human cardiac beta1- or beta2-adrenergic receptor stimulation and the negative chronotropic effect of low-dose pirenzepine. (United States)

    Jakubetz, J; Schmuck, S; Wochatz, G; Ruhland, B; Poller, U; Radke, J; Brodde, O E


    The M1-muscarinic receptor antagonist pirenzepine in low doses (pirenzepine differ in volunteers with activated cardiac beta1-adrenergic receptors versus activated cardiac beta2-adrenergic receptors. In 17 male volunteers (25 +/- 1 years) we studied effects of pirenzepine infusion (0.5 mg intravenous bolus followed by continuous infusion of 0.15 microg/kg/min) on heart rate and heart rate-corrected duration of electromechanical systole (QS2c, as a measure of inotropism) that had been stimulated by activation of cardiac beta1-adrenergic receptors (bicycle exercise in the supine position for 60 minutes at 25 W) or cardiac beta2-adrenergic receptors (continuous intravenous infusion of 100 ng/kg/min terbutaline). Bicycle exercise and terbutaline infusion significantly increased heart rate and shortened QS2c. When pirenzepine was infused 20 minutes after the beginning of the exercise or terbutaline infusion, heart rate decreased in both settings by approximately the same extent (approximately -10 to -14 beats/min), although exercise and terbutaline infusion continued; however, QS2c was not affected. Pirenzepine (0.05 to 1 mg intravenous bolus)-induced decrease in heart rate was abolished after 6 days of transdermal scopolamine treatment of volunteers. Low-dose pirenzepine decreased heart rate by muscarinic receptor stimulation, because this was blocked by scopolamine. Moreover, low-dose pirenzepine did not differentiate between cardiac beta1- or beta2-adrenergic receptor stimulation; however, low-dose pirenzepine did not affect cardiac contractility as measured by QS2c. Low-dose pirenzepine therefore exerted a unique pattern of action in the human heart: it decreased heart rate (basal and beta1- and/or beta2-adrenergic receptor-stimulated) without affecting contractility.

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

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


    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. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost;


    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...... physiology in mammals. Here, we show that compared to β1 and β3, β2 stabilizes the Na(+)-occluded E1P state relative to the outward-open E2P state, and that the effect is mediated by its transmembrane domain. Molecular dynamics simulations further demonstrate that the tilt angle of the β transmembrane helix...

  3. Development and validation of fluorescent receptor assays based on the human recombinant estrogen receptor subtypes alpha and beta

    NARCIS (Netherlands)

    de boer, T; Otjens, D; Muntendam, A; Meulman, E; van Oostijen, M; Ensing, K


    This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a cla

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

    Directory of Open Access Journals (Sweden)

    Megan K Mulligan

    Full Text Available 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

  5. The brain-specific Beta4 subunit downregulates BK channel cell surface expression. (United States)

    Shruti, Sonal; Urban-Ciecko, Joanna; Fitzpatrick, James A; Brenner, Robert; Bruchez, Marcel P; Barth, Alison L


    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  6. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Directory of Open Access Journals (Sweden)

    Sonal Shruti

    Full Text Available The large-conductance K(+ channel (BK channel can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  7. Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits. (United States)

    Senior, A E; Langman, L; Cox, G B; Gibson, F


    To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.

  8. Expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated and nuclear transfer derived bovine pregnancies. (United States)

    Ravelich, S R; Shelling, A N; Wells, D N; Peterson, A J; Lee, R S F; Ramachandran, A; Keelan, J A


    Bovine nuclear transfer pregnancies are characterized by a high incidence of placental abnormalities, notably, increased placentome size and deficiencies in trophoblast cell function and establishment of placental vasculature. Alterations in gene expression during placental growth and development may contribute to the appearance of large placentomes in pregnancies derived from nuclear transfer. The placenta synthesizes a number of cytokines and growth factors, including the transforming growth factor-betas (TGF-betas) that are involved in the establishment, maintenance and/or regulation of pregnancy. All forms of TGF-beta and their receptors are present at the fetal-maternal interface of the bovine placentome, where they are thought to play an important role in regulating growth, differentiation, and function of the placenta. Using real-time RT-PCR, we have examined the expression of TGF-beta1, TGF-beta2, TGF-beta3 and the receptors TGF-betaRI and TGF-betaRII in placentomes of artificially inseminated (AI) and nuclear transfer (NT)-derived bovine pregnancies at days 50, 100 and 150 of gestation. TGF-beta1, TGF-beta2 and TGF-beta3 mRNA expression increased by 2.0-2.8-fold, while TGF-betaRI and TGF-betaRII mRNA expression decreased by 1.7-2.0-fold in NT placentomes compared to AI controls at all gestational ages examined. These findings indicate that NT placentomes may be resistant to the growth suppressive effects of TGF-betas and could contribute to the placental proliferative abnormalities observed in NT-derived placentas. Alternatively, deficiencies in placentation may provide a mechanism whereby TGF-betas are dysregulated in NT pregnancies.

  9. Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /



    G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequ...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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 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.

  11. Efficient autophosphorylation and phosphorylation of the beta-subunit by casein kinase-2 require the integrity of an acidic cluster 50 residues downstream from the phosphoacceptor site

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;


    -64 are also involved in the process of autophosphorylation, possibly by means of a loop formation. The results obtained with the COOH-terminal-deleted mutants support the view that reconstitution of a functional holoenzyme must occur to allow efficient autophosphorylation. Polylysine prevents...... mutants reconstituting a tetrameric holoenzyme. Only with the three largest COOH-terminal deletion mutants beta delta 150-215, beta delta 171-215, and beta delta 181-215 is no significant alpha-subunit autophosphorylation observed. The phosphorylation of the beta-subunit mutants added in large molar...... excess to CK-2 holoenzyme (either native or recombinant) is also severely impaired by Ala for Glu/Asp substitutions at position 5,6 and in the 55-64 region and by the deletion of the COOH-terminal segments 150-215 and 171-215. Such a phosphorylation is inhibited by polylysine, with the exception...

  12. In vivo affinity label of a protein expressed in Escherichia coli. Coenzyme A occupied the AT(D)P binding site of the mutant F1-ATPase beta subunit (Y307C) through a disulfide bond. (United States)

    Odaka, M; Kiribuchi, K; Allison, W S; Yoshida, M


    When Tyr-307 of the beta subunit of F1-ATPase from a thermophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant beta subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant beta subunit can be reconstituted into the alpha 3 beta 3 complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the beta subunit, Coenzyme A binds to the mutant beta subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

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

    Directory of Open Access Journals (Sweden)

    Gersende Maugars

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


    Directory of Open Access Journals (Sweden)

    A.B.L. Ishak


    Full Text Available The aim of study was to identify the association of FSH beta-subunit gene polymorphisms withsperm quality traits. A total of 470 samples of normal mature bull from several breeds were used forpopulation study and 127 bulls from National and Regional AI centre of Indonesia for association study.To amplify, a PCR-RFLP method was used and digested with Pst1 restriction enzyme. The allelefrequency of the A and B in Bali cattle were (0.000 and (1.000, respectively. The absence of otherallele A suggested that the Bali cattle was monomorphic, while Brahman, FH, Simmental and Limousinewere polymorphic. The highest observed heterozygosity were found in Limousine (0.318 and thehighest expected heterozygosity were in Simmental (0.420. The higher incident of percentage of spermabnormalities were found in Simmental, Limousin, Brahman compared to Bali and FH. Among all typesof sperm abnormalities, the abaxial and microcephalus were found in highest number.

  15. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D


    Cell-cycle transition from the G(2) phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC...... interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2beta causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1-Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found...... regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis.Oncogene advance online publication, 12 May 2008; doi:10.1038/onc.2008.146....

  16. Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

    Institute of Scientific and Technical Information of China (English)

    Frank D.Rinkevich; Jeffrey G.Scott


    Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor.The α6 nicotinic acetyl-choline receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6.RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Ga14-UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants.These Dα6-deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity.The expression of Dα6 was reduced 60%-75% as verified by quantitative polymerase chain reaction.However,there was no change in spinosad sensitivity in D.melanogaster.We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6.RNAi of Tcasα6 did not result in changes in spinosad sensitivity,similar to results obtained with D.melanogaster.The lack of change in spinosad sensitivity in both D.melanogaster and T.castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species.Potential causes for the lack of change in spinosad sensitivity are discussed.

  17. Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition

    Directory of Open Access Journals (Sweden)

    Megan S. Wyeth


    Full Text Available Although Netos are considered auxiliary subunits critical for kainate receptor (KAR function, direct evidence for their regulation of native KARs is limited. Because Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type-specific contexts. We demonstrate Neto1/2 expression in somatostatin (SOM-, cholecystokinin/cannabinoid receptor 1 (CCK/CB1-, and parvalbumin (PV-containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 because kainate yields comparable effects in Neto2 knockouts and wild-types but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation, dampening CCK/CB1 interneuron output, and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediated inhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

  18. Dual actions of enflurane on postsynaptic currents abolished by the gamma-aminobutyric acid type A receptor beta3(N265M) point mutation. (United States)

    Drexler, Berthold; Jurd, Rachel; Rudolph, Uwe; Antkowiak, Bernd


    At concentrations close to 1 minimum alveolar concentration (MAC)-immobility, volatile anesthetics display blocking and prolonging effects on gamma-aminobutyric acid type A receptor-mediated postsynaptic currents. It has been proposed that distinct molecular mechanisms underlie these dual actions. The authors investigated whether the blocking or the prolonging effect of enflurane is altered by a point mutation (N265M) in the beta3 subunit of the gamma-aminobutyric acid type A receptor. Furthermore, the role of the beta3 subunit in producing the depressant actions of enflurane on neocortical neurons was elucidated. Spontaneous inhibitory postsynaptic currents were sampled from neocortical neurons in cultured slices derived from wild-type and beta3(N265M) mutant mice. The effects of 0.3 and 0.6 mm enflurane on decay kinetics, peak amplitude, and charge transfer were quantified. Furthermore, the impact of enflurane-induced changes in spontaneous action potential firing was evaluated by extracellular recordings in slices from wild-type and mutant mice. In slices derived from wild-type mice, enflurane prolonged inhibitory postsynaptic current decays and decreased peak amplitudes. Both effects were almost absent in slices from beta3(N265M) mutant mice. At clinically relevant concentrations between MAC-awake and MAC-immobility, the anesthetic was less effective in depressing spontaneous action potential firing in slices from beta3(N265M) mutant mice compared with wild-type mice. At concentrations between MAC-awake and MAC-immobility, beta3-containing gamma-aminobutyric acid type A receptors contribute to the depressant actions of enflurane in the neocortex. The beta3(N265M) mutation affects both the prolonging and blocking effects of enflurane on gamma-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents in neocortical neurons.

  19. Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families (United States)

    Kyrpides, N. C.; Woese, C. R.


    As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.

  20. AMPA receptor subunit mRNAs and intracellular [Ca(2+)] in cultured mouse and rat cerebellar granule cells

    DEFF Research Database (Denmark)

    Mogensen, Helle Smidt; Jørgensen, Ole Steen


    +) chelator, Fluo-3, and the relative concentrations of mRNAs for the four AMPA receptor subunits, GluR1-4. GluR1-4 mRNAs were measured by restriction enzyme analysis of a PCR product containing cDNA with a composition proportional to the four subunit mRNAs. We found that the [Ca(2+)](i)-response to AMPA...

  1. Beta-Adrenergic Receptor Expression in Muscle Cells (United States)

    Young, Ronald B.; Bridge, K.; Vaughn, J. R.


    beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

  2. Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Anna R Karataeva

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

  4. Endoglin structure and function - Determinants of endoglin phosphorylation by transforming growth factor-beta receptors

    NARCIS (Netherlands)

    Koleva, Rositsa I.; Conley, Barbara A.; Romero, Diana; Riley, Kristin S.; Marto, Jarrod A.; Lux, Andreas; Vary, Calvin P. H.


    Determination of the functional relationship between the transforming growth factor-beta(TGF beta) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGF beta 1 caused recruitment of ALK1 into a complex with end

  5. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors. (United States)

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba


    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus.

  6. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABAA Receptors (United States)

    Reddy, Sandesh D.; Younus, Iyan; Clossen, Bryan L.


    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABAA receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABAA receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABAA receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABAA receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam’s use for controlling acute seizures and status epilepticus. PMID:25784648

  7. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;


    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

  8. Ab initio study of the {sup 57}Fe quadrupole splitting in the heme models of {alpha}- and {beta}-subunits in tetrameric deoxyhemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Yuryeva, E. I. [Ural Branch of the Russian Academy of Sciences, Institute of Solid State Chemistry (Russian Federation); Oshtrakh, M. I., E-mail: [Ural State Technical University - UPI, Division of Applied Biophysics, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation)


    Ab initio X{alpha} discrete variation method was used for calculation of quadrupole splitting for the rough heme models in {alpha}- and {beta}-subunits of tetrameric deoxyhemoglobin accounting small stereochemical variations. The differences of theoretical values of quadrupole splitting for these heme models were obtained.


    NARCIS (Netherlands)



    The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyani

  10. scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis

    NARCIS (Netherlands)

    Brondijk, THC; Durand, R; vanderGiezen, M; Gottschal, JC; Prins, RA; Fevre, M


    A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counterparts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degr

  11. The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting beta-subunit of neuronal K(V)4.2 channels. (United States)

    Dougherty, Kevin; Tu, Liwei; Deutsch, Carol; Covarrubias, Manuel


    Auxiliary beta-subunits dictate the physiological properties of voltage-gated K(+) (K(V)) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific beta-subunit of neuronal K(V)4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel's voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated K(V)4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and K(V)1.3 channels or the K(V)4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the K(V)4.2-VSD is unique among the known actions of K(V) channel beta-subunits. This study shows that the modular VSD of a K(V) channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic beta-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.

  12. Do specific NMDA receptor subunits act as gateways for addictive behaviors? (United States)

    Hopf, F W


    Addiction to alcohol and drugs is a major social and economic problem, and there is considerable interest in understanding the molecular mechanisms that promote addictive drives. A number of proteins have been identified that contribute to expression of addictive behaviors. NMDA receptors (NMDARs), a subclass of ionotropic glutamate receptors, have been of particular interest because their physiological properties make them an attractive candidate for gating induction of synaptic plasticity, a molecular change thought to mediate learning and memory. NMDARs are generally inactive at the hyperpolarized resting potentials of many neurons. However, given sufficient depolarization, NMDARs are activated and exhibit long-lasting currents with significant calcium permeability. Also, in addition to stimulating neurons by direct depolarization, NMDARs and their calcium signaling can allow strong and/or synchronized inputs to produce long-term changes in other molecules (such as AMPA-type glutamate receptors) which can last from days to years, binding internal and external stimuli in a long-term memory trace. Such memories could allow salient drug-related stimuli to exert strong control over future behaviors and thus promote addictive drives. Finally, NMDARs may themselves undergo plasticity, which can alter subsequent neuronal stimulation and/or the ability to induce plasticity. This review will address recent and past findings suggesting that NMDAR activity promotes drug- and alcohol-related behaviors, with a particular focus on GluN2B subunits as possible central regulators of many addictive behaviors, as well as newer studies examining the importance of non-canonical NMDAR subunits and endogenous NMDAR cofactors. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  13. Novel O-palmitolylated beta-E1 subunit of pyruvate dehydrogenase is phosphorylated during ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Barr Amy J


    Full Text Available Abstract Background During and following myocardial ischemia, glucose oxidation rates are low and fatty acids dominate as a source of oxidative metabolism. This metabolic phenotype is associated with contractile dysfunction during reperfusion. To determine the mechanism of this reliance on fatty acid oxidation as a source of ATP generation, a functional proteomics approach was utilized. Results 2-D gel electrophoresis of mitochondria from working rat hearts subjected to 25 minutes of global no flow ischemia followed by 40 minutes of aerobic reperfusion identified 32 changes in protein abundance compared to aerobic controls. Of the five proteins with the greatest change in abundance, two were increased (long chain acyl-coenzyme A dehydrogenase (48 ± 1 versus 39 ± 3 arbitrary units, n = 3, P In silico analysis identified the putative kinases as the insulin receptor kinase for the more basic form and protein kinase Cζ or protein kinase A for the more acidic form. These modifications of pyruvate dehydrogenase are associated with a 35% decrease in glucose oxidation during reperfusion. Conclusions Cardiac ischemia/reperfusion induces significant changes to a number of metabolic proteins of the mitochondrial proteome. In particular, ischemia/reperfusion induced the post-translational modification of pyruvate dehydrogenase, the rate-limiting step of glucose oxidation, which is associated with a 35% decrease in glucose oxidation during reperfusion. Therefore these post-translational modifications may have important implications in the regulation of myocardial energy metabolism.

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

    Waldvogel, H J; Faull, R L M


    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. © 2015 Elsevier Inc. All rights reserved.

  15. [Regulation of G protein-coupled receptor kinase activity]. (United States)

    Haga, T; Haga, K; Kameyama, K; Nakata, H


    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  16. Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES. (United States)

    Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K


    A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.

  17. Beta-adrenergic receptors are differentially expressed in distinct interneuron subtypes in the rat hippocampus. (United States)

    Cox, David J; Racca, Claudia; LeBeau, Fiona E N


    Noradrenaline (NA) acting via beta-adrenergic receptors (betaARs) plays an important role in the modulation of memory in the hippocampus. betaARs have been shown to be expressed in principal cells, but their distribution across different interneuron classes is unknown. We have used specific interneuron markers including calcium binding proteins (parvalbumin, calbindin, and calretinin) and neuropeptides (somatostatin, neuropeptide Y, and cholecystokinin) together with either beta1AR or beta2AR to determine the distribution of these receptors in all major subfields of the hippocampus. We found that beta1AR-expressing interneurons were more prevalent in the CA3 and CA1 regions of the hippocampus than in the dentate gyrus, where they were relatively sparse. beta2AR-expressing interneurons were more uniformly distributed between all three regions of the hippocampus. A high proportion of neuropeptide Y-containing interneurons in the dentate gyrus co-expressed beta2AR. beta1AR labeling was common in interneurons expressing somatostatin and parvalbumin in the CA3 and CA1 regions, particularly in the stratum oriens of these regions. beta2AR labeling was more likely to be found than beta1AR labeling in cholecystokinin-expressing interneurons. In contrast, calretinin-containing interneurons were virtually devoid of beta1AR or beta2AR labeling. These regional and interneuron type-specific differences suggest functionally distinct roles for NA in modulating hippocampal activity via activation of betaARs.

  18. Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively

    DEFF Research Database (Denmark)

    Collins, C; Duff, C; Duncan, A M;


    A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor...... subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1...... that the gene for torsion dystonia has also been localized by genetic studies to 9q34.3, the same regional map location as NMDAR1....

  19. Function of Partially Duplicated Human α7 Nicotinic Receptor Subunit CHRFAM7A Gene (United States)

    de Lucas-Cerrillo, Ana M.; Maldifassi, M. Constanza; Arnalich, Francisco; Renart, Jaime; Atienza, Gema; Serantes, Rocío; Cruces, Jesús; Sánchez-Pacheco, Aurora; Andrés-Mateos, Eva; Montiel, Carmen


    The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response. PMID:21047781

  20. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells. (United States)

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


    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.

  1. The paracaspase MALT1 cleaves the LUBAC subunit HOIL1 during antigen receptor signaling. (United States)

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas


    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.

  2. A Presynaptic Glutamate Receptor Subunit Confers Robustness to Neurotransmission and Homeostatic Potentiation

    Directory of Open Access Journals (Sweden)

    Beril Kiragasi


    Full Text Available Homeostatic signaling systems are thought to interface with other forms of plasticity to ensure flexible yet stable levels of neurotransmission. The role of neurotransmitter receptors in this process, beyond mediating neurotransmission itself, is not known. Through a forward genetic screen, we have identified the Drosophila kainate-type ionotropic glutamate receptor subunit DKaiR1D to be required for the retrograde, homeostatic potentiation of synaptic strength. DKaiR1D is necessary in presynaptic motor neurons, localized near active zones, and confers robustness to the calcium sensitivity of baseline synaptic transmission. Acute pharmacological blockade of DKaiR1D disrupts homeostatic plasticity, indicating that this receptor is required for the expression of this process, distinct from developmental roles. Finally, we demonstrate that calcium permeability through DKaiR1D is necessary for baseline synaptic transmission, but not for homeostatic signaling. We propose that DKaiR1D is a glutamate autoreceptor that promotes robustness to synaptic strength and plasticity with active zone specificity.

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

    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


    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.

  4. Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit* (United States)

    Mahalingam, Mohana; Perez, Claudio F.; Fessenden, James D.


    The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S. These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling. PMID:27129199

  5. Fluorescence Resonance Energy Transfer-based Structural Analysis of the Dihydropyridine Receptor α1S Subunit Reveals Conformational Differences Induced by Binding of the β1a Subunit. (United States)

    Mahalingam, Mohana; Perez, Claudio F; Fessenden, James D


    The skeletal muscle dihydropyridine receptor α1S subunit plays a key role in skeletal muscle excitation-contraction coupling by sensing membrane voltage changes and then triggering intracellular calcium release. The cytoplasmic loops connecting four homologous α1S structural domains have diverse functions, but their structural arrangement is poorly understood. Here, we used a novel FRET-based method to characterize the relative proximity of these intracellular loops in α1S subunits expressed in intact cells. In dysgenic myotubes, energy transfer was observed from an N-terminal-fused YFP to a FRET acceptor, ReAsH (resorufin arsenical hairpin binder), targeted to each α1S intracellular loop, with the highest FRET efficiencies measured to the α1S II-III loop and C-terminal tail. However, in HEK-293T cells, FRET efficiencies from the α1S N terminus to the II-III and III-IV loops and the C-terminal tail were significantly lower, thus suggesting that these loop structures are influenced by the cellular microenvironment. The addition of the β1a dihydropyridine receptor subunit enhanced FRET to the II-III loop, thus indicating that β1a binding directly affects II-III loop conformation. This specific structural change required the C-terminal 36 amino acids of β1a, which are essential to support EC coupling. Direct FRET measurements between α1S and β1a confirmed that both wild type and truncated β1a bind similarly to α1S These results provide new insights into the role of muscle-specific proteins on the structural arrangement of α1S intracellular loops and point to a new conformational effect of the β1a subunit in supporting skeletal muscle excitation-contraction coupling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells. (United States)

    Bousquet-Mélou, A; Muñoz, C; Galitzky, J; Berlan, M; Lafontan, M


    The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Muñoz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.

  7. Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse. (United States)

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


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

  8. Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits. (United States)

    Alzayady, Kamil J; Wagner, Larry E; Chandrasekhar, Rahul; Monteagudo, Alina; Godiska, Ronald; Tall, Gregory G; Joseph, Suresh K; Yule, David I


    Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

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

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    Florian Wegner

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

  10. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits. (United States)

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes


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

  11. Identification of proteasome subunit beta type 6 (PSMB6) associated with deltamethrin resistance in mosquitoes by proteomic and bioassay analyses. (United States)

    Sun, Linchun; Ye, Yuting; Sun, Haibo; Yu, Jing; Zhang, Li; Sun, Yan; Zhang, Donghui; Ma, Lei; Shen, Bo; Zhu, Changliang


    Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.

  12. Identification of proteasome subunit beta type 6 (PSMB6 associated with deltamethrin resistance in mosquitoes by proteomic and bioassay analyses.

    Directory of Open Access Journals (Sweden)

    Linchun Sun

    Full Text Available Deltamethrin (DM insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE and mass spectrometry (MS. Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6 is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.

  13. Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its beta1 subunit. (United States)

    Maltsev, Victor A; Kyle, John W; Undrovinas, Albertas


    Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I(NaL)) for the function of both normal and, especially, failing myocardium, in which I(NaL) is reportedly increased. While recent molecular studies identified the cardiac Na(+) channel (NaCh) alpha subunit isoform (Na(v)1.5) as a major contributor to I (NaL), the molecular mechanisms underlying alterations of I(NaL) in heart failure (HF) are still unknown. Here we tested the hypothesis that I(NaL) is modulated by the NaCh auxiliary beta subunits. tsA201 cells were transfected simultaneously with human Na(v)1.5 (former hH1a) and cardiac beta(1) or beta(2) subunits, and whole-cell patch-clamp experiments were performed. We found that I(NaL) decay kinetics were significantly slower in cells expressing alpha + beta(1) (time constant tau = 0.73 +/- 0.16 s, n = 14, mean +/- SEM, P < 0.05) but remained unchanged in cells expressing alpha + beta(2) (tau = 0.52 +/- 0.09 s, n = 5), compared with cells expressing Na(v)1.5 alone (tau = 0.54 +/- 0.09 s, n = 20). Also, beta(1), but not beta(2), dramatically increased I(NaL) relative to the maximum peak current, I(NaT) (2.3 +/- 0.48%, n = 14 vs. 0.48 +/- 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary beta(1) subunit modulates I(NaL), produced by the human cardiac Na(+) channel Na(v)1.5 by slowing its decay and increasing I(NaL) amplitude relative to I(NaT). Because expression of Na(v)1.5 reportedly decreases but beta(1) remains unchanged in chronic HF, the relatively higher expression of beta(1) may contribute to the known I(NaL) increase in HF via the modulation mechanism found in this study.

  14. Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells. (United States)

    Cappelletti, Vera; Miodini, Patrizia; Di Fronzo, Giovanni; Daidone, Maria Grazia


    High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

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

    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


    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.

  16. Molecular cloning of the cDNA encoding follicle-stimulating hormone beta subunit of the Chinese soft-shell turtle Pelodiscus sinensis, and its gene expression. (United States)

    Chien, Jung-Tsun; Shen, San-Tai; Lin, Yao-Sung; Yu, John Yuh-Lin


    Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSHbeta in reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae) FSHbeta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned Chinese soft-shell turtle FSHbeta cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 206-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHbeta subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the Chinese soft-shell turtle FSHbeta subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSHbeta shares identities of 97% with Reeves's turtle (Family of Bataguridae), 83-89% with birds, 61-70% with mammals, 63-66% with amphibians and 40-58% with fish. By contrast, when comparing the FSHbeta with the beta-subunits of the Chinese soft-shell turtle luteinizing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree including reptilian species of FSHbeta subunits, is presented for the first time. Out of various tissues examined, FSHbeta mRNA was only expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by fluorescence real-time PCR analysis.

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

    NARCIS (Netherlands)

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


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

  18. MAP1B binds to the NMDA receptor subunit NR3A and affects NR3A protein concentrations

    DEFF Research Database (Denmark)

    Eriksson, Maria; Samuelsson, Helena; Björklund, Stefan;


    Incorporation of the N-methyl-d-aspartate receptor (NMDAR) subunit NR3A into functional NMDARs results in reduced channel conductance and Ca(2+) permeability. To further investigate the function of NR3A, we have set out to characterize its intracellular binding partners. Here, we report a novel p...

  19. Deletion of the GluA1 AMPA Receptor Subunit Alters the Expression of Short-Term Memory (United States)

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


    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.…

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

    NARCIS (Netherlands)

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


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

  1. Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.T.; Lee, Y.C.


    The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which (43 kilodalton (kDa)) exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an /sup 125/I-labeled high-affinity reagent the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. The authors postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work.

  2. Suppression of the inflammatory response in experimental arthritis is mediated via estrogen receptor alpha but not estrogen receptor beta

    NARCIS (Netherlands)

    Dulos, John; Vijn, Peter; van Doorn, Cindy; Hofstra, Claudia L.; Veening-Griffioen, Desiree; de Graaf, Jan; Dijcks, Fred A.; Boots, Annemieke M. H.


    Introduction: The immune modulatory role of estrogens in inflammation is complex. Both pro- and anti-inflammatory effects of estrogens have been described. Estrogens bind both estrogen receptor (ER)alpha and beta. The contribution of ER alpha and ER beta to ER-mediated immune modulation was studied

  3. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M


    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

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

    Martin, Jessica A; Garczynski, Stephen F


    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.

  5. Spinal D1-like dopamine receptors modulate NMDA receptor-induced hyperexcitability and NR1 subunit phosphorylation at serine 889. (United States)

    Aira, Zigor; Barrenetxea, Teresa; Buesa, Itsaso; Martínez, Endika; Azkue, Jon Jatsu


    Activation of the N-methyl-d-aspartate receptor (NMDAR) in dorsal horn neurons is recognized as a fundamental mechanism of central sensitization and pathologic pain. This study assessed the influence of dopaminergic, D1-like receptor-mediated input to the spinal dorsal horn on NMDAR function. Spinal superfusion with selective NMDAR agonist cis-ACPD significantly increased C-fiber-evoked field potentials in rats subjected to spinal nerve ligation (SNL), but not in sham-operated rats. Simultaneous application of D1LR antagonist SCH 23390 dramatically reduced hyperexcitability induced by cis-ACPD. Furthermore, cis-ACPD-induced hyperexcitability seen in nerve-ligated rats could be mimicked in unin-jured rats during stimulation of D1LRs by agonist SKF 38393 at subthreshold concentration. Phosphorylation of NMDAR subunit NR1 at serine 889 at postsynaptic sites was found to be increased in dorsal horn neurons 90 min after SNL, as assessed by increased co-localization with postsynaptic marker PSD-95. Increased NR1 phosphorylation was attenuated in the presence of SCH 23390 in the spinal superfusate. The present results support that D1LRs regulate most basic determinants of NMDAR function in dorsal horn neurons, suggesting a potential mechanism whereby dopaminergic input to the dorsal horn can modulate central sensitization and pathologic pain.

  6. Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus. (United States)

    Standaert, D G; Landwehrmeyer, G B; Kerner, J A; Penney, J B; Young, A B


    NMDA receptors are composed of proteins from two families: NMDAR1, which are required for channel activity, and NMDAR2, which modulate properties of the channels. The mRNA encoding the NMDAR2D subunit has a highly restricted pattern of expression: in the forebrain, it is found in only a small subset of cortical, neostriatal and hippocampal neurons. We have used a quantitative double-label in situ hybridization method to examine the expression of NMDAR2D mRNA in neurochemically defined populations of neurons. In the neostriatum, NMDAR2D was expressed by the interneuron populations marked by preprosomatostatin (SOM), the 67-kDa form of glutamic acid decarboxylase (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) mRNAs but not by the projection neurons expressing beta-preprotachykinin (SP) or preproenkephalin (ENK) mRNAs. In the neocortex, NMDAR2D expression was observed in only a small number of neurons, but these included almost all of the SOM-, GAD67-, and PARV-expressing interneurons. In the hippocampus, NMDAR2D was not present in pyramidal or granule cells, but was abundant in SOM-, GAD67-, and PARV-positive interneurons. NMDAR2D expression appears to be a property shared by interneurons in several regions of the brain. The unique electrophysiological characteristics conveyed by this subunit, which include resistance to blockade by magnesium ion and long channel offset latencies, may be important for the integrative functions of these neurons. NMDAR2D-containing receptor complexes may prove to be important therapeutic targets in human disorders of movement. In addition, the presence of NMDAR2D subunits may contribute to the differential vulnerability of interneurons to excitotoxic injury.

  7. Translation initiation factor (iso) 4E interacts with BTF3, the beta subunit of the nascent polypeptide-associated complex. (United States)

    Freire, Miguel Angel


    A two-hybrid screen with the translation initiation factor, eIF(iso)4E from Arabidopsis, identified a clone encoding a lipoxygenase type 2 [Freire, M.A., et al., 2000. Plant lipoxygenase 2 is a translation initiation factor-4E-binding protein. Plant Molecular Biology 44, 129-140], and three cDNA clones encoding the homologue of the mammalian BTF3 factor, the beta subunit of the nascent polypeptide-associated complex (NAC). Here we report on the interaction between the translation initiation factor eIF(iso)4E and AtBTF3. AtBTF3 protein is able to interact with the wheat initiation factors eIF4E and eIF(iso)4E. AtBTF3 contains a sequence related to the prototypic motif found on most of the 4E-binding proteins, and competes with the translation initiation factor eIF(iso)4G for eIF4(iso)4E binding, in a two hybrid interference assay. These findings provide a molecular link between the translation initiation mechanism and the emergence of the nascent polypeptide chains.

  8. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin. (United States)

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela


    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile.

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

    Directory of Open Access Journals (Sweden)

    Amanda Lorraine Wright


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

  10. TGF-beta isoforms and TGF-beta receptors in drug-induced and hereditary gingival overgrowth. (United States)

    Wright, H J; Chapple, I L; Matthews, J B


    Drug therapy and hereditary factors are two of the main causes of gingival overgrowth (GO). Both of these forms of GO are associated with increased extracellular matrix production by fibroblasts. Transforming growth factor beta (TGF-beta) is an important mediator of wound healing and tissue regeneration, which stimulates fibroblasts to produce extracellular matrix materials. The aim of this immunohistochemical study was to determine whether there is any altered expression of TGF-beta isoforms or its receptors in tissue from patients with drug-induced GO (DIGO; n=10) and hereditary gingival fibromatosis (n=10) when compared to non-overgrowth tissue (n=10). Compared to control tissues, significantly more fibroblasts expressed TGF-beta1 in both DIGO and hereditary gingival fibromatosis tissues (Pfibroblast densities between groups, there was a proportional increase in TGF-beta3 as well as TGF-beta1 expressing cells within both overgrowth populations (Preceptor-positive cells in the total cell population analysed in overgrowth tissues (Pisoform and receptor expression by fibroblasts in gingival overgrowth that may contribute to disease pathogenesis.

  11. Integrin alpha(3)-subunit expression modulates alveolar epithelial cell monolayer formation. (United States)

    Lubman, R L; Zhang, X L; Zheng, J; Ocampo, L; Lopez, M Z; Veeraraghavan, S; Zabski, S M; Danto, S I; Borok, Z


    We investigated expression of the alpha(3)-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the alpha(3)-integrin subunit on AEC monolayer formation. alpha(3)-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. alpha(3)- and beta(1)-integrin subunits coprecipitated in immunoprecipitation experiments with alpha(3)- and beta(1)-subunit-specific antibodies, consistent with their association as the alpha(3)beta(1)-integrin receptor at the cell membrane. Treatment with blocking anti-alpha(3) monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that alpha(3)- and beta(1)-integrin subunits are expressed in AEC monolayers where they form the heterodimeric alpha(3)beta(1)-integrin receptor at the cell membrane. Blockade of the alpha(3)-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the alpha(3)-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the alpha(3)beta(1)-integrin receptor in AEC adhesion.

  12. Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats. (United States)

    Pellegrini-Giampietro, D E; Zukin, R S; Bennett, M V; Cho, S; Pulsinelli, W A


    Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.

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

    Directory of Open Access Journals (Sweden)

    Liisa A. Tremere


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

  14. Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W


    associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage...

  15. A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF. (United States)

    Tavernier, J; Devos, R; Cornelis, S; Tuypens, T; Van der Heyden, J; Fiers, W; Plaetinck, G


    cDNA clones encoding two receptor proteins involved in the binding of human interleukin 5 (hIL5) have been isolated. A first class codes for an IL5-specific chain (hIL5R alpha). The major transcript of this receptor gene, as analyzed in both HL-60 eosinophilic cells and eosinophilic myelocytes grown from cord blood, encodes a secreted form of this receptor. This soluble hIL5R alpha has antagonistic properties. A second component of the hIL5R is found to be identical to the beta chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) high affinity receptor. The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each other's binding and have highly overlapping biological activities on eosinophils.

  16. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C


    of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha......The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  17. Molecular cloning of cDNAs and structural model analysis of two gonadotropin beta-subunits of snakehead fish (Channa maculata). (United States)

    Chatterjee, Abhijit; Shen, San-Tai; Yu, John Yuh-Lin


    The cDNAs encoding beta-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been cloned from the pituitary of snakehead fish, Channa maculata, and the three-dimensional structural models of the encoded FSH and LH were investigated. The cloned cDNAs, including 5'-untranslated region (UTR), open-reading frame, and 3'-UTR followed by a poly(A) tail, were obtained by reverse transcription-polymerase chain reaction and rapid amplification of cDNA end methods. The open-reading frames of FSH-beta cDNA encodes a 120-amino acid protein with a signal peptide of 18 amino acids and a mature protein of 102 amino acids; while LH-beta cDNA encodes a 140-amino acid protein with a signal peptide of 33 amino acids and a mature protein of 115 amino acids. The amino acid sequence identities of snakehead fish FSH-beta and LH-beta in comparison with other fish are 27.8-81.9% and 45.2-88.8%, respectively; while in comparison with tetrapods are 26.2-28.9% and 37.5-51.2%, respectively. Both FSH-beta and LH-beta of snakehead fish resemble most to those of Perciformes, implying their closer phylogenetic relationship. All 12 cysteine residues are conserved in snakehead fish LH-beta; while 11 cysteine residues are conserved in its FSH-beta. The third cysteine is absent in snakehead fish FSH-beta; instead, a positionally shifted cysteine residue is present at the N-terminus, as found in some phylogenetic related fish. The structure models of snakehead fish FSH and LH, constructed by using the crystal structures of human FSH and human chorionic gonadotropin as respective template, showed that the positionally shifted N-terminal cysteine residue of snakehead fish FSH-beta likely can substitute the third cysteine to form a disulfide bond with the 12th cysteine.

  18. PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-d-aspartate receptor subunit NR2A



    Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-d-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in fyn-mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpre...

  19. Normotensive sodium loading in conscious dogs: Regulation of renin secretion during beta receptor blockade

    DEFF Research Database (Denmark)

    Bie, Peter; Mølstrøm, Simon; Wamberg, Søren


    irrespective of diet. In conclusion, PRC depended on dietary sodium and beta1-adrenergic control as expected; however, the acute sodium-driven decrease in PRC at constant MAP and GFR was unaffected by beta1-receptor blockade demonstrating that renin may be regulated without changes in MAP, GFR, or beta1......Renin secretion is regulated in part by renal nerves operating through beta1-receptors of the renal juxtaglomerular cells. Slow sodium loading may decrease plasma renin (PRC) and cause natriuresis at constant mean arterial blood pressure (MAP) and glomerular filtration rate (GFR). We hypothesized...... that in this setting, renin secretion and renin-dependent sodium excretion are controlled by via the renal nerves and therefore eliminated or reduced by blocking the action of norepinephrine on the juxtaglomerular cells with the beta1-receptor antagonist metoprolol. This was tested in conscious dogs by infusion of Na...

  20. The benzodiazepine receptor in rat brain and its interaction with ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Martin, I.L.; Doble, A.


    (3H)Ethyl beta-carboline-3-carboxylate ((3H) beta-CCE) binds to a homogeneous population of recognition sites in rat whole brain membranes with high affinity. The (3H)beta-CCE binding is completely displaceable by low concentrations of a number of benzodiazepines with similar potencies found when using a 3H-benzodiazepine as the ligand. This suggests that the recognition sites for beta-CCE and the benzodiazepines are identical or that they are involved in a close interaction. The binding of (3H)beta-CCE does not obey simple mass-action kinetics. (3H)Flunitrazepam dissociation from its receptor population is biphasic, and different methods of initiation of this dissociation indicate that cooperative interactions take place within the receptor population. We conclude that the benzodiazepine receptor is a single entity that can exist in two conformations, the equilibrium between which may be controlled by some as yet unidentified factor.

  1. Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures. (United States)

    Gonzalez, R R; Leavis, P


    Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

  2. Anxiolytics not acting at the benzodiazepine receptor: beta blockers. (United States)

    Tyrer, P


    1. Although there is clear evidence for many controlled trials in the past 25 years that beta blockers are effective in anxiety disorders clear indications for their use are lacking. 2. The balance of evidence suggests that the mechanism of action of beta-blocking drugs is through peripheral blockade of beta-mediated symptoms. 3. Most evidence to the efficacy of beta-blockers comes from study of their use in generalized anxiety and in acute stress. 4. Because beta-blockers carry no risks of pharmacological dependence they may be preferred to many other anti-anxiety drugs.

  3. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis. (United States)

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


    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

  4. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus. (United States)

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


    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. Copyright © 2015. Published by Elsevier B.V.

  5. AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus. (United States)

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


    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.

  6. Characterization of a novel monoclonal antibody with restricted specificity to the free beta 2 integrin alpha M CD11b subunit. (United States)

    Tanfous, Naouel Guedel-Ben; Essafi, Makram; Larguech, Beya; Barbouche, Ridha; Fathallah, Dahmani M


    Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.

  7. Lurasidone and fluoxetine reduce novelty-induced hypophagia and NMDA receptor subunit and PSD-95 expression in mouse brain. (United States)

    Stan, Tiberiu Loredan; Sousa, Vasco Cabral; Zhang, Xiaoqun; Ono, Michiko; Svenningsson, Per


    Lurasidone, a novel second-generation antipsychotic agent, exerts antidepressant actions in patients suffering from bipolar type I disorder. Lurasidone acts as a high affinity antagonist at multiple monoamine receptors, particularly 5-HT2A, 5-HT7, D2 and α2 receptors, and as a partial agonist at 5-HT1A receptors. Accumulating evidence indicates therapeutic actions by monoaminergic antidepressants are mediated via alterations of glutamate receptor-mediated neurotransmission. Here, we used mice and investigated the effects of chronic oral administration of vehicle, lurasidone (3 or 10mg/kg) or fluoxetine (20mg/kg) in the novelty induced hypophagia test, a behavioral test sensitive to chronic antidepressant treatment. We subsequently performed biochemical analyses on NMDA receptor subunits and associated proteins. Both lurasidone and fluoxetine reduced the latency to feed in the novelty-induced hypophagia test. Western blotting experiments showed that both lurasidone and fluoxetine decreased the total levels of NR1, NR2A and NR2B subunits of NMDA receptors and PSD-95 (PostSynaptic Density-95) in hippocampus and prefrontal cortex. Taken together, these data indicate that antidepressant/anxiolytic-like effects of lurasidone, as well as fluoxetine, could involve reduced NMDA receptor-mediated signal transduction, particularly in pathways regulated by PSD-95, in hippocampus and prefrontal cortex.

  8. Synthesis and characterization of human recombinant thyrotropin (rec-hTSH) with a chimeric {beta}-subunit (rec-hTSH{beta}-CTPE hCG{beta}); Sintese e caracterizacao do hormonio tireotrofico humano recombinante (rec-hTSH) contendo uma subunidade {beta} quimerica (rec-hTSH{beta}-CTPE hCG{beta})

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Yoko


    Recombinant hTSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of hTSH by fusing the carboxy terminal extension peptide (CTEP) of hCG{beta} onto hTSH{beta}. When coexpressed either with {alpha}-subunit complementary DNA or {alpha}-minigene in African green monkey (Cos-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 29 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild type (WT) and chimeric hTSH secreted by Cos-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the difference in the terminal sialic acid and sulfate of their oligosaccharide chains. Chimeric and WT hTSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [{sup 3} H]-thymidine incorporation. Chimeric hTSH secreted by Cos-7 appears to be more active than that secreted by 293 cells, as judged by growth assay. Cos-7 produced chimeric hTSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly {beta} and to a lesser extent {alpha}, appears to be responsible, at least in part, for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby in the in vivo bioactivity. (author). 104 refs., 23 figs., 3 tabs.

  9. Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase. (United States)

    Peebles, Christie A M; Hong, Seung-Beom; Gibson, Susan I; Shanks, Jacqueline V; San, Ka-Yiu


    Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

  10. The transforming growth factor-beta receptor genes and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Ynte M.; Baas, Annette F.; Medic, Jelena; Wijmenga, Cisca; Rinkel, Gabriel J. E.


    Background Mutations in the receptor genes of the transforming growth factor beta pathway, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysms, while genetic variants in TGFBR1 and TGFBR2 are associated with abdominal aortic aneurysms. The transforming growth factor-beta pathway may be

  11. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Cherezov, Vadim; Hanson, Michael A


    crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context...

  12. Crystal structure of the human beta2 adrenergic G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; Choi, Hee-Jung; Rosenbaum, Daniel M;


    Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which...

  13. Habenular expression of rare missense variants of the β4 nicotinic receptor subunit alters nicotine consumption (United States)

    Ślimak, Marta A.; Ables, Jessica L.; Frahm, Silke; Antolin-Fontes, Beatriz; Santos-Torres, Julio; Moretti, Milena; Gotti, Cecilia; Ibañez-Tallon, Inés


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

  14. Genetic ablation of NMDA receptor subunit NR3B in mouse reveals motoneuronal and nonmotoneuronal phenotypes. (United States)

    Niemann, Stephan; Kanki, Hiroaki; Fukui, Yasuyuki; Takao, Keizo; Fukaya, Masahiro; Hynynen, Meri N; Churchill, Michael J; Shefner, Jeremy M; Bronson, Roderick T; Brown, Robert H; Watanabe, Masahiko; Miyakawa, Tsuyoshi; Itohara, Shigeyoshi; Hayashi, Yasunori


    NR3B is a modulatory subunit of the NMDA receptor, abundantly expressed in both cranial and spinal somatic motoneurons and at lower levels in other regions of the brain as well. Recently, we found the human NR3B gene (GRIN3B) to be highly genetically heterogeneous, and that approximately 10% of the normal European-American population lacks NR3B due to homozygous occurrence of a null allele in the gene. Therefore, it is especially important to understand the phenotypic consequences of the genetic loss of NR3B in both humans and animal models. We here provide results of behavioral analysis of mice genetically lacking NR3B, which is an ideal animal model due to homogeneity in genetic and environmental background. The NR3B(-/-) mice are viable and fertile. Consistent with the expression of NR3B in somatic motoneurons, the NR3B(-/-) mice showed a moderate but significant impairment in motor learning or coordination, and decreased activity in their home cages. Remarkably, the NR3B(-/-) mice showed a highly increased social interaction with their familiar cage mates in their home cage but moderately increased anxiety-like behaviour and decreased social interaction in a novel environment, consistent with the inhibitory role of NR3B on the functions of NMDA receptors. This work is the first reporting of the functional significance of NR3B in vivo and may give insight into the contribution of genetic variability of NR3B in the phenotypic heterogeneity among human population.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  16. N-methyl-D-aspartate receptor subunit changes after traumatic injury to the developing brain. (United States)

    Giza, Christopher C; Maria, Naomi S Santa; Hovda, David A


    Traumatic brain injury (TBI) is a major cause of disability in the pediatric population and can result in abnormal development. Experimental studies conducted in animals have revealed impaired plasticity following developmental TBI, even in the absence of significant anatomical damage. The N-methyl-D-aspartate receptor (NMDAR) is clearly involved in both normal development and in the pathophysiology of TBI. Following lateral fluid percussion injury in postnatal day (PND) 19 rats, we tested the hypothesis that TBI sustained at an early age would result in impaired NMDAR expression. Using immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR), protein and RNA levels of NMDAR subunits were measured in the cerebral cortex and hippocampus on post-injury days (PID) 1, 2, 4, and 7 (though the PID7 analysis was only for protein) and compared with age-matched shams. Significant effects of hemisphere (analysis of variance [ANOVA], pPID1, PID2, PID4, and PID7, respectively. Within the cortex, there was a significant effect of injury (ANOVA, pPID1. It is known that NR2A expression levels increase during normal development, and in response to environmental stimuli. Our data suggest that injury-induced reduction in the expression of NR2A is one likely mechanism for the impaired experience-dependent neuroplasticity seen following traumatic injury to the immature brain.

  17. TGF beta-1 dependent fast stimulation of ATM and p53 phosphorylation following exposure to ionizing radiation does not involve TGF beta-receptor I signalling

    NARCIS (Netherlands)

    Wiegman, Erwin M.; Blaese, Marcet A.; Loeffler, Heidi; Coppes, Rob P.; Rodemann, H. Peter


    Background and purpose: It has been proposed that radiation induced stimulation of ATM and downstream components involves activation of TGF beta-1 and that this may be due to TGF beta-1-receptor I-Smad signalling. Therefore, the aim of this study was to clarify the distinct role of TGF beta-1-recept

  18. Reinforcing Effects Of Compounds Lacking Intrinsic Efficacy At α1 Subunit-Containing GABAA Receptor Subtypes in Midazolam- But Not Cocaine-Experienced Rhesus Monkeys (United States)

    Shinday, Nina M; Sawyer, Eileen K; Fischer, Bradford D; Platt, Donna M; Licata, Stephanie C; Atack, John R; Dawson, Gerard R; Reynolds, David S; Rowlett, James K


    Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors (‘α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration. PMID:23303046

  19. Ser49Gly of beta1-adrenergic receptor is associated with effective beta-blocker dose in dilated cardiomyopathy. (United States)

    Magnusson, Yvonne; Levin, Malin C; Eggertsen, Robert; Nyström, Ernst; Mobini, Reza; Schaufelberger, Maria; Andersson, Bert


    Our objective was to evaluate the influence of polymorphisms at codons 49 and 389 of the beta1-adrenergic receptor (beta1-AR) on the response to beta-blockers and outcome in patients with dilated cardiomyopathy. We genotyped both codons of the beta1-AR in 375 patients with dilated cardiomyopathy and 492 control subjects. Neither of the polymorphisms was associated with susceptibility for dilated cardiomyopathy. In a retrospective analysis of patients receiving beta-blockers, there was a significant association between long-term survival rate and codon 49 (P = .014) but not codon 389 (P = .08). Despite a similar mean heart rate (69 beats/min), patients with the Ser49 genotype tended to have higher doses of beta-blockade compared with Gly49 carriers (P = .065). In patients receiving a low dose of beta-blockade (< or = 50% of targeted full dose), the 5-year mortality rate was lower among Gly49 carriers than Ser49 patients (risk ratio [RR], 0.24; 95% confidence interval [CI], 0.07-0.80; P = .020). In patients receiving high doses of beta-blockers, there was no significant difference in outcome between genotypes (P = .20), which was attributable to a better outcome for Ser49 patients treated with a high dose of beta-blockade as compared with a low dose. Gly49 carriers had a similar survival rate with different doses of beta-blockers. With low-dose beta-blockers, both codon 49 (RR, 0.26; 95% CI, 0.08-0.89; P = .029) and codon 389 (RR, 2.42; 95% CI, 1.04-5.63, P = .039) were related to 5-year mortality rate. In patients with heart failure, the influence of codon 49 on the outcome and effect of beta-blockers appeared to be more pronounced than that of codon 389. The more common Ser49Ser genotype responded less beneficially to beta-blockade and would motivate genotyping to promote higher doses for the best outcome effect.

  20. Characterization and regulation of. beta. /sub 2/-adrenergic receptors in rat vas deferens

    Energy Technology Data Exchange (ETDEWEB)

    May, J.M.


    ..beta../sub 2/-Adrenergic receptors in rat vas deferens were examined by measuring the binding of /sup 125/I-pindolol (/sup 125/IPIN) to membrane preparations and the inhibition of evoked contractions in intact tissues. /sup 125/IPIN labeled a single class of binding sites with mass action kinetics. Affinity constants for ..beta..-adrenergic receptor antagonists calculated from both binding and functional experiments agreed well, suggesting that /sup 125/IPIN labels the functional ..beta../sub 2/-adrenergic receptor. n-Bromoacetylalprenololmenthane (BAAM) was used to decrease receptor density so that agonist affinity constants could be determined functionally. Treatment of tissues with BAAM decreased the functional potencies of agonists. Higher concentrations of BAAM decreased the maximum tissue response. Affinity constants for agonists calculated after BAAM treatment were compared to affinity constants determined from binding studies done under conditions designed to promote high or low affinity agonist binding. Functional affinity constants for isoproterenol and salbutamol agreed with the low affinity binding constants, suggesting that the low affinity form of the receptor initiates the functional response. Because acute denervation of vasa deferentia did not alter the density of /sup 125/IPIN binding sites, the sites are probably post-junctional. Chronic infusion of isoproterenol reduced the potency of isoproterenol, the maximum tissue response, and the receptor density. These results suggest that ..beta..-adrenergic receptor density and responsiveness in rat vas deferens are not affected by removing catecholamine sources, but receptor density and responsiveness can be decreased by increasing agonist concentration at the receptor.

  1. Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells. (United States)

    James, Sharon Y; Lin, Feng; Kolluri, Siva Kumar; Dawson, Marcia I; Zhang, Xiao-kun


    The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.

  2. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.


    {gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

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

    Directory of Open Access Journals (Sweden)

    Keith B Hengen

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

  4. Platelets possess functional TGF-beta receptors and Smad2 protein. (United States)

    Lev, P R; Salim, J P; Marta, R F; Osorio, M J Mela; Goette, N P; Molinas, F C


    TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

  5. Expression profile of nicotinic acetylcholine receptor subunits in the brain of HIV-1 transgenic rats given chronic nicotine treatment. (United States)

    Cao, Junran; Nesil, Tanseli; Wang, Shaolin; Chang, Sulie L; Li, Ming D


    Abuse of addictive substances, including cigarettes, is much greater in HIV-1-infected individuals than in the general population and challenges the efficiency of highly active anti-retroviral therapy (HAART). The HIV-1 transgenic (HIV-1Tg) rat, an animal model used to study drug addiction in HIV-1-infected patients on HAART, displays abnormal neurobehavioral responses to addictive substances. Given that the cholinergic system plays an essential part in the central reward circuitry, we evaluated the expression profile of nine nicotinic acetylcholine receptor (nAChR) subunit genes in the central nervous system (CNS) of HIV-1Tg rats. We found that nAChR subunits were differentially expressed in various brain regions in HIV-1Tg rats compared to F344 control rats, with more subunits altered in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of the HIV-1Tg rats than in other brain regions. We also found that chronic nicotine treatment (0.4 mg/kg/day) decreased the mRNA expression of nAChR subunits α6, β3, and β4 in the VTA of HIV-1Tg rats, whereas expression of α4 and α6 subunits in the NAc increased. No such changes were observed in F344 rats. Together, our data suggest that HIV-1 proteins alter the expression of nAChRs, which may contribute to the vulnerability to cigarette smoking addiction in HIV-1 patients.

  6. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B


    signals were observed for lung, liver and testicles. In the case of CK2beta mRNA the highest signals were found for testicles, kidney, brain and liver. The amount of CK2beta mRNA in testicles was estimated to be about 6-fold higher than in brain. The strongest CK2beta signals in the Western blot were...... in brain and testicles. By contrast, Northern blot analyses of the CK2alpha mRNA revealed a somewhat different picture. Here, the strongest signals were obtained for brain, liver, heart and lung. In kidney, spleen and testicles mRNAs were only weakly detectable. For CK2alpha' mRNA distribution strong...

  7. Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients. (United States)

    Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho


    Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

  8. Functional and molecular plasticity of gamma and alpha-1 GABAA receptor subunits in the dorsal motor nucleus of the vagus after experimentally-induced diabetes. (United States)

    Boychuk, Carie R; Smith, Katalin Cs; Smith, Bret N


    Chronic experimentally-induced hyperglycemia augments subunit specific gamma-aminobutyric acid A (GABAA) receptor-mediated inhibition of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV). However, the contribution of α1 or γ GABAA receptor subunits, which are ubiquitously expressed on central nervous system neurons, to this elevation in inhibitory tone have not been determined. This study investigated the effect of chronic hyperglycemia/hypoinsulinemia on α1- and γ-subunit specific GABAA receptor-mediated inhibition using electrophysiological recordings in vitro and quantitative (q)RT-PCR. DMV neurons from streptozotocin-treated mice demonstrated enhancement of both phasic and tonic inhibitory currents in response to application of the α1-subunit selective GABAA receptor positive allosteric modulator, zolpidem. Responses to low concentrations of the GABAA receptor antagonist, gabazine suggested an additional increased contribution of γ-subunit-containing receptors to tonic currents in DMV neurons. Consistent with the functional elevation in α1- and γ-subunit-dependent activity, transcription of both the α1- and γ2-subunits was increased in the dorsal vagal complex of streptozotocin-treated mice. Overall these findings suggest an increased sensitivity to both zolpidem and gabazine after several days of hyperglycemia/hypoinsulinemia, which could contribute to altered parasympathetic output from DMV neurons in diabetes. Copyright © 2017, Journal of Neurophysiology.

  9. Increased Sensitivity of the Neuronal Nicotinic Receptor α2 Subunit Causes Familial Epilepsy with Nocturnal Wandering and Ictal Fear


    Aridon, Paolo; Marini, Carla; Di Resta, Chiara; Brilli, Elisa; De Fusco, Maurizio; Politi, Fausta; Parrini, Elena; Manfredi, Irene; Pisano, Tiziana; Pruna, Dario; Curia, Giulia; Cianchetti, Carlo; Pasqualetti, Massimo; Becchetti, Andrea; Guerrini, Renzo


    Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the α4 and β2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular gene...

  10. The disintegrin and metalloproteinase ADAM12 contributes to TGF-beta signaling through interaction with the type II receptor

    DEFF Research Database (Denmark)

    Atfi, Azeddine; Dumont, Emmanuelle; Colland, Frédéric;


    Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-beta type I receptor and the TGF-beta type II receptor (TbetaRII). Upon ligand binding, TGF-beta type I receptor activated by TbetaRII propagates...... signals to Smad proteins, which mediate the activation of TGF-beta target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-beta signaling pathway that acts through association with TbetaRII. We found that ADAM12 functions by a mechanism...... independent of its protease activity to facilitate the activation of TGF-beta signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TbetaRII in early endosomal vesicles and stabilizes the Tbeta...

  11. Reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP, with E. coli F1-ATPase and its subunits: the roles of the high affinity binding site in the alpha subunit and the low affinity binding site in the beta subunit. (United States)

    Matsuoka, I; Takeda, K; Futai, M; Tonomura, Y


    We performed kinetic studies on the reactions of a fluorescent ATP analog, 2'-(5-dimethyl-aminonaphthalene-1-sulfonyl) amino-2'-deoxyATP (DNS-ATP), with E. coli F1-ATPase (EF1) and its subunits, to clarify the role of each subunit in the ATPase reaction. The following results were obtained. 1. One mol of EF1, which contains nonexchangeable 2 mol ATP and 0.5 mol ADP, binds 3 mol of DNS-ATP. The apparent dissociation constant, in the presence of Mg2+, was 0.23 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased exponentially with t1/2 of 35 s, and reached 3.5 times the original fluorescence level. Following the fluorescence increase, DNS-ATP was hydrolyzed, and the fluorescence intensity maintained its enhanced level. 2. The addition of an excess of ATP over the EF1-DNS-nucleotide complex, in the presence of Mg2+, decreased the fluorescence intensity rapidly, indicating the acceleration of DNS-nucleotide release from EF1. ADP and GTP also decreased the fluorescence intensity. 3. DCCD markedly inhibited the accelerating effect of ATP on DNS-nucleotide release from EF1 and the EF1-DNS-ATPase or -ATPase activity in a steady state. On the other hand, DCCD only slightly inhibited the fluorescence increase of DNS-ATP, due to its binding to EF1, and the rate of single cleavage of 1 mol of DNS-ATP per mol of alpha subunit of EF1. 4. In the presence of Mg2+, 0.65-0.82 mol of DNS-ATP binds to 1 mol of the isolated alpha subunit of EF1 with an apparent dissociation constant of 0.06-0.07 microM. Upon binding, the fluorescence intensity of DNS-ATP at 520 nm increased 1.55 fold very rapidly (t1/2 less than 1 s). No hydrolysis of DNS-ATP was observed upon the addition of the isolated alpha subunit. The fluorescence intensity of DNS-ATP was unaffected by the addition of the isolated beta subunit. DNS-ATP was also unhydrolyzed by the isolated beta subunit. 5. EF1-ATPase was reconstituted from alpha, beta, and gamma subunits in the presence of Mg2+ and ATP

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

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


    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…

  13. Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI. (United States)

    Huusko, N; Pitkänen, A


    Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague-Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM-VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p0.05). Also, the volume of the VPM-VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate

  14. [Beta-3 adrenergic receptor--structure and role in obesity and metabolic disorders]. (United States)

    Wiejak, J; Wyroba, E


    Structure and essential motifs of beta 3-adrenergic receptor (known previously as atypical beta-AR), which plays a central role in regulation of lipid metabolism have been described. Obesity results from an imbalance between caloric intake and energy expenditure. The consequence of catecholamine activation of beta 3-AR is increased mobilization of fatty acids from triglyceride stores (lipolysis) in brown and white adipose tissue as well as increased fatty acid beta-oxidation and heat-production via UCP-1 (thermogenesis) in brown adipose tissue. A pharmacokinetic effects of beta 3-agonists and putative involvement of Trp/Arg mutation in beta 3-AR gene in obesity and another metabolic disorders have been discussed.

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

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    A Mobascher

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

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

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    Lansdell Stuart J


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

  17. Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression. (United States)

    Friess, H; Yamanaka, Y; Büchler, M; Berger, H G; Kobrin, M S; Baldwin, R L; Korc, M


    We have recently found that human pancreatic adenocarcinomas exhibit strong immunostaining for the three mammalian transforming growth factor beta (TGF-beta) isoforms. These important growth-regulating polypeptides bind to a number of proteins, including the type I TGF-beta receptor (T beta R-I), type II TGF-beta receptor (T beta R-II), and the type III TGF-beta receptor (T beta R-III). In the present study we sought to determine whether T beta R-II and T beta R-III expression is altered in pancreatic cancer. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas exhibited a 4.6-fold increase (P beta R-II. In contrast, mRNA levels encoding T beta R-III were not increased. In situ hybridization showed that T beta R-II mRNA was expressed in the majority of cancer cells, whereas mRNA grains encoding T beta R-III were detectable in only a few cancer cells and were present mainly in the surrounding stroma. These findings suggest that enhanced levels of T beta R-II may have a role in regulating human pancreatic cancer cell growth, while T beta R-III may function in the extracellular matrix.

  18. Association of nicotinic acetylcholine receptor subunit alpha-4 polymorphisms with smoking behaviors in Chinese male smokers

    Institute of Scientific and Technical Information of China (English)

    CHU Cheng-jing; YANG Yan-chun; WEI Jin-xue; ZHANG Lan


    Background It has been reported that the nicotinic acetylcholine receptor subunit a4 gene (CHRNA4) might be associated with smoking behaviors in the previous studies. Up to now, there are few reports on the relationship between CHRNA4 and smoking initiation. In this study, we tried to explore the role of two polymorphisms in CHRNA4 (rs 1044396 and rs 1044397) in smoking initiation and nicotine dependence in Chinese male smokers.Methods Nine hundred and sixty-six Chinese male lifetime nonsmokers and smokers were assessed by the Fagerstr(o)m test for nicotine dependence (FTND), smoking quantity (SQ) and the heaviness of smoking index (HSI). All subjects were divided into four groups based on their tobacco use history and the FTND scores. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find two polymorphisms of CHRNA4 in these subjects.Results The x2 test showed that rs1044396 was significantly associated with smoking initiation (x2=4.65, P=0.031),while both rs1044396 and rs1044397 were significantly associated with nicotine dependence (x2=5.42, P=0.020; x2=758,P=0.005). Furthermore, the T-G (3.9%) haplotype of rs1044396-rs1044397 showed significant association with smoking initiation (x2=6.30, P=0.012) and the C-G haplotype (58.9%) remained positive association with nicotine dependence (x2=8.64, P=0.003) after Bonferroni correction. The C-G haplotype also significantly increased the HSI (P=0.002) and FTND scores (P=0.001) after Bonferroni correction.Conclusion These findings suggest that CHRNA4 may be associated with smoking initiation and the C-G haplotype of rs1044396-rs1044397 might increase the vulnerability to nicotine dependence in Chinese male smokers.

  19. Glycine receptor α3 and α2 subunits mediate tonic and exogenous agonist-induced currents in forebrain. (United States)

    McCracken, Lindsay M; Lowes, Daniel C; Salling, Michael C; Carreau-Vollmer, Cyndel; Odean, Naomi N; Blednov, Yuri A; Betz, Heinrich; Harris, R Adron; Harrison, Neil L


    Neuronal inhibition can occur via synaptic mechanisms or through tonic activation of extrasynaptic receptors. In spinal cord, glycine mediates synaptic inhibition through the activation of heteromeric glycine receptors (GlyRs) composed primarily of α1 and β subunits. Inhibitory GlyRs are also found throughout the brain, where GlyR α2 and α3 subunit expression exceeds that of α1, particularly in forebrain structures, and coassembly of these α subunits with the β subunit appears to occur to a lesser extent than in spinal cord. Here, we analyzed GlyR currents in several regions of the adolescent mouse forebrain (striatum, prefrontal cortex, hippocampus, amygdala, and bed nucleus of the stria terminalis). Our results show ubiquitous expression of GlyRs that mediate large-amplitude currents in response to exogenously applied glycine in these forebrain structures. Additionally, tonic inward currents were also detected, but only in the striatum, hippocampus, and prefrontal cortex (PFC). These tonic currents were sensitive to both strychnine and picrotoxin, indicating that they are mediated by extrasynaptic homomeric GlyRs. Recordings from mice deficient in the GlyR α3 subunit (Glra3(-/-)) revealed a lack of tonic GlyR currents in the striatum and the PFC. In Glra2(-/Y) animals, GlyR tonic currents were preserved; however, the amplitudes of current responses to exogenous glycine were significantly reduced. We conclude that functional α2 and α3 GlyRs are present in various regions of the forebrain and that α3 GlyRs specifically participate in tonic inhibition in the striatum and PFC. Our findings suggest roles for glycine in regulating neuronal excitability in the forebrain.

  20. The anxioselective agent 7-(2-chloropyridin-4-yl)pyrazolo-[1,5-a]-pyrimidin-3-yl](pyridin-2-yl)methanone (DOV 51892) is more efficacious than diazepam at enhancing GABA-gated currents at alpha1 subunit-containing GABAA receptors. (United States)

    Popik, Piotr; Kostakis, Emmanuel; Krawczyk, Martyna; Nowak, Gabriel; Szewczyk, Bernadeta; Krieter, Philip; Chen, Zhengming; Russek, Shelley J; Gibbs, Terrell T; Farb, David H; Skolnick, Phil; Lippa, Arnold S; Basile, Anthony S


    Studies using mice with point mutations of GABA(A) receptor alpha subunits suggest that the sedative and anxiolytic properties of 1,4-benzodiazepines are mediated, respectively, by GABA(A) receptors bearing the alpha(1) and alpha(2) subunits. This hypothesis predicts that a compound with high efficacy at GABA(A) receptors containing the alpha(1) subunit would produce sedation, whereas an agonist acting at alpha(2) subunit-containing receptors (with low or null efficacy at alpha(1)-containing receptors) would be anxioselective. Electrophysiological studies using recombinant GABA(A) receptors expressed in Xenopus oocytes indicate that maximal potentiation of GABA-stimulated currents by the pyrazolo-[1,5-a]-pyrimidine, DOV 51892, at alpha(1)beta(2)gamma(2S) constructs of the GABA(A) receptor was significantly higher (148%) than diazepam. In contrast, DOV 51892 was considerably less efficacious and/or potent than diazepam in enhancing GABA-stimulated currents mediated by constructs containing alpha(2), alpha(3), or alpha(5) subunits. In vivo, DOV 51892 increased punished responding in the Vogel conflict test, an effect blocked by flumazenil, and increased the percentage of time spent in the open arms of the elevated plus-maze. However, DOV 51892 had no consistent effects on motor function or muscle relaxation at doses more than 1 order of magnitude greater than the minimal effective anxiolytic dose. Although the mutant mouse data predict that the high-efficacy potentiation of GABA(A1a) receptor-mediated currents by DOV 51892 would be sedating, behavioral studies demonstrate that DOV 51892 is anxioselective, indicating that GABA potentiation mediated by alpha(1) subunit-containing GABA(A) receptors may be neither the sole mechanism nor highly predictive of the sedative properties of benzodiazepine recognition site modulators.

  1. Rat neuronal nicotinic acetylcholine receptors containing a7 subunit: pharmacological properties of ligand binding and function

    Institute of Scientific and Technical Information of China (English)



    Aim: To compare pharmacological properties of heterologously expressed homomeric a7 nicotinic acetylcholine receptors (a.7 nAChRs) with those of native nAChRs containing a.7 subunit (a.7* nAChRs) in rat hippocampus and cerebral cortex. Methods: We established a stably transfected HEK-293 cell line that expresses homomeric rat a7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat a.7* nAChRs in rat hippocampus and cerebral cortex. We used [125IJ-a-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the a.7 nAChRs expressed in this cell line were studied using whole-cell current recording.Results: The newly established cell line, KXa7Rl, expresses homomeric a7 nAChRs that bind [125I]-a-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kj values of native rat a.7* nAChRs from hippocampus (Kd=0.28±0.03 nmol/L) and cerebral cortex (Kd=0.33±0.05 nmol/L). Using whole-cell current recording, the homomeric a7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC50 values of 280±19 nmol/L and 180±40 nmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of a.7 nAChRs, a-bungarotoxin (IC5o=19±2 nmol/L) and methyllycaconitine (IC50=100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric a.7 nAChRs and native a.7* receptors in rat brain, but it also revealed several notable differences.Conclusion: This newly established stable cell line should be very useful for studying the properties of homomeric a7 nAChRs and comparing these properties to native a.7* nAChRs.

  2. Increase of AMPA receptor glutamate receptor 1 subunit and B-cell receptor-associated protein 31 gene expression in hippocampus of fatigued mice. (United States)

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


    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.

  3. Cloning and Characterization of an mRNA Encoding F1-ATPase Beta-Subunit Abundant in Epithelial Cells of Mantle and Gill of Pearl Oyster, Pinctadafucata

    Institute of Scientific and Technical Information of China (English)


    In oyster biomineralization, large amounts of calcium are absorbed from external media, transported to the mineralization site, and finally deposited via a matrix-mediated process. All these activities are very energy intensive; therefore, investigations of the energy metabolism pathways of different oyster tissues will facilitate understanding of oyster biomineralization physiology. A full-length cDNA encoding the F1-ATPase beta-subunit (the F1-β-subunit, a major calalytic subunit of F-ATPase) from the pearl oyster (Pinctads fucata) was cloned using the homology strategy with a pair of degenerated primers based on the conserved regions of other animals' F1-β-subunit genes. Sequencing and structural analyses showed that the obtained sequence shared high identity with other animals' F1-β-subunits, and had a unique phosphorylation site of PKC and CK Ⅱ on the external surface of the putative protein. Results from semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization demonstrated this oyster F1-β-subunit mRNA is abundant in the gill and mantle, and distributed widely in the periostracal groove, the outer folder,and the dorsal region of the mantle and in the gill epithelial cells. These tissues were the main regions that participate in biomineralization processes such as calcium uptake, transport, and matrix secretion. The results indicate that tissues involved in biomineralization have stronger energy metabolic processes and that F1-ATPase might play an important role in oyster biomineralization by providing energy transport.

  4. Differential dendritic targeting of AMPA receptor subunit mRNAs in adult rat hippocampal principal neurons and interneurons. (United States)

    Cox, David J; Racca, Claudia


    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.

  5. The effects of NR2 subunit-dependent NMDA receptor kinetics on synaptic transmission and CaMKII activation.

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    David M Santucci


    Full Text Available N-Methyl-D-aspartic acid (NMDA receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future

  6. Cytisine binds with similar affinity to nicotinic alpha4beta2 receptors on the cell surface and in homogenates. (United States)

    Zhang, Jessie; Steinbach, Joe Henry


    Cytisine and nicotine bound to specific sites in homogenates prepared from HEK 293 cells which stably express human neuronal nicotinic alpha4 and beta2 subunits. The number of sites was the same for both ligands and nicotine was a full competitive inhibitor of cytisine binding. However, when binding was done to intact cells the number of binding sites per cell for nicotine was approximately 4-fold the number of sites for cytisine. Nicotine fully blocked cytisine binding, but cytisine only partially blocked nicotine binding to intact cells. When cells were permeabilized with saponin, the number of sites for nicotine was unchanged, while the number of sites for cytisine was increased, and cytisine was able to fully block nicotine binding. These data indicate that cytisine binds only to surface receptors on intact cells. The apparent affinity of cytisine for surface receptors (K(d)=0.8 nM) was not significantly different from that for receptors in the cell homogenate (0.3 nM).

  7. Induction of interleukin 2 receptor beta chain expression by self-recognition in the thymus. (United States)

    Hanke, T; Mitnacht, R; Boyd, R; Hünig, T


    1-2% of adult mouse thymocytes express the T cell receptor alpha/beta (TCR-alpha/beta) together with the interleukin (IL) 2R beta (p70), but not the alpha (p 55) chain. We show that the previously described alpha/beta-TCR +CD4-8- and the partially overlapping Ly6C+ thymocytes are contained within this subset. Most IL-2R beta+ alpha/beta-TCR+ cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44high, CD69+) phenotype. Overrepresentation of V beta 8.2 in both CD4-8- and CD4 and/or CD8+ IL-2R beta+ thymocytes suggests that IL-2R beta expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2Kb-specific TCR, IL-2R beta+ cells were abundant under conditions of mainstream negative selection, i.e., in the presence of Kb, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, self-recognition by immature thymocytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R beta. IL-2-deficient mice contain normal numbers of IL-2R beta+ alpha/beta-TCR+ thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2R beta+ thymocytes is skewed towards CD4-8+, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.

  8. Core-binding factor subunit beta is not required for non-primate lentiviral Vif-mediated APOBEC3 degradation. (United States)

    Ai, Youwei; Zhu, Dantong; Wang, Cuihui; Su, Chao; Ma, Jian; Ma, Jianzhang; Wang, Xiaojun


    Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-β) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-β is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-β. In addition, CBF-β did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-β silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-β was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-β knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-β to degrade APOBEC3. CBF-β did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and

  9. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus. (United States)

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


    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.

  10. Immunoanalogue of vertebrate beta-adrenergic receptor in the unicellular eukaryote Paramecium. (United States)

    Wiejak, Jolanta; Surmacz, Liliana; Wyroba, Elzbieta


    Cell fractionation, SDS-PAGE, quantitative Western blot, confocal immunolocalization and immunogold labelling were performed to find an interpretation of the physiological response of the unicellular eukaryote Paramecium to beta-adrenergic ligands. The 69 kDa polypeptide separated by SDS-PAGE in S2 and P2 Paramecium subcellular fractions cross-reacted with antibody directed against human beta2-adrenergic receptor. This was detected by Western blotting followed by chemiluminescent detection. Quantitative image analysis showed that beta-selective adrenergic agonist (-)-isoproterenol--previously shown to enhance phagocytic activity--evoked redistribution of the adrenergic receptor analogue from membraneous (P2) to cytosolic (S2) fraction. The relative increase in immunoreactive band intensity in S2 reached 80% and was paralleled by a 59% decrease in P2 fraction. Confocal immunofluorescence revealed beta2-adrenergic receptor sites on the cell surface and at the ridge of the cytopharynx--where nascent phagosomes are formed. This localization was confirmed by immunoelectron microscopy. These results indicate that the 69 kDa Paramecium polypeptide immunorelated to vertebrate beta2-adrenergic receptor appeared in this evolutionary ancient cell as a nutrient receptor.

  11. Induction and identification of disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 from Escherichia coli O157:H7 using MALDI-TOF-TOF-MS/MS and top-down proteomics (United States)

    The disulfide-intact and disulfide-reduced beta-subunit of Shiga toxin 2 (beta-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic an...

  12. Effect of developmental lead exposure on synaptic plasticity and N—methyl—D—aspartate receptor subunit in rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    RuanDY; SuiL


    Chronic lead(Pb) exposure is known to be associated with learning and memory,and cognitive dysfunction in children.Previous studies have demonstrated that Pb exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N-methyl-D-aspartate (NMDA) receptors(NMDARs).The studies described here were carried out to investigate effect of developmental Pb exposure on long-term potentiation(LTP),long-tern depression(LTD) and NMDAs subunits in rat hippocampus.The results are listed as follows:(1)low-level Pb exposture can impair the induction and maintenance of LTP in vivo and in vitro;(2)the Pb-induced impairment of LTD magnitude was an age-related decline in area CA1 of rat hippocampus;(3)chronic Pb exposure affected two components,voltage-gated calcium channel-dependent LTD and NMDARs-dependent LTD,of LTD induction in area CA1 of rat hippocampus;(4)different effects of developmental Pb exposure on NMDA receptor NR1,NR2A,NR2B,NR2C,NR2D and NR3A subunits in area CA1,CA2,CA3 and CA4 of rat hippocampus were observed;(5)the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors enriched in area CA1,CA3 and dentate gyrus and kainite receptors enriched in area CA1 and dentate gyrus of rat hippocampus were impaired by Pb exposure.

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

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


    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.

  14. Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors. (United States)

    Houlihan, L M; Slater, Y; Guerra, D L; Peng, J H; Kuo, Y P; Lukas, R J; Cassels, B K; Bermudez, I


    Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.

  15. Distinct regions within the GluN2C subunit regulate the surface delivery of NMDA receptors

    Directory of Open Access Journals (Sweden)

    Katarina eLichnerova


    Full Text Available N-methyl-D-aspartate (NMDA receptors mediate fast excitatory synaptic transmission in the mammalian central nervous system. The activation of NMDA receptors plays a key role in brain development, synaptic plasticity, and memory formation, and is a major contributor to many neuropsychiatric disorders. Here, we investigated the mechanisms that underlie the trafficking of GluN1/GluN2C receptors. Using an approach combining molecular biology, microscopy, and electrophysiology in mammalian cell lines and cultured cerebellar granule cells, we found that the surface delivery of GluN2C-containing receptors is reduced compared to GluN2A- and GluN2B-containing receptors. Furthermore, we identified three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits that are required for proper intracellular processing and surface delivery of NMDA receptors. These results shed new light on the regulation of NMDA receptor trafficking, and these findings can be exploited to develop new strategies for treating some forms of neuropsychiatric disorders.

  16. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;


    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  17. Receptor density is key to the alpha2/beta interferon differential activities. (United States)

    Moraga, Ignacio; Harari, Daniel; Schreiber, Gideon; Uzé, Gilles; Pellegrini, Sandra


    Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.

  18. Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABAA Receptor γ2 Subunit in the Cerebellum (United States)

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


    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

  19. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site. (United States)

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R


    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  20. Acetylcholine release in mouse hippocampal CA1 preferentially activates inhibitory-selective interneurons via alpha4 beta2* nicotinic receptor activation

    Directory of Open Access Journals (Sweden)

    L. Andrew Bell


    Full Text Available Acetylcholine (ACh release onto nicotinic receptors directly activates subsets of inhibitory interneurons in hippocampal CA1. However, the specific interneurons activated and their effect on the hippocampal network is not completely understood. Therefore, we investigated subsets of hippocampal CA1 interneurons that respond to ACh release through the activation of nicotinic receptors and the potential downstream effects this may have on hippocampal CA1 network function. ACh was optogenetically released in mouse hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated viral mediated transfection. The actions of optogenetically released ACh were assessed on both pyramidal neurons and different interneuron subtypes via whole cell patch clamp methods. Vasoactive intestinal peptide (VIP-expressing interneurons that selectively innervate other interneurons (VIP/IS were excited by ACh through the activation of nicotinic receptors containing alpah4 and beta2 subunits (alpha4 beta2*. ACh release onto VIP/IS was presynaptically inhibited by M2 muscarinic autoreceptors. ACh release produced spontaneous inhibitory postsynaptic current (sIPSC barrages blocked by dihydro-beta-erythroidine in interneurons but not pyramidal neurons. Optogenetic suppression of VIP interneurons did not inhibit these sIPSC barrages suggesting other interneuron-selective interneurons were also excited by 42* nicotinic receptor activation. In contrast, interneurons that innervate pyramidal neuron perisomatic regions were not activated by ACh release onto nicotinic receptors. Therefore, we propose ACh release in CA1 facilitates disinhibition through activation of 42* nicotinic receptors on interneuron-selective interneurons whereas interneurons that innervate pyramidal neurons are less affected by nicotinic receptor activation.

  1. AT1-IR-beta Association: A New Mechanism for the Inhibition of Insulin Receptor Function in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Lakshmi Pulakat


    Full Text Available Epidemiological evidence show that increased mortality in breast cancer is linked to hypertension and insulin resistance. Because Angiotensin II (Ang II, a hormone implicated in hypertension and insulin resistance, is a normal mitogen for breast tissue and elevated expression of the Ang II receptor AT1 is seen in breast cancer, we analyzed the effects of Ang II exposure on the functions of IR in human breast cancer cell line MCF-7. Exposure of MCF-7 to Ang II for 2 hours a significantly reduced 125I-insulin binding to IR, and b induced co-immuno-precipitation of the AT1 with IR-beta subunit. These Ang II-mediated effects on IR were inhibited by the AT1 antagonist losartan, and were not observed when exposure time was below 1-hour. These observations suggest extended exposure to Ang II have detrimental effects on insulin binding to IR that were not discovered in the previous studies where Ang II-exposure of insulin responsive cells was performed for periods less than one hour. In addition, they suggest a novel mechanism that involves AT1-IR-beta association for the inhibition of insulin binding to IR in response to extended exposure (2-hours of breast cancer cells to elevated levels of Ang II (as seen in hypertensive patients, and provides a molecular link for the inhibition of normal IR signaling by Ang II in breast cancer.

  2. Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Tao Bo; Jiang Li; Jian Li; Xingfang Li; Kaihui Xing


    We used RNA interference (RNAi) to disrupt synthesis of the cortical neuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

  3. A molecular dynamics approach to receptor mapping: application to the 5HT3 and beta 2-adrenergic receptors. (United States)

    Gouldson, P R; Winn, P J; Reynolds, C A


    A molecular dynamics-based approach to receptor mapping is proposed, based on the method of Rizzi (Rizzi, J. P.; et al. J. Med. Chem. 1990, 33, 2721). In Rizzi's method, the interaction energy between a series of drug molecules and probe atoms (which mimic functional groups on the receptor, such as hydrogen bond donors) was calculated. These interactions were calculated on a three-dimensional grid within a molecular mechanics parameters, were placed at these minima. The distances between the dummy atom sites were monitored during molecular dynamics simulations and plotted as distance distribution functions. Important distances within the receptor became apparent, as drugs with a common mode of binding share similar peaks in the distance distribution functions. In the case of specific 5HT3 ligands, the important donor--acceptor distance within the receptor has a range of ca. 7.9--8.9 A. In the case of specific beta 2-adrenergic ligands, the important donor--acceptor distances within the receptor lie between ca. 7--9 A and between 8 and 10 A. These distances distribution functions were used to assess three different models of the beta 2-adrenergic G-protein-coupled receptor. The comparison of the distance distribution functions for the simulation with the actual donor--acceptor distances in the receptor models suggested that two of the three receptor models were much more consistent with the receptor-mapping studies. These receptor-mapping studies gave support for the use of rhodopsin, rather than the bacteriorhodopsin template, for modeling G-protein-coupled receptors but also sounded a warning that agreement with binding data from site-directed mutagenesis experiments does not necessarily validate a receptor model.

  4. Reconstitution of the functional receptors for murine and human interleukin 5



    The murine interleukin 5 receptor (mIL-5R) is composed of two distinct subunits, alpha and beta. The alpha subunit (mIL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (mIL-5R beta) does not bind IL-5 by itself, but forms the high-affinity receptor with mIL-5R alpha. mIL-5R beta has been revealed to be the mIL-3R-like protein, AIC2B which is shared with receptors for IL-3 and granulocyte/macrophage colony-stimulating factor. We demonstrated here the reconstitution of the...

  5. Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin. (United States)

    Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan; Ramsden, Sarah L; Ramerstorfer, Joachim; Saiepour, Leila; Maric, Hans-Michael; Moss, Stephen J; Schindelin, Hermann; Harvey, Robert J; Sieghart, Werner; Harvey, Kirsten


    The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.

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

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


    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.

  7. Characterization of beta-adrenergic receptors in dispersed rat testicular interstitial cells

    Energy Technology Data Exchange (ETDEWEB)

    Poyet, P.; Labrie, F.


    Recent studies have shown that beta-adrenergic agents stimulate steroidogenesis and cyclic AMP formation in mouse Leydig cells in culture. To obtain information about the possible presence and the characteristics of a beta-adrenergic receptor in rat testicular interstitial cells, the potent beta-adrenergic antagonist (/sup 125/I)cyanopindolol (CYP) was used as ligand. Interstitial cells prepared by collagenase dispersion from rat testis were incubated with the ligand for 2 h at room temperature. (/sup 125/I)cyanopindolol binds to a single class of high affinity sites at an apparent KD value of 15 pM. A number of sites of 6,600 sites/cell is measured when 0.1 microM (-) propranolol is used to determine non-specific binding. The order of potency of a series of agonists competing for (/sup 125/I)cyanopindolol binding is consistent with the interaction of a beta 2-subtype receptor: zinterol greater than (-) isoproterenol greater than (-) epinephrine = salbutamol much greater than (-) norepinephrine. In addition, it was observed that the potency of a large series of specific beta 1 and beta 2 synthetic compounds for displacing (/sup 125/I)cyanopindolol in rat interstitial cells is similar to the potency observed for these compounds in a typical beta 2-adrenergic tissue, the rat lung. For example, the potency of zinterol, a specific beta 2-adrenergic agonist, is 10 times higher in interstitial cells and lung than in rat heart, a typical beta 1-adrenergic tissue. Inversely, practolol, a typical beta 1-antagonist, is about 50 times more potent in rat heart than in interstitial cells and lung.

  8. [Expression of beta-catenin and estrogen receptor in desmoid-type fibromatosis]. (United States)

    Zhang, Hong-Ying; Ke, Qi; Zhang, Zhang; Zhang, Rui; Fu, Jing; Chen, Hui-Jiao; Wei, Bing; Bu, Hong


    To detect the expression of beta-catenin and Estrogen Receptor in desmoid-type fibromatosis. Nuclear beta-catenin expression was detected by immunohistochemistry in 77 lesions with desmoid-type fibromatosis and 171 other spindle cell lesions, including superficial fibromatosis (n = 18), nodular fasciitis (n = 36), keloid (n = 16), scar (n = 10), granulation tissue (n = 9), synovial sarcoma (n = 38), neufibroma (n = 13), solitary fibrous tumor (n =12), gastrointestinal stromal tumor (n = 10), low-grade myxofibrosarcoma (n = 3), low-grade fibromyxoid sarcoma (n = 3), and smooth muscle tumor (n = 10). In addition, the immunohistochemical expressions of ER-alpha, ER-beta and Ki-67 were examined in all of the lesions with desmoid-type fibromatosis. The nuclear immunohistochemical staining for nuclear beta-catenin and ER-beta was graded as high level ( > or = 25% of cells), low level (5%-25%) or none. High-level nuclear beta-catenin staining was detected in a very limited subset of tissue types, which included 70.1% of lesions with desmoid-type fibromatosis (54/77) and 6.3% of lesions with keloid (1/16). No high-level nuclear beta-catenin staining was seen in any of the other lesions. None of the lesions with desmoid-type fibromatosis expressed ER-alpha. However, 62 (80.5%) of the lesions with desmoids-type fibromatosis were positive in ER-beta, which included 52 (67.5%) with high-level expression, and 10 (13%) with low-level expression. The Spearman correlation analysis suggested that the expression of beta-catenin was positively correlated (r = 0.867, P fibromatosis had very low Ki-67 positive rate. The recurrence of desmoids-type fibromatosis was not correlated independently with beta-catenin, ER-beta or Ki-67. High-level nuclear beta-catenin staining serves as a useful diagnostic tool for desmoid-type fibromatosis. The high expression of ER-beta in desmoid-type fibromatosis provides a biological mechanism for the antiestrogenic compounds to treat fibromatosis. There

  9. Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating. (United States)

    Stephan, Gabriele; Kowalski-Jahn, Maria; Zens, Christopher; Schmalzing, Günther; Illes, Peter; Hausmann, Ralf


    P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify inter-subunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gating-induced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Charge-reversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

  10. Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata. (United States)

    Kouyanou-Koutsoukou, Sophia; Baier, Andrea; Kolaitis, Regina-Maria; Maniatopoulou, Evanthia; Thanopoulou, Konstantina; Szyszka, Ryszard


    The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (β) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2β subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2β subunit, that also possess the conserved features of regulatory CK2β subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2β proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

  11. Removal of GABA(A receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations.

    Directory of Open Access Journals (Sweden)

    Elli Leppä

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

  12. Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium. (United States)

    Wiejak, Jolanta; Surmacz, Liliana; Wyroba, Elzbieta


    Evidence that dynamin is associated with the sequestration of the Paramecium beta(2)-adrenergic receptor (betaAR) immunoanalogue is presented. We previously reported a dramatic change in the distribution of betaAR analogue in the subcellular fractions upon isoproterenol treatment: it is redistributed from the membraneous to the cytosolic fraction, as revealed by quantitative image analysis of western blots. Here we confirm and extend this observation by laser scanning confocal and immunogold electron microscopy. In the presence of isoproterenol (10 micro mol l(-1)) betaAR translocated from the cell surface into dynamin-positive vesicles in the cytoplasmic compartment, as observed by dual fluorochrome immunolabeling in a series of the confocal optical sections. Colocalization of betaAR and dynamin in the tiny endocytic vesicles was detected by further electron microscopic studies. Generally receptor sequestration follows its desensitization, which is initiated by receptor phosphorylation by G-protein-coupled receptor kinase. We cloned and sequenced the gene fragment of 407 nucleotides homologous to the beta-adrenergic receptor kinase (betaARK): its deduced amino acid sequence shows 51.6% homology in 126 amino acids that overlap with the human betaARK2 (GRK3), and may participate in Paramecium betaAR desensitization. These results suggest that the molecular machinery for the desensitization/sequestration of the receptor immunorelated to vertebrate betaAR exists in unicellular PARAMECIUM:

  13. Cross-linking of the beta-glucan receptor on human monocytes results in interleukin-1 receptor antagonist but not interleukin-1 production

    NARCIS (Netherlands)

    Poutsiaka, D D; Mengozzi, M; Vannier, E; Sinha, B; Dinarello, C A


    The beta-glucan receptor, found on monocytes and neutrophils, binds glucose polymers derived from fungi. Ligands for the receptor have various immunomodulatory effects, including increased microbicidal killing activity. We have investigated the effect of beta-glucans on the production of

  14. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R


    Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins...... with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin...... activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals...

  15. Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Thomas Sunil


    Full Text Available Abstract Background The complement system is thought to be involved in the pathogenesis of numerous neurological diseases. We previously reported that pre-treatment of murine cortico-hippocampal neuronal cultures with the complement derived anaphylatoxin C5a, protects against glutamate mediated apoptosis. Our present study with C5a receptor knock out (C5aRKO mice corroborates that the deficiency of C5a renders C5aRKO mouse more susceptible to apoptotic injury in vivo. In this study we explored potential upstream mechanisms involved in C5a mediated neuroprotection in vivo and in vitro. Methods Based on evidence suggesting that reduced expression of glutamate receptor subunit 2 (GluR2 may influence apoptosis in neurons, we studied the effect of human recombinant C5a on GluR2 expression in response to glutamate neurotoxicity. Glutamate analogs were injected into C5aRKO mice or used to treat in vitro neuronal culture and GluR2 expression were assessed in respect with cell death. Results In C5aRKO mice we found that the neurons are more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor compared to WT control mice. Our results suggest that C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor expression. Conclusion Complement C5a neuroprotects through regulation of GluR2 receptor subunit.

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

    Directory of Open Access Journals (Sweden)

    Zhe eJin


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

  17. Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

    DEFF Research Database (Denmark)

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


    ) toxicity mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H...... nedioxy-5H-2,3-benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate- and domoate-mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization...

  18. beta-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations

    DEFF Research Database (Denmark)

    Sanni, S J; Hansen, J T; Bonde, M M;


    The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often...

  19. Identification of Tctex2 beta, a novel dynein light chain family member that interacts with different transforming growth factor-beta receptors

    NARCIS (Netherlands)

    Meng, QingJun; Lux, Andreas; Holloschi, Andreas; Li, Jian; Hughes, John M. X.; Foerg, Tassilo; McCarthy, John E. G.; Heagerty, Anthony M.; Kioschis, Petra; Hafner, Mathias; Garland, John M.


    Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-beta(TGF-beta) receptor family and has been identified as the gene involved in hereditary hemorrha

  20. Cardiac beta-receptors in experimental Chagas' disease Receptores beta cardíacos na doença de Chagas experimental

    Directory of Open Access Journals (Sweden)

    Julio E. Enders


    Full Text Available Experimental Chagas' disease (45 to 90 days post-infection showed serious cardiac alterations in the contractility and in the pharmacological response to beta adrenergic receptors in normal and T. cruzi infected mice (post-acute phase. Chagasic infection did not change the beta receptors density (78.591 ± 3.125 fmol/mg protein and 73.647 ± 2.194 fmol/mg protein for controls but their affinity was significantly diminished (Kd = 7.299 ± 0.426 nM and Kd = 3.759 ± 0.212 nM for the control p Estudaram-se os receptores beta cardíacos de camundongos infectados pelo Trypanosoma cruzi na fase pós-aguda da doença de Chagas para estabelecer em que medida os mesmos contribuem a gerar respostas anômalas às catecolaminas observadas nestes miocardios. Utilizara-se 3-H/DHA para a marcação dos receptores beta cardíacos dos camundongos normais e dos infectados na fase pós-aguda (45 a 90 dias pós-infecção. O número dos sítios de fixação foi similar nos dois grupos, 78.591 ± 3.125 fmol/mg. Proteína nos chagásicos e 73.647 ± 2.194 fmol/mg. Proteína no grupo controle. Em vez disso, a afinidade verificou-se significativamente diminuida no grupo chagásico (Kd = 7.299 ± 0.426 nM respeito do controle (Kd = 3.759 ± 0.212 nM p < 0.001. Os resultados obtidos demonstram que as modificações observadas na estimulação adrenérgica do miocárdio chagásico se correlacionam com a menor afinidade dos receptores beta cardíacos e que estas alterações exerceriam uma parte determinante para as consequências funcionais que são detectadas na fase crônica.

  1. Transforming growth factor-beta1 induces transforming growth factor-beta1 and transforming growth factor-beta receptor messenger RNAs and reduces complement C1qB messenger RNA in rat brain microglia. (United States)

    Morgan, T E; Rozovsky, I; Sarkar, D K; Young-Chan, C S; Nichols, N R; Laping, N J; Finch, C E


    Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth

  2. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S


    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...

  3. Ischemia- and agonist-induced changes in. alpha. - and. beta. -adrenergic receptor traffic in guinea pig hearts

    Energy Technology Data Exchange (ETDEWEB)

    Maisel, A.S.; Motulsky, H.J.; Ziegler, M.G.; Insel, P.A. (Univ. of California, La Jolla (USA))


    The authors have used radioligand binding techniques and subcellular fraction to assess whether changes in expression of myocardial {alpha}{sub 1}- and {beta}-adrenergic receptors are mediated by a redistribution of receptors between various membrane fractions. Three fractions were prepared from the left ventricles of guinea pigs that underwent either 1 h of ischemia or injection of epinephrine a crude membrane, a purified sarcolemma, and a light vesicle fraction. In control animals {alpha}{sub 1}-adrenergic receptors (({sup 3}H)prazosin binding) in light vesicles was only 25% of the total {alpha}{sub 1}-receptor density found in sarcolemmal and light vesicle fractions as compared with 50% for {beta}-adrenergic receptors (({sup 125}I)iodocyanopindolol binding sites). Although ischemia was associated with a 53% decrease in the number of light vesicle {beta}-adrenergic receptors and a 42% increase in the number of sarcolemma {beta}-receptors there was no change in the number of light vesicle {alpha}{sub 1}-receptors, even though the number of sarcolemmal {alpha}{sub 1}-receptors increased 34%. Epinephrine treatment promoted internalization of {beta}-adrenergic receptors. These results indicate that {alpha}{sub 1} and {beta}{sub 1}-adrenergic receptors may undergo a different cellular itinerary in guinea pig myocardium. Agonist and ischemia-induced changes in surface {beta}-receptors, but not {alpha}{sub 1}-receptors, appear to result from entry and exit of receptors from an intracellular pool that can be isolated in a light vesicle fraction. Changes in expression of {alpha}{sub 1}-adrenergic receptors may represent changes in the properties of receptors found in the sarcolemma or in a membrane fraction other than the light vesicle fraction that they have isolated.

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

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


    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

    Energy Technology Data Exchange (ETDEWEB)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.


    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  6. Acetylcholine receptor subunit and P-glycoprotein transcription patterns in levamisole-susceptible and -resistant Haemonchus contortus (United States)

    Sarai, Ranbir S.; Kopp, Steven R.; Coleman, Glen T.; Kotze, Andrew C.


    The mechanism of resistance to the anthelmintic levamisole in parasitic nematodes is poorly understood, although there is some evidence implicating changes in expression of nicotinic acetylcholine receptor (nAChR) subunit genes. Hence, in order to define levamisole resistance mechanisms in some Australian field-derived isolates of Haemonchus contortus we examined gene expression patterns and SNPs in nAChR subunit genes, as well as expression levels for P-glycoprotein (P-gp) and receptor ancillary protein genes, in various life stages of one levamisole-sensitive and three levamisole-resistant isolates of this species. Larvae of two isolates showed high-level resistance to levamisole (resistance ratios at the IC50 > 600) while the third isolate showed a degree of heterogeneity, with a resistance factor of only 1.1-fold at the IC50 alongside the presence of a resistant subpopulation. Transcription patterns for nAChR subunit genes showed a great degree of variability across the different life stages and isolates. The most consistent observation was the down-regulation of Hco-unc-63a in adults of all resistant isolates. Transcription of this gene was also reduced in the L3 stage of the two most resistant isolates, highlighting its potential as a resistance marker in the readily accessible free-living stages. There was down regulation of all four Hco-unc-29 paralogs in adults of one resistant isolate. There were no consistent changes in expression of P-gps or ancillary protein genes across the resistant isolates. The present study has demonstrated a complex pattern of nAChR subunit gene expression in H. contortus, and has highlighted several instances where reduced expression of subunit genes (Hco-unc-63a, Hco-unc-29) may be associated with the observed levamisole resistance. The data also suggests that it will be difficult to detect resistance using gene transcription-based methods on pooled larval samples from isolates containing only a resistant subpopulation due to

  7. The atypical alpha2beta2 IGF receptor expressed in inducible c2.7 myoblasts is derived from post-translational modifications of the mouse IGF-I receptor. (United States)

    Navarro, Magali; Joulia, Dominique; Fedon, Yann; Levin, Jonathan; Barenton, Bruno; Bernardi, Henri


    Unlike parental permissive C2.7 myoblasts, inducible C2.7 myoblasts require IGF-I or IGF-II to differentiate and expression of MyoD is not constitutive. Our previous studies indicated that inducible myoblasts express an atypical alpha2beta2 IGF receptor that differs from the classical IGF-I receptor by its higher affinity for IGF-II compared with IGF-I and the higher molecular weight of its alpha and beta subunits. Expression of this atypical IGF-I receptor is developmentally regulated; hence this receptor is lost upon terminal differentiation. Muscle cell differentiation is a system in which IGF-II plays an essential role and developmentally regulated atypical IGF-I receptor may represent a candidate for mediating differentiation signals provided by IGF-II. To further understand the structure and the role of the atypical IGF-I receptor, (i) we investigated for a putative IGF-I receptor transcript polymorphism by extensive sequencing of RT-PCR products; (ii) we overexpressed cloned mouse IGF-I receptor in permissive and inducible C2.7 myoblasts and characterized the binding and structural properties of overexpressed IGF-I receptor and (iii) we analysed the effects of this overexpression on myoblasts differentiation. Cultured mouse myoblasts C2.7 and subclone variant inducible C2.7 cell lines were used. Mouse IGF-I receptor cDNA was cloned by cDNA library screening. Gene expression was measured by semi-quantitative RT-PCR analysis and receptor affinity by ligand binding. Receptor protein autophosphorylation of IGF-IR was analysed by immunoprecipitation and Western blot. Myoblastic differentiation was accessed by myogenic factors expression and immunofluorescence study. Atypical IGF-I receptor may correspond to a new receptor belonging to the insulin/IGF-I receptor family, or it may also derive from alternate splicing of the gene of the insulin/IGF-I receptors and/or post-translational modifications of the insulin/IGF-I receptors. Our results exclude the existence of

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

    DEFF Research Database (Denmark)

    Tretter, Verena; Kerschner, Bernd; Milenkovic, Ivan


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

  9. Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors. (United States)

    Sirisoma, N S; Ratra, G S; Tomizawa, M; Casida, J E


    Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.

  10. Brain beta-adrenergic receptor binding in rats with obesity induced by a beef tallow diet. (United States)

    Matsuo, T; Suzuki, M


    We have previously reported that compared with safflower oil diet, feeding a beef tallow diet leads to a greater accumulation of body fat by reducing sympathetic activities. The present study examined the effects of dietary fats consisting of different fatty acids on alpha1- and beta-adrenergic receptor binding in the hypothalamus and cerebral cortex. Male Sprague-Dawley rats were meal-fed isoenergetic diets based on safflower oil (rich in n-6 polyunsaturated fatty acids) or beef tallow (rich in saturated fatty acids) for 8 weeks. Binding affinities of the beta-adrenergic receptor in the hypothalamus and cortex were significantly lower in the beef tallow diet group, but those of the alpha1-receptor did not differ between the two groups. The polyunsaturated to saturated fatty acid (P/S) ratio and fluidities of plasma membranes in the hypothalamus and cortex were lower in the beef tallow diet group than in the safflower oil diet group. These results suggest that the beef tallow diet decreases membrane fluidity by altering the fatty acid composition of plasma membranes in the hypothalamus and cerebral cortex of rat. Consequently, beta-adrenergic receptor binding affinities in the brain were lower in rats fed the beef tallow diet than in rats fed the safflower oil diet. We recognized that there is possible link between the membrane fluidity and the changes in affinity of beta-adrenoceptors in rat brain.

  11. Glucagon-like peptide-1 receptor agonist treatment reduces beta cell mass in normoglycaemic mice

    NARCIS (Netherlands)

    Ellenbroek, J.H.; Tons, H.A.; Westerouen van Meeteren, M.J.; de Graaf, N.; Hanegraaf, M.A.; Rabelink, T.J.; Carlotti, F.; de Koning, E.J.


    AIMS/HYPOTHESIS: Incretin-based therapies improve glycaemic control in patients with type 2 diabetes. In animal models of diabetes, glucagon-like peptide-1 receptor agonists (GLP-1RAs) increase beta cell mass. GLP-1RAs are also evaluated in non-diabetic individuals with obesity and cardiovascular di

  12. Estrogen receptors alpha and beta and the risk of open-angle glaucoma

    NARCIS (Netherlands)

    de Voogd, Simone; Wolfs, Roger C. W.; Jansonius, Nomdo M.; Uitterlinden, Andre G.; Pols, Huibert A. P.; Hofman, Albert; de Jong, Paulus T. V. M.


    Objective: To investigate whether polymorphisms in the estrogen receptor alpha (ESR1) and beta (ESR2) genes were a risk factor for open-angle glaucoma (OAG). Methods: Participants 55 years and older from the population-based Rotterdam Study underwent, at baseline and at follow-up, the same ophthalmi

  13. Thyroid Hormone Receptor beta Mediates Acute Illness-Induced Alterations in Central Thyroid Hormone Metabolism

    NARCIS (Netherlands)

    A. Boelen; J. Kwakkel; O. Chassande; E. Fliers


    Acute illness in mice profoundly affects thyroid hormone metabolism in the hypothalamus and pituitary gland. It remains unknown whether the thyroid hormone receptor (TR)-beta is involved in these changes. In the present study, we investigated central thyroid hormone metabolism during lipopolysacchar

  14. Age-associated alterations in hepatic. beta. -adrenergic receptor/adenylate cyclase complex

    Energy Technology Data Exchange (ETDEWEB)

    Graham, S.M.; Herring, P.A.; Arinze, I.J.


    The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 22-mo-old rats. The Mn/sup 2 +/-dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and approx. 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the ..beta..-adrenergic receptor, as measured by the binding of (/sup 125/I)-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase in the proportion of ..beta..-receptors in the high-affinity state. These observations suggest that ..beta..-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of ..beta..-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase.

  15. Estrogen Receptor beta 2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1 alpha in Prostate Cancer


    Prasenjit Dey; Velazquez-Villegas, Laura A.; Michelle Faria; Anthony Turner; Philp Jonsson; Paul Webb; Cecilia Williams; Jan-Åke Gustafsson; Ström, Anders M.


    The estrogen receptor (ER) beta variant ER beta 2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ER beta 2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ER beta 2 interacts with and stabilizes HIF-1 alpha protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1 alpha is known to stimulate met...

  16. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine. (United States)

    Savignac, Helene M; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P E; Tzortzis, George; Burnet, Philip W J


    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and

  17. In vitro gamma oscillations following partial and complete ablation of δ subunit-containing GABAA receptors from parvalbumin interneurons. (United States)

    Ferando, Isabella; Mody, Istvan


    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.

  18. GABA(A receptors containing the α2 subunit are critical for direction-selective inhibition in the retina.

    Directory of Open Access Journals (Sweden)

    Olivia Nicola Auferkorte

    Full Text Available Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS retinal circuits include several subtypes of ganglion cells (GCs and inhibitory interneurons, such as starburst amacrine cells (SACs. Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL of mouse and rabbit retina, GABA(A receptor subunit α2 (GABA(AR α2 aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(AR α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(AR α2 knock-out (KO mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(ARs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.

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

    KAUST Repository

    Kiraly, D. D.


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  1. Proteolytic fragments of laminin promote excitotoxic neurodegeneration by up-regulation of the KA1 subunit of the kainate receptor. (United States)

    Chen, Zu-Lin; Yu, Huaxu; Yu, Wei-Ming; Pawlak, Robert; Strickland, Sidney


    Degradation of the extracellular matrix (ECM) protein laminin contributes to excitotoxic cell death in the hippocampus, but the mechanism of this effect is unknown. To study this process, we disrupted laminin gamma1 (lamgamma1) expression in the hippocampus. Lamgamma1 knockout (KO) and control mice had similar basal expression of kainate (KA) receptors, but the lamgamma1 KO mice were resistant to KA-induced neuronal death. After KA injection, KA1 subunit levels increased in control mice but were unchanged in lamgamma1 KO mice. KA1 levels in tissue plasminogen activator (tPA)-KO mice were also unchanged after KA, indicating that both tPA and laminin were necessary for KA1 up-regulation after KA injection. Infusion of plasmin-digested laminin-1 into the hippocampus of lamgamma1 or tPA KO mice restored KA1 up-regulation and KA-induced neuronal degeneration. Interfering with KA1 function with a specific anti-KA1 antibody protected against KA-induced neuronal death both in vitro and in vivo. These results demonstrate a novel pathway for neurodegeneration involving proteolysis of the ECM and KA1 KA receptor subunit up-regulation.

  2. Repression of estrogen receptor {beta} function by putative tumor suppressor DBC1

    Energy Technology Data Exchange (ETDEWEB)

    Koyama, Satoshi [Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan); Wada-Hiraike, Osamu, E-mail: [Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan); Nakagawa, Shunsuke; Tanikawa, Michihiro; Hiraike, Haruko; Miyamoto, Yuichiro; Sone, Kenbun; Oda, Katsutoshi [Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan); Fukuhara, Hiroshi [Department of Urology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan); Nakagawa, Keiichi [Department of Radiology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan); Kato, Shigeaki [SORST, Japan Science and Technology, Honcho 4-1-8, Kawaguchi, Saitama 332-0012 (Japan); Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1 Bunkyo-ku, Tokyo 113-0034 (Japan); Yano, Tetsu; Taketani, Yuji [Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655 (Japan)


    It has been well established that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor (ER) {alpha} appears to promote the proliferation of cancer tissues, while ER{beta} can protect against the mitogenic effect of estrogen in breast tissue. The expression status of ER{alpha} and ER{beta} may greatly influence on the development, treatment, and prognosis of breast cancer. Previous studies have indicated that the deleted in breast cancer 1 (DBC1/KIAA1967) gene product has roles in regulating functions of nuclear receptors. The gene encoding DBC1 is a candidate for tumor suppressor identified by genetic search for breast cancer. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of the endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. In addition, DBC1 modulates ER{alpha} expression and promotes breast cancer cell survival by binding to ER{alpha}. Here we report an ER{beta}-specific repressive function of DBC1. Immunoprecipitation and immunofluorescence studies show that ER{beta} and DBC1 interact in a ligand-independent manner similar to ER{alpha}. In vitro pull-down assays revealed a direct interaction between DBC1 amino-terminus and activation function-1/2 domain of ER{beta}. Although DBC1 shows no influence on the ligand-dependent transcriptional activation function of ER{alpha}, the expression of DBC1 negatively regulates the ligand-dependent transcriptional activation function of ER{beta}in vivo, and RNA interference-mediated depletion of DBC1 stimulates the transactivation function of ER{beta}. These results implicate the principal role of DBC1 in regulating ER{beta}-dependent gene expressions.

  3. Identification of a single cytosine base insertion mutation at Arg-597 of the beta subunit of the human epithelial sodium channel in a family with Liddle's disease. (United States)

    Inoue, T; Okauchi, Y; Matsuzaki, Y; Kuwajima, K; Kondo, H; Horiuchi, N; Nakao, K; Iwata, M; Yokogoshi, Y; Shintani, Y; Bando, H; Saito, S


    We describe a family with Liddle's disease caused by a novel mutation of the beta subunit of the human epithelial sodium channel (ENaC). A 15-year-old Japanese female was referred to our outclinic because of hypertension. The physical examination showed no abnormal findings except mild hypertension, but the laboratory data revealed low levels of plasma renin activity, plasma aldosterone and serum potassium. A comprehensive analysis of steroid hormones showed only high levels of urinary free cortisol and 17-hydroxycorticosteroids. During loading tests, blood pressure and serum potassium responded well to triamterene and slightly to spironolactone, but did not respond to dexamethasone. In addition, the normal ratio of tetrahydrocortisol plus 5alpha-tetrahydrocortisol to tetrahydrocortisone in a 24 h urinary excretion test strongly suggested a diagnosis of Liddle's disease rather than apparent mineralocorticoid excess syndrome. DNA sequence analysis of members of this family revealed a single cytosine base insertion at Arg-597 of the beta human ENaC in the proband and her mother, leading to a loss of the last 34 amino acids from the normally encoded protein as the result of a frameshift. We conclude that a de novo cytosine insertion into the final exon of the C-terminus of the beta human ENaC is responsible for Liddle's disease in this Japanese family.

  4. Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle


    Ahern, Chris A; Powers, Patricia A.; Biddlecome, Gloria H; Roethe, Laura; Vallejo, Paola; Mortenson, Lindsay; Strube, Caroline; Campbell, Kevin P.; Coronado, Roberto; Gregg, Ronald G.


    Background The multisubunit (α1S,α2-δ, β1a and γ1) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca2+ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca2+ channel. To more fully investigate the function of the γ1 subunit in these two processes, we produced mice lacking this subunit by gene targeting. Results Mice lacking the DHPR γ1 subunit (γ1 null) survive to adulthood, are fertile and have no obvious gross phenotypic abno...

  5. The Roles of TGF-Beta and TGF-Beta Signaling Receptors in Breast Carcinogenesis. (United States)


    cell surface while HT1080 human fibrosarcoma cells used as a...expression.Monolayer confluent cultures of MCF-7 cells and human fibrosarcoma tions (Fig. 2). Therefore, the lack of cell surface RI and RII HT1080 cells ...examining several more human cell lines which are known to response to TGF-B and thus presumably express the type II receptor. If this antibody

  6. Complete inhibition of the tentoxin-resistant F1-ATPase from Escherichia coli by the phytopathogenic inhibitor tentoxin after substitution of critical residues in the alpha - and beta -subunit. (United States)

    Schnick, Claudia; Körtgen, Nicole; Groth, Georg


    Substitution of critical residues in the alpha- and beta-subunit can turn the typically resistant ATP synthase from the bacterium Escherichia coli into an enzyme showing high sensitivity to the phytopathogenic inhibitor tentoxin, which usually affects only certain sensitive plant species. In contrast to recent results obtained with the thermophilic F(1) (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119), substitution of a critical serine in the beta-subunit (betaSer(59)), which is supposed to provide an important intermolecular hydrogen bond in the binding site, was not sufficient on its own for conferring tentoxin sensitivity to the E. coli F(1) complex. Superimposition of the chloroplast F(1)-tentoxin inhibitor complex on a homology model of the E. coli F(1) complex provided detailed information on the critical residues in the alpha-subunit of the binding cleft and allowed us to model the binding site according to the steric requirements of the inhibitor. Substitution of the highly conserved residue alphaLeu(64) seems to be most important for allowing access of the inhibitor to the binding site. Combining this substitution with either additional replacements in the alpha-subunit (Q49A, L95A, E96Q, I273M) or the replacement of Ser(59) in the beta-subunit enhanced the sensitivity to the inhibitor and resulted in a complete inhibition of the E. coli F(1)-ATPase by the plant-specific inhibitor tentoxin.

  7. DHEA metabolites activate estrogen receptors alpha and beta


    Michael Miller, Kristy K.; AL-RAYYAN, NUMAN; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M; Prough, Russell A.


    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, andro...

  8. GABAB receptor subunit GB1 at the cell surface independently activates ERK1/2 through IGF-1R transactivation.

    Directory of Open Access Journals (Sweden)

    Guillaume A Baloucoune

    Full Text Available BACKGROUND: Functional GABA(B receptor is believed to require hetero-dimerization between GABA(B1 (GB1 and GABA(B2 (GB2 subunits. The GB1 extracellular domain is required for ligand binding, and the GB2 trans-membrane domain is responsible for coupling to G proteins. Atypical GABA(B receptor responses observed in GB2-deficient mice suggested that GB1 may have activity in the absence of GB2. However the underlying mechanisms remain poorly characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here, by using cells overexpressing a GB1 mutant (GB1asa with the ability to translocate to the cell surface in the absence of GB2, we show that GABA(B receptor agonists, such as GABA and Baclofen, can induce ERK1/2 phosphorylation in the absence of GB2. Furthermore, we demonstrate that GB1asa induces ERK1/2 phosphorylation through Gi/o proteins and PLC dependent IGF-1R transactivation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that GB1 may form a functional receptor at the cell surface in the absence of GB2.

  9. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

    Directory of Open Access Journals (Sweden)

    Incilay Sinici

    Full Text Available The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # (encoded by the HEXA and HEXB genes, respectively, and the GM2-activator protein (GM2AP, encoded by the GM2A gene. Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750. Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1 and a new more extensive hybrid (H2, with our documented in cellulo (live cell- based assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

  10. Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening (United States)

    Talukder, Iehab


    The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel–forming domain, composed of M1–M4 segments, are connected by three linkers: S1–M1, M3–S2, and S2–M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3–S2 and S2–M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process. PMID:21746848

  11. A Trp474Cys mutation in the alpha-subunit of beta-hexosaminidase causes a subacute encephalopathic form of G{sub M2} gangliosidosis, type 1

    Energy Technology Data Exchange (ETDEWEB)

    Petroulakis, E.; Cao, Z.; Salo, T. [Univ. of Manitoba, Winnipeg (Canada)] [and others


    Mutations in the HEXA gene that encodes the {alpha}-subunit of the heterodimeric lysosomal enzyme {beta}-hexosaminidase A, or Hex A ({alpha}{beta}), cause G{sub M2} gangliosidosis, type 1. The infantile form (Tay-Sachs disease) results when there is no residual Hex A activity, while less severe and more variable clinical phenotypes result when residual Hex A activity is present. A non-Jewish male who presented with an acute psychotic episode at age 16 was diagnosed with a subacute encephalopathic form of G{sub M2} gangliosidosis. At age 19, chronic psychosis with intermittent acute exacerbations remains the most disabling symptom in this patient and his affected brother although both exhibit some ataxia and moderately severe dysarthria. We have found a 4 bp insertion (+TATC 1278) associated with infantile Tay-Sachs disease on one allele; no previously identified mutation was found on the second allele. SSCP analysis detected a shift in exon 13 and sequencing revealed a G1422C mutation in the second allele that results in a Trp474Cys substitution. The presence of the mutation was confirmed by the loss of HaeIII and ScrFI sites in exon 13 PCR products from the subjects and their father. The mutation was introduced into the {alpha}-subunit cDNA and Hex S ({alpha}{alpha}) and Hex A ({alpha}{beta}) were transiently expressed in monkey COS-7 cells. The Trp474Cys mutant protein had approximately 5% and 12% of wild-type Hex S and Hex A activity, respectively. Western blot analysis revealed a small amount of residual mature {alpha}-subunit and a normal level of precursor protein. We conclude that the Trp474Cys mutation is the cause of the Hex A deficiency associated with a subacute (juvenile-onset) phenotype in this patient. Like other mutations in exon 13 of HEXA, it appears to affect intracellular processing. Studies of the defect in intracellular processing are in progress.

  12. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity. (United States)

    de Fiebre, Nancyellen C; de Fiebre, Christopher M


    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  13. GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

    Directory of Open Access Journals (Sweden)

    De Blas Angel L


    Full Text Available Abstract Background Gamma-aminobutyric acid type A receptors (GABAA-Rs are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1 insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably

  14. Beta-Adrenergic Receptors and Mechanisms in Asthma: The New Long-Acting Beta-Agonists

    Directory of Open Access Journals (Sweden)

    Robert G Townley


    Full Text Available The objective is to review β-adrenergic receptors and mechanisms in the immediate and late bronchial reaction in asthma and the new long-acting β-agonist. This will be discussed in light of the controversy of the potential adverse effect of regular use of long-acting β-agonists. We studied the effect of formoterol on the late asthmatic response (LAR and airway inflammation in guinea-pigs. Formoterol suppressed the LAR, antigen-induced airway inflammation and hyperresponsiveness, although isoproterenol failed to inhibit these parameters. β-Adrenergic hyporesponsiveness, and cholinergic and a- adrenergic hyperresponsiveness have been implicated in the pathogenesis of asthma. A decrease in β-adrenoreceptor function can result either from exogenously administered β-agonist or from exposure to allergens resulting in a late bronchial reaction. There is increasing evidence that eosinophils, macrophages, and lymphocytes which are of primary importance in the late bronchial reaction are also modulated by β2- adrenoreceptors. In functional studies of guinea-pig or human isolated trachea and lung parenchyma, PAF and certain cytokines significantly reduced the potency of isoproterenol to reverse methacholine- or histamine-induced contraction. The effect of glucocorticoids on pulmonary β-adrenergic receptors and responses suggests an important role for glucocorticoids to increase β-adrenergic receptors and responsiveness.

  15. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

    NARCIS (Netherlands)

    Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.


    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where i

  16. AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2/3 subunits in most interneurons. (United States)

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


    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.

  17. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))


    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  18. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)


    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  19. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

    Directory of Open Access Journals (Sweden)

    Morten Skøtt Thomsen

    Full Text Available 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.

  20. DHEA metabolites activate estrogen receptors alpha and beta (United States)

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M.; Prough, Russell A.


    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβ in vitro, modulating estrogen target genes in vivo. PMID:23123738

  1. Impact of rs361072 in the phosphoinositide 3-kinase p110beta gene on whole-body glucose metabolism and subunit protein expression in skeletal muscle

    DEFF Research Database (Denmark)

    Ribel-Madsen, Rasmus; Poulsen, Pernille; Holmkvist, Johan


    . The aim was to investigate the influence of rs361072 on in vivo glucose metabolism, skeletal muscle PI3K subunit protein levels, and type 2 diabetes. RESEARCH DESIGN AND METHODS: The functional role of rs361072 was studied in 196 Danish healthy adult twins. Peripheral and hepatic insulin sensitivity...... was assessed by a euglycemic-hyperinsulinemic clamp. Basal and insulin-stimulated biopsies were taken from the vastus lateralis muscle, and tissue p110beta and p85alpha proteins were measured by Western blotting. The genetic association with type 2 diabetes and quantitative metabolic traits was investigated...... in 9,316 Danes with glucose tolerance ranging from normal to overt type 2 diabetes. RESULTS: While hepatic insulin resistance was similar in the fasting state, carriers of the minor G allele had lower hepatic glucose output (per-allele effect: -16%, P(add) = 0.004) during high physiological insulin...

  2. Subcellular distribution of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells within subventricular zone of adult rats

    Institute of Scientific and Technical Information of China (English)

    Zhining Li; Wenlong Lü; Hongyan Dong; Hongbin Fan; Ruiguo Dong; Tiejun Xu


    The subcellular localization of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells of the subventricular zone of adult rats was detected using electron microscopy, following immunohistochemistry and immunogold-silver double staining. Results confirmed the presence of neural stem cells in the subventricular zone, which is a key neurogenic region in the central nervous system of adult mammals. The expression of N-methyl-D-aspartic acid receptor subunit 1 was higher than that of nestin and mainly distributed in the cell membrane, cytoplasm, rough endoplasmic reticulum and Golgi complex of neural stem cells.

  3. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    Directory of Open Access Journals (Sweden)

    Zhao Xueqiang


    Full Text Available Abstract Background In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p. Results We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain α-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p. Conclusion Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61β in processes unlinked to protein translocation.

  4. Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction. (United States)

    Christensen, Mark H; Kohlmeier, Kristi A


    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. © 2014 Society for the Study of Addiction.

  5. Ligand-dependent inhibition of beta-catenin/TCF signaling by androgen receptor. (United States)

    Chesire, Dennis R; Isaacs, William B


    Beta-catenin signaling may contribute to prostate cancer (CaP) progression. Although beta-catenin is known to upregulate T cell factor (TCF) target gene expression in CaP cells, recent evidence demonstrates its capacity to enhance ligand-dependent androgen receptor (AR) function. Thus, we wished to further understand the interaction between these two pathways. We find in both CaP cells (CWR22-Rv1, LAPC-4, DU145) and non-CaP cells (HEK-293, TSU, SW480, HCT-116) that beta-catenin/TCF-related transcription (CRT), as measured by activation of a synthetic promoter and that of cyclin D1, is inhibited by androgen treatment. This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Additional analyses convey that the ligand-dependent nature of CRT suppression depends on transactivation-competent AR in the nucleus, but not on indirect effects stemming from AR target gene expression. Given the recent work identifying an AR/beta-catenin interaction, and from our finding that liganded AR does not prompt gross changes in the constitutive nuclear localization of TCF4 or mutant beta-catenin, we hypothesized that transcription factor (i.e. AR and TCF) competition for beta-catenin recruitment may explain, in part, androgen-induced suppression of CRT. To address this idea, we expressed an AR mutant lacking its DNA-binding domain (DBD). This receptor could not orchestrate ligand-dependent CRT repression, thereby providing support for those recent data implicating the AR DBD/LBD as necessary for beta-catenin interaction. Further supporting this hypothesis, TCF/LEF over-expression counteracts androgen-induced suppression of CRT, and requires beta-catenin binding activity to do so. Interestingly, TCF4 over-expression potently antagonizes AR function; however, this inhibition may occur independently of beta-catenin/TCF4 interaction. These results from TCF4 over-expression analyses, taken together, provide

  6. The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation. (United States)

    Poulsen, Lisbeth Rosager; López-Marqués, Rosa Laura; McDowell, Stephen C; Okkeri, Juha; Licht, Dirk; Schulz, Alexander; Pomorski, Thomas; Harper, Jeffrey F; Palmgren, Michael Gjedde


    Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

  7. Delta-subunit containing GABAA-receptors mediate tonic inhibition in paracapsular cells of the mouse amygdala

    Directory of Open Access Journals (Sweden)

    Anne eMarowsky


    Full Text Available 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 postsynaptic 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.

  8. Blockade of NMDA receptors 2A subunit in the dorsal striatum impairs the learning of a complex motor skill. (United States)

    Lemay-Clermont, Julie; Robitaille, Christine; Auberson, Yves P; Bureau, Geneviève; Cyr, Michel


    Accumulating evidence proposes that the striatum, known to control voluntary movement, may also play a role in learning and memory. Striatum learning is thought to require long-lasting reorganization of striatal circuits and changes in the strength of synaptic connections during the memorization of a complex motor task. Whether the ionotropic glutamate receptor N-methyl-D-aspartate (NMDAR) contributes to the molecular mechanisms of these memory processes is still unclear. The aim of the present study was to investigate the role of striatal NMDAR and its subunit composition during the learning of the accelerating rotarod task in mice. To this end, we injected directly into the dorsal striatum of mice, via chronically implanted cannula, the NMDAR channel blocker MK-801 as well as the NR2A and NR2B subunit-selective antagonists NVP-AAM077 and Ro 25-6981, respectively, before rotarod training. There was no effect in the motor performances of mice treated with 1.0 μg/side of MK-801, 0.1 μg/side of NVP-AAM077, or 5 and 10 μg/side of Ro 25-6981. In contrast, injections of 2.5 and 5 μg/side of MK-801 or 0.5 and 1 μg/side of NVP-AAM077 impaired motor learning at Day 3 and 8. Interestingly, treatments with MK-801 and NVP-AAM077 did not alter the general motor capacities of mice as revealed by the stepping, wire suspension, and pole tests. Our study demonstrates that the NMDAR of the dorsal striatum contributes to motor learning, especially during the slow acquisition phase, and that NR2A subunits play a critical role in this process.

  9. {beta}1-Adrenergic receptor activation induces mouse cardiac myocyte death through both L-type calcium channel-dependent and -independent pathways. (United States)

    Wang, Wei; Zhang, Hongyu; Gao, Hui; Kubo, Hajime; Berretta, Remus M; Chen, Xiongwen; Houser, Steven R


    Cardiac diseases persistently increase the contractility demands of cardiac myocytes, which require activation of the sympathetic nervous system and subsequent increases in myocyte Ca(2+) transients. Persistent exposure to sympathetic and/or Ca(2+) stress is associated with myocyte death. This study examined the respective roles of persistent beta-adrenergic receptor (beta-AR) agonist exposure and high Ca(2+) concentration in myocyte death. Ventricular myocytes (VMs) were isolated from transgenic (TG) mice with cardiac-specific and inducible expression of the beta(2a)-subunit of the L-type Ca(2+) channel (LTCC). VMs were cultured, and the rate of myocyte death was measured in the presence of isoproterenol (ISO), other modulators of Ca(2+) handling and the beta-adrenergic system, and inhibitors of caspases and reactive oxygen species generation. The rate of myocyte death was greater in TG vs. wild-type myocytes and accelerated by ISO in both groups, although ISO did not increase LTCC current (I(Ca-L)) in TG-VMs. Nifedipine, an LTCC antagonist, only partially prevented myocyte death. These results suggest both LTCC-dependent and -independent mechanisms in ISO induced myocyte death. ISO increased the contractility of wild type and TG-VMs by enhancing sarcoplasmic reticulum function and inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger, and CaMKII partially protected myocyte from death induced by both Ca(2+) and ISO. Caspase and reactive oxygen species inhibitors did not, but beta(2)-AR activation did, reduce myocyte death induced by enhanced I(Ca-L) and ISO stimulation. Our results suggest that catecholamines induce myocyte necrosis primarily through beta(1)-AR-mediated increases in I(Ca-L), but other mechanisms are also involved in rodents.

  10. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    DEFF Research Database (Denmark)

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis


    (A) receptors in mouse neurons in vitro and in vivo. Whole-cell patch-clamp recordings were carried out in the dentate gyrus in mouse brain slices. In granule cells, AA29504 (1 μM) caused a 4.2-fold potentiation of a tonic current induced by THIP (1 μM), while interneurons showed a potentiation of 2.6-fold......, and possibly recruits perisynaptic δ-containing receptors to participate in synaptic phasic inhibition in dentate gyrus....

  11. Mutations in Two Genes Encoding Different Subunits of a Receptor Signaling Complex Result in an Identical Disease Phenotype (United States)

    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


    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

  12. Traumatic brain injury and the effects of diazepam, diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

    Directory of Open Access Journals (Sweden)

    Meyer Rebecca C


    Full Text Available Abstract Background Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood. Methods We used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits α1, α2, α3, α5, β3, and γ2 at 3 h, 6 h, 24 h, and 7 days post-injuy. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltage-gated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of α1, α2, α3, and γ2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors. Results Western blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor α2 or α5 subunits at any time point post-injury. Significant time-dependent changes in α1, α3, β3, and γ2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI. Conclusions These studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function.

  13. GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

    Directory of Open Access Journals (Sweden)

    Yang Yongguang


    Full Text Available Abstract Background The proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF, a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1, RET tyrosine kinase, and neural cell adhesion molecule (NCAM have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs. However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs. Results In the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs. Conclusions GDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

  14. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P;


    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  15. Bilaterian phylogeny based on analyses of a region of the sodium-potassium ATPase beta-subunit gene. (United States)

    Anderson, Frank E; Córdoba, Alonso J; Thollesson, Mikael


    Molecular investigations of deep-level relationships within and among the animal phyla have been hampered by a lack of slowly evolving genes that are amenable to study by molecular systematists. To provide new data for use in deep-level metazoan phylogenetic studies, primers were developed to amplify a 1.3-kb region of the alpha subunit of the nuclear-encoded sodium-potassium ATPase gene from 31 bilaterians representing several phyla. Maximum parsimony, maximum likelihood, and Bayesian analyses of these sequences (combined with ATPase sequences for 23 taxa downloaded from GenBank) yield congruent trees that corroborate recent findings based on analyses of other data sets (e.g., the 18S ribosomal RNA gene). The ATPase-based trees support monophyly for several clades (including Lophotrochozoa, a form of Ecdysozoa, Vertebrata, Mollusca, Bivalvia, Gastropoda, Arachnida, Hexapoda, Coleoptera, and Diptera) but do not support monophyly for Deuterostomia, Arthropoda, or Nemertea. Parametric bootstrapping tests reject monophyly for Arthropoda and Nemertea but are unable to reject deuterostome monophyly. Overall, the sodium-potassium ATPase alpha-subunit gene appears to be useful for deep-level studies of metazoan phylogeny.

  16. Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

    Directory of Open Access Journals (Sweden)

    Huitfeldt Henrik S


    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. Results We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. Conclusion Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

  17. Deletion of the NMDA receptor GluN2A subunit significantly decreases dendritic growth in maturing dentate granule neurons.

    Directory of Open Access Journals (Sweden)

    Timal S Kannangara

    Full Text Available It is known that NMDA receptors can modulate adult hippocampal neurogenesis, but the contribution of specific regulatory GluN2 subunits has been difficult to determine. Here we demonstrate that mice lacking GluN2A (formerly NR2A do not show altered cell proliferation or neuronal differentiation, but present significant changes in neuronal morphology in dentate granule cells. Specifically, GluN2A deletion significantly decreased total dendritic length and dendritic complexity in DG neurons located in the inner granular zone. Furthermore, the absence of GluN2A also resulted in a localized increase in spine density in the middle molecular layer, a region innervated by the medial perforant path. Interestingly, alterations in dendritic morphology and spine density were never seen in dentate granule cells located in the outer granular zone, a region that has been hypothesized to contain older, more mature, neurons. These results indicate that although the GluN2A subunit is not critical for the cell proliferation and differentiation stages of the neurogenic process, it does appear to play a role in establishing synaptic and dendritic morphology in maturing dentate granule cells localized in the inner granular zone.

  18. Targeted deletion of the mouse α2 nicotinic acetylcholine receptor subunit gene (Chrna2) potentiates nicotine-modulated behaviors. (United States)

    Lotfipour, Shahrdad; Byun, Janet S; Leach, Prescott; Fowler, Christie D; Murphy, Niall P; Kenny, Paul J; Gould, Thomas J; Boulter, Jim


    Baseline and nicotine-modulated behaviors were assessed in mice harboring a null mutant allele of the nicotinic acetylcholine receptor (nAChR) subunit gene α2 (Chrna2). Homozygous Chrna2(-/-) mice are viable, show expected sex and Mendelian genotype ratios, and exhibit no gross neuroanatomical abnormalities. A broad range of behavioral tests designed to assess genotype-dependent effects on anxiety (elevated plus maze and light/dark box), motor coordination (narrow bean traverse and gait), and locomotor activity revealed no significant differences between mutant mice and age-matched wild-type littermates. Furthermore, a panel of tests measuring traits, such as body position, spontaneous activity, respiration, tremors, body tone, and startle response, revealed normal responses for Chrna2-null mutant mice. However, Chrna2(-/-) mice do exhibit a mild motor or coordination phenotype (a decreased latency to fall during the accelerating rotarod test) and possess an increased sensitivity to nicotine-induced analgesia in the hotplate assay. Relative to wild-type, Chrna2(-/-) mice show potentiated nicotine self-administration and withdrawal behaviors and exhibit a sex-dependent enhancement of nicotine-facilitated cued, but not trace or contextual, fear conditioning. Overall, our results suggest that loss of the mouse nAChR α2 subunit has very limited effects on baseline behavior but does lead to the potentiation of several nicotine-modulated behaviors.

  19. Ethanol activation of protein kinase A regulates GABA-A receptor subunit expression in the cerebral cortex and contributes to ethanol-induced hypnosis

    Directory of Open Access Journals (Sweden)

    Sandeep eKumar


    Full Text Available Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC and A (PKA are involved in regulation of γ-aminobutyric acid type A (GABA-A receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

  20. Recruitment of beta-arrestin2 to the dopamine D2 receptor: insights into anti-psychotic and anti-parkinsonian drug receptor signaling

    DEFF Research Database (Denmark)

    Klewe, Ib V; Nielsen, Søren M; Tarpø, Louise


    Drugs acting at dopamine D2-like receptors play a pivotal role in the treatment of both schizophrenia and Parkinson's disease. Recent studies have demonstrated a role for G-protein independent D2 receptor signaling pathways acting through beta-arrestin. In this study we describe the establishment...... of a Bioluminescence Resonance Energy Transfer (BRET) assay for measuring dopamine induced recruitment of human beta-arrestin2 to the human dopamine D2 receptor. Dopamine, as well as the dopamine receptor agonists pramipexole and quinpirole, acted as full agonists in the assay as reflected by their ability to elicit...... marked concentration dependent increases in the BRET signal signifying beta-arrestin2 recruitment to the D2 receptor. As expected from their effect on G-protein coupling and cAMP levels mediated through the D2 receptor RNPA, pergolide, apomorphine, ropinirole, bromocriptine, 3PPP, terguride, aripiprazole...

  1. Conversion of agonist site to metal-ion chelator site in the beta(2)-adrenergic receptor

    DEFF Research Database (Denmark)

    Elling, C E; Thirstrup, K; Holst, Birgitte


    in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic......Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the beta(2)-adrenergic receptor......, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III-or a His residue introduced at this position-and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated...

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

    Directory of Open Access Journals (Sweden)

    Sean C. Piantadosi


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

  3. Glucagon and plasma catecholamines during beta-receptor blockade in exercising man

    DEFF Research Database (Denmark)

    Galbo, H; Holst, Janett; Christensen, N J


    Seven men ran at 60% of individual maximal oxygen uptake to exhaustion during beta-adrenergic blockade with propranolol (P), during lipolytic blockade with nicotinic acid (N), or without drugs (C). The total work times (83 +/- 9 (P), 122 +/- 8 (N), 166 +/- 10 (C) min, mean and SE) differed...... decrease glucagon concentrations increased progressively in parallel with declining plasma glucose and were at exhaustion always three times preexercise values. Thus beta-adrenergic blockade did not diminish the glucagon response. Nor was this response increased when alpha-receptor stimulation in P...... experiments was intensified. Carbohydrate combustion was smaller and NEFA and glycerol concentrations in serum larger during C experiments. Alanine concentrations were never raised at exhaustion. Accordingly, neither stimulation of adrenergic receptors nor NEFA and alanine concentrations are major...

  4. Photoaffinity labeling of alpha- and beta- scorpion toxin receptors associated with rat brain sodium channel. (United States)

    Darbon, H; Jover, E; Couraud, F; Rochat, H


    Azido nitrophenylaminoacetyl [125I]iodo derivative of toxin II from Centruroides suffusus suffusus, a beta-toxin, and azido nitrophenylaminoacetyl [125I]iodo derivative of toxin V from Leiurus quinquestriatus quinquestriatus, an alpha-toxin, have been covalently linked after binding to their receptor sites that are related to the voltage sensitive sodium channel present in rat brain synaptosomes. Both derivatives labeled two polypeptides of 253000 +/- 20000 and 35000 +/- 2000 mol. wt. Labeling was blocked for each derivative by a large excess of the corresponding native toxin but no cross inhibition was obtained. These results suggest that both alpha - and beta - scorpion toxin receptors are located on or near the same two membrane polypeptides which may be part of the voltage dependent sodium channel.

  5. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte


    with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable......Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor...... Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding...

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

    DEFF Research Database (Denmark)

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


    The circadian rhythms of mammals are generated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Its intrinsic period is entrained to a 24 h cycle by external cues, mainly by light. Light impinging on the SCN at night causes either advancing or delaying phase...... shifts of the circadian clock. N-methyl-d-aspartate receptors (NMDAR) are the main glutamate receptors mediating the effect of light on the molecular clockwork in the SCN. They are composed of multiple subunits, each with specific characteristics whose mutual interactions strongly determine properties...... of the receptor. In the brain, the distribution of NMDAR subunits depends on the region and developmental stage. Here, we report the circadian expression of the NMDAR1 subunit in the adult rat SCN and depict its splice variants that may constitute the functional receptor channel in the SCN. During ontogenesis...

  7. Chemokine receptor expression on B cells and effect of interferon-beta in multiple sclerosis

    DEFF Research Database (Denmark)

    Sørensen, Torben Lykke; Roed, Hanne; Sellebjerg, Finn


    We investigated the B-cell expression of chemokine receptors CXCR3, CXCR5 and CCR5 in the blood and cerebrospinal fluid (CSF) from patients in relapse of multiple sclerosis (MS) and in neurological controls. Chemokine receptor expression was also studied in interferon-beta-treated patients...... with relapsing-remitting or secondary progressive MS. We observed significantly higher expression of CXCR3 on B cells in the CSF in active MS than in controls. Patients with active MS also had higher B-cell expression of CCR5 in blood. No major differences between RRMS and SPMS patients were detected...

  8. Polimorfismos del receptor adrenérgico beta-1 y sus implicaciones farmacodinámicas


    Ignacio Rodríguez; Jesualdo Fuentes; Valery Valencia; Fanny Cuesta González; Gabriel Bedoya Berrío; Sergio Parra


    Los betabloqueadores son fármacos que han demostrado eficacia
    clínica al disminuir tanto la morbilidad como la mortalidad de múltiples enfermedades cardiovasculares. Como común denominador todos los bloqueadores beta adrenérg