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Sample records for gaba-a receptor subunit

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

    PubMed

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

    2000-12-01

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

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

    PubMed

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

    2010-10-15

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

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

    PubMed

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

    2003-10-01

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

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

    PubMed

    Waldvogel, H J; Faull, R L M

    2015-01-01

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

  5. Altered gamma oscillations during pregnancy through loss of δ subunit-containing GABA(A) receptors on parvalbumin interneurons.

    PubMed

    Ferando, Isabella; Mody, Istvan

    2013-01-01

    Gamma (γ) oscillations (30-120 Hz), an emergent property of neuronal networks, correlate with memory, cognition and encoding. In the hippocampal CA3 region, locally generated γ oscillations emerge through feedback between inhibitory parvalbumin-positive basket cells (PV+BCs) and the principal (pyramidal) cells. PV+BCs express δ-subunit-containing GABA(A)Rs (δ-GABA(A)Rs) and NMDA receptors (NMDA-Rs) that balance the frequency of γ oscillations. Neuroactive steroids (NS), such as the progesterone-derived (3α,5α)-3-hydroxy-pregnan-20-one (allopregnanolone; ALLO), modulate the expression of δ-GABA(A)Rs and the tonic conductance they mediate. Pregnancy produces large increases in ALLO and brain-region-specific homeostatic changes in δ-GABA(A)Rs expression. Here we show that in CA3, where most PV+ interneurons (INs) express δ-GABA(A)Rs, expression of δ-GABA(A)Rs on INs diminishes during pregnancy, but reverts to control levels within 48 h postpartum. These anatomical findings were corroborated by a pregnancy-related increase in the frequency of kainate-induced CA3 γ oscillations in vitro that could be countered by the NMDA-R antagonists D-AP5 and PPDA. Mimicking the typical hormonal conditions during pregnancy by supplementing 100 nM ALLO lowered the γ frequencies to levels found in virgin or postpartum mice. Our findings show that states of altered NS levels (e.g., pregnancy) may provoke perturbations in γ oscillatory activity through direct effects on the GABAergic system, and underscore the importance of δ-GABA(A)Rs homeostatic plasticity in maintaining constant network output despite large hormonal changes. Inaccurate coupling of NS levels to δ-GABA(A)R expression may facilitate abnormal neurological and psychiatric conditions such as epilepsy, post-partum depression, and post-partum psychosis, thus providing insights into potential new treatments.

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

    PubMed Central

    Gangisetty, Omkaram; Reddy, Doodipala Samba

    2009-01-01

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

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

    PubMed

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

    2002-05-01

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

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

    PubMed

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

    2002-01-01

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

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

    PubMed

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

    2012-08-01

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

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

    PubMed

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

    2000-01-01

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

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

    PubMed

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

    2016-07-01

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

  12. GABA(A) receptor epsilon-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat.

    PubMed

    Kasparov, S; Davies, K A; Patel, U A; Boscan, P; Garret, M; Paton, J F

    2001-11-01

    1. Benzodiazepines (BZ) and barbiturates both potentiate chloride currents through GABA(A) receptors to enhance inhibition. However, unlike barbiturates BZ do not impair autonomic control of heart rate. We hypothesised that BZ might not significantly potentiate GABAergic transmission in the caudal nucleus of the solitary tract (cNTS), which is critically important for mediating the baroreceptor reflex. 2. In rat brain slices the BZ agonists chlordiazepoxide and midazolam (2 and 50 microM) did not significantly enhance currents evoked by GABA in voltage-clamped cNTS neurones. Chlordiazepoxide (50 microM) reversibly increased electrically evoked IPSPs in 5/10 rostral NTS (rNTS) neurones but only in 2/10 cNTS neurones. Pentobarbitone (50-100 microM) was effective in enhancing GABA(A)-mediated responses in all NTS neurones. An inverse BZ agonist, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM; 1 or 10 microM), failed to depress GABA-induced currents in the cNTS. 3. Microinjections of midazolam (10 and 100 microM solutions) into the cNTS did not affect the baroreceptor reflex (P > 0.2) while pentobarbitone (100 microM) significantly and reversibly depressed it (gain decrease to 53 +/- 11 % of control, P < 0.01). 4. Reverse transcriptase polymerase chain reaction revealed the presence of alpha(1), alpha(2), beta(2), beta(3) and gamma(2) GABA(A) receptor subunit mRNA in the cNTS. No alternatively spliced variants of the alpha(1)- and gamma(2)-subunits were revealed. Moreover, GABA(A) epsilon-unit mRNA was found in both the cNTS and rNTS as two alternatively spliced transcripts. 5. Immunocytochemical analysis revealed numerous GABA(A) epsilon-subunit-positive neurones within the cNTS with significantly fewer epsilon-subunit-positive cells in the rNTS. 6. As incorporation of the epsilon-subunit in recombinant GABA(A) receptors may confer BZ insensitivity we propose that the paucity of BZ actions in the cNTS is due to a high level of epsilon-subunit

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

    PubMed

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

    2014-07-01

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

  14. GABA-A and NMDA receptor subunit mRNA expression is altered in the caudate but not the putamen of the postmortem brains of alcoholics.

    PubMed

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

    2014-01-01

    Chronic consumption of alcohol by humans has been shown to lead to impairment of executive and cognitive functions. Here, we have studied the mRNA expression of ion channel receptors for glutamate and GABA in the dorsal striatum of post-mortem brains from alcoholics (n = 29) and normal controls (n = 29), with the focus on the caudate nucleus that is associated with the frontal cortex executive functions and automatic thinking and on the putamen area that is linked to motor cortices and automatic movements. The results obtained by qPCR assay revealed significant changes in the expression of specific excitatory ionotropic glutamate and inhibitory GABA-A receptor subunit genes in the caudate but not the putamen. Thus, in the caudate we found reduced levels of mRNAs encoding the GluN2A glutamate receptor and the δ, ε, and ρ2 GABA-A receptor subunits, and increased levels of the mRNAs encoding GluD1, GluD2, and GABA-A γ1 subunits in the alcoholics as compared to controls. Interestingly in the controls, 11 glutamate and 5 GABA-A receptor genes were more prominently expressed in the caudate than the putamen (fold-increase varied from 1.24 to 2.91). Differences in gene expression patterns between the striatal regions may underlie differences in associated behavioral outputs. Our results suggest an altered balance between caudate-mediated voluntarily controlled and automatic behaviors in alcoholics, including diminished executive control on goal-directed alcohol-seeking behavior.

  15. Drug interactions at GABA(A) receptors.

    PubMed

    Korpi, Esa R; Gründer, Gerhard; Lüddens, Hartmut

    2002-06-01

    Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the gamma-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16-19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABA(A) receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors' various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABA(A) receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABA(A) receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABA(A) receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man.

  16. Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy.

    PubMed

    Bouilleret, V; Loup, F; Kiener, T; Marescaux, C; Fritschy, J M

    2000-01-01

    Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of

  17. Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the alpha4 subunit of GABA(A) receptors in an animal model of epilepsy.

    PubMed

    Sun, Chengsan; Mtchedlishvili, Zakaria; Erisir, Alev; Kapur, Jaideep

    2007-11-14

    In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABA(A) receptors on dentate granule cells (DGCs) is diminished; the molecular mechanism underlying this phenomenon remains unclear. The current study investigated a mechanism for loss of neurosteroid sensitivity of synaptic GABA(A) receptors in TLE. Synaptic currents recorded from DGCs of epileptic animals (epileptic DGCs) were less frequent, larger in amplitude, and less sensitive to allopregnanolone modulation than those recorded from DGCs of control animals (control DGCs). Synaptic currents recorded from epileptic DGCs were less sensitive to diazepam and had altered sensitivity to benzodiazepine inverse agonist RO 15-4513 (ethyl-8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) and furosemide than those recorded from control DGCs. Properties of synaptic currents recorded from epileptic DGCs appeared similar to those of recombinant receptors containing the alpha4 subunit. Expression of the alpha4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the alpha4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The alpha4 immunogold labeling was present more commonly within the synapse in epileptic DGCs compared with control DGCs, in which the subunit was extrasynaptic. These studies demonstrate that, in epileptic DGCs, the neurosteroid modulation of synaptic currents is diminished and alpha4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

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

    PubMed Central

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

    2012-01-01

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

  19. Localization of two major GABA(A) receptor subunits in the dentate gyrus of the rat and cell type-specific up-regulation following entorhinal cortex lesion.

    PubMed

    Simbürger, E; Plaschke, M; Fritschy, J M; Nitsch, R

    2001-01-01

    GABA(A) receptor subunits show a specific regional distribution in the CNS during development and in the adult animal. In the hippocampal formation, individual subsets of GABAergic interneurons are highly immunoreactive for the alpha1-subunit, whereas granule and pyramidal cells show a strong expression of the alpha2-subunit. Using confocal microscopy and digital image analysis, we demonstrate that in the dentate gyrus the alpha1-subunit immunolabeling appears in differently sized clusters. The large clusters, which are confined to dendrites of interneurons, show no alpha2 labeling, whereas the smaller ones coincide with alpha2-subunit-positive clusters. In the molecular layer, the clusters of both alpha-subunits co-localize with the anchoring protein gephyrin. In the granule cell layer and hilus, we found alpha1- and alpha2-subunit-positive clusters which were devoid of gephyrin labeling. Lesions of the medial entorhinal cortex led to the deafferentation of dendrites in the middle molecular layer of the dentate gyrus. This resulted in a significantly increased concentration of alpha2-subunit-positive clusters. We also observed an increase of alpha1-subunit immunolabeling in the deafferented area. We found no change in the co-localization between alpha1 and alpha2, and no significant change in the number of large alpha1-positive clusters along individual dendritic segments of interneurons. In a previous study, we demonstrated that calbindin-immunoreactive dendrites of granule cells revealed a significant increase in gephyrin immunoreactivity following lesion, whereas parvalbumin-positive dendrites showed no such alterations. The predominant localization of small gephyrin clusters in dendrites of granule cells, which was also described in this study, leads to the conclusion that the increase of the alpha2-subunit-positive clusters, demonstrated in the present study, indicates that, following entorhinal cortex lesion, new GABAergic synapses may be formed and that

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

    PubMed

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

    2009-04-21

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

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

    PubMed

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    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. 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.

    PubMed

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

    1998-07-01

    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

  3. The reciprocal regulation of stress hormones and GABA(A) receptors.

    PubMed

    Mody, Istvan; Maguire, Jamie

    2011-01-01

    Stress-derived steroid hormones regulate the expression and function of GABA(A) receptors (GABA(A)Rs). Changes in GABA(A)R subunit expression have been demonstrated under conditions of altered steroid hormone levels, such as stress, as well as following exogenous steroid hormone administration. In addition to the effects of stress-derived steroid hormones on GABA(A)R subunit expression, stress hormones can also be metabolized to neuroactive derivatives which can alter the function of GABA(A)Rs. Neurosteroids allosterically modulate GABA(A)Rs at concentrations comparable to those during stress. In addition to the actions of stress-derived steroid hormones on GABA(A)Rs, GABA(A)Rs reciprocally regulate the production of stress hormones. The stress response is mediated by the hypothalamic-pituitary-adrenal (HPA) axis, the activity of which is governed by corticotropin releasing hormone (CRH) neurons. The activity of CRH neurons is largely controlled by robust GABAergic inhibition. Recently, it has been demonstrated that CRH neurons are regulated by neurosteroid-sensitive, GABA(A)R δ subunit-containing receptors representing a novel feedback mechanism onto the HPA axis. Further, it has been demonstrated that neurosteroidogenesis and neurosteroid actions on GABA(A)R δ subunit-containing receptors on CRH neurons are necessary to mount the physiological response to stress. Here we review the literature describing the effects of steroid hormones on GABA(A)Rs as well as the importance of GABA(A)Rs in regulating the production of steroid hormones. This review incorporates what we currently know about changes in GABA(A)Rs following stress and the role in HPA axis regulation.

  4. Benzodiazepine-dependent stabilization of GABA(A) receptors at synapses.

    PubMed

    Gouzer, Géraldine; Specht, Christian G; Allain, Laure; Shinoe, Toru; Triller, Antoine

    2014-11-01

    GABA(A) receptors constitutively enter and exit synapses by lateral diffusion in the plane of the neuronal membrane. They are trapped at synapses through their interactions with gephyrin, the main scaffolding protein at inhibitory post-synaptic densities. Previous work has shown that the synaptic accumulation and diffusion dynamics of GABA(A)Rs are controlled via excitatory synaptic activity. However, it remains unknown whether GABA(A)R activity can itself impact the surface trafficking of the receptors. Here we report the effects of GABA(A)R agonists, antagonists and allosteric modulators on the receptor's surface dynamics. Using immunocytochemistry and single particle tracking experiments on mouse hippocampal neurons, we show that the agonist muscimol decreases GABA(A)R and gephyrin levels at synapses and accelerates the receptor's lateral diffusion within 30–120 min of treatment. In contrast, the GABA(A)R antagonist gabazine increased GABA(A)R amounts and slowed down GABA(A)R diffusion at synapses. The response to GABA(A)R activation or inhibition appears to be an adaptative regulation of GABAergic synapses. Surprisingly, the positive allosteric modulator diazepam abolished the regulation induced by muscimol, and this effect was observed on α1, α2, α5 and γ2 GABA(A)R subunits. Altogether these results indicate that diazepam stabilizes synaptic GABA(A)Rs and thus prevents the agonist-induced regulation of GABA(A)R levels at synapses. This occurred independently of neuronal activity and intracellular calcium and involved GABA(A)R–gephyrin interactions, suggesting that the changes in GABA(A)R diffusion depend on conformational changes of the receptor. Our study provides a new molecular mechanism involved in the adaptative response to changes in GABA(A)R activity and benzodiazepine treatments.

  5. The role of GABA(A) receptors in the development of alcoholism.

    PubMed

    Enoch, Mary-Anne

    2008-07-01

    Alcoholism is a common, heritable, chronic relapsing disorder. GABA(A) receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABA(A) receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABA(A) receptors: tolerance is associated with generally decreased GABA(A) receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABA(A) receptors may be implicated in the switch from heavy drinking to dependence. GABA(A) receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABA(A) receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABA(A) receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review.

  6. Testing the silence of mutations: Transcriptomic and behavioral studies of GABA(A) receptor α1 and α2 subunit knock-in mice.

    PubMed

    Harris, R A; Osterndorff-Kahanek, E; Ponomarev, I; Homanics, G E; Blednov, Y A

    2011-01-13

    Knock-in mice were constructed with mutations in the α1 (H(270), A(277)) and α2 (H(270), A(277)) subunits of the GABAA receptor, which resulted in receptors that lacked modulation by ethanol but retained normal responses to GABA in vitro. A key question is whether these mutant receptors also function normally in vivo. Perturbation of brain function was evaluated by gene expression profiling in the cerebral cortex and by behavioral pharmacology experiments with GABAergic drugs. Analysis of individual transcripts found only six transcripts that were changed in α1 knock-in mice and three in the α2 mutants (p<0.05, corrected for multiple comparisons). Two transcripts that are sensitive to neuronal activity, Arc and Fos, increased about 250% in the α2 mutants, and about 50% in the α1 mutants. Behavioral effects (loss of righting reflex, rotarod) of flurazepam and pentobarbital were not different between α2 mutants and wild-type, but they were enhanced for α1 knock-in mice. These results indicate that introduction of these mutations in the α2 subunit of the GABAA receptor does not produce marked perturbation of brain function, as measured by gene expression and GABAergic behavioral responses, but the same mutations in the α1 subunit produce more pronounced changes, especially in GABAergic function.

  7. The effect of chronic administration of corticosterone on anxiety- and depression-like behavior and the expression of GABA-A receptor alpha-2 subunits in brain structures of low- and high-anxiety rats.

    PubMed

    Skórzewska, Anna; Lehner, Małgorzata; Wisłowska-Stanek, Aleksandra; Krząścik, Paweł; Ziemba, Andrzej; Płaźnik, Adam

    2014-01-01

    The aim of this study was to examine changes in rat emotional behavior and determine differences in the expression of GABA-A receptor alpha-2 subunits in brain structures of low- (LR) and high-anxiety (HR) rats after the repeated corticosterone administration. The animals were divided into LR and HR groups based on the duration of their conditioned freezing in a contextual fear test. Repeated daily administration of corticosterone (20 mg/kg) for 21 days decreased activity in a forced swim test, reduced body weight and decreased prefrontal cortex corticosterone concentration in both the LR and HR groups. These effects of corticosterone administration were stronger in the HR group in comparison with the appropriate control group, and compared to LR treated and LR control animals. Moreover, in the HR group, chronic corticosterone administration increased anxiety-like behavior in the open field and elevated plus maze tests. The behavioral effects in HR rats were accompanied by a decrease in alpha-2 subunit density in the medial prefrontal cortex (prelimbic cortex and frontal association cortex) and by an increase in the expression of alpha-2 subunits in the basolateral amygdala. These studies have shown that HR rats are more susceptible to anxiogenic and depressive effects of chronic corticosterone administration, which are associated with modification of GABA-A receptor function in the medial prefrontal cortex and basolateral amygdala. The current data may help to better understand the neurobiological mechanisms responsible for individual differences in changes in mood and emotions induced by repeated administration of high doses of glucocorticoids or by elevated levels of these hormones associated with chronic stress or affective pathology.

  8. Aging of whiskey increases the potentiation of GABA(A) receptor response.

    PubMed

    Koda, Hirofumi; Hossain, Sheikh Julfikar; Kiso, Yoshinobu; Aoshima, Hitoshi

    2003-08-27

    It is known that the target of most mood-defining compounds such as ethanol is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activities in the human brain. Because both extracts of whiskey by pentane and fragrant components in whiskey potentiate the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting cRNAs prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors in order to study the effects of whiskey itself on the GABA(A) receptor-mediated response. Whiskey itself also potentiated the electrical responses of GABA(A) receptors generally more than ethanol at the same concentration as that of the whiskey. The potentiation of the GABA(A) receptor-mediated response increased with the aging period of the whiskey. Inhalation of whiskey to mice increased the sleeping time induced by pentobarbital more than that of the same concentration of ethanol as the whiskey. These results suggest that not only ethanol but also minor components in whiskey play an important role in the potentiation of GABA(A) receptor-mediated response and possibly the sedative effect of whiskey. Although the minor components are present in extremely small quantities compared with ethanol in alcoholic beverages, they may modulate the mood or consciousness of humans through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic compounds are easily absorbed into the brain across the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

  9. PR-independent neurosteroid regulation of α2-GABA-A receptors in the hippocampus subfields.

    PubMed

    Reddy, Doodipala Samba; Gangisetty, Omkaram; Wu, Xin

    2017-03-15

    Progesterone (P) binding to the intracellular progesterone receptors (PRs) plays a key role in epilepsy via modulation of GABA-A receptor plasticity in the brain. This is thought to occur via conversion of P to neurosteroids such as allopregnanolone, an allosteric modulator of GABA-A receptors. In the female brain, the composition of GABA-A receptors is not static and undergoes dynamic spatial changes in response to fluctuations in P and neurosteroid levels. Synaptic α2-containing GABA-A receptors contribute to phasic neuronal excitability and seizure susceptibility. However, the mechanisms underlying α2-subunit plasticity remain unclear. Here, we utilized the neurosteroid synthesis inhibitor finasteride and PR knockout mice to investigate the role of PRs in α2-subunit in the hippocampus. α2-Subunit expression was significantly upregulated during the high-P state of diestrous stage and with P treatment in wildtype and PR knockout mice. In contrast, there was no change in α2-subunit expression when metabolism of P into neurosteroids was blocked by finasteride in both genotypes. These findings suggest that ovarian cycle-related P and neurosteroids regulate α2-GABA-A receptor expression in the hippocampus via a non-PR pathway, which may be relevant to menstrual-cycle related brain conditions.

  10. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

    PubMed

    Dixon, C I; Rosahl, T W; Stephens, D N

    2008-07-01

    Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

  11. Tolerance to allopregnanolone with focus on the GABA-A receptor

    PubMed Central

    Turkmen, Sahruh; Backstrom, Torbjorn; Wahlstrom, Goran; Andreen, Lotta; Johansson, Inga-Maj

    2011-01-01

    Many studies have suggested a relationship between stress, sex steroids, and negative mental and mood changes in humans. The progesterone metabolite allopregnanolone is a potent endogenous ligand of the γ-amino butyric acid –A (GABA-A) receptor, and the most discussed neuroactive steroid. Variations in the levels of neuroactive steroids that influence the activity of the GABA-A receptor cause a vulnerability to mental and emotional pathology. There are physiological conditions in which allopregnanolone production increases acutely (e.g. stress) or chronically (e.g. menstrual cycle, pregnancy), thus exposing the GABA-A receptor to high and continuous allopregnanolone concentrations. In such conditions, tolerance to allopregnanolone may develop. We have shown that both acute and chronic tolerances can develop to the effects of allopregnanolone. Following the development of acute allopregnanolone tolerance, there is a decrease in the abundance of the GABA-A receptor α4 subunit and the expression of the α4 subunit mRNA in the ventral-posteriomedial nucleus of the thalamus. Little is known about the mechanism behind allopregnanolone tolerance and its effects on assembly of the GABA-A receptor composition. The exact mechanism of the allopregnanolone tolerance phenomena remains unclear. The purpose of this review is to summarize certain aspects of current knowledge concerning allopregnanolone tolerance and changes in the GABA-A receptors. PMID:20883478

  12. Neurosteroids and GABA-A Receptor Function

    PubMed Central

    Wang, Mingde

    2011-01-01

    Neurosteroids represent a class of endogenous steroids that are synthesized in the brain, the adrenals, and the gonads and have potent and selective effects on the GABAA-receptor. 3α-hydroxy A-ring reduced metabolites of progesterone, deoxycorticosterone, and testosterone are positive modulators of GABAA-receptor in a non-genomic manner. Allopregnanolone (3α-OH-5α-pregnan-20-one), 5α-androstane-3α, 17α-diol (Adiol), and 3α5α-tetrahydrodeoxycorticosterone (3α5α-THDOC) enhance the GABA-mediated Cl- currents acting on a site (or sites) distinct from the GABA, benzodiazepine, barbiturate, and picrotoxin binding sites. 3α5α-P and 3α5α-THDOC potentiate synaptic GABAA-receptor function and activate δ-subunit containing extrasynaptic receptors that mediate tonic currents. On the contrary, 3β-OH pregnane steroids and pregnenolone sulfate (PS) are GABAA-receptor antagonists and induce activation-dependent inhibition of the receptor. The activities of neurosteroid are dependent on brain regions and types of neurons. In addition to the slow genomic action of the parent steroids, the non-genomic, and rapid actions of neurosteroids play a significant role in the GABAA-receptor function and shift in mood and memory function. This review describes molecular mechanisms underlying neurosteroid action on the GABAA-receptor, mood changes, and cognitive functions. PMID:22654809

  13. Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs.

    PubMed

    Reyes-Haro, Daniel; Cabrera-Ruíz, Elizabeth; Estrada-Mondragón, Argel; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2014-11-01

    Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation-π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl(-) channel.

  14. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    PubMed

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  15. Activation of GABA(A) receptors in subthalamic neurons in vitro: properties of native receptors and inhibition mechanisms.

    PubMed

    Baufreton, J; Garret, M; Dovero, S; Dufy, B; Bioulac, B; Taupignon, A

    2001-07-01

    The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABA(A) receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABA(A) receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified epsilon subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABA(A) receptors contain epsilon proteins. Applications of increasing concentrations of muscimol, a selective GABA(A) agonist, induced Cl(-) and HCO currents with an EC(50) of 5 microM. Currents induced by muscimol were fully blocked by the GABA(A) receptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an epsilon subunit-specific antibody showed that the epsilon protein was not expressed within the STN. Native subthalamic GABA(A) receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising epsilon. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABA(A) receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in

  16. Taurine is a potent activator of extrasynaptic GABA(A) receptors in the thalamus.

    PubMed

    Jia, Fan; Yue, Minerva; Chandra, Dev; Keramidas, Angelo; Goldstein, Peter A; Homanics, Gregg E; Harrison, Neil L

    2008-01-02

    Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (> or = 100 microM), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (> or = 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA(A)-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10-100 microM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 microM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 microM Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha4beta2delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha4beta2delta GABA(A)-Rs than at alpha1beta2gamma2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

  17. The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

    PubMed

    Johnson, W David; Howard, Rebecca J; Trudell, James R; Harris, R Adron

    2012-02-01

    Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

  18. Local and global ligand-induced changes in the structure of the GABA(A) receptor.

    PubMed

    Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B

    2006-06-13

    Ligand-gated channels mediate synaptic transmission through conformational transitions triggered by the binding of neurotransmitters. These transitions are well-defined in terms of ion conductance, but their structural basis is poorly understood. To probe these changes in structure, GABA(A) receptors were expressed in Xenopus oocytes and labeled at selected sites with environment-sensitive fluorophores. With labels at two different residues in the alpha1 subunit in loop E of the GABA-binding pocket, GABA elicited fluorescence changes opposite in sign. This pattern of fluorescence changes is consistent with a closure of the GABA-binding cavity at the subunit interface. The competitive antagonist SR-95531 inverted this pattern of fluorescence change, but the noncompetitive antagonist picrotoxin failed to elicit optical signals. In response to GABA (but not SR-95531), labels at the homologous residues in the beta2 subunit showed the same pattern of fluorescence change as the alpha1-subunit labels, indicating a global transition with comparable movements in homologous regions of different subunits. Incorporation of the gamma2 subunit altered the fluorescence changes of alpha1-subunit labels and eliminated them in beta2-subunit labels. Thus, the ligand-induced structural changes in the GABA(A) receptor can extend over considerable distances or remain highly localized, depending upon subunit composition and ligand.

  19. [Autoimmune encephalitis induced by antibodies against GABA-A receptor].

    PubMed

    González R, Pablo; Hudson A, Lorena; Basáez M, Esteban; Miranda C, Marcelo

    2016-11-01

    Among autoimmune encephalitides, a prevalent group are those associated with antibodies against the N-Methyl-D-aspartate receptor, which present with behavior abnormalities, psychosis, seizures and abnormal movements. A new variant, mediated by antibodies against the GABA-A receptor, was recen-tly described. We report a 66-years-old female with this form of encephalitis whose main manifestation was the presence of severe seizures leading to status epilepticus. The patient had a good response to immunomodulatory therapy with intravenous methylprednisolone, azathioprine and anticonvulsants. The laboratory tests initially detected anti-thyroid peroxidase antibodies which lead to the misdiagnosis of Hashimoto Encephalitis, which was ruled out after the detection of antibodies against GABA-A receptor. No malignancy was detected.

  20. Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) receptor subtypes indicate a different binding mode for GABA at these receptors.

    PubMed

    Karim, Nasiara; Wellendorph, Petrine; Absalom, Nathan; Bang, Line Haunstrup; Jensen, Marianne Lerbech; Hansen, Maja Michelle; Lee, Ho Joon; Johnston, Graham A R; Hanrahan, Jane R; Chebib, Mary

    2012-08-15

    Ionotropic GABA(A) receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA(A) receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA(A) receptors. α4/δ-Containing GABA(A) receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀=24 nM) and α4β3δ (EC₅₀=12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1)=16 nM; EC₅₀(2)=1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀=1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA(A) receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA(A) receptors compared to their synaptic counterparts.

  1. Neurosteroids shift partial agonist activation of GABA(A) receptor channels from low- to high-efficacy gating patterns.

    PubMed

    Bianchi, Matt T; Macdonald, Robert L

    2003-11-26

    Although GABA activates synaptic (alphabetagamma) GABA(A) receptors with high efficacy, partial agonist activation of alphabetagamma isoforms and GABA activation of the primary extrasynaptic (alphabetadelta) GABA(A) receptors are limited to low-efficacy activity, characterized by minimal desensitization and brief openings. The unusual sensitivity of alphabetadelta receptor channels to neurosteroid modulation prompted investigation of whether this high sensitivity was dependent on the delta subunit or the low-efficacy channel function that it confers. We show that the isoform specificity (alphabetadelta > alphabetagamma) of neurosteroid modulation could be reversed by conditions that reversed isoform-specific activity modes, including the use of beta-alanine to achieve increased efficacy with alphabetadelta receptors and taurine to render alphabetagamma receptors low efficacy. We suggest that neurosteroids preferentially enhance low-efficacy GABA(A) receptor activity independent of subunit composition. Allosteric conversion of partial to full agonism may be a general mechanism for reversibly scaling the efficacy of GABA(A) receptors to endogenous partial agonists.

  2. Thiocolchicoside inhibits the activity of various subtypes of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes.

    PubMed

    Mascia, Maria Paola; Bachis, Elisabetta; Obili, Nicola; Maciocco, Elisabetta; Cocco, Giovanni Antonio; Sechi, Gian Pietro; Biggio, Giovanni

    2007-03-08

    Thiocolchicoside is a myorelaxant drug with anti-inflammatory and analgesic properties as well as pronounced convulsant activity. To characterize the mechanisms of action of this drug at the molecular level, we examined its effects on the function of various recombinant neurotransmitter receptors expressed in Xenopus oocytes. Electrophysiological recordings from recombinant human gamma-aminobutyric acid type A (GABA(A)) receptors consisting of alpha1beta1gamma2L, alpha1beta2gamma2L, or alpha2beta2gamma2L subunit combinations revealed that thiocolchicoside inhibited GABA-evoked Cl(-) currents with similar potencies (median inhibitory concentrations of 0.13 to 0.2 microM) and in a competitive manner. Consistent with previous observations, thiocolchicoside also inhibited the binding of GABA to rat cerebral cortical membranes. Thiocolchicoside inhibited the function of recombinant human strychnine-sensitive glycine receptors composed of the alpha1 subunit with a potency (median inhibitory concentration of 47 microM) lower than that apparent with recombinant GABA(A) receptors. It also inhibited the function of human nicotinic acetylcholine receptors composed of the alpha4 and beta2 subunits, but this effect was only partial and apparent at high concentrations. In contrast, thiocolchicoside had no effect on the function of 5-HT(3A) serotonin receptors. Our results thus provide molecular evidence that the epileptogenic activity of thiocolchicoside might be due to inhibition of the function of inhibitory receptors in the central nervous system, especially that of GABA(A) receptors.

  3. An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons.

    PubMed

    Toulmé, Estelle; Blais, Dominique; Léger, Claire; Landry, Marc; Garret, Maurice; Séguéla, Philippe; Boué-Grabot, Eric

    2007-08-01

    Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.

  4. GABA A/Bz receptor subtypes as targets for selective drugs.

    PubMed

    Da Settimo, F; Taliani, S; Trincavelli, M L; Montali, M; Martini, C

    2007-01-01

    The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory neuronal receptors in the mammalian brain. Their activation by GABA opens the intrinsic ion channel, enabling chloride flux into the cell with subsequent hyperpolarization. Several GABA(A) receptor subunit isoforms have been cloned, the major isoform containing alpha, beta, and gamma subunits, and a regional heterogeneity associated with distinct physiological effects has been suggested. As a variety of allosteric ligands can modulate GABA-gated conductance changes through binding to distinct sites, the development of subtype-selective ligands may lead to the selective treatment of GABA system-associated pathology. In particular, the best characterized binding site is the benzodiazepine site (BzR), localized at the alpha/gamma subunit interface, in which the alpha subunit is the main determinant of BzR ligand action selectivity. The alpha1-containing BzR have been proposed to be responsible for the sedative action; the alpha2 and/or the alpha3 subtypes have been suggested to mediate the anxiolytic activity and the myorelaxation effects, and the alpha5 subtype has been associated with cognition processes. The discovery of alpha-selective subtype ligands may help in the specific treatment of anxiety, sleep disorders, convulsions and memory deficits with fewer side effects. Selectivity may be achieved by two approaches: selective affinity or selective efficacy. Selective affinity needs a compound to bind with a higher affinity to one receptor subtype compared with another, whereas subtype-selective efficacy relies on a compound binding to all subtypes, but having different efficacies at various subtypes. The status of BzR ligands, subdivided on the basis of their main chemical structural features, is reviewed in relation to structure-activity relationships which determine their affinity or efficacy selectivity for a certain BzR subtype.

  5. GABA(A) receptor physiology and its relationship to the mechanism of action of the 1,5-benzodiazepine clobazam.

    PubMed

    Sankar, Raman

    2012-03-01

    Clobazam was initially developed in the early 1970s as a nonsedative anxiolytic agent, and is currently available as adjunctive therapy for epilepsy and anxiety disorders in more than 100 countries. In October 2011, clobazam (Onfi™; Lundbeck Inc., Deerfield, IL, USA) was approved by the US FDA for use as adjunctive therapy for the treatment of seizures associated with Lennox-Gastaut syndrome in patients aged 2 years and older. It is a long-acting 1,5-benzodiazepine whose structure distinguishes it from the classic 1,4-benzodiazepines, such as diazepam, lorazepam and clonazepam. Clobazam is well absorbed, with peak concentrations occurring linearly 1-4 hours after administration. Both clobazam and its active metabolite, N-desmethylclobazam, are metabolized in the liver via the cytochrome P450 pathway. The mean half-life of N-desmethylclobazam (67.5 hours) is nearly double the mean half-life of clobazam (37.5 hours). Clobazam was synthesized with the anticipation that its distinct chemical structure would provide greater efficacy with fewer benzodiazepine-associated adverse effects. Frequently reported adverse effects of clobazam therapy include dizziness, sedation, drowsiness and ataxia. Evidence gathered from approximately 50 epilepsy clinical trials in adults and children indicated that the sedative effects observed with clobazam treatment were less severe than those reported with 1,4-benzodiazepines. In several studies of healthy volunteers and patients with anxiety, clobazam appeared to enhance participants' performance in cognitive tests, further distinguishing it from the 1,4-benzodiazepines. The anxiolytic and anticonvulsant effects of clobazam are associated with allosteric activation of the ligand-gated GABA(A) receptor. GABA(A) receptors are found extensively throughout the CNS, occurring synaptically and extrasynaptically. GABA(A) receptors are composed of five protein subunits, two copies of a single type of α subunit, two copies of one type of

  6. α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

    PubMed

    Absalom, Nathan; Eghorn, Laura F; Villumsen, Inge S; Karim, Nasiara; Bay, Tina; Olsen, Jesper V; Knudsen, Gitte M; Bräuner-Osborne, Hans; Frølund, Bente; Clausen, Rasmus P; Chebib, Mary; Wellendorph, Petrine

    2012-08-14

    γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA(A) receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA(A) receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC(50) = 140 nM) over α4β(2/3)δ (EC(50) = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [(3)H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA(A) receptors and postulate a role for extrasynaptic α4δ-containing GABA(A) receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

  7. The modulation of synaptic GABA(A) receptors in the thalamus by eszopiclone and zolpidem.

    PubMed

    Jia, Fan; Goldstein, Peter A; Harrison, Neil L

    2009-03-01

    Eszopiclone (Lunesta; Sepracor, Marlborough, MA) and zolpidem [N,N,6-trimethyl-2-(4-methylphenyl)-imidazo(1,2-a)pyridine-3-acetamide] are among the most commonly prescribed hypnotics in use in the United States. The thalamus plays a pivotal role in sleep regulation and rhythmicity. Two distinct subtypes of synaptic GABA(A) receptors (GABA(A)-Rs), alpha(1)beta(2)gamma(2) and alpha(3)beta(3)gamma(2), are expressed in thalamocortical relay neurons and in interneurons of the RTN (reticular thalamic nucleus), respectively. Thalamocortical neurons also express extrasynaptic GABA(A)-Rs composed of alpha(4)beta(2)delta subunits. In this study, we compared the effects of eszopiclone and zolpidem on miniature inhibitory postsynaptic currents (IPSCs), spontaneous IPSCs, and tonic inhibition in the mouse thalamus. Eszopiclone (0.1-1 microM) slowed the decay phase of IPSCs recorded from RTN neurons, whereas zolpidem was less effective and increased the decay time constant only at > or = 0.3 microM. IPSCs of RTN neurons were more sensitive to eszopiclone than zolpidem at all concentrations tested. On the other hand, IPSCs of relay neurons in the ventrobasal nucleus (VB) were more sensitive to zolpidem than eszopiclone. Zolpidem (0.1-1 microM) prolonged the decay of IPSCs from VB neurons, whereas eszopiclone increased the decay time constant only at > or = 0.3 microM. Neither of these two hypnotics affected tonic inhibition in relay neurons. Our results demonstrate that eszopiclone has greater efficacy at synaptic GABA(A)-Rs of RTN neurons than in relay neurons, whereas zolpidem exerts bigger effects on relay neurons than RTN neurons. This distinct pattern of activity on thalamic neurons may contribute to some of the observed differences in the clinical effects of these two hypnotics.

  8. Regulation of cognition and symptoms of psychosis: focus on GABA(A) receptors and glycine transporter 1.

    PubMed

    Möhler, Hanns; Rudolph, Uwe; Boison, Detlev; Singer, Philipp; Feldon, Joram; Yee, Benjamin K

    2008-07-01

    Adaptive purposeful behaviour depends on appropriate modifications of synaptic connectivity that incorporate an organism's past experience. At least some forms of such synaptic plasticity are believed to be mediated by NMDA receptors (NMDARs). Complementary interaction with inhibitory neurotransmission mediated by GABA(A) receptors, and upstream control of the excitability of NMDARs by glycine availability can greatly influence the efficacy of NMDAR mediated neuroplasticity, and thereby exert significant effects on cognition. Memory, selective attention or sensorimotor gating functions can be modified in mice with a reduction of alpha(5)GABA(A) receptors in the hippocampus or a selective deletion of glycine transporter 1 (GlyT1) in the forebrain. Both genetic manipulations altered the formation or persistence of associative links leading to distinct phenotypes on trace conditioning, extinction learning, latent inhibition, working memory, and object recognition. Behavioural assays of latent inhibition, prepulse inhibition, working memory, and sensitivity to psychostimulants in particular suggest that alpha(3) and alpha(5) subunit-containing GABA(A) receptors as well as GlyT1 are potential sites for ameliorating psychotic-like behaviour. Taken together, these results qualify distinct GABA-A receptor subtypes and GlyT1 as molecular targets for the development of a new pharmacology in the treatment of cognitive decline and psychotic symptoms.

  9. Glycine and GABA(A) ultra-sensitive ethanol receptors as novel tools for alcohol and brain research.

    PubMed

    Naito, Anna; Muchhala, Karan H; Asatryan, Liana; Trudell, James R; Homanics, Gregg E; Perkins, Daya I; Davies, Daryl L; Alkana, Ronald L

    2014-12-01

    A critical obstacle to developing effective medications to prevent and/or treat alcohol use disorders is the lack of specific knowledge regarding the plethora of molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To identify the role of individual receptor subunits in ethanol-induced behaviors, we developed a novel class of ultra-sensitive ethanol receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as four residues in the extracellular loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABA(A)Rs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode voltage clamp, demonstrated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABA(A)Rs. Collectively, our studies set the stage for using USER technology in genetically engineered animals as a unique tool to increase understanding of the neurobiological basis of the behavioral effects of ethanol.

  10. The supramammillary nucleus mediates primary reinforcement via GABA(A) receptors.

    PubMed

    Ikemoto, Satoshi

    2005-06-01

    The supramammillary nucleus (SUM), a dorsal layer of the mammillary body, has recently been implicated in positive reinforcement. The present study examined whether GABA(A) receptors in the SUM or adjacent regions are involved in primary reinforcement using intracranial self-administration procedures. Rats learned quickly to lever-press for infusions of the GABA(A) antagonist picrotoxin into the SUM. Although picrotoxin was also self-administered into the posterior hypothalamic nuclei and anterior ventral tegmental area, these regions were less responsive to lower doses of picrotoxin than the SUM. The finding that rats learned to respond selectively on the lever triggering drug infusions is consistent with picrotoxin's reinforcing effect. Coadministration of the GABA(A) agonist muscimol disrupted picrotoxin self-administration, and another GABA(A) antagonist, bicuculline, was also self-administered into the SUM; thus, the reinforcing effect of picrotoxin is mediated by GABA(A) receptors. Since rats did not self-administer the GABA(B) antagonist 2-hydroxysaclofen into the SUM, the role of GABA(B) receptors may be distinct from that of GABA(A) receptors. Pretreatment with the dopamine receptor antagonist SCH 23390 (0.05 mg/kg, i.p.) extinguished picrotoxin self-administration into the SUM, suggesting that the reinforcing effects of GABA(A) receptor blockade depend on normal dopamine transmission. In conclusion, the blockade of GABA(A) receptors in the SUM is reinforcing, and the brain 'reward' circuitry appears to be tonically inhibited via supramammillary GABA(A) receptors and more extensive than the meso-limbic dopamine system.

  11. Excitatory Synaptic Responses Mediated by GABA_A Receptors in the Hippocampus

    NASA Astrophysics Data System (ADS)

    Michelson, Hillary B.; Wong, Robert K. S.

    1991-09-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABA_A receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABA_A receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABA_A receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABA_A receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

  12. GABA A receptor abnormalities in Prader-Willi syndrome assessed with positron emission tomography and [11C]flumazenil.

    PubMed

    Lucignani, Giovanni; Panzacchi, Andrea; Bosio, Laura; Moresco, Rosa Maria; Ravasi, Laura; Coppa, Isabella; Chiumello, Giuseppe; Frey, Kirk; Koeppe, Robert; Fazio, Ferruccio

    2004-05-01

    Prader-Willi syndrome (PWS) is a multi-system disorder characterized clinically by abnormal mental and physical development. PWS patients have a deletion in an imprinted region on paternal chromosome 15 (15q11-13), maternal disomy for this segment, or rarely, a chromosomal imprinting center deletion that gives rise to suppression of the equivalent paternal genes. Within the affected segment of chromosome 15 are genes encoding the alpha(5), beta(3) and gamma(3) subunits of the gamma-aminobutyric acid type-A (GABA(A)) receptor. Therefore, altered neurobehavioral function could arise in PWS due directly to altered GABA(A) receptor composition and expression, or alternatively, from brain developmental and maturational effects of these or other genes in the imprinted region. The aim of the present study was to assess cerebral GABA(A) receptors in PWS with the use of positron emission tomography of the benzodiazepine binding site employing [11C]flumazenil ([11C]FMZ). A reduction in [11C]FMZ binding was found predominantly in the cingulate, frontal and temporal neocortices and insula in six adult PWS patients compared to nine normal subjects. A possible role for the deleted beta(3) subunit gene in PWS is supported in part by the wide cortical distribution of its mRNA expression and the effects of experimental knockouts on benzodiazepine binding described in prior studies. Altered GABA(A) receptor composition or number in these cortical regions may account for neurobehavioral abnormalities in PWS including mild mental retardation, poor impulse control, and impaired responses to somatic pain.

  13. Time-course of SKF-81297-induced increase in glutamic acid decarboxylase 65 and 67 mRNA levels in striatonigral neurons and decrease in GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, in adult rats with a unilateral 6-hydroxydopamine lesion.

    PubMed

    Yamamoto, N; Soghomonian, J-J

    2008-06-26

    Striatal projection neurons use GABA as their neurotransmitter and express the rate-limiting synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter vGAT. The chronic systemic administration of an agonist of dopamine D1/D5-preferring receptors is known to alter GAD mRNA levels in striatonigral neurons in intact and dopamine-depleted rats. In the present study, the effects of a single or subchronic systemic administration of the dopamine D1/D5-preferring receptor agonist SKF-81297 on GAD65, GAD67, PPD and vGAT mRNA levels in the striatum and GABA(A) receptor alpha1 subunit mRNA levels in the substantia nigra, pars reticulata, were measured in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. After a single injection of SKF-81297, striatal GAD65 mRNA levels were significantly increased at 3 but not 72 h. In contrast, striatal GAD67 mRNA levels were increased and nigral alpha1 mRNA levels were decreased at 72 but not 3 h. Single cell analysis on double-labeled sections indicated that increased GAD or vGAT mRNA levels after acute SKF-81297 occurred in striatonigral neurons identified by their lack of preproenkephalin expression. Subchronic SKF-81297 induced significant increases in striatal GAD67, GAD65, preprodynorphin and vGAT mRNA levels and decreases in nigral alpha1 mRNA levels. In the striatum contralateral to the 6-OHDA lesion, subchronic but not acute SKF-81297 induced a significant increase in GAD65 mRNA levels. The other mRNA levels were not significantly altered. Finally, striatal GAD67 mRNA levels were negatively correlated with nigral alpha1 mRNA levels in the dopamine-depleted but not dopamine-intact side. The results suggest that different signaling pathways are involved in the modulation by dopamine D1/D5 receptors of GAD65 and GAD67 mRNA levels in striatonigral neurons. They also suggest that the down-regulation of nigral GABA(A) receptors is linked to the increase in striatal GAD67 mRNA levels in the dopamine

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

    PubMed

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-07

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

  15. Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

    PubMed

    Trigo, Federico F; Chat, Mireille; Marty, Alain

    2007-11-14

    Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

  16. Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations

    PubMed Central

    Zaugg, Janine; Ebrahimi, Samad Nejad; Smiesko, Martin; Baburin, Igor; Hering, Steffen; Hamburger, Matthias

    2011-01-01

    A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (IGABA) by 122.5 ± 0.3% (n = 2) when tested at 100 μg/ml in Xenopus laevis oocytes expressing GABA A receptors (α1β2γ2S subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated IGABA through GABA A receptors composed of α1β2γ2S subunits in a concentration-dependent manner. Potencies ranged from 12.8 ± 3.1 to 135.6 ± 85.7 μM, and efficiencies ranged from 129.7 ± 36.8% to 885.8 ± 291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1 ± 2.6% potentiation of IGABA at 100 μg/ml, n = 2), and Schisandra chinensis fruits (inactive at 100 μg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata. PMID:21889177

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

    PubMed

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

    2004-08-18

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

  18. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site

    PubMed Central

    Snell, Heather D.

    2015-01-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride’s positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound’s potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ. PMID:25829529

  19. Amiloride and GMQ Allosteric Modulation of the GABA-A ρ1 Receptor: Influences of the Intersubunit Site.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2015-06-01

    Amiloride, a diuretic used in the treatment of hypertension and congestive heart failure, and 2-guanidine-4-methylquinazoline (GMQ) are guanidine compounds that modulate acid-sensing ion channels. Both compounds have demonstrated affinity for a variety of membrane proteins, including members of the Cys-loop family of ligand-gated ion channels, such as the heteromeric GABA-A αβγ receptors. The actions of these guanidine compounds on the homomeric GABA-A ρ1 receptor remains unclear, especially in light of how many GABA-A αβγ receptor modulators have different effects in the GABA-A ρ1 receptors. We sought to characterize the influence of amiloride and GMQ on the human GABA-A ρ1 receptors using whole-cell patch-clamp electrophysiology. The diuretic amiloride potentiated the human GABA-A ρ1 GABA-mediated current, whereas GMQ antagonized the receptor. Furthermore, a GABA-A second transmembrane domain site, the intersubunit site, responsible for allosteric modulation in the heteromeric GABA-A receptors mediated amiloride's positive allosteric actions. In contrast, the mutation did not remove GMQ antagonism but only changed the guanidine compound's potency within the human GABA-A ρ1 receptor. Through modeling and introduction of point mutations, we propose that the GABA-A ρ1 intersubunit site plays a role in mediating the allosteric effects of amiloride and GMQ.

  20. Phasic, Nonsynaptic GABA-A Receptor-Mediated Inhibition Entrains Thalamocortical Oscillations

    PubMed Central

    Rovó, Zita; Mátyás, Ferenc; Barthó, Péter; Slézia, Andrea; Lecci, Sandro; Pellegrini, Chiara; Astori, Simone; Dávid, Csaba; Hangya, Balázs

    2014-01-01

    GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations. PMID:24849349

  1. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site.

    PubMed

    Zaugg, Janine; Baburin, Igor; Strommer, Barbara; Kim, Hyun-Jung; Hering, Steffen; Hamburger, Matthias

    2010-02-26

    A plant extract library was screened for GABA(A) receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 microg/mL] potentiated GABA-induced chloride currents through GABA(A) receptors (composed of alpha(1), beta(2), and gamma(2S) subunits) by 169.1 +/- 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1-4, 6-13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 +/- 26.5% with an EC(50) of 52.4 +/- 9.4 microM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABA(A) receptor modulation. The stimulation of chloride currents through GABA(A) receptors by compound 5 was not antagonized by flumazenil (10 microM). These data show that piperine (5) represents a new scaffold of positive allosteric GABA(A) receptor modulators targeting a benzodiazepine-independent binding site.

  2. Semisynthetic preparation of amentoflavone: A negative modulator at GABA(A) receptors.

    PubMed

    Hanrahan, Jane R; Chebib, Mary; Davucheron, Neil L M; Hall, Belinda J; Johnston, Graham A R

    2003-07-21

    Amentoflavone is found in a number of plants with medicinal properties, including Ginkgo biloba and Hypericum perforatum (St. John's Wort). We have developed a rapid and economic semi-synthetic preparation of amentoflavone from biflavones isolated from autumnal Ginkgo biloba leaves. Several studies have shown that amentoflavone binds to benzodiazepine receptors. Using two electrode voltage-clamp methodology, amentoflavone has been shown to be a negative modulator of GABA at GABA(A) alpha(1)beta(2)gamma(2L) receptors expressed in Xenopus laevis oocytes This action appears to be independent of the flumazenil-sensitive benzodiazepine modulatory sites on the GABA(A) receptor.

  3. A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

    PubMed Central

    Clayton, Terry; Poe, Michael M.; Rallapalli, Sundari; Biawat, Poonam; Savić, Miroslav M.; Rowlett, James K.; Gallos, George; Emala, Charles W.; Kaczorowski, Catherine C.; Stafford, Douglas C.; Arnold, Leggy A.; Cook, James M.

    2015-01-01

    An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors. PMID:26682068

  4. Dual Modulators of GABA-A and Alpha7 Nicotinic Receptors for Treating Autism

    DTIC Science & Technology

    2014-08-01

    and Alpha7 Nicotinic Receptors for Treating Autism PRINCIPAL INVESTIGATOR: Kelvin W. Gee RECIPIENT: University of California Irvine...Aug 2014 4. TITLE AND SUBTITLE Dual Modulators of GABA-A and Alpha7 Nicotinic Receptors for Treating Autism 5a. CONTRACT NUMBER 5b. GRANT NUMBER...DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism

  5. Spontaneous thermal motion of the GABA(A) receptor M2 channel-lining segments.

    PubMed

    Bera, Amal K; Akabas, Myles H

    2005-10-21

    The gamma-aminobutyric acid type A (GABA(A)) receptor channel opening involves translational and rotational motions of the five channel-lining, M2 transmembrane segments. The M2 segment's extracellular half is loosely packed and undergoes significant thermal motion. To characterize the extent of the M2 segment's motion, we used disulfide trapping experiments between pairs of engineered cysteines. In alpha1beta1 gamma2S receptors the single gamma subunit is flanked by an alpha and beta subunit. The gamma2 M2-14' position is located in the alpha-gamma subunit interface. Gamma2 13' faces the channel lumen. We expressed either the gamma2 14' or the gamma2 13' cysteine substitution mutants with alpha1 cysteine substitution mutants between 12' and 16' and wild-type beta1. Disulfide bonds formed spontaneously between gamma2 14'C and both alpha1 15'C and alpha1 16'C and also between gamma2 13'C and alpha1 13'C. Oxidation by copper phenanthroline induced disulfide bond formation between gamma2 14'C and alpha1 13'C. Disulfide bond formation rates with gamma2 14'C were similar in the presence and absence of GABA, although the rate with alpha1 13'C was slower than with the other two positions. In a homology model based on the acetylcholine receptor structure, alphaM2 would need to rotate in opposite directions by approximately 80 degrees to bring alpha1 13' and alpha1 15' into close proximity with gamma2 14'. Alternatively, translational motion of alphaM2 would reduce the extent of rotational motion necessary to bring these two alpha subunit residues into close proximity with the gamma2 14' position. These experiments demonstrate that in the closed state the M2 segments undergo continuous spontaneous motion in the region near the extracellular end of the channel gate. Opening the gate may involve similar but concerted motions of the M2 segments.

  6. GABA(A) receptor subtype-selectivity of novel bicuculline derivatives.

    PubMed

    Ramerstorfer, Joachim; Foppa, Verena; Thiery, Hanna; Hermange, Philippe; Janody, Simon; Berger, Michael L; Dodd, Robert H; Sieghart, Werner

    2015-01-01

    GABA(A) receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are targets of clinically important drugs modulating GABA induced ion flux by interacting with distinct allosteric binding sites. ROD 185 is a previously investigated structural analogue of the GABA site antagonist bicuculline, and a positive allosteric modulator acting via the benzodiazepine binding site. Here, we investigated 13 newly synthesized structural analogues of ROD 185 for their interaction with rat GABA(A) receptors. Using [(3)H]flunitrazepam binding assays, we identified four compounds exhibiting a higher affinity for the benzodiazepine binding site than ROD 185. Two electrode voltage clamp electrophysiology at recombinant GABA(A) receptors indicated that most of these compounds positively modulated GABA-induced currents at these receptors. Additionally, these experiments revealed that this compound class not only interacts with the benzodiazepine binding site at αβγ receptors but also with a novel, so far unidentified binding site present in αβ receptors. Compounds with a high affinity for the benzodiazepine binding site stimulated GABA-induced currents stronger at αβγ than at αβ receptors and preferred α3β3γ2 receptors. Compounds showing equal or smaller effects at αβγ compared to αβ receptors differentially interacted with various αβ or αβγ receptor subtypes. Surprisingly, five of these compounds interacting with αβ receptors showed a strong stimulation at α6β3γ2 receptors. The absence of any direct effects at GABA(A) receptors, as well as their potential selectivity for receptor subtypes not being addressed by benzodiazepines, make this compound class to a starting point for the development of drugs with a possible clinical importance.

  7. Coupling between agonist and chloride ionophore sites of the GABA(A) receptor: agonist/antagonist efficacy of 4-PIOL.

    PubMed

    Rabe, H; Picard, R; Uusi-Oukari, M; Hevers, W; Lüddens, H; Korpi, E R

    2000-12-15

    Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

  8. Using an α-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking.

    PubMed

    Brady, Megan L; Moon, Charles E; Jacob, Tija C

    2014-03-28

    It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

  9. Potentiating effect of eszopiclone on GABA(A) receptor-mediated responses in pedunculopontine neurons.

    PubMed

    Ye, Meijun; Garcia-Rill, Edgar

    2009-07-01

    The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

  10. 5-(N, N-Hexamethylene) amiloride is a GABA-A ρ1 receptor positive allosteric modulator.

    PubMed

    Snell, Heather D; Gonzales, Eric B

    2016-11-01

    Guanidine compounds act as ion channel modulators. In the case of Cys-loop receptors, the guanidine compound amiloride antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors. However, amiloride exhibits characteristics consistent with a positive allosteric modulator for the human GABA-A (hGABA-A) ρ1 receptor. Site-directed mutagenesis revealed that the positive allosteric modulation was influenced by the GABA-A ρ1 second transmembrane domain 15' position, a site implicated in ligand allosteric modulation of Cys-loop receptors. There are a variety of amiloride derivatives that provide opportunities to assess the significance of amiloride functional groups (e.g., the guanidine group, the pyrazine ring, etc.) in the modulation of the GABA-A ρ1 receptor activity. We utilized 3 amiloride derivatives (benzamil, phenamil, and 5-(N, N-Hexamethylene) amiloride) to assess the contribution of these groups toward the potentiation of the GABA-A ρ1 receptor. Benzamil and phenamil failed to potentiate on the wild type GABA-A ρ1 GABA-mediated current while HMA demonstrated efficacy only at the highest concentration studied. The hGABA-A ρ1 (I15'N) mutant receptor activity was potentiated by lower HMA concentrations compared to the wild type receptor. Our findings suggest that an exposed guanidine group on amiloride and amiloride derivatives is critical for modulating the GABA-A ρ1 receptor. The present study provides a conceptual framework for predicting which amiloride derivatives will demonstrate positive allosteric modulation of the GABA-A ρ1 receptor.

  11. Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway

    PubMed Central

    TYAGI, NEETU; GILLESPIE, WILLIAM; VACEK, JONATHAN C.; SEN, UTPAL; TYAGI, SURESH C.; LOMINADZE, DAVID

    2010-01-01

    Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase-9 (MMP-9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ-aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA-A receptor in Hcy-induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP-9 activity via the extracellular signal-regulated kinase (ERK) signaling pathway and by inhibition of GABA-A receptors, thus behaving as an inhibitory neurotransmitter. Hcy-induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′-dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate-oxidase-4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP-9, measured by gelatin zymography. The GABA-A receptor agonist, muscimol ameliorated the Hcy-mediated MMP-9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase-4 (TIMP-4). Treatment of the endothelial cell with muscimol restored the levels of TIMP-4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy-induced MMP-9 via ERK signaling pathway. These results suggest that Hcy competes with GABA-A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. PMID:19308943

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

    PubMed

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-03-02

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

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

    PubMed

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

    2007-07-01

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

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

    PubMed

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

    2001-10-01

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

  15. Diminished GABA(A) receptor-binding capacity and a DNA base substitution in a patient with treatment-resistant depression and anxiety.

    PubMed

    Kosel, Markus; Rudolph, Uwe; Wielepp, Peter; Luginbühl, Martin; Schmitt, Wolfgang; Fisch, Hans U; Schlaepfer, Thomas E

    2004-02-01

    In this report, we describe the case of a caucasian male patient, aged 42 years, suffering from severe treatment-resistant generalized anxiety disorder with panic attacks and from severe major depression, for which he was treated with a course of electroconvulsive therapy. During electroconvulsive treatment, anesthesia was difficult to induce with etomidate and, once, propofol. Bispectral indices recordings (assessing the depth of anesthesia) revealed a much shorter duration of loss of responsiveness compared to a control patient receiving also a course of electroconvulsive therapy. Since GABA receptor-mediated regulation of cortical excitability is important with respect to general anesthesia, we investigated the density of GABA(A) receptors with (123)I-iomazenil SPECT and found a clearly diminished binding of the radiotracer in the right frontal and orbitotemporal regions compared to the recordings in a 38-year-old healthy male control. Genetic analysis of the exons 7 and 8 of the GABRB1-3 genes coding for the beta1-3 subunits of the GABA(A) receptors revealed a silent G to A substitution in the third position of amino acid 257 of the beta1-subunit. To our knowledge, this is the first report of a link between insensitivity to anesthetic agents and altered GABA(A) receptor function in a clinical case. Whereas reduced GABA(A) receptor-binding capacity has been investigated in anxiety disorders, this has not been the case in depressive disorders. This case illustrates how clinical observations in psychiatry can prompt investigation by modern techniques and potentially link clinics and basic sciences. No conclusion can, however, be made about casual links in this single case [corrected].

  16. GABA(A) receptor activation is involved in noncontingent shock inhibition of instrumental conditioning in spinal rats.

    PubMed

    Ferguson, Adam R; Washburn, Stephanie N; Crown, Eric D; Grau, James W

    2003-08-01

    Previous work has demonstrated that the spinal cord, isolated from higher neural structures, can support a simple form of instrumental learning. Furthermore, preexposure to uncontrollable (noncontingent) shock to the leg or tail inhibits this form of learning. The present study explores the role of GABA(A) receptor modulation on this inhibitory effect in spinal cord-transected rats. Intrathecal administration of the GABA(A) receptor antagonist bicuculline blocked induction and expression of the inhibition. The GABA(A) receptor agonist muscimol inhibited learning in a dose-dependent manner. However, this effect was transient and showed no additivity with shock. The findings suggest that GABA(A) receptor activation may work like a pharmacological switch that is activated by noncontingent shock to inhibit instrumental conditioning within the spinal cord.

  17. GABA-A receptors regulate neocortical neuronal migration in vitro and in vivo.

    PubMed

    Heck, Nicolas; Kilb, Werner; Reiprich, Petra; Kubota, Hisahiko; Furukawa, Tomonori; Fukuda, Atsuo; Luhmann, Heiko J

    2007-01-01

    The cortical migration process depends on a number of trophic factors and on the activation of different voltage- and ligand-gated channels. We investigated the role of gamma-aminobutyric acid (GABA) type A receptors in the neuronal migration process of the newborn rat parietal cortex in vivo and in vitro. Local in vivo application of the GABA-A antagonist bicuculline methiodide (BMI) or the agonist muscimol via cortical surface Elvax implants induced prominent alterations in the cortical architecture when compared with untreated or sham-operated controls. BMI- and muscimol-treated animals revealed heterotopic cell clusters in the upper layers and a complete loss of the cortical lamination in the region underlying the Elvax implant. Immunocytochemical staining for glial fibrillary acidic protein, N-methyl-D-aspartate receptors, and GABA demonstrated that heterotopia was not provoked by glial proliferation and confirmed the presence of both glutamatergic and GABAergic neurons. In organotypic neocortical slices from embryonic day 18-19 embryos, application of BMI and to a lesser extent also muscimol induced an increase in the migration speed and an accumulation of neurons in the upper cortical layers. Spontaneous intracellular calcium ([Ca2+]i) oscillations in neocortical slices from newborn rats were abolished by BMI (5 and 20 microM) and muscimol (1 and 10 microM), indicating that both compounds interfere with [Ca2+]i signaling required for normal neuronal migration. Electrophysiological recordings from migrating neurons in newborn rat neocortical slices indicate that long-term application of muscimol causes a pronounced reduction (1 microM muscimol) or blockade (10 microM) in the responsiveness of postsynaptic GABA-A receptors due to a pronounced receptor desensitization. Our results indicate that modulation of GABA-A receptors by compounds acting as agonists or antagonists may profoundly influence the neuronal migration process in the developing cerebral cortex.

  18. Effects of Antecedent GABA A Receptor Activation on Counterregulatory Responses to Exercise in Healthy Man.

    PubMed

    Hedrington, Maka S; Tate, Donna B; Younk, Lisa M; Davis, Stephen N

    2015-09-01

    The aim of this study was to determine whether antecedent stimulation of γ-aminobutyric acid (GABA) A receptors with the benzodiazepine alprazolam can blunt physiologic responses during next-day moderate (90 min) exercise in healthy man. Thirty-one healthy individuals (16 male/15 female aged 28 ± 1 year, BMI 23 ± 3 kg/m(2)) were studied during separate, 2-day protocols. Day 1 consisted of morning and afternoon 2-h hyperinsulinemic-euglycemic or hypoglycemic clamps with or without 1 mg alprazolam given 30 min before a clamp. Day 2 consisted of 90-min euglycemic cycling exercise at 50% VO2max. Despite similar euglycemia (5.3 ± 0.1 mmol/L) and insulinemia (46 ± 6 pmol/L) during day 2 exercise studies, GABA A activation with alprazolam during day 1 euglycemia resulted in significant blunting of plasma epinephrine, norepinephrine, glucagon, cortisol, and growth hormone responses. Lipolysis (glycerol, nonesterified fatty acids) and endogenous glucose production during exercise were also reduced, and glucose infusion rates were increased following prior euglycemia with alprazolam. Prior hypoglycemia with alprazolam resulted in further reduction of glucagon and cortisol responses during exercise. We conclude that prior activation of GABA A pathways can play a significant role in blunting key autonomous nervous system, neuroendocrine, and metabolic physiologic responses during next-day exercise in healthy man.

  19. Catamenial-like seizure exacerbation in mice with targeted ablation of extrasynaptic δGABA-a receptors in the brain.

    PubMed

    Clossen, Bryan L; Reddy, Doodipala Samba

    2017-02-25

    Neurosteroids play a key role in catamenial epilepsy, a menstrual cycle-related seizure clustering in women with epilepsy. While neurosteroids act on all GABA-A receptor isoforms, they cause greater effects on extrasynaptic δGABA-A receptors that mediate tonic inhibition in the brain. Previously, we identified a potential GABA-A receptor mechanism for catamenial epilepsy. However, the precise functional role of extrasynaptic δGABA-A receptors in the pathophysiology of catamenial epilepsy remains unclear. In this study, we utilized mice lacking extrasynaptic δGABA-A receptors (δKO) to investigate whether reduction of tonic inhibition affects catamenial seizure susceptibility or intensity. Intact female wildtype (WT) and δKO mice were subjected to hippocampus kindling until they exhibited stage 5 seizures. Elevated gonadal hormone-based neurosteroid levels were induced by standard gonadotropin regimen and neurosteroid withdrawal (NSW) was triggered by finasteride. NSW increased susceptibility to, as well the intensity of evoked catamenial-like seizures in WT and δKO mice. However, fully kindled δKO mice exhibited an accelerated and augmented response to NSW, with a more rapid increase in seizure susceptibility and intensity than WT mice undergoing the NSW paradigm. Moreover, δKO mice in NSW showed reduced benzodiazepine sensitivity, but in stark contrast to the increased neurosteroid sensitivity observed in WT animals, δKO mice displayed no change in neurosteroid sensitivity in response to NSW. The increased catamenial seizure exacerbation and alterations in antiseizure drug responses are consistent with NSW-induced changes in the abundance of δGABA-A receptors. Collectively, these findings provide evidence of a potential protective role for extrasynaptic δGABA-A receptors in catamenial-like seizures. © 2017 Wiley Periodicals, Inc.

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

    PubMed

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

    2009-10-01

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

  1. Effects of central histamine receptors blockade on GABA(A) agonist-induced food intake in broiler cockerels.

    PubMed

    Morteza, Zendehdel; Vahhab, Babapour; Hossein, Jonaidi

    2008-02-01

    In this study, the effect of intracerebroventricular (i.c.v) injection of H1, H2 and H3 antagonists on feed intake induced by GABA(A) agonist was evaluated. In Experiment 1, the animals received chloropheniramine, a H1 antagonist and then muscimol, a GABA(A) agonist. In Experiment 2, chickens received famotidine, a H2 receptor antagonist, prior to injection of muscimol. Finally in Experiment 3, the birds were injected with thioperamide, a H3 receptor antagonist and muscimol. Cumulative food intake was measured 15, 30, 45, 60, 90, 120, 150 and 180 min after injections. The results of this study indicated that effects of muscimol on food intake inhibited by pretreatment with chloropheneramine maleate (p < or = 0.05), significantly, while the famotidine and thioperamide were ineffective. These results suggest the existence of H1-receptor mediated histamine-GABA(A) receptor interaction on food intake in broiler cockerels.

  2. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons.

    PubMed

    Govindaiah, G; Wang, Yanyan; Cox, Charles L

    2010-02-01

    Substance P (SP) is co-localized and co-released with gamma-amino butyric acid (GABA) from approximately 50% of GABAergic medium spiny neurons (MSNs) in the striatum. MSNs innervate several cellular targets including neighboring MSNs and cholinergic interneurons via collaterals. However, the functional role of SP release onto striatal interneurons is unknown. Here we examined SP-mediated actions on inhibitory synaptic transmission in cholinergic interneurons using whole-cell recordings in mouse corticostriatal slices. We found that SP selectively suppressed GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), but not excitatory post-synaptic currents (EPSCs) in cholinergic interneurons. In contrast, SP did not alter IPSCs in fast-spiking interneurons and MSNs. SP suppressed IPSC amplitude in a concentration-dependent and reversible manner, and the NK1 receptor antagonist RP67580 attenuated the SP-mediated suppression. In addition, RP67580 alone enhanced the evoked IPSC amplitude in cholinergic interneurons, suggesting an endogenous action of SP on regulation of inhibitory synaptic transmission. SP did not alter the paired-pulse ratio, but reduced the amplitudes of GABA(A) agonist muscimol-induced outward currents and miniature IPSCs in cholinergic interneurons, suggesting SP exerts its effects primarily at the post-synaptic site. Our results indicate that the physiological effects of SP are to enhance the activity of striatal cholinergic interneurons and provide a rationale for designing potential new antiparkinsonian agents.

  3. Attenuating GABA(A) receptor signaling in dopamine neurons selectively enhances reward learning and alters risk preference in mice.

    PubMed

    Parker, Jones G; Wanat, Matthew J; Soden, Marta E; Ahmad, Kinza; Zweifel, Larry S; Bamford, Nigel S; Palmiter, Richard D

    2011-11-23

    Phasic dopamine (DA) transmission encodes the value of reward-predictive stimuli and influences both learning and decision-making. Altered DA signaling is associated with psychiatric conditions characterized by risky choices such as pathological gambling. These observations highlight the importance of understanding how DA neuron activity is modulated. While excitatory drive onto DA neurons is critical for generating phasic DA responses, emerging evidence suggests that inhibitory signaling also modulates these responses. To address the functional importance of inhibitory signaling in DA neurons, we generated mice lacking the β3 subunit of the GABA(A) receptor specifically in DA neurons (β3-KO mice) and examined their behavior in tasks that assessed appetitive learning, aversive learning, and risk preference. DA neurons in midbrain slices from β3-KO mice exhibited attenuated GABA-evoked IPSCs. Furthermore, electrical stimulation of excitatory afferents to DA neurons elicited more DA release in the nucleus accumbens of β3-KO mice as measured by fast-scan cyclic voltammetry. β3-KO mice were more active than controls when given morphine, which correlated with potential compensatory upregulation of GABAergic tone onto DA neurons. β3-KO mice learned faster in two food-reinforced learning paradigms, but extinguished their learned behavior normally. Enhanced learning was specific for appetitive tasks, as aversive learning was unaffected in β3-KO mice. Finally, we found that β3-KO mice had enhanced risk preference in a probabilistic selection task that required mice to choose between a small certain reward and a larger uncertain reward. Collectively, these findings identify a selective role for GABA(A) signaling in DA neurons in appetitive learning and decision-making.

  4. Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

    PubMed

    Cordato, D J; Chebib, M; Mather, L E; Herkes, G K; Johnston, G A

    1999-09-01

    1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.

  5. Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area.

    PubMed

    del Olmo, N; Bustamante, J; del Río, R M; Solís, J M

    2000-05-12

    We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.

  6. Effects of halothane on GABA(A) receptor kinetics: evidence for slowed agonist unbinding.

    PubMed

    Li, X; Pearce, R A

    2000-02-01

    Many anesthetics, including the volatile agent halothane, prolong the decay of GABA(A) receptor-mediated IPSCs at central synapses. This effect is thought to be a major factor in the production of anesthesia. A variety of different kinetic mechanisms have been proposed for several intravenous agents, but for volatile agents the kinetic mechanisms underlying this change remain unknown. To address this question, we used rapid solution exchange techniques to apply GABA to recombinant GABA(A) receptors (alpha(1)beta(2)gamma(2s)) expressed in HEK 293 cells, in the absence and presence of halothane. To differentiate between different microscopic kinetic steps that may be altered by the anesthetic, we studied a variety of measures, including peak concentration-response characteristics, macroscopic desensitization, recovery from desensitization, maximal current activation rates, and responses to the low-affinity agonist taurine. Experimentally observed alterations were compared with predictions based on a kinetic scheme that incorporated two agonist binding steps, and open and desensitized states. We found that, in addition to slowing deactivation after a brief pulse of GABA, halothane increased agonist sensitivity and slowed recovery from desensitization but did not alter macroscopic desensitization or maximal activation rate and only slightly slowed rapid deactivation after taurine application. This pattern of responses was found to be consistent with a reduction in the microscopic agonist unbinding rate (k(off)) but not with changes in channel gating steps, such as the channel opening rate (beta), closing rate (alpha), or microscopic desensitization. We conclude that halothane slows IPSC decay by slowing dissociation of agonist from the receptor.

  7. Synthesis and GABA(A) receptor activity of 2,19-sulfamoyl analogues of allopregnanolone.

    PubMed

    Durán, Fernando J; Edelsztein, Valeria C; Ghini, Alberto A; Rey, Mariana; Coirini, Héctor; Dauban, Philippe; Dodd, Robert H; Burton, Gerardo

    2009-09-15

    The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the beta-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3alpha-substituted analogues such as the 3alpha-fluoro derivative. GABA(A) receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [(3)H]flunitrazepam and [(3)H]muscimol. The 3alpha-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [(3)H]flunitrazepam. For the binding of [(3)H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC(50). The 3alpha-fluoro derivative was inactive in both assays.

  8. GABA-A and GABA-B receptors in the cuneate nucleus of the rat in vivo.

    PubMed

    Orviz, P; Cecchini, B G; Andrés-Trelles, F

    1986-09-01

    Electric stimulation of the rat forepaw evokes a negative potential (N-wave) at the ipsilateral cuneate nucleus. The responses of the N-wave to microiontophoretically applied GABA agonists and antagonists have been studied. Applications of GABA-A agonists (3-amino-propanesulfonic acid and muscimol) reduce the amplitude of the N-wave. This effect decreases during prolonged application, suggesting a desensitization of GABA-A receptors. In addition the effect of muscimol is reduced by (-)-bicuculline methiodide. Baclofen (a GABA-B agonist) also depresses the N-wave but its action lasts longer, is less reversible, shows no desensitization and is not blocked by (-)-bicuculline methiodide. The different responses of the N-wave to GABA-A and GABA-B agonists are compatible with the existence of different types of functional receptors for them in the cuneate nucleus of the rat. The receptors activated by muscimol (GABA-A) are clearly not the same as the ones activated by baclofen (conceivably GABA-B).

  9. Ethanol, not metabolized in brain, significantly reduces brain metabolism, probably via specific GABA(A) receptors

    PubMed Central

    Rae, Caroline D.; Davidson, Joanne E.; Maher, Anthony D.; Rowlands, Benjamin D.; Kashem, Mohammed A.; Nasrallah, Fatima A.; Rallapalli, Sundari K.; Cook, James M; Balcar, Vladimir J.

    2014-01-01

    Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we used an indirect approach, measuring the effect of alcohol on metabolism of [3-13C]pyruvate in the adult Guinea pig brain cortical tissue slice and comparing the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-13C]ethanol. Ethanol (10, 30 and 60 mM) significantly reduced metabolic flux into all measured isotopomers and reduced all metabolic pool sizes. The metabolic profiles of these three concentrations of ethanol were similar and clustered with that of the α4β3δ positive allosteric modulator DS2 (4-Chloro-N-[2-(2-thienyl)imidazo[1,2a]-pyridin-3-yl]benzamide). Ethanol at a very low concentration (0.1 mM) produced a metabolic profile which clustered with those from inhibitors of GABA uptake, and ligands showing affinity for α5, and to a lesser extent, α1-containing GABA(A)R. There was no measureable metabolism of [1,2-13C]ethanol with no significant incorporation of 13C from [1,2-13C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabeled ethanol. The reduction in metabolism seen in the presence of ethanol is therefore likely to be due to its actions at neurotransmitter receptors, particularly α4β3δ receptors, and not because ethanol is substituting as a substrate or because of the effects of ethanol catabolites acetaldehyde or acetate. We suggest that the stimulatory effects of very low concentrations of ethanol are due to release of GABA via GAT1 and the subsequent interaction of this GABA with local α5-containing, and to a lesser extent, α1-containing GABA(A)R. PMID:24313287

  10. The effects of agonists of ionotropic GABA(A) and metabotropic GABA(B) receptors on learning.

    PubMed

    Zyablitseva, Evgeniya A; Kositsyn, Nikolay S; Shul'gina, Galina I

    2009-05-01

    The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes.

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

    PubMed

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

    2007-08-24

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

  12. Loss of functional GABA(A) receptors in the Alzheimer diseased brain.

    PubMed

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

    2012-06-19

    The cholinergic and glutamatergic neurotransmission systems are known to be severely disrupted in Alzheimer's disease (AD). GABAergic neurotransmission, in contrast, is generally thought to be well preserved. Evidence from animal models and human postmortem tissue suggest GABAergic remodeling in the AD brain. Nevertheless, there is no information on changes, if any, in the electrophysiological properties of human native GABA receptors as a consequence of AD. To gain such information, we have microtransplanted cell membranes, isolated from temporal cortices of control and AD brains, into Xenopus oocytes, and recorded the electrophysiological activity of the transplanted GABA receptors. We found an age-dependent reduction of GABA currents in the AD brain. This reduction was larger when the AD membranes were obtained from younger subjects. We also found that GABA currents from AD brains have a faster rate of desensitization than those from non-AD brains. Furthermore, GABA receptors from AD brains were slightly, but significantly, less sensitive to GABA than receptors from non-AD brains. The reduction of GABA currents in AD was associated with reductions of mRNA and protein of the principal GABA receptor subunits normally present in the temporal cortex. Pairwise analysis of the transcripts within control and AD groups and analyses of the proportion of GABA receptor subunits revealed down-regulation of α1 and γ2 subunits in AD. In contrast, the proportions of α2, β1, and γ1 transcripts were up-regulated in the AD brains. Our data support a functional remodeling of GABAergic neurotransmission in the human AD brain.

  13. Antiplasmodial and GABA(A)-benzodiazepine receptor binding activities of five plants used in traditional medicine in Mali, West Africa.

    PubMed

    Bah, Sekou; Jäger, Anna K; Adsersen, Anne; Diallo, Drissa; Paulsen, Berit Smestad

    2007-04-04

    Extracts of five medicinal plants: Boscia angustifolia, Cissus quadrangularis, Securidaca longipedunculata, Stylosanthes erecta and Trichilia emetica, used traditionally in Malian traditional medicine were screened for in vitro antiplasmodial activity and GABA(A)-benzodiazepine receptor binding activity. Four extracts showed significant antiplasmodial activities, with the dichloromethane extract of leaf of Securidaca longipedunculata being the most active (IC(50) of 7 microg/ml [95% CI: 5-9]). The dichloromethane extract of leaf of Trichilia emetica, in addition to its antiplasmodial activity (IC(50): 12 microg/ml [95% CI: 12-14]), exhibited a good binding activity to the GABA(A)-benzodiazepine receptor, while water and methanol extracts of the same plant did not show any activity. A strong GABA(A)-receptor complex binding activity was observed in the methanol extract of aerial part of Stylosanthes erecta. The results in this study justify some of the traditional indications of the plants investigated and may thus be candidates for Improved Traditional Medicines in Mali.

  14. GABA-A Receptor Modulation and Anticonvulsant, Anxiolytic, and Antidepressant Activities of Constituents from Artemisia indica Linn

    PubMed Central

    Khan, Imran; Karim, Nasiara; Ahmad, Waqar; Abdelhalim, Abeer; Chebib, Mary

    2016-01-01

    Artemisia indica, also known as “Mugwort,” has been widely used in traditional medicines. However, few studies have investigated the effects of nonvolatile components of Artemisia indica on central nervous system's function. Fractionation of Artemisia indica led to the isolation of carnosol, ursolic acid, and oleanolic acid which were evaluated for their effects on GABA-A receptors in electrophysiological studies in Xenopus oocytes and were subsequently investigated in mouse models of acute toxicity, convulsions (pentylenetetrazole induced seizures), depression (tail suspension and forced swim tests), and anxiety (elevated plus maze and light/dark box paradigms). Carnosol, ursolic acid, and oleanolic acid were found to be positive modulators of α1β2γ2L GABA-A receptors and the modulation was antagonized by flumazenil. Carnosol, ursolic acid, and oleanolic acid were found to be devoid of any signs of acute toxicity (50–200 mg/kg) but elicited anticonvulsant, antidepressant, and anxiolytic activities. Thus carnosol, ursolic acid, and oleanolic acid demonstrated CNS activity in mouse models of anticonvulsant, antidepressant, and anxiolysis. The anxiolytic activity of all three compounds was ameliorated by flumazenil suggesting a mode of action via the benzodiazepine binding site of GABA-A receptors. PMID:27143980

  15. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    PubMed

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  16. Striatal Serotonin 2C receptors decrease nigrostriatal dopamine release by increasing GABA-A receptor tone in the substantia nigra

    PubMed Central

    Burke, M.V.; Nocjar, C.; Sonneborn, A.J.; McCreary, A.C.

    2017-01-01

    Drugs acting at the serotonin-2C (5-HT2C) receptor subtype have shown promise as therapeutics in multiple syndromes including obesity, depression, and Parkinson’s disease. While it is established that 5-HT2C receptor stimulation inhibits DA release, the neural circuits and the localization of the relevant 5-HT2C receptors remain unknown. The present study used dual-probe in vivo microdialysis to investigate the relative contributions of 5-HT2C receptors localized in the rat substantia nigra (SN) and caudate-putamen (CP) in the control of nigrostriatal DA release. Systemic administration (3.0 mg/kg) of the 5-HT2C receptor selective agonist Ro 60-0175 [(α S )-6-Chloro-5-fluoro-α-methyl-1 H-indole-1-ethanamine fumarate] decreased, whereas intrastriatal infusions of the selective 5-HT2C antagonist SB 242084 [6-Chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1 H-indole-1-carboxyamide; 1.0 µM] increased, basal DA in the CP. Depending on the site within the SN pars reticulata (SNpr), infusions of SB 242084 had more modest but significant effects. Moreover, infusions of the GABA-A receptor agonist muscimol (10 µM) into the SNpr completely reversed the increases in striatal DA release produced by intrastriatal infusions of SB 242084. These findings suggest a role for 5-HT2C receptors regulating striatal DA release that is highly localized. 5-HT2C receptors localized in the striatum may represent a primary site of action that is mediated by actions on GABAergic activity in the SN. PMID:25073477

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

    PubMed

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

    2010-02-15

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

  18. Chronic anabolic-androgenic steroid treatment affects brain GABA(A) receptor-gated chloride ion transport.

    PubMed

    Bitran, D; Hilvers, R J; Frye, C A; Erskine, M S

    1996-01-01

    Previous research in this laboratory has shown that chronic treatment of adult male rats with an anabolic-androgenic steroid (AAS) produced anxiolytic behavior and increased the functional response of cortical gamma-aminobutyric acid(A) (GABA(A)) receptors. The experiments reported here were aimed at further characterizing the effect of chronic AAS exposure on cerebral cortical GABA(A) receptors. Adult male rats were injected with dianabol (1,4-androstadien-17alpha-methyl-17beta-ol-3-one; 10 mg/kg/day, SC) for 4 weeks. A significant decrease in ventral prostate gland weight was found after 2 weeks of dianabol, and returned to control levels 3 and 10 days after steroid discontinuation. Testicular weights decreased throughout the treatment period but reached statistical significance only during the withdrawal period. Serum 3alpha-androstanediol level was marginally increased afer 2 weeks of dianabol injection, and was significantly decreased at 3 and 10 days after withdrawal. GABA-stimulated 36chloride (Cl-) influx in cortical synaptoneurosomes was increased in animals treated with dianabol for 2 and 4 weeks, and remained elevated 3 days after dianabol withdrawal, returning to control levels at withdrawal day 10. The increase in receptor efficacy was associated with a transient increase in receptor sensitivity (inverse of EC50), apparent after 2 weeks of AAS treatment and at withdrawal day 3. In a follow-up experiment, metabolites of dianabol were tested for the in vitro efficacy in potentiating GABA-stimulated Cl- transport. Only 3alpha-androstanedial and androsterone were found to have potent stimulatory effects. The 3beta-reduced metabolites were inactive, as were metabolites that contained a methyl group at the 17alpha position. These results point to significant facilitative effects of dianabol treatment on brain GABA(A) receptors via the metabolic formation of neuroactive steroids.

  19. Role of the amygdala GABA-A receptors in ACPA-induced deficits during conditioned fear learning.

    PubMed

    Nasehi, Mohammad; Roghani, Farnaz; Ebrahimi-Ghiri, Mohaddeseh; Zarrindast, Mohammad-Reza

    2017-04-04

    The basolateral amygdala (BLA) is a key structure for the emotional processing and storage of memories associated with emotional events, especially fear. On the other hand, endocannabinoids and CB1 receptors play a key role in learning and memory partly through long-term synaptic depression of GABAergic synapses in the BLA. The aim of this study was to explore the effects of GABA-A receptor agonist and antagonist in the fear-related memory acquisition deficits induced by ACPA (a selective CB1 cannabinoid receptor agonist). This study used context and tone fear conditioning paradigms to assess fear-related memory in male NMRI mice. Our results showed that the pre-training intraperitoneal administration of ACPA (0.5mg/kg) or (0.1 and 0.5mg/kg) decreased the percentage of freezing time in the contextual and tone fear conditioning, respectively. This indicated an impaired context- or tone-dependent fear memory acquisition. Moreover, the pre-training intra-BLA microinjection of GABA-A receptor agonist, muscimol, at 0.05 and 0.5μg/mouse impaired context-dependent fear memory, while the same doses of GABA-A antagonist, bicuculline, impaired tone-dependent fear memory. However, a subthreshold dose of muscimol or bicuculline increased the effect of ACPA at 0.1 and 0.5 or 0.05mg/kg on context- or tone-dependent fear memory, respectively. In addition, bicuculline at the lower dose increased the ACPA response on locomotor activity compared to its respective group. Such findings highlighted an interaction between BLA GABAergic and cannabinoidergic systems during the acquisition phase of conditioned fear memories.

  20. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    PubMed

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development.

  1. Interaction between taurine and GABA(A)/glycine receptors in neurons of the rat anteroventral cochlear nucleus.

    PubMed

    Song, Ning-Ying; Shi, Hai-Bo; Li, Chun-Yan; Yin, Shan-Kai

    2012-09-07

    Taurine, one of the most abundant endogenous amino acids in the mammalian central nervous system (CNS), is involved in neural development and many physiological functions. In this study, the interaction between taurine and GABA(A)/glycine receptors was investigated in young rat (P13-P15) anteroventral cochlear nucleus (AVCN) neurons using the whole-cell patch-clamp method. We found that taurine at low (0.1mM) and high (1mM) concentrations activated both GABA(A) and glycine receptors, but not AMPA and NMDA receptors. The reversal potentials of taurine-, GABA- or glycine-evoked currents were close to the expected chloride equilibrium potential, indicating that receptors activated by these agonists were mediating chloride conductance. Moreover, our results showed that the currents activated by co-application of GABA and glycine were cross-inhibitive. Sequential application of GABA and glycine or vice versa also reduced the glycine or GABA evoked currents. There was no cross-inhibition when taurine and GABA or taurine and glycine were applied simultaneously, but the response was larger than that evoked by GABA or glycine alone. These results suggest that taurine can serve as a neuromodulator to strengthen GABAergic and glycinergic neurotransmission in the rat AVCN.

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

    PubMed

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

    2005-01-05

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

  3. Snake neurotoxin α-bungarotoxin is an antagonist at native GABA(A) receptors.

    PubMed

    Hannan, Saad; Mortensen, Martin; Smart, Trevor G

    2015-06-01

    The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β-α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus.

  4. Recruitment of GABA(A) receptors and fearfulness in chicks: modulation by systemic insulin and/or epinephrine.

    PubMed

    Cid, Mariana Paula; Toledo, Carolina Maribel; Salvatierra, Nancy Alicia

    2013-02-01

    One-day-old chicks were individually assessed on their latency to peck pebbles, and categorized as low latency (LL) or high latency (HL) according to fear. Interactions between acute stress and systemic insulin and epinephrine on GABA(A) receptor density in the forebrain were studied. At 10 days of life, LL and HL chicks were intraperitoneally injected with insulin, epinephrine or saline, and immediately after stressed by partial water immersion for 15 min and killed by decapitation. Forebrains were dissected and the GABA(A) receptor density was measured ex vivo by the (3)[H]-flunitrazepam binding assay in synaptosomes. In non-stressed chicks, insulin (non-hypoglycemic dose) at 2.50 IU/kg of body weight incremented the Bmax by 40.53% in the HL chicks compared to saline group whereas no significant differences were observed between individuals in the LL subpopulation. Additionally, insulin increased the Bmax (23.48%) in the HL group with respect to the LL ones, indicating that the insulin responses were different according to the anxiety of each category. Epinephrine administration (0.25 and 0.50mg/kg) incremented the Bmax in non-stressed chicks, in the LL group by about 37% and 33%, respectively, compared to ones injected with saline. In the stressed chicks, 0.25mg/kg bw epinephrine increased the Bmax significantly in the HL group by about 24% compared to saline, suggesting that the effect of epinephrine was only observed in the HL group under acute stress conditions. Similarly, the same epinephrine doses co-administered with insulin increased the receptor density in both subpopulations and also showed that the highest dose of epinephrine did not further increase the maximum density of GABA(A)R in HL chicks. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulated the increase in the forebrain GABA(A) receptor recruitment induced by both insulin and stress in different ways depending on the subpopulation

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

    PubMed

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

    2000-12-01

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

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

    PubMed

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

    2003-03-28

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

  7. Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons.

    PubMed

    Porcher, Christophe; Hatchett, Caroline; Longbottom, Rebecca E; McAinch, Kristina; Sihra, Talvinder S; Moss, Stephen J; Thomson, Alex M; Jovanovic, Jasmina N

    2011-06-17

    During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

  8. Proteomic and systems biology analysis of monocytes exposed to securinine, a GABA(A) receptor antagonist and immune adjuvant.

    PubMed

    Shipman, Matt; Lubick, Kirk; Fouchard, David; Guram, Rajani; Grieco, Paul; Jutila, Mark; Dratz, Edward A

    2012-01-01

    Securinine, a GABA(A) receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABA(A) receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5'-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca(2+) binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.

  9. Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice

    PubMed Central

    Braudeau, J; Delatour, B; Duchon, A; Pereira, P Lopes; Dauphinot, L; de Chaumont, F; Olivo-Marin, J-C; Dodd, RH; Hérault, Y; Potier, M-C

    2011-01-01

    An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals. PMID:21693554

  10. Altered expression of GABAA receptors (α4, γ2 subunit), potassium chloride cotransporter 2 and astrogliosis in tremor rat hippocampus.

    PubMed

    Mao, Xiaoyuan; Ma, Ping; Cao, Danfeng; Sun, Chunyan; Ji, Zhong; Min, Dongyu; Sun, Hongli; Xie, Ni; Cai, Jiqun; Cao, Yonggang

    2011-11-25

    Impaired GABAergic inhibitory neurotransmission plays an essential role in the pathogenesis of epilepsy. GABA(A) receptor (GABA(A)R), potassium chloride cotransporter 2 (KCC2) and astrocytes are of particular importance to GABAergic transmission and thus involved in the development of increased seizure susceptibility. The tremor rat (TRM: tm/tm), a genetic mutant discovered in a Kyoto-Wistar colony, can manifest both absence-like seizures and tonic convulsions without any external stimuli. So far, there are no reports that can elucidate the effects of GABA(A)R (α4, γ2 subunit), KCC2 and astrocytes on TRMs. The present study was undertaken to detect the expressions of GABA(A)R α4, GABA(A)R γ2 and KCC2 in TRMs hippocampus at mRNA and protein levels. In this work, mRNA and protein expressions of GABA(A)R α4 were significantly elevated while GABA(A)R γ2 and KCC2 were both evidently decreased in TRMs hippocampus by real-time RT-PCR and western blot, respectively. Furthermore, a dramatic elevation of KCC2 protein level was found after cerebroventricular injection with K252a to TRMs than that in the DMSO-treated TRMs. Besides, our present study also demonstrated that GFAP (a major component of astrocyte) immunoreactivity was much more intense in TRMs hippocampal CA1, CA3 and DG regions than that in control group with immnohistochemistry and confocal microscopic analyses. The protein expression of GFAP was also markedly elevated in TRMs hippocampus, suggesting that astrogliosis appeared in the TRM model. These data demonstrate that altered expressions of GABA(A)R (α4, γ2) and KCC2 and astrogliosis observed in TRMs hippocampus may provide us good therapeutic targets for the treatment of genetic epilepsy.

  11. Exploring QSARs of the interaction of flavonoids with GABA (A) receptor using MLR, ANN and SVM techniques.

    PubMed

    Deeb, Omar; Shaik, Basheerulla; Agrawal, Vijay K

    2014-10-01

    Quantitative Structure-Activity Relationship (QSAR) models for binding affinity constants (log Ki) of 78 flavonoid ligands towards the benzodiazepine site of GABA (A) receptor complex were calculated using the machine learning methods: artificial neural network (ANN) and support vector machine (SVM) techniques. The models obtained were compared with those obtained using multiple linear regression (MLR) analysis. The descriptor selection and model building were performed with 10-fold cross-validation using the training data set. The SVM and MLR coefficient of determination values are 0.944 and 0.879, respectively, for the training set and are higher than those of ANN models. Though the SVM model shows improvement of training set fitting, the ANN model was superior to SVM and MLR in predicting the test set. Randomization test is employed to check the suitability of the models.

  12. Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ.

    PubMed

    Trigo, Federico F; Papageorgiou, George; Corrie, John E T; Ogden, David

    2009-07-30

    Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

  13. Analysis of GABA(A)- and GABA(B)-receptor mediated effects on intracellular Ca(2+) in DRG hybrid neurones.

    PubMed

    Yokogawa, T; Kim, S U; Krieger, C; Puil, E

    2001-09-01

    1. Using pharmacological analysis and fura-2 spectrofluorimetry, we examined the effects of gamma-aminobutyric acid (GABA) and related substances on intracellular Ca(2+) concentration ([Ca(2+)]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone. 2. MD3 cells exhibited DRG neurone-like properties, such as immunoreactivity to microtubule-associated protein-2 and neurofilament proteins. Bath applications of capsaicin and alpha, beta-methylene adenosine triphosphate reversibly increased [Ca(2+)]i. However, repeated applications of capsaicin were much less effective. 3. Pressure applications of GABA (100 microM), (Z)-3-[(aminoiminomethyl) thio] prop-2-enoic acid sulphate (ZAPA; 100 microM), an agonist at low affinity GABA(A)-receptors, or KCl (25 mM), transiently increased [Ca(2+)]i. 4. Bath application of bicuculline (100 nM - 100 microM), but not picrotoxinin (10 - 25 microM), antagonized GABA-induced increases in [Ca(2+)]i in a concentration-dependent manner (IC(50)=9.3 microM). 5. Ca(2+)-free perfusion reversibly abolished GABA-evoked increases in [Ca(2+)]i. Nifedipine and nimodipine eliminated GABA-evoked increases in [Ca(2+)]i. These results imply GABA response dependence on extracellular Ca(2+). 6. Baclofen (500 nM - 100 microM) activation of GABA(B)-receptors reversibly attenuated KCl-induced increases in [Ca(2+)]i in a concentration-dependent manner (EC(50)=1.8 microM). 2-hydroxy-saclofen (1 - 20 microM) antagonized the baclofen-depression of the KCl-induced increase in [Ca(2+)]i. 7. In conclusion, GABA(A)-receptor activation had effects similar to depolarization by high external K(+), initiating Ca(2+) influx through high voltage-activated channels, thereby transiently elevating [Ca(2+)]i. GABA(B)-receptor activation reduced Ca(2+) influx evoked by depolarization, possibly at Ca(2+)-channel sites in MD3 cells.

  14. GABA(A) receptors in the pontine reticular formation of C57BL/6J mouse modulate neurochemical, electrographic, and behavioral phenotypes of wakefulness.

    PubMed

    Flint, RaShonda R; Chang, Theresa; Lydic, Ralph; Baghdoyan, Helen A

    2010-09-15

    Drugs that potentiate transmission at GABA(A) receptors are widely used to enhance sleep and to cause general anesthesia. The mechanisms underlying these effects are unknown. This study tested the hypothesis that GABA(A) receptors in the pontine reticular nucleus, oral part (PnO) of mouse modulate five phenotypes of arousal: sleep and wakefulness, cortical electroencephalogram (EEG) activity, acetylcholine (ACh) release in the PnO, breathing, and recovery time from general anesthesia. Microinjections into the PnO of saline (vehicle control), the GABA(A) receptor agonist muscimol, muscimol with the GABA(A) receptor antagonist bicuculline, and bicuculline alone were performed in male C57BL/6J mice (n = 33) implanted with EEG recording electrodes. Muscimol caused a significant increase in wakefulness and decrease in rapid eye movement (REM) and non-REM (NREM) sleep. These effects were reversed by coadministration of bicuculline. Bicuculline administered alone caused a significant decrease in wakefulness and increase in NREM sleep and REM sleep. Muscimol significantly increased EEG power in the delta range (0.5-4 Hz) during wakefulness and in the theta range (4-9 Hz) during REM sleep. Dialysis delivery of bicuculline to the PnO of male mice (n = 18) anesthetized with isoflurane significantly increased ACh release in the PnO, decreased breathing rate, and increased anesthesia recovery time. All drug effects were concentration dependent. The effects on phenotypes of arousal support the conclusion that GABA(A) receptors in the PnO promote wakefulness and suggest that increasing GABAergic transmission in the PnO may be one mechanism underlying the phenomenon of paradoxical behavioral activation by some benzodiazepines.

  15. Recovery from ketamine-induced amnesia by blockade of GABA-A receptor in the medial prefrontal cortex of mice.

    PubMed

    Farahmandfar, Maryam; Akbarabadi, Ardeshir; Bakhtazad, Atefeh; Zarrindast, Mohammad-Reza

    2017-03-06

    Ketamine and other noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists are known to induce deficits in learning and cognitive performance sensitive to prefrontal cortex (PFC) functions. The interaction of a glutamatergic and GABAergic systems is essential for many cognitive behaviors. In order to understand the effect of γ-aminobutyric acid (GABA)/glutamate interactions on learning and memory, we investigated the effects of intra medial prefrontal cortex (mPFC) injections of GABAergic agents on ketamine-induced amnesia using a one-trial passive avoidance task in mice. Pre-training systemic administration of ketamine (5, 10 and 15mg/kg, i.p.) dose-dependently decreased the memory acquisition of a one-trial passive avoidance task. Pre-training intra-mPFC injection of muscimol, GABAA receptor agonist (0.05, 0.1 and 0.2μg/mouse) and baclofen GABAB receptor agonist (0.05, 0.1, 0.5 and 1μg/mouse), impaired memory acquisition. However, co-pretreatment of different doses of muscimol and baclofen with a lower dose of ketamine (5mg/kg), which did not induce amnesia by itself, caused inhibition of memory formation. Our data showed that sole pre-training administration of bicuculline, GABA-A receptor antagonist and phaclofen GABA-B receptor antagonist into the mPFC, did not affect memory acquisition. In addition, the amnesia induced by pre-training ketamine (15mg/kg) was significantly decreased by the pretreatment of bicuculline (0.005, 0.1 and 0.5μg/mouse). It can be concluded that GABAergic system of the mPFC is involved in the ketamine-induced impairment of memory acquisition.

  16. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

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

  17. Imprinting in the schizophrenia candidate gene GABRB2 encoding GABA(A) receptor β(2) subunit.

    PubMed

    Pun, F W; Zhao, C; Lo, W-S; Ng, S-K; Tsang, S-Y; Nimgaonkar, V; Chung, W S; Ungvari, G S; Xue, H

    2011-05-01

    Schizophrenia is a complex genetic disorder, the inheritance pattern of which is likely complicated by epigenetic factors yet to be elucidated. In this study, transmission disequilibrium tests with family trios yielded significant differences between paternal and maternal transmissions of the disease-associated single-nucleotide polymorphism (SNP) rs6556547 and its haplotypes. The minor allele (T) of rs6556547 was paternally undertransmitted to male schizophrenic offsprings, and this parent-of-origin effect strongly suggested that GABRB2 is imprinted. 'Flipping' of allelic expression in heterozygotes of SNP rs2229944 (C/T) in GABRB2 or rs2290732 (G/A) in the neighboring GABRA1 was compatible with imprinting effects on gene expression. Clustering analysis of GABRB2 mRNA expressions suggested that imprinting brought about the observed two-tiered distribution of expression levels in controls with heterozygous genotype at the disease-associated SNP rs1816071 (A/G). The deficit of upper-tiered expressions accounted for the lowered expression levels in the schizophrenic heterozygotes. The occurrence of a two-tiered distribution furnished support for imprinting, and also pointed to the necessity of differentiating between two kinds of heterozygotes of different parental origins in disease association studies on GABRB2. Bisulfite sequencing revealed hypermethylation in the neighborhood of SNP rs1816071, and methylation differences between controls and schizophrenia patients. Notably, the two schizophrenia-associated SNPs rs6556547 and rs1816071 overlapped with a CpG dinucleotide, thereby opening the possibility that CpG methylation status of these sites could have an impact on the risk of schizophrenia. Thus multiple lines of evidence pointed to the occurrence of imprinting in the GABRB2 gene and its possible role in the development of schizophrenia.

  18. Alcohol use disorders and current pharmacological therapies: the role of GABA(A) receptors.

    PubMed

    Liang, Jing; Olsen, Richard W

    2014-08-01

    Alcohol use disorders (AUD) are defined as alcohol abuse and alcohol dependence, which create large problems both for society and for the drinkers themselves. To date, no therapeutic can effectively solve these problems. Understanding the underlying mechanisms leading to AUD is critically important for developing effective and safe pharmacological therapies. Benzodiazepines (BZs) are used to reduce the symptoms of alcohol withdrawal syndrome. However, frequent use of BZs causes cross-tolerance, dependence, and cross-addiction to alcohol. The FDA-approved naltrexone and acamprosate have shown mixed results in clinical trials. Naltrexone is effective to treat alcohol dependence (decreased length and frequency of drinking bouts), but its severe side effects, including withdrawal symptoms, are difficult to overcome. Acamprosate showed efficacy for treating alcohol dependence in European trials, but two large US trials have failed to confirm the efficacy. Another FDA-approved medication, disulfiram, does not diminish craving, and it causes a peripheral neuropathy. Kudzu is the only natural medication mentioned by the National Institute on Alcohol Abuse and Alcoholism, but its mechanisms of action are not yet established. It has been recently shown that dihydromyricetin, a flavonoid purified from Hovenia, has unique effects on GABAA receptors and blocks ethanol intoxication and withdrawal in alcoholic animal models. In this article, we review the role of GABAA receptors in the treatment of AUD and currently available and potentially novel pharmacological agents.

  19. GABA(A)- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis.

    PubMed

    Moccia, Francesco; Di Cristo, Carlo; Winlow, William; Di Cosmo, Anna

    2009-03-01

    To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either "active" or "silent" preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABA(A) receptors, excited silent cells and reversed GABA-induced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABA(A) receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs.

  20. Maternal separation increases GABA(A) receptor-mediated modulation of norepinephrine release in the hippocampus of a rat model of ADHD, the spontaneously hypertensive rat.

    PubMed

    Sterley, Toni-Lee; Howells, Fleur M; Russell, Vivienne A

    2013-02-25

    Experiencing early life stress increases the risk of developing a psychiatric disorder later in life, possibly by altering neural networks, such as the locus-coeruleus norepinephrine (LC-NE) system. Whether early life stress affects the LC-NE system directly, or whether the effects are via changes in glutamate and GABA modulation of the LC-NE system, is unclear. Early life stress has been shown to alter glutamate and GABA transmission, and in particular, to alter GABA(A) receptor expression. The LC-NE system has been implicated in attention-deficit/hyperactivity disorder (ADHD), amongst other disorders, and is over-responsive to glutamate stimulation in a validated rat model of ADHD, the spontaneously hypertensive rat (SHR). It is plausible that the LC-NE system, or glutamate and GABA modulation thereof, in an individual already genetically predisposed to develop ADHD, or in SHR, may respond in a unique way to early life stress. To investigate this we applied a mild developmental stressor, maternal separation, onto SHR, and onto their control strain, Wistar-Kyoto rats (WKY), from post-natal day (P)2-14. On P50-52, in early adulthood, we assayed glutamate and potassium stimulated release of radio-actively labelled NE ((3)[H]NE) from hippocampal slices using an in vitro superfusion technique, in the presence or absence of a GABA(A) receptor antagonist, bicuculline. Our results show that maternal separation altered GABA(A) receptor-mediated modulation of NE release in the hippocampus of the two strains in opposite directions, increasing it in SHR and decreasing it in WKY. Our findings indicate that effects of early life stress are highly dependent on genetic predisposition, since opposite changes in GABA(A) receptor-mediated modulation of NE release were observed in the rat model of ADHD, SHR, and their control strain, WKY.

  1. Receptor subtype-dependent positive and negative modulation of GABA(A) receptor function by niflumic acid, a nonsteroidal anti-inflammatory drug.

    PubMed

    Sinkkonen, Saku T; Mansikkamäki, Salla; Möykkynen, Tommi; Lüddens, Hartmut; Uusi-Oukari, Mikko; Korpi, Esa R

    2003-09-01

    In addition to blocking cyclooxygenases, members of the fenamate group of nonsteroidal anti-inflammatory drugs have been proposed to affect brain GABAA receptors. Using quantitative autoradiography with GABAA receptor-associated ionophore ligand [35S]t-butylbicyclophosphorothionate (TBPS) on rat brain sections, one of the fenamates, niflumate, at micromolar concentration was found to potentiate GABA actions in most brain areas, whereas being in the cerebellar granule cell layer an efficient antagonist similar to furosemide. With recombinant GABAA receptors expressed in Xenopus laevis oocytes, we found that niflumate potentiated 3 microM GABA responses up to 160% and shifted the GABA concentration-response curve to the left in alpha1beta2gamma2 receptors, the predominant GABAA receptor subtype in the brain. This effect needed the gamma2 subunit, because on alpha1beta2 receptors, niflumate exhibited solely an antagonistic effect at high concentrations. The potentiation was not abolished by the specific benzodiazepine site antagonist flumazenil. Niflumate acted as a potent antagonist of alpha6beta2 receptors (with or without gamma2 subunit) and of alphaXbeta2gamma2 receptors containing a chimeric alpha1 to alpha6 subunit, which suggests that niflumate antagonism is dependent on the same transmembrane domain 1- and 2-including fragment of the alpha6 subunit as furosemide antagonism. This antagonism was noncompetitive because the maximal GABA response, but not the potency, was reduced by niflumate. These data show receptor subtype-dependent positive and negative modulatory actions of niflumate on GABAA receptors at clinically relevant concentrations, and they suggest the existence of a novel positive modulatory site on alpha1beta2gamma2 receptors that is dependent on the gamma2 subunit but not associated with the benzodiazepine binding site.

  2. Activation of GABA(A) receptors by taurine and muscimol blocks the neurotoxicity of beta-amyloid in rat hippocampal and cortical neurons.

    PubMed

    Paula-Lima, Andréa C; De Felice, Fernanda G; Brito-Moreira, Jordano; Ferreira, Sérgio T

    2005-12-01

    The beta-amyloid peptide (Abeta) is centrally related to the pathogenesis of Alzheimer's disease (AD) and is potently neurotoxic to central nervous system neurons. The neurotoxicity of Abeta has been partially related to the over activation of glutamatergic transmission and excitotoxicity. Taurine is a naturally occurring beta-amino acid present in the mammalian brain. Due to its safety and tolerability, taurine has been clinically used in humans in the treatment of a number of non-neurological disorders. Here, we show that micromolar doses of taurine block the neurotoxicity of Abeta to rat hippocampal and cortical neurons in culture. Moreover, taurine also rescues central neurons from the excitotoxicity induced by high concentrations of extracellular glutamate. Neuroprotection by taurine is abrogated by picrotoxin, a GABA(A) receptor antagonist. GABA and muscimol, an agonist of the GABA(A) receptor, also block neuronal death induced by Abeta in rat hippocampal and cortical neurons. These results suggest that activation of GABA(A) receptors protects neurons against Abeta toxicity in AD-affected regions of the mammalian brain and that taurine should be investigated as a novel therapeutic tool in the treatment of AD and of other neurological disorders in which excitotoxicity plays a relevant role.

  3. Activation of glycine and extrasynaptic GABA(A) receptors by taurine on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis.

    PubMed

    Nguyen, Thi Thanh Hoang; Bhattarai, Janardhan Prasad; Park, Soo Joung; Han, Seong Kyu

    2013-01-01

    The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been known for the processing and transmission of orofacial nociceptive information. Taurine, one of the most plentiful free amino-acids in humans, has proved to be involved in pain modulation. In this study, using whole-cell patch clamp technique, we investigated the direct membrane effects of taurine and the action mechanism behind taurine-mediated responses on the SG neurons of the Vc. Taurine showed non-desensitizing and repeatable membrane depolarizations and inward currents which remained in the presence of amino-acid receptors blocking cocktail (AARBC) with tetrodotoxin, indicating that taurine acts directly on the postsynaptic SG neurons. Further, application of taurine at different doses (10  μM to 3 mM) showed a concentration dependent depolarizations and inward currents with the EC50 of 84.3  μM and 723  μM, respectively. Taurine-mediated responses were partially blocked by picrotoxin (50  μM) and almost completely blocked by strychnine (2  μM), suggesting that taurine-mediated responses are via glycine receptor (GlyR) activation. In addition, taurine (1 mM) activated extrasynaptic GABA(A) receptor (GABA(A)R)-mediated currents. Taken together, our results indicate that taurine can be a target molecule for orofacial pain modulation through the activation of GlyRs and/or extrasynaptic GABA(A)Rs on the SG neurons.

  4. Decreased density of GABA-A receptors in the left sensorimotor cortex in akinetic catatonia: investigation of in vivo benzodiazepine receptor binding

    PubMed Central

    Northoff, G.; Steinke, R.; Czcervenka, C.; Krause, R.; Ulrich, S.; Danos, P.; Kropf, D.; Otto, H.; Bogerts, B.

    1999-01-01

    OBJECTIVES—Catatonia is a psychomotor syndrome with concomittant akinesia and anxiety which both respond almost immediately to benzodiazepines such as lorazepam. The benzodiazepine receptor distribution was therefore investigated in akinetic catatonia with single photon emission tomography (SPECT) using iodine-123-iomazenil (123 I Iomazenil).
METHODS—Ten akinetic catatonic patients, 10 psychiatric controls (similar age, sex, medication, and underlying psychiatric diagnosis but without catatonic syndrome), and 20 healthy controls were investigated with SPECT 2 hours after injection of 123 I Iomazenil. To exclude potential effects of cerebral perfusion (r-CBF) r-CBF was additionally investigated with Tc-99mECD SPECT.
RESULTS—Catatonic patients showed significantly lower iomazenil binding and altered right-left relations in the left sensorimotor cortex compared with psychiatric (p<0.001) and healthy (p<0.001) controls. In addition, there was significantly lower r-CBF in the right lower prefrontal and parietal cortex in catatonia whereas in the left sensorimotor cortex no differences in r-CBF between groups were found. Catatonic motor and affective symptoms showed significant correlations (p<0.05) with benzodiazepine binding in the left sensorimotor cortex as well as with right parietal r-CBF.
CONCLUSIONS—Reduced iomazenil binding suggests decreased density of GABA-A receptors in the left sensorimotor cortex in akinetic catatonia. In addition to reduced GABA-A receptor density in the left sensorimotor cortex the parietal cortex seems to be involved in pathophysiology of catatonic symptoms. It is concluded that, considering results from correlation analyses, both emotional and motor symptoms in catatonia seem to be closely related to left sensorimotor and right parietal alterations.

 PMID:10486389

  5. Neuroprotection by estradiol: a role of aromatase against spine synapse loss after blockade of GABA(A) receptors.

    PubMed

    Zhou, Lepu; Lehan, Nadine; Wehrenberg, Uwe; Disteldorf, Erik; von Lossow, Richard; Mares, Ute; Jarry, Hubertus; Rune, Gabriele M

    2007-01-01

    Estrogen has been suggested to be pro-epileptic by reducing GABA synthesis, resulting in increased spine density and a decreased threshold for seizures in the hippocampus, which, once they occur, are characterized by a dramatic spine loss in the affected brain areas. As considerable amounts of estradiol are synthesized in the hippocampus, in this study we focused on aromatase, the rate-limiting enzyme in estrogen synthesis in order to examine the role of locally synthesized estrogens in epilepsy. To this end, we first examined the effects of letrozole, a potent aromatase inhibitor, on GABA metabolism in single interneurons of hippocampal dispersion cultures. Letrozole downregulated estradiol release into the medium, as well as glutamate decarboxylase (GAD) expression and GABA synthesis, and decreased the number of GAD positive cells in the cultures. Next, we counted spine synapses and measured estradiol release of hippocampal slice cultures, in which GABA(A) receptors had been blocked by bicuculline, in order to mimic epileptic activity. Treatment of slice cultures with bicuculline resulted in a dramatic decrease in the number of spine synapses and in a significant suppression of estrogen synthesis. The decrease in synapse number in response to bicuculline was restored by combined application of estradiol and bicuculline. Surprisingly, estradiol alone had no effect on either spine synapse number or on GAD expression and GABA synthesis. "Rescue" of synapse number in "epileptic slices" by estradiol and maintenance of GABA metabolism by hippocampus-derived estradiol points to a neuroprotective role of aromatase in epilepsy. Re-filling of estradiol stores after their depletion due to overexcitation may therefore add to therapeutical strategies in epilepsy.

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

    PubMed

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

    2007-02-09

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

  7. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    PubMed

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  8. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

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

    2011-09-01

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

  9. GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating.

    PubMed

    Bera, Amal K; Chatav, Maya; Akabas, Myles H

    2002-11-08

    The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.

  10. Embryonic exposure to ethanol disturbs regulation of mitotic spindle orientation via GABA(A) receptors in neural progenitors in ventricular zone of developing neocortex.

    PubMed

    Tochitani, Shiro; Sakata-Haga, Hiromi; Fukui, Yoshihiro

    2010-03-19

    Neural progenitors in the ventricular zone of the developing neocortex divide oriented either parallel or perpendicular to the ventricular surface based on their mitotic spindle orientation. It has been shown that the cleavage plane orientation is developmentally regulated and plays a crucial role in cell fate determination of neural progenitors or the maintenance of the proliferative ventricular zone during neocortical development. We tested if fetal exposure to ethanol, the most widely used psychoactive agent and a potent teratogen that may cause malformation in the central nervous system, alters mitotic cleavage orientation of the neural progenitors at the apical surface of the ventricular zone in the developing neocortex. Fetal exposure to ethanol on E10.5 and 11.5 increased the occurrence frequency of a horizontal cleavage plane that is parallel to the ventricular surface on E 12.5. Administration of picrotoxin, a GABA(A) receptor antagonist, prior to ethanol administration canceled the effect of ethanol with the frequency of horizontal division similar to the control level, although picrotoxin itself did not show any effect on cleavage plane orientation. Phenobarbital, a GABA(A) receptor agonist, induced horizontal cleavage to an extent similar to that induced by ethanol administration. (+)MK801, an antagonist of NMDA receptor that is another major target of ethanol in neural cells, did not affect the cleavage plane of dividing progenitors. These results suggest that fetal ethanol exposure induced alterations in the cleavage plane orientation of neural progenitors in the ventricular zone of the neocortex via the enhancement of the function of GABA(A) receptors.

  11. Functional characterization of ivermectin binding sites in α1β2γ2L GABA(A) receptors

    PubMed Central

    Estrada-Mondragon, Argel; Lynch, Joseph W.

    2015-01-01

    GABAA receptors (GABAARs) are the major inhibitory neurotransmitter receptors in the brain and are therapeutic targets for many indications including sedation, anesthesia and anxiolysis. There is, however, considerable scope for the development of new therapeutics with improved beneficial effects and reduced side-effect profiles. The anthelminthic drug, ivermectin, activates the GABAAR although its binding site is not known. The molecular site of action of ivermectin has, however, been defined by crystallography in the homologous glutamate-gated chloride channel. Resolving the molecular mechanisms of ivermectin binding to α1β2γ2L GABAARs may provide insights into the design of improved therapeutics. Given that ivermectin binds to subunit interfaces, we sought to define (1) which subunit interface sites it binds to, (2) whether these sites are equivalent in terms of ivermectin sensitivity or efficacy, and (3) how many must be occupied for maximal efficacy. Our approach involved precluding ivermectin from binding to particular interfaces by introducing bulky M3 domain 36′F sidechains to the “+” side of those interfaces. We thereby demonstrated that ivermectin produces irreversible channel activation only when it binds to the single γ2L-β2 interface site. When it binds to α1-β2 sites it elicits potentiation of GABA-gated currents but has no irreversible activating effect. Ivermectin cannot bind to the β2-α1 interface site due to its endogenous bulky 36′ methionine. Replacing this with an alanine creates a functional site at this interface, but surprisingly it is inhibitory. Molecular docking simulations reveal that the γ2L-β2 interface forms more contacts with ivermectin than the other interfaces, possibly explaining why ivermectin appears to bind irreversibly at this interface. This study demonstrates unexpectedly stark pharmacological differences among GABAAR ivermectin binding sites. PMID:26441518

  12. Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

    PubMed

    Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

    2005-11-01

    Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

  13. GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception.

    PubMed

    Freitas, Renato Leonardo; Uribe-Mariño, Andrés; Castiblanco-Urbina, Maria Angélica; Elias-Filho, Daoud Hibraim; Coimbra, Norberto Cysne

    2009-12-11

    Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1998-03-01

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

  16. Ethanol attenuates sensory stimulus-evoked responses in cerebellar granule cells via activation of GABA(A) receptors in vivo in mice.

    PubMed

    Wu, Guang; Liu, Heng; Jin, Juan; Hong, Lan; Lan, Yan; Chu, Chun-Ping; Qiu, De-Lai

    2014-02-21

    Acute alcohol intoxication affects cerebellar motor regulation possibly by altering the transfer and integration of external information in cerebellar cortical neurons, resulting in a dysfunction of cerebellar motor regulation or a cerebellar atexia. However, the synaptic mechanisms of ethanol induced impairments of sensory information processing in cerebellar cortical neurons are not fully understand. In the present study, we used electrophysiological and pharmacological methods to study the effects of ethanol on the sensory stimulation-evoked responses in cerebellar granule cells (GCs) in vivo in urethane anesthetized mice. Air-puff stimulation of the ipsilateral whisker-pad evoked stimulus-on (P1) and stimulus-off responses (P2) in GCs of cerebellar Crus II. Cerebellar surface perfusion of ethanol did not alter the onset latency of the sensory stimulation-evoked responses, but reversible reduced the amplitude of P1 and P2. The ethanol-induced reduction of the GCs sensory responses was concentration-dependent. In the presence of ethanol, the mean half-width, area under curve, rise Tau and decay Tau of P1 were significantly decreased. Blockade of gamma-aminobutyric acid type A (GABA(A)) receptors activity induced an increase in amplitude of P1, and abolished the ethanol induced inhibition of the GCs sensory responses. These results indicate that ethanol inhibits the tactile evoked responses in cerebellar GCs through enhancement of GABA(A) receptors activity.

  17. Roles of hippocampal GABA(A) and muscarinic receptors in consolidation of context memory and context-shock association in contextual fear conditioning: a double dissociation study.

    PubMed

    Chang, Shih-Dar; Liang, K C

    2012-07-01

    Contextual fear conditioning involves forming a context representation and associating it to a shock, both of which involved the dorsal hippocampus (DH) according to our recent findings. This study tested further whether the two processes may rely on different neurotransmitter systems in the DH. Male Wistar rats with cannula implanted into the DH were subjected to a two-phase training paradigm of contextual fear conditioning to separate context learning from context-shock association in two consecutive days. Immediately after each training phase, different groups of rats received bilateral intra-DH infusion of the GABA(A) agonist muscimol, 5HT(1A) agonist 8-OH-DPAT, NMDA antagonist APV or muscarinic antagonist scopolamine at various doses. On the third day, freezing behavior was tested in the conditioning context. Results showed that intra-DH infusion of muscimol impaired conditioned freezing only if it was given after context learning. In contrast, scopolamine impaired conditioned freezing only if it was given after context-shock training. Posttraining infusion of 8-OH-DPAT or APV had no effect on conditioned freezing when the drug was given at either phase. These results showed double dissociation for the hippocampal GABAergic and cholinergic systems in memory consolidation of contextual fear conditioning: forming context memory required deactivation of the GABA(A) receptors, while forming context-shock memory involved activation of the muscarinic receptors.

  18. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

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

  19. Modulation of horizontal cell function by GABA(A) and GABA(C) receptors in dark- and light-adapted tiger salamander retina.

    PubMed

    Yang, X L; Gao, F; Wu, S M

    1999-01-01

    The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark-and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABA(C) receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABA(A) receptors, and the other half is mediated by GABA(C) receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABA(A) and GABA(C) receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.

  20. Regional mRNA expression of GABAergic receptor subunits in brains of C57BL/6J and 129P3/J mice: Strain and heroin effects

    PubMed Central

    Brownstein, A.J.; Zhang, Y.; Ho, A.; Kreek, M.J.

    2014-01-01

    C57BL/6J and 129 substrains of mice are known to differ in their basal levels of anxiety and behavioral response to drugs of abuse. We have previously shown strain differences in heroin-induced conditioned place preference (CPP) between C57BL/6J (C57) and 129P3/J (129) mice, and in the regional expression of several receptor and peptide mRNAs. In this study, we examined the contribution of the GABAergic system in the cortex, nucleus accumbens (NAc), caudate putamen (CPu) and the region containing the substantia nigra and ventral tegmental area (SN/VTA) to heroin reward by measuring mRNA levels of 7 of the most commonly expressed GABA-A receptor subunits, and both GABA-B receptor subunits, in these same mice following saline (control) or heroin administration in a CPP design. Using real-time PCR, we studied the effects of strain and heroin administration on GABA-A α1, α2, α3, β2, and γ2 subunits, which typically constitute synaptic GABA-A receptors, GABA-A α4 and δ subunits, which typically constitute extrasynaptic GABA-A receptors, and GABA-B R1 and R2 subunits. In saline-treated animals, we found an experiment-wise significant strain difference in GABA-Aα2 mRNA expression in the SN/VTA. Point-wise significant strain differences were also observed in GABA-Aα2, GABA-Aα3, and GABA-Aα4 mRNA expression in the NAc, as well as GABA-BR2 mRNA expression in the NAc and CPu, and GABA-BR1 mRNA expression in the cortex. For all differences, 129 mice had higher mRNA expression compared to C57 animals, with the exception of GABA-BR1 mRNA in the cortex where we observed lower levels in 129 mice. Therefore, it may be possible that known behavioral differences between these two strains are, in part, due to differences in their GABAergic systems. While we did not find heroin dose-related changes in mRNA expression levels in C57 mice, we did observe dose-related differences in 129 mice. These results may relate to our earlier behavioral finding that 129 mice are

  1. Taurine-induced attenuation of MPP+ neurotoxicity in vitro: a possible role for the GABA(A) subclass of GABA receptors.

    PubMed

    O'Byrne, M B; Tipton, K F

    2000-05-01

    Taurine is a sulphur-containing beta-amino acid found in high (millimolar) concentrations in excitable tissues such as brain and heart. Its suggested roles include osmoregulator, thermoregulator, neuromodulator, and potential neurotransmitter. This amino acid has also been shown to be released in large concentrations during ischaemia and excitotoxin-induced neuronal damage. Here we report a protective effect of taurine against MPP(+)-induced neurotoxicity in coronal slices from rat brain. Significant protective effects were observed at taurine concentrations of 20 and 1 mM, suggesting a potential role for taurine in cases of neuronal insult. Studies with the synthetic taurine analogues taurine phosphonate, guanidinoethane sulphonate, and trimethyltaurine suggested the observed effect to be mediated via an extracellular mechanism. The use of GABA receptor ligands muscimol and bicuculline indicated the effect to be mediated through activation of GABA(A) receptors.

  2. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2010-04-01

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

  4. Ethanol, not detectably metabolized in brain, significantly reduces brain metabolism, probably via action at specific GABA(A) receptors and has measureable metabolic effects at very low concentrations.

    PubMed

    Rae, Caroline D; Davidson, Joanne E; Maher, Anthony D; Rowlands, Benjamin D; Kashem, Mohammed A; Nasrallah, Fatima A; Rallapalli, Sundari K; Cook, James M; Balcar, Vladimir J

    2014-04-01

    Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we measured the effect of alcohol on metabolism of [3-¹³C]pyruvate in the adult Guinea pig brain cortical tissue slice and compared the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-¹³C]ethanol. Analyses of metabolic profile clusters suggest that the significant reductions in metabolism induced by ethanol (10, 30 and 60 mM) are via action at neurotransmitter receptors, particularly α4β3δ receptors, whereas very low concentrations of ethanol may produce metabolic responses owing to release of GABA via GABA transporter 1 (GAT1) and the subsequent interaction of this GABA with local α5- or α1-containing GABA(A)R. There was no measureable metabolism of [1,2-¹³C]ethanol with no significant incorporation of ¹³C from [1,2-¹³C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabelled ethanol.

  5. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

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

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

    PubMed

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

    2014-12-01

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

  7. Expression of glutamate receptor subunits in human cancers.

    PubMed

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

    2009-10-01

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

  8. The effect of BLA GABA(A) receptors in anxiolytic-like effect and aversive memory deficit induced by ACPA

    PubMed Central

    Kangarlu-Haghighi, Katayoon; Oryan, Shahrbanoo; Nasehi, Mohammad; Zarrindast, Mohammad-Reza

    2015-01-01

    The roles of GABAergic receptors of the Basolateral amygdala (BLA) in the cannabinoid CB1 receptor agonist (arachydonilcyclopropylamide; ACPA)-induced anxiolytic-like effect and aversive memory deficit in adult male mice were examined in elevated plus-maze task. Results showed that pre-test intra-peritoneal injection of ACPA induced anxiolytic-like effect (at dose of 0.05 mg/kg) and aversive memory deficit (at doses of 0.025 and 0.05 mg/kg). The results revealed that Pre-test intra-BLA infusion of muscimol (GABAA receptor agonist; at doses of 0.1 and 0.2 µg/mouse) or bicuculline (GABAA receptor antagonist; at all doses) impaired and did not alter aversive memory, respectively. All previous GABA agents did not have any effects on anxiety-like behaviors. Interestingly, pretreatment with a sub-threshold dose of muscimol (0.025 µg/mouse) and bicuculline (0.025 µg/mouse) did not alter anxiolytic-like behaviors induced by ACPA, while both drugs restored ACPA-induced amnesia. Moreover, muscimol or bicuculline increased and decreased ACPA-induced locomotor activity, respectively. Finally the data may indicate that BLA GABAA receptors have critical and different roles in anxiolytic-like effect, aversive memory deficit and locomotor activity induced by ACPA. PMID:26648818

  9. p-Coumaric acid activates the GABA-A receptor in vitro and is orally anxiolytic in vivo.

    PubMed

    Scheepens, Arjan; Bisson, Jean-Francois; Skinner, Margot

    2014-02-01

    The increasing prevalence and social burden of subclinical anxiety in the western world represents a significant psychosocial and financial cost. Consumers are favouring a more natural and nonpharmacological approach for alleviating the effects of everyday stress and anxiety. The gamma-aminobutyric acid (GABA) receptor is the primary mediator of central inhibitory neurotransmission, and GABA-receptor agonists are well known to convey anxiolytic effects. Using an in vitro screening approach to identify naturally occurring phytochemical GABA agonists, we discovered the plant secondary metabolite p-coumaric acid to have significant GABAergic activity, an effect that could be blocked by co-administration of the specific GABA-receptor antagonist, picrotoxin. Oral administration of p-coumaric acid to rodents induced a significant anxiolytic effect in vivo as measured using the elevated plus paradigm, in line with the effects of oral diazepam. Given that p-coumaric acid is reasonably well absorbed following oral consumption in man and is relatively nontoxic, it may be suitable for the formulation of a safe and effective anxiolytic functional food.

  10. [Effects of intra-amygdala injection of GABA(A)-receptor agonist and antagonist on respiratory and cardiac components of conditioned fear in rabbits].

    PubMed

    Pavlova, I V; Rysakova, M P

    2013-01-01

    The role of GABAergic system of amygdala in vegetative and behavioral manifestations of conditioned fear was studied in groups of active and passive rabbits, selected upon testing in open field and light-dark chamber. During training, the light (4 s) was paired with electrodermal stimulation of hind limbs(1 s, 10 Hz). Conditioned fear was judged by heartbeats and respiratory rates before and in the process of conditioned and unconditioned stimuli presentation. Solutions of GABA(A)-receptor agonist (muscimol, 0.1 μg/μL) or antagonist (bicuculline methbromide, 0.5 μg/μL) were applied to the right or left basal nucleus of amygdala after conditioning. Muscimol injection evoked more powerful vegetative changes in passive rabbits than in the active ones; respiratory deceleration during freezing in response to conditioned stimuli was eliminated and the reaction to electrodermal stimulation was reduced or altered. Activation of the left amygdala by bicuculline resulted in the increased probability of active locomotor reactions in active rabbits, activation of the right amygdale, on the contrary, increased probability of freezing. The results evidence the non-equivalence of amygdalar GABAergic system in active and passive animals.

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

    PubMed

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

    2013-03-08

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

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

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  14. GABA-A receptor modulators alter emotionality and hippocampal theta rhythm in an animal model of long-lasting anxiety.

    PubMed

    Hoeller, Alexandre Ademar; Duzzioni, Marcelo; Duarte, Filipe Silveira; Leme, Leandro Rinaldi; Costa, Ana Paula Ramos; Santos, Evelyn Cristina da Silva; de Pieri, Claudini Honório; dos Santos, Alessandra Antunes; Naime, Aline Aita; Farina, Marcelo; de Lima, Thereza Christina Monteiro

    2013-09-26

    The cholinergic system is implicated in emotional regulation. The injection of non-convulsant doses of the muscarinic receptor agonist pilocarpine (PILO) induces long-lasting anxiogenic responses in rats evaluated at different time-points (24h to 3 months). To investigate the underlying mechanisms, rats treated with PILO (150mg/kg) were injected 24h or 1 month later with an anxiolytic (diazepam, 1mg/kg, DZP) or anxiogenic (pentylenetetrazole, 15mg/kg, PTZ) drug and evaluated in the elevated plus-maze (EPM). Prefrontal cortex (PFC) and hippocampal (HIP) electroencephalographic recordings and acetylcolinesterase (AChE) activity were also analyzed after PILO treatment. Anxiogenic responses observed in the EPM 24h or 1 month after PILO treatment (e.g., decreased time spent and number of entries into the open arms of the maze) were blocked by DZP but not affected by PTZ. No epileptiform events were registered in the HIP or PFC at 24h or 1 month after PILO injection, but enhanced theta activity was observed in the HIP. DZP decreased hippocampal theta of PILO-treated rats in contrast with PTZ, which increased this parameter in saline- and PILO-treated rats. The HIP and PFC AChE activity did not change after PILO treatment. Our findings demonstrate that the long-term effects on the emotionality of rats induced by PILO are associated with electrophysiological changes in the HIP and sensitive to pharmacological manipulation of the GABAergic system. The present work may support this new research model of long-lasting anxiety, while also highlighting the muscarinic system as a potential target involved in anxiety disorders.

  15. Interactions between opioid-peptides-containing pathways and GABA(A)-receptors-mediated systems modulate panic-like-induced behaviors elicited by electric and chemical stimulation of the inferior colliculus.

    PubMed

    Calvo, Fabrício; Coimbra, Norberto Cysne

    2006-08-09

    Aiming to clarify the effect of interactive interconnections between the endogenous opioid peptides-neural links and GABAergic pathways on panic-like responses, in the present work, the effect of the peripheral and central administration of morphine or the non-specific opioid receptors antagonist naloxone was evaluated on the fear-induced responses (defensive attention, defensive immobility and escape behavior) elicited by electric and chemical stimulation of the inferior colliculus. Central microinjections of opioid drugs in the inferior colliculus were also performed followed by local administration of the GABA(A)-receptor antagonist bicuculline. The defensive behavior elicited by the blockade of GABAergic receptors in the inferior colliculus had been quantitatively analyzed, recording the number of crossing, jump, rotation and rearing, in each minute, during 30 min, in the open-field test. The opioid receptors stimulation with morphine decreased the defensive attention, the defensive immobility and escape behavior thresholds, and the non-specific opioid receptors blockade caused opposite effects, enhancing the defensive behavior thresholds. These effects were corroborated by either the stimulation or the inhibition of opioid receptors followed by the GABA(A) receptor blockade with bicuculline, microinjected into the inferior colliculus. There was a significant increase in the diverse fear-induced responses caused by bicuculline with the pretreatment of the inferior colliculus with morphine, and the opposite effect was recorded after the pretreatment of the inferior colliculus nuclei with naloxone followed by bicuculline local administration. These findings suggest an interaction between endogenous opioid-peptides-containing connections and GABA(A)-receptor-mediated system with direct influence on the organization of the panic-like or fear-induced responses elaborated in the inferior colliculus during critical emotional states.

  16. Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes.

    PubMed

    Ciechanowska, Magdalena; Lapot, Magdalena; Malewski, Tadeusz; Mateusiak, Krystyna; Misztal, Tomasz; Przekop, Franciszek

    2009-04-01

    The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.

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

    SciTech Connect

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

    2010-07-13

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

  18. Changes in ventral respiratory column GABAaR ε- and δ-subunits during hibernation mediate resistance to depression by EtOH and pentobarbital.

    PubMed

    Hengen, K B; Gomez, T M; Stang, K M; Johnson, S M; Behan, M

    2011-02-01

    During hibernation in the 13-lined ground squirrel, Ictidomys tridecemlineatus, the cerebral cortex is electrically silent, yet the brainstem continues to regulate cardiorespiratory function. Previous work showed that neurons in slices through the medullary ventral respiratory column (VRC) but not the cortex are insensitive to high doses of pentobarbital during hibernation, leading to the hypothesis that GABA(A) receptors (GABA(A)R) in the VRC undergo a seasonal modification in subunit composition. To test whether alteration of GABA(A)R subunits are responsible for hibernation-associated pentobarbital insensitivity, we examined an array of subunits using RT-PCR and Western blots and identified changes in ε- and δ-subunits in the medulla but not the cortex. Using immunohistochemistry, we confirmed that during hibernation, the expression of ε-subunit-containing GABA(A)Rs nearly doubles in the VRC. We also identified a population of δ-subunit-containing GABA(A)Rs adjacent to the VRC that were differentially expressed during hibernation. As δ-subunit-containing GABA(A)Rs are particularly sensitive to ethanol (EtOH), multichannel electrodes were inserted in slices of medulla and cortex from hibernating squirrels and EtOH was applied. EtOH, which normally inhibits neuronal activity, excited VRC but not cortical neurons during hibernation. This excitation was prevented by bicuculline pretreatment, indicating the involvement of GABA(A)Rs. We propose that neuronal activity in the VRC during hibernation is unaffected by pentobarbital due to upregulation of ε-subunit-containing GABA(A)Rs on VRC neurons. Synaptic input from adjacent inhibitory interneurons that express δ-subunit-containing GABA(A)Rs is responsible for the excitatory effects of EtOH on VRC neurons during hibernation.

  19. Comparison of Steroid Modulation of Spontaneous Inhibitory Postsynaptic Currents in Cultured Hippocampal Neurons and Steady-State Single-Channel Currents from Heterologously Expressed α1β2γ2L GABA(A) Receptors.

    PubMed

    Chakrabarti, Sampurna; Qian, Mingxing; Krishnan, Kathiresan; Covey, Douglas F; Mennerick, Steven; Akk, Gustav

    2016-04-01

    Neuroactive steroids are efficacious modulators of γ-aminobutyric acid type A receptor (GABA(A)) receptor function. The effects of steroids on the GABA(A) receptor are typically determined by comparing steady-state single-channel open probability or macroscopic peak responses elicited by GABA in the absence and presence of a steroid. Due to differences in activation conditions (exposure duration, concentration of agonist), it is not obvious whether modulation measured using typical experimental protocols can be used to accurately predict the effect of a modulator on native receptors under physiologic conditions. In the present study, we examined the effects of 14 neuroactive steroids and analogs on the properties of spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured rat hippocampal neurons. The goal was to determine whether the magnitude of modulation of the decay time course of sIPSCs correlates with the extent of modulation and kinetic properties of potentiation as determined in previous single-channel studies. The steroids were selected to cover a wide range of efficacy on heterologously expressed rat α1β2γ2L GABA(A) receptors, ranging from essentially inert to highly efficacious (strong potentiators of single-channel and macroscopic peak responses). The data indicate a strong correlation between prolongation of the decay time course of sIPSCs and potentiation of single-channel open probability. Furthermore, changes in intracluster closed time distributions were the single best predictor of prolongation of sIPSCs. We infer that the information obtained in steady-state single-channel recordings can be used to forecast modulation of synaptic currents.

  20. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

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

  1. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

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

  2. Affinities and densities of high-affinity (/sup 3/H)muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    SciTech Connect

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-09-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using (/sup 3/H)muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using (/sup 3/H)flunitrazepam and (/sup 3/H)Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of (/sup 3/H)muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy.

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

    PubMed

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

    2013-03-20

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

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

    PubMed

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

    2017-03-01

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

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

    PubMed Central

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

    2017-01-01

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

  6. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

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

    2017-01-01

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

  7. α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations.

    PubMed

    Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

    2013-02-15

    GABA(A) receptors are critically involved in hippocampal oscillations. GABA(A) receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABA(A) receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABA(A) receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABA(A) receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABA(A) receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations.

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

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2012-01-01

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

  9. GABA-A RECEPTOR ACTIVITY IN THE NORADRENERGIC LOCUS COERULEUS DRIVES TRIGEMINAL NEUROPATHIC PAIN IN THE RAT; CONTRIBUTION OF NAα1 RECEPTORS IN THE MEDIAL PREFRONTAL CORTEX

    PubMed Central

    KAUSHAL, R.; TAYLOR, B. K.; JAMAL, A. B.; ZHANG, L.; MA, F.; DONAHUE, R.; WESTLUND, K. N.

    2017-01-01

    Trigeminal neuropathic pain is described as constant excruciating facial pain. The study goal was to investigate the role of nucleus locus coeruleus (LC) in a model of chronic orofacial neuropathic pain (CCI-ION). The study examines LC’s relationship to both the medullary dorsal horn receiving trigeminal nerve sensory innervation and the medial prefrontal cortex (mPFC). LC is a major source of CNS noradrenaline (NA) and a primary nucleus involved in pain modulation. Although descending inhibition of acute pain by LC is well established, contribution of the LC to facilitation of chronic neuropathic pain is also reported. In the present study, a rat orofacial pain model of trigeminal neuropathy was induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Orofacial neuropathic pain was indicated by development of whisker pad mechanical hypersensitivity. Hypersensitivity was alleviated by selective elimination of NA neurons, including LC (A6 cell group), with the neurotoxin anti-dopamine-β-hydroxylase saporin (anti-DβH-saporin) microinjected either intracerebroventricularly (i.c.v.) or into trigeminal spinal nucleus caudalis (spVc). The GABAA receptor antagonist, bicuculline, administered directly into LC (week 8) inhibited hypersensitivity. This indicates a valence shift in which increased GABAA signaling ongoing in LC after trigeminal nerve injury paradoxically produces excitatory facilitation of the chronic pain state. Microinjection of NAα1 receptor antagonist, benoxathian, into mPFC attenuated whisker pad hypersensitivity, while NAα2 receptor antagonist, idazoxan, was ineffective. Thus, GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC. These data indicate LC is a chronic pain generator. Published by Elsevier Ltd on behalf of IBRO. PMID:27520081

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

    PubMed

    Schmieden, V; Kuhse, J; Betz, H

    1993-10-08

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

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

    PubMed

    Marchetti, Carla; Gavazzo, Paola

    2003-10-07

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

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

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

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

  13. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats

    PubMed Central

    Blacktop, Jordan M.; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A.; Mantsch, John R.

    2015-01-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 hrs/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5” duration, average every 40 sec; range 10–70 sec) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 µg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. PMID:26596556

  14. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats.

    PubMed

    Blacktop, Jordan M; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A; Mantsch, John R

    2016-03-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 h/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5" duration, average every 40 s; range 10-70 s) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 μg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts.

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

    PubMed Central

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

    2012-01-01

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

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

    SciTech Connect

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

    1988-03-01

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

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

    PubMed

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

    1997-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    1996-11-15

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

  1. Putative TRP channel antagonists, SKF 96365, flufenamic acid and 2-APB, are non-competitive antagonists at recombinant human α1β2γ2 GABA(A) receptors.

    PubMed

    Rae, M G; Hilton, J; Sharkey, J

    2012-05-01

    Although transient receptor potential (TRP) channel biology research has expanded rapidly in recent years, the field is hampered by the widely held, but relatively poorly investigated, belief that most of the pharmacological tools used to investigate TRP channel function may not be particularly selective for their intended targets. The objective of this study was therefore to determine if this was indeed the case by systematically evaluating the effects of three routinely used putative TRP channel antagonists, SKF 96365, flufenamic acid (FF) and 2-aminoethoxydiphenyl borate (2-APB) against one of the most widely expressed CNS receptor subtypes CNS, the human α1β2γ2 GABA(A) receptor. Using whole cell patch-clamp recording to record responses to rapidly applied GABA in the absence and presence of the three putative antagonists in turn we found that SKF 96365 (1-100 μM) and FF (1-100 μM) significantly inhibited GABA responses of recombinant human α1β2γ2 GABA(A) receptor stably expressed in HEK293 cells with IC(50) values of 13.4 ± 5.1 and 1.9 ± 1.4 μM, respectively, suppressing the maximal response to GABA at all concentrations used in a manner consistent with a non-competitive mode of action. SKF 96365 and FF also both significantly reduced desensitisation and prolonged the deactivation kinetics of the receptors to GABA (1mM; P<0.05). 2-APB (10-1000 μM) also inhibited responses to GABA at all concentrations used with an IC(50) value of 16.7 ± 5.4 μM (n=3-5) but had no significant effect on the activation, desensitisation or deactivation kinetics of the GABA responses. Taken together this investigation revealed that these widely utilised TRP channel antagonists display significant 'off-target' effects at concentrations that are routinely used for the study of TRP channel function in numerous biological systems and as such, data which is obtained utilising these compounds should be interpreted with caution.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2012-11-01

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

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

    PubMed Central

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

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

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

    PubMed

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

    1997-08-01

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

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

    PubMed Central

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

    1999-01-01

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

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

    SciTech Connect

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

    2008-03-07

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

  9. Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse.

    PubMed

    Yin, Wenyuan; Majumder, Samarpan; Clayton, Terry; Petrou, Steven; VanLinn, Michael L; Namjoshi, Ojas A; Ma, Chunrong; Cromer, Brett A; Roth, Bryan L; Platt, Donna M; Cook, James M

    2010-11-01

    A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at α(x)β(3)γ(2) GABA(A)/benzodiazepine receptor subtypes by radioligand binding assays in search of α(1) subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (L(Di) region) of GABA(A)/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L(1), while N(2) presumably underwent a hydrogen bonding interaction with H(1). Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were 'near GABA neutral antagonists'. Based on the SAR, the most potent (in vitro) α(1) selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.(1-3) Moreover, these two β-carbolines were orally active, and in addition, were

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Copits, Bryan A.; Swanson, Geoffrey T.

    2012-01-01

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

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

    PubMed

    Gerrow, K; Triller, A

    2014-05-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

    Lentz, T L

    1990-04-01

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

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

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2008-07-01

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

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

    PubMed

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

    2014-01-13

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

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

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2014-01-01

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

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

    PubMed

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

    1997-04-01

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

  20. GABAA receptors are located in cholinergic terminals in the nucleus pontis oralis of the rat: implications for REM sleep control.

    PubMed

    Liang, Chang-Lin; Marks, Gerald A

    2014-01-16

    The oral pontine reticular formation (PnO) of rat is one region identified in the brainstem as a rapid eye movement (REM) sleep induction zone. Microinjection of GABA(A) receptor antagonists into PnO induces a long lasting increase in REM sleep, which is similar to that produced by cholinergic agonists. We previously showed that this REM sleep-induction can be completely blocked by a muscarinic antagonist, indicating that the REM sleep-inducing effect of GABA(A) receptor antagonism is dependent upon the local cholinergic system. Consistent with these findings, it has been reported that GABA(A) receptor antagonists microdialyzed into PnO resulted in increased levels of acetylcholine. We hypothesize that GABA(A) receptors located on cholinergic boutons in the PnO are responsible for the REM sleep induction by GABA(A) receptor antagonists through blocking GABA inhibition of acetylcholine release. Cholinergic, varicose axon fibers were studied in the PnO by immunofluorescence and confocal, laser scanning microscopy. Immunoreactive cholinergic boutons were found to be colocalized with GABA(A) receptor subunit protein γ2. This finding implicates a specific subtype and location of GABA(A) receptors in PnO of rat in the control of REM sleep.

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

    PubMed Central

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

    1998-01-01

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

  2. Gene expression changes in serotonin, GABA-A receptors, neuropeptides and ion channels in the dorsal raphe nucleus of adolescent alcohol-preferring (P) rats following binge-like alcohol drinking.

    PubMed

    McClintick, Jeanette N; McBride, William J; Bell, Richard L; Ding, Zheng-Ming; Liu, Yunlong; Xuei, Xiaoling; Edenberg, Howard J

    2015-02-01

    Alcohol binge-drinking during adolescence is a serious public health concern with long-term consequences. We used RNA sequencing to assess the effects of excessive adolescent ethanol binge-drinking on gene expression in the dorsal raphe nucleus (DRN) of alcohol preferring (P) rats. Repeated binges across adolescence (three 1h sessions across the dark-cycle per day, 5 days per week for 3 weeks starting at 28 days of age; ethanol intakes of 2.5-3 g/kg/session) significantly altered the expression of approximately one-third of the detected genes. Multiple neurotransmitter systems were altered, with the largest changes in the serotonin system (21 of 23 serotonin-related genes showed decreased expression) and GABA-A receptors (8 decreased and 2 increased). Multiple neuropeptide systems were also altered, with changes in the neuropeptide Y and corticotropin-releasing hormone systems similar to those associated with increased drinking and decreased resistance to stress. There was increased expression of 21 of 32 genes for potassium channels. Expression of downstream targets of CREB signaling was increased. There were also changes in expression of genes involved in inflammatory processes, axonal guidance, growth factors, transcription factors, and several intracellular signaling pathways. These widespread changes indicate that excessive binge drinking during adolescence alters the functioning of the DRN and likely its modulation of many regions of the central nervous system, including the mesocorticolimbic system.

  3. Expression Levels of GABA-A Receptor Subunit Alpha 3, Gabra3 and Lipoprotein Lipase, Lpl Are Associated with the Susceptibility to Acetaminophen-Induced Hepatotoxicity

    PubMed Central

    Kim, Minjeong; Yun, Jun-Won; Shin, Kyeho; Cho, Yejin; Yang, Mijeong; Nam, Ki Taek; Lim, Kyung-Min

    2017-01-01

    Drug-induced liver injury (DILI) is the serious and fatal drug-associated adverse effect, but its incidence is very low and individual variation in severity is substantial. Acetaminophen (APAP)-induced liver injury accounts for >50% of reported DILI cases but little is known for the cause of individual variations in the severity. Intrinsic genetic variation is considered a key element but the identity of the genes was not well-established. Here, pre-biopsy method and microarray technique was applied to uncover the key genes for APAP-induced liver injury in mice, and a cause and effect experiment employing quantitative real-time PCR was conducted to confirm the correlation between the uncovered genes and APAP-induced hepatotoxicity. We identified the innately and differentially expressed genes of mice susceptible to APAP-induced hepatotoxicity in the pre-biopsied liver tissue before APAP treatment through microarray analysis of the global gene expression profiles (Affymetrix GeneChip® Mouse Gene 1.0 ST for 28,853 genes). Expression of 16 genes including Gdap10, Lpl, Gabra3 and Ccrn4l were significantly different (t-test: FDR <10%) more than 1.5 fold in the susceptible animals than resistant. To confirm the association with the susceptibility to APAP-induced hepatotoxicity, another set of animals were measured for the expression level of selected 4 genes (higher two and lower two genes) in the liver pre-biopsy and their sensitivity to APAP-induced hepatotoxicity was evaluated by post hoc. Notably, the expressions of Gabra3 and Lpl were significantly correlated with the severity of liver injury (p<0.05) demonstrating that these genes may be linked to the susceptibility to APAP-induced hepatotoxicity. PMID:27530116

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

    PubMed

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

    2007-02-23

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

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

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

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

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

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

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

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

    PubMed Central

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

    2012-01-01

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

  11. Allosteric modulation of GABA(A) receptor subtypes:effects on visual recognition and visuospatial working memory in rhesus monkeys [corrected].

    PubMed

    Soto, Paul L; Ator, Nancy A; Rallapalli, Sundari K; Biawat, Poonam; Clayton, Terry; Cook, James M; Weed, Michael R

    2013-10-01

    Non-selective positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) are known to impair anterograde memory. The role of the various GABAAR subtypes in the memory-impairing effects of non-selective GABAAR PAMs has not been fully elucidated. The current study assessed, in rhesus monkeys, effects of modulation of α1, α2/3, and α5GABAARs on visual recognition and spatial working memory using delayed matching-to-sample (DMTS) and self-ordered spatial search (SOSS) procedures, respectively. The DMTS procedure (n=8) involved selecting a previously presented 'sample' image from a set of multiple images presented after a delay. The SOSS procedure (n=6) involved touching a number of boxes without repeats. The non-selective GABAAR PAM triazolam and the α1GABAA preferential PAMS zolpidem and zaleplon reduced accuracy in both procedures, whereas the α5GABAA preferential PAMs SH-053-2'F-R-CH3 and SH-053-2'F-S-CH3, and the α2/3GABAA preferential PAM TPA023B were without effects on accuracy or trial completion. The low-efficacy α5GABAAR negative allosteric modulator (NAM) PWZ-029 slightly increased only DMTS accuracy, whereas the high-efficacy α5GABAAR NAMs RY-23 and RY-24 did not affect accuracy under either procedure. Finally, the slopes of the accuracy dose-effect curves for triazolam, zolpidem, and zaleplon increased with box number in the SOSS procedure, but were equivalent across DMTS delays. The present results suggest that (1) α1GABAARs, compared with α2/3 and α5GABAARs, are primarily involved in the impairment, by non-selective GABAAR PAMs, of visual recognition and visuospatial working memory in nonhuman primates; and (2) relative cognitive impairment produced by positive modulation of GABAARs increases with number of locations to be remembered, but not with the delay for remembering.

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2013-01-01

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

  14. A Negative Allosteric Modulator for α5 Subunit-Containing GABA Receptors Exerts a Rapid and Persistent Antidepressant-Like Action without the Side Effects of the NMDA Receptor Antagonist Ketamine in Mice

    PubMed Central

    Nelson, Mackenzie E.; Krimmel, Samuel R.; Georgiou, Polymnia; Gould, Todd D.

    2017-01-01

    Abstract New antidepressant pharmacotherapies that provide rapid relief of depressive symptoms are needed. The NMDA receptor antagonist ketamine exerts rapid antidepressant actions in depressed patients but also side effects that complicate its clinical utility. Ketamine promotes excitatory synaptic strength, likely by producing high-frequency correlated activity in mood-relevant regions of the forebrain. Negative allosteric modulators of GABA-A receptors containing α5 subunits (α5 GABA-NAMs) should also promote high-frequency correlated electroencephalogram (EEG) activity and should therefore exert rapid antidepressant responses. Because α5 subunits display a restricted expression in the forebrain, we predicted that α5 GABA-NAMs would produce activation of principle neurons but exert fewer side effects than ketamine. We tested this hypothesis in male mice and observed that the α5 GABA-NAM MRK-016 exerted an antidepressant-like response in the forced swim test at 1 and 24 h after administration and an anti-anhedonic response after chronic stress in the female urine sniffing test (FUST). Like ketamine, MRK-016 produced a transient increase in EEG γ power, and both the increase in γ power and its antidepressant effects in the forced swim test were blocked by prior administration of the AMPA-type glutamate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). Unlike ketamine, however, MRK-016 produced no impairment of rota-rod performance, no reduction of prepulse inhibition (PPI), no conditioned-place preference (CPP), and no change in locomotion. α5 GABA-NAMs, thus reproduce the rapid antidepressant-like actions of ketamine, perhaps via an AMPA receptor (AMPAR)-dependent increase in coherent neuronal activity, but display fewer potential negative side effects. These compounds thus demonstrate promise as clinically useful fast-acting antidepressants. PMID:28275719

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1996-05-24

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

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

    ERIC Educational Resources Information Center

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

    2016-01-01

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

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

    PubMed Central

    Qian, H; Ripps, H

    1999-01-01

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

  1. GABAρ subunits confer a bicuculline-insensitive component to GFAP+ cells of cerebellum

    PubMed Central

    Pétriz, Adriana; Reyes-Haro, Daniel; González-González, María Alejandra; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2014-01-01

    GABA-A receptors mediating synaptic or extrasynaptic transmission are molecularly and functionally distinct, and glial cells are known to express a plethora of GABA-A subunits. Here we demonstrate that GFAP+ cells of the granular layer of cerebellum express GABAρ subunits during early postnatal development, thereby conferring peculiar pharmacologic characteristics to GABA responses. Electron microscopy revealed the presence of GABAρ in the plasma membrane of GFAP+ cells. In contrast, expression in the adult was restricted to Purkinje neurons and a subset of ependymal cells. Electrophysiological studies in vitro revealed that astrocytes express functional receptors with an EC50 of 52.2 ± 11.8 μM for GABA. The evoked currents were inhibited by bicuculline (100 μM) and TPMPA (IC50, 5.9 ± 0.6 μM), indicating the presence of a GABAρ component. Coimmunoprecipitation demonstrated protein–protein interactions between GABAρ1 and GABAα1, and double immunofluorescence showed that these subunits colocalize in the plasma membrane. Three populations of GABA-A receptors in astrocytes were identified: classic GABA-A, bicuculline-insensitive GABAρ, and GABA-A–GABAρ hybrids. Clusters of GABA-A receptors were distributed in the perinuclear space and along the processes of GFAP+ cells. Time-lapse microscopy showed GABAρ2-GFP accumulation in clusters located in the soma and along the processes. The clusters were relatively immobile, with mean displacement of 9.4 ± 0.9 μm and a net distance traveled of 1–2 μm, owing mainly to directional movement or simple diffusion. Modulation of GABAρ dynamics may be a novel mechanism of extrasynaptic transmission regulating GABAergic control of GFAP+ cells during early postnatal development. PMID:25422464

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

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

    PubMed

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

    2012-08-01

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

  4. Lungfish aestivating activities are locked in distinct encephalic γ-aminobutyric acid type A receptor α subunits.

    PubMed

    Giusi, Giuseppina; Crudo, Michele; Di Vito, Anna; Facciolo, Rosa Maria; Garofalo, Filippo; Chew, Shit Fun; Ip, Yuen Kwong; Canonaco, Marcello

    2011-03-01

    Ammonia in dipnoans plays a crucial role on neuronal homeostasis, especially for those brain areas that maintain torpor and awakening states in equilibrium. In the present study, specific α subunits of the major neuroreceptor inhibitory complex (GABA(A) R), which predominated during some phases of aestivation of the lungfish Protopterus annectens, turned out to be key adaptive factors of this species. From the isolation, for the first time, of the encoding sequence for GABA(A) R α₁, α₄ , and α₅ subunits in Protopterus annectens, qPCR and in situ hybridization levels of α₄ transcript in thalamic (P < 0.001) and mesencephalic (P < 0.01) areas proved to be significantly higher during long aestivating maintenance states. Very evident α₅ mRNA levels were detected in diencephalon during short inductive aestivating states, whereas an α₄ /α₁ turnover characterized the arousal state. Contextually, the recovery of physiological activities appeared to be tightly related to an evident up-regulation of α₁ transcripts in telencephalic and cerebellar sites. Surprisingly, TUNEL and amino cupric silver methods corroborated apoptotic and neurodegenerative cellular events, respectively, above all in telencephalon and cerebellum of lungfish exposed to long maintenance aestivating conditions. Overall, these results tend to underlie a novel GABAergic-related ON/OFF molecular switch operating during aestivation of the lungfish, which might have a bearing on sleeping disorders.

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

    PubMed

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

    2013-01-24

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

  6. NKCC1 knockdown decreases neuron production through GABA(A)-regulated neural progenitor proliferation and delays dendrite development.

    PubMed

    Young, Stephanie Z; Taylor, M Morgan; Wu, Sharon; Ikeda-Matsuo, Yuri; Kubera, Cathryn; Bordey, Angélique

    2012-09-26

    Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.

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

    PubMed

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

    2000-07-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

    PubMed

    Gondré-Lewis, Marjorie C; Warnock, Kaitlin T; Wang, Hong; June, Harry L; Bell, Kimberly A; Rabe, Holger; Tiruveedhula, Veera Venkata Naga Phani Babu; Cook, James; Lüddens, Hartmut; Aurelian, Laure; June, Harry L

    2016-01-01

    Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the

  11. Potentiation of the ionotropic GABA receptor response by whiskey fragrance.

    PubMed

    Hossain, Sheikh Julfikar; Aoshima, Hitoshi; Koda, Hirofumi; Kiso, Yoshinobu

    2002-11-06

    It is well-known that the target of most mood-defining compounds is an ionotropic gamma-aminobutyric acid receptor (GABA(A) receptor). The potentiation of the response of these inhibitory neurotransmitter receptors induces anxiolytic, sedative, and anesthetic activity in the human brain. To study the effects of whiskey fragrance on the GABA(A) receptor-mediated response, GABA(A) receptors were expressed in Xenopus oocyte by injecting rat whole brain mRNA or cRNA prepared from the cloned cDNA for the alpha(1) and beta(1) subunits of the bovine receptors. Most whiskey components such as phenol, ethoxy, and lactone derivatives potentiated the electrical responses of GABA(A) receptors, especially ethyl phenylpropanoate (EPP), which strongly potentiated the response. When this compound was applied to mice through respiration, the convulsions induced by pentetrazole were delayed, suggesting that EPP was absorbed by the brain, where it could potentiate the GABA(A) receptor responses. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated the responses to varying degrees. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness of the human through the potentiation of the GABA(A) receptor response after absorption into the brain, because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of the GABA(A) receptor-mediated response.

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2006-11-01

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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2008-05-01

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

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

    PubMed

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

    1997-12-01

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

  18. Expression variations of chromogranin A and α1,2,4 GABA(A)Rs in discrete limbic and brainstem areas rescue cardiovascular alterations.

    PubMed

    Avolio, Ennio; Facciolo, Rosa Maria; Alò, Raffaella; Mele, Maria; Carelli, Antonio; Canonaco, Alessia; Mosciaro, Lucia; Talani, Giuseppe; Biggio, Giovanni; Sanna, Enrico; Mahata, Sushil K; Canonaco, Marcello

    2013-01-01

    Recent interferences of hemodynamic functions via modified brain neuronal mechanisms have proven to be major causes of dementia and sleeping disorders. In this work, cerebral expression differences of the neuroactive vesicular chromogranin A (CgA) and distinct α GABA(A)R subunits were detected in the facultative hibernating hamster. In particular, damaged neuronal fields of hypotensive torpor (TORP) state were correlated to elevated CgA and GABA(A)R α1, α4 mRNA levels in the paraventricular hypothalamic nucleus (PVN), central amygdalar nucleus (CeA) plus solitary tractus nucleus (NTS). Conversely, few neurodegeneration signals of hypertensive arousal (AROU) state, accounted for mostly lower CgA levels in the same areas. This state also provided increased α2-containing sites in amygdala, hippocampal and NTS neurons together with elevated α4-containing receptors in the periventricular hypothalamic nucleus (Pe). Interestingly in our hibernating model, CgA appeared to preferentially feature inhibitory neurosignals as indicated by preliminary perfusion of amygdalar sites with its highly specific antihypertensive derived peptide (catestatin) promoting GABA-dependent sIPSCs. Overall, evident neuronal damages plus altered expression capacities of CgA and α1-, α2-, α4-GABA(A)Rs in CeA, Pe, PVN as well as NTS during both hibernating states corroborate for the first time key molecular switching events guaranteeing useful cardiovascular rescuing abilities of neurodegenerative disorders.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-04-16

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

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

    PubMed

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

    2003-07-01

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

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

    SciTech Connect

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

    1985-11-15

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

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

    PubMed

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

    2013-03-26

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

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

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

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

    1994-09-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2006-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed Central

    1989-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2017-01-18

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

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

    PubMed Central

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

    1989-01-01

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

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

    PubMed

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

    1989-09-01

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

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

    PubMed Central

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

    2014-01-01

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

  19. Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

    NASA Technical Reports Server (NTRS)

    Moffitt, Julia A.; Heesch, Cheryl M.; Hasser, Eileen M.

    2002-01-01

    Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.

  20. Waking action of ursodeoxycholic acid (UDCA) involves histamine and GABAA receptor block.

    PubMed

    Yanovsky, Yevgenij; Schubring, Stephan R; Yao, Quiaoling; Zhao, Yan; Li, Sha; May, Andrea; Haas, Helmut L; Lin, Jian-Sheng; Sergeeva, Olga A

    2012-01-01

    Since ancient times ursodeoxycholic acid (UDCA), a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer's disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA(A)R antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC(50) = 70 µM) and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA(A) receptors composed of α1, β1-3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA(A) receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA(A)R potentiation by several neurosteroids, had no effect on GABA(A)R inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA(A) receptors.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed Central

    McGee, Thomas P.; Bats, Cécile

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Breer, H; Kleene, R; Hinz, G

    1985-12-01

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

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

    PubMed

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

    2007-05-15

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

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

    PubMed

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

    2017-02-17

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

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

    PubMed Central

    1996-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    1986-05-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2008-05-01

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

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

    SciTech Connect

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

    1986-03-05

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

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

    PubMed

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

    2001-09-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

    1993-01-01

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

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

    PubMed Central

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

    2014-01-01

    Thyroid-stimulating hormone (TSH) is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated. PMID:25386660

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

    PubMed

    Wondolowski, Joyce; Frerking, Matthew

    2009-01-14

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

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

    PubMed

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

    2013-09-03

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

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

    PubMed Central

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

    2015-01-01

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

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

    DOE PAGES

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

    2015-10-12

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

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

    SciTech Connect

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

    2015-10-12

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

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

    PubMed

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

    2015-11-03

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2006-02-16

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

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

    PubMed Central

    Gibson, Scott K.; Gilman, Alfred G.

    2006-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2008-02-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed Central

    Wright, Amanda; Vissel, Bryce

    2012-01-01

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

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

    PubMed

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

    2002-06-25

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

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

    PubMed

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

    2017-03-01

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

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

    PubMed

    Douanne, Tiphaine; Gavard, Julie; Bidère, Nicolas

    2016-05-01

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

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

    PubMed

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

    2017-01-31

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

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

    PubMed

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

    2000-01-01

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

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1996-03-01

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

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

    PubMed

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

    2004-09-01

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

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

    NASA Astrophysics Data System (ADS)

    Maksay, Gábor; Simonyi, Miklós; Bikádi, Zsolt

    2004-10-01

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

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

    PubMed

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

    2008-02-12

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

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

    PubMed Central

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

    2016-01-01

    Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity. PMID:27901080

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

    PubMed

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

    2008-11-10

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

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

    PubMed

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

    2001-02-15

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

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

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

    PubMed

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

    2017-03-05

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Lin, Da-Ting; Huganir, Richard L

    2007-12-12

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

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

    PubMed Central

    Miyazaki, J; Nakanishi, S; Jingami, H

    1999-01-01

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

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

    PubMed

    Martin, Jessica A; Garczynski, Stephen F

    2016-04-01

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

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

    PubMed

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

    1988-12-15

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

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

    PubMed

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

    2009-05-01

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

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    1995-05-01

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

  2. Paradoxical effects of GABA-A modulators may explain sex steroid induced negative mood symptoms in some persons.

    PubMed

    Bäckström, T; Haage, D; Löfgren, M; Johansson, I M; Strömberg, J; Nyberg, S; Andréen, L; Ossewaarde, L; van Wingen, G A; Turkmen, S; Bengtsson, S K

    2011-09-15

    Some women have negative mood symptoms, caused by progestagens in hormonal contraceptives or sequential hormone therapy or by progesterone in the luteal phase of the menstrual cycle, which may be attributed to metabolites acting on the GABA-A receptor. The GABA system is the major inhibitory system in the adult CNS and most positive modulators of the GABA-A receptor (benzodiazepines, barbiturates, alcohol, GABA steroids), induce inhibitory (e.g. anesthetic, sedative, anticonvulsant, anxiolytic) effects. However, some individuals have adverse effects (seizures, increased pain, anxiety, irritability, aggression) upon exposure. Positive GABA-A receptor modulators induce strong paradoxical effects including negative mood in 3%-8% of those exposed, while up to 25% have moderate symptoms. The effect is biphasic: low concentrations induce an adverse anxiogenic effect while higher concentrations decrease this effect and show inhibitory, calming properties. The prevalence of premenstrual dysphoric disorder (PMDD) is also 3%-8% among women in fertile ages, and up to 25% have more moderate symptoms of premenstrual syndrome (PMS). Patients with PMDD have severe luteal phase-related symptoms and show changes in GABA-A receptor sensitivity and GABA concentrations. Findings suggest that negative mood symptoms in women with PMDD are caused by the paradoxical effect of allopregnanolone mediated via the GABA-A receptor, which may be explained by one or more of three hypotheses regarding the paradoxical effect of GABA steroids on behavior: (1) under certain conditions, such as puberty, the relative fraction of certain GABA-A receptor subtypes may be altered, and at those subtypes the GABA steroids may act as negative modulators in contrast to their usual role as positive modulators; (2) in certain brain areas of vulnerable women the transmembrane Cl(-) gradient may be altered by factors such as estrogens that favor excitability; (3) inhibition of inhibitory neurons may promote

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-04-15

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2007-07-01

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

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

    PubMed

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

    2004-09-03

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

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

    PubMed

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

    1998-11-01

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

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

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

    SciTech Connect

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

    1989-02-09

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

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

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

    2008-09-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2008-09-09

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

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

    PubMed Central

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

    2009-01-01

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

  1. Phenibut (beta-phenyl-GABA): a tranquilizer and nootropic drug.

    PubMed

    Lapin, I

    2001-01-01

    Phenibut (beta-phenyl-gamma-aminobutyric acid HCl) is a neuropsychotropic drug that was discovered and introduced into clinical practice in Russia in the 1960s. It has anxiolytic and nootropic (cognition enhancing) effects. It acts as a GABA-mimetic, primarily at GABA(B) and, to some extent, at GABA(A) receptors. It also stimulates dopamine receptors and antagonizes beta-phenethylamine (PEA), a putative endogenous anxiogenic. The psychopharmacological activity of phenibut is similar to that of baclofen, a p-Cl-derivative of phenibut. This article reviews the structure-activity relationship of phenibut and its derivatives. Emphasis is placed on the importance of the position of the phenyl ring, the role of the carboxyl group, and the activity of optical isomers. Comparison of phenibut with piracetam and diazepam reveals similarities and differences in their pharmacological and clinical effects. Phenibut is widely used in Russia to relieve tension, anxiety, and fear, to improve sleep in psychosomatic or neurotic patients; as well as a pre- or post-operative medication. It is also used in the therapy of disorders characterized by asthenia and depression, as well as in post-traumatic stress, stuttering and vestibular disorders.

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    2003-04-01

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

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

    PubMed Central

    Doherty, Faye C; Sladek, Celia D

    2011-01-01

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

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

    PubMed

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

    2004-12-10

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

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

    PubMed

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

    1998-04-17

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

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

    PubMed Central

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

    1982-01-01

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

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

    SciTech Connect

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

    1996-02-15

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

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    1986-12-01

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

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

    PubMed

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

    2017-04-21

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

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

    ERIC Educational Resources Information Center

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

    2014-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2005-10-14

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

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

    SciTech Connect

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

    2005-01-01

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

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

    PubMed

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

    2012-01-11

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

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

    PubMed

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

    2010-12-15

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

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

    PubMed

    Cox, David J; Racca, Claudia

    2013-06-15

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

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

    PubMed

    Dederer, Helene; Werr, Margaret; Ilg, Thomas

    2011-01-01

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

  20. Kindling alters neurosteroid-induced modulation of phasic and tonic GABAA receptor-mediated currents: role of phosphorylation.

    PubMed

    Kia, Arash; Ribeiro, Fabiola; Nelson, Renee; Gavrilovici, Cezar; Ferguson, Stephen S G; Poulter, Michael O

    2011-03-01

    We have previously shown that after kindling (a model of temporal lobe epilepsy), the neuroactive steroid tetrahydrodeoxycorticosterone (THDOC) was unable to augment GABA type A receptor (GABA(A))-mediated synaptic currents occurring on pyramidal cells of the piriform cortex. Phosphorylation of GABA(A) receptors has been shown previously to alter the activity of THDOC, so we tested the hypothesis that kindling induces changes in the phosphorylation of GABA(A) receptors and this accounts for the loss in efficacy. To assay whether GABA(A) receptors are more phosphorylated after kindling, we examined the phosphorylation state of the β3 subunit and found that it was increased. Incubation of brain slices with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (100 nM) also increased phosphorylation in the same assay. In patch clamp, recordings from non-kindled rat brain slices PMA also reduced the activity of THDOC in a manner that was identical to what is observed after kindling. We also found that the tonic current was no longer augmented by THODC after kindling and PMA treatment. The protein kinase C (PKC) antagonist bisindolylmaleimide I blocked the effects PMA on the synaptic but not the tonic currents. However, the broad spectrum PKC antagonist staurosporine blocked the effects of PMA on the tonic currents, implying that different PKC isoforms phosphorylate GABA(A) receptors responsible for phasic and tonic currents. The phosphatase activator Li(+) palmitate restored the 'normal' activity of THDOC on synaptic currents in kindled brain slices but not the tonic currents. These data demonstrate that kindling enhances the phosphorylation state of GABA(A) receptors expressed in pyramidal neurons reducing THDOC efficacy.

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

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2003-11-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2013-12-19

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2012-03-17

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

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

    PubMed Central

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

    1985-01-01

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

  9. GABA(A) Receptor Alpha5 Subunit as a Candidate Gene for Autism and Bipolar Disorder: A Proposed Endophenotype with Parent-of-Origin and Gain-of-Function Features, with or without Oculocutaneous Albinism

    ERIC Educational Resources Information Center

    Delong, Robert

    2007-01-01

    Our earlier family history studies of individuals with autism found a high incidence of major affective disorder, especially bipolar disorder, and unusual talents or intellectual abilities among family members. We now describe a subgroup of such families, selected from a large clinical experience, illustrating specific features of major affective…

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed Central

    1996-01-01

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

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

    PubMed

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

    2016-01-01

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

  13. Design, Synthesis and Subtype Selectivity of 3, 6-Disubstituted β-Carbolines at Bz/GABA(A)ergic Receptors. SAR and Studies Directed Toward Agents For Treatment of Alcohol Abuse

    PubMed Central

    Yin, Wenyuan; Majumder, Samarpan; Clayton, Terry; Petrou, Steven; VanLinn, Michael L.; Namjoshi, Ojas A.; Ma, Chunrong; Cromer, Brett A.; Roth, Bryan L.; Platt, Donna M.; Cook, James M.

    2010-01-01

    A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at αxβ3γ2 GABAA/benzodiazepine receptor subtypes by radioligand binding assays in search of α1 subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position -6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (LDi region) of GABAA/Bz receptors (see 32 and 33). Moreover, substituents located at position -3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L1, while N(2) presumably underwent a hydrogen bonding interaction with H1. Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were “near GABA neutral antagonists”. Based on the SAR, the most potent (in vitro) α1 selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.1–3 These data prompted the synthesis of the β-carbolines presented here. PMID:20888240

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-11-01

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

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

    PubMed

    Safieddine, S; Wenthold, R J

    1997-10-01

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

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

    PubMed Central

    Klarsfeld, A; Changeux, J P

    1985-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2011-09-01

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

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

    PubMed

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

    2014-07-05

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

    Ru, Wenjuan; Tang, Shao-Jun

    2015-01-01

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

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

    PubMed

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

    1996-10-01

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

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

    PubMed

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

    2014-02-01

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

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

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Baur, Roland; Sigel, Erwin

    2017-01-01

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

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

    PubMed

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

    2006-08-01

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

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

    PubMed Central

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

    2011-01-01

    The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor (GEF) localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins including PSD-95, DISC-1, AF-6 and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal LTP as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell-surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity. PMID:21880917

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

    PubMed

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

    2011-08-31

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

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

    PubMed

    Ferando, Isabella; Mody, Istvan

    2015-01-01

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

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

    PubMed Central

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

    2001-01-01

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

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

    SciTech Connect

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

    2014-05-01

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

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

    PubMed

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

    1995-11-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed

    Sabirzhanov, Boris; Keifer, Joyce

    2011-07-01

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