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1.
Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acidA (GABAA) receptor α5-subunit. These anti-peptide α5(2–10) or anti-peptide α5(427–433) antibodies reacted specifically with GABAA receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α5(2–10) and the anti-peptide α5(427–433) antibody. These results indicate the existence of at least two different α5-sub-units of the GABAA receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABAA receptors so far investigated, at least one of these two α5-subunits contains O-linked carbohydrates.  相似文献   

2.
Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABAA receptors that show diazepam (or clonazepam)-insensitive [3H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α6, α1, γ2S, γ2L, and β2 or β3 subunits colocalizing in the same receptor complex.  相似文献   

3.
Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABAA) receptor-gated Cl channel response is a common action of structurally diverse anesthetics, suggesting that the GABAA receptor plays an important role in anesthesia. To determine if GABAA receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABAA receptor protein subunits; the order of sensitivity was α1β1 > α1β1γ2s1β1γ2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABAA receptor protein complex to modulate the Cl channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.  相似文献   

4.
Abstract: The γ2 subunit of the GABAA receptor (GABAA-R) is alternatively spliced. The long variant (γ2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336–351 of the intracellular loop of the γ2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The K m values for PKC-and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 μM , respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the γ2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.  相似文献   

5.
Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acidA (GABAA) receptor α4-subunit. These anti-peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti-peptide α4 (517–523) antibodies and could then be identified by the anti-α4-antibodies as well as by the GABAA receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABAA receptors. Intact GABAA receptors appeared to be retained by the immunoaffinity column because other GABAA receptor subunit proteins like the β2/β3-subunits and the γ2-subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67-kDa protein, a 51-kDa protein could be detected by the antibody bd-28 and the anti-peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1-subunit-specific anti-peptide α1 (1–9) antibody. In contrast to the α1-subunit, the 51-kDa protein identified by the anti-α4 antibody could not be deglycosylated by N -Glycanase. The identity of the 51-kDa protein identified by the anti-α4-antibodies thus must be further investigated.  相似文献   

6.
Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABAA receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABAA receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α1 and α5 subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α1 and α5 subunit immunoreactivity was reduced. In the VTA, levels of α1 subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α1 subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α5 mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABAA receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.  相似文献   

7.
GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of α1S205C and α1T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (–NCS). We also provide new data that identify α1H101C and α1N102C as exclusive sites of the reaction of a diazepam derivative where the –Cl atom is replaced by a –NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of α1β2γ2 receptors.  相似文献   

8.
Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human α2, β1, and γ2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α1 or α3 for the α2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α2 and β1 alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.  相似文献   

9.
Abstract: We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk) cells stably transfected with GABAA receptor α1β1γ2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.  相似文献   

10.
Abstract: GABAA and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABAA receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk cells stably transfected with GABAA receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [3H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K D and B max of [3H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t 1/2 of ∼30 min. The EC50 for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as benzodiazepine tolerance.  相似文献   

11.
Abstract: The multisubunit γ-aminobutyric acid type A (GABAA) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABAA-receptor ionophore ligand tert -[35S]butylbicyclophosphorothionate ([35S]TBPS) and its modulation by GABA and the GABAA antagonists bicuculline and 2'-(3'-carboxy-2',3'-propyl)-3-amino-6- p -methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABAA subunits in brain as reported previously. In some brain regions SR 95531 reduced [35S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [35S]TBPS binding. This ratio correlated positively with α2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit-containing GABAA receptors. The data aid in developing GABAA-receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABAA antagonists.  相似文献   

12.
Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the α1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1-subunit-containing GABAA receptors. Results of a biochemical characterization of these G ABAA receptor- t ubulin complex- a ssociated p roteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.  相似文献   

13.
Abstract: Recombinant GABAA receptors, expressed from α-, β-, and γ2-subunits, are diazepam-insensitive when the α-subunit is either α4 or α6. In situ, diazepam-insensitive receptors containing the α6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABAA receptors in various brain areas containing the α4-subunit. GABAA receptors immunoprecipitated with a newly developed α4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2-recombinant receptors and diazepam-insensitive [3H]Ro 15-4513 binding sites in rat brain membranes. In addition, α4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4-subunit containing receptors revealed that the α4-subunit contributes to several receptor subtypes. Depending on the brain region, the α4-subunit can be coassembled with a second type of α4-subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [3H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.  相似文献   

14.
Abstract: Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABAA receptor α1- and β3-subunit mRNA was conducted using synthetic 3'- end 35S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of α1-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of β3-subunit mRNA were not affected. Dramatically increased levels of GABAA receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the α1 subunit and in cerebral cortex only for the β3-subunit. These data provide further support to the structural and pharmacological GABAA receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.  相似文献   

15.
Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ1, γ1, and δ1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.  相似文献   

16.
Abstract: This study examined γ-aminobutyric acidA (GABAA) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [3H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced ( p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate ( E max) (16.5 ± 1.2% and 11.3 ± 1.0%, 2-day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7-day control and treated cells, respectively; best-fit E max± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC50) (1.2 ± 1.7 and 2.3 ± 1.8 µ M , 2-day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µ M , 7-day control and treated cells, respectively; best-fit EC50± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABAA receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [3H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABAA receptor subunit protein expression, rather than loss of [3H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABAA receptor function.  相似文献   

17.
Abstract: The expression of six mRNA species (α2, α3, α5, β2, β3, and γ2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. α3, α5, and β3 subunit expression declined, α2 expression increased, whereas β2 and γ2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert -butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the α3, α5, or β3 subunit mRNA expression declined and prevented the increase in α2 subunit mRNA expression. As well, allopregnanolone down-regulated β2 subunit mRNA expression. TBPS, on the other hand, up-regulated α3, α5, β2, and β3 subunit mRNA expression. It also down-regulated the expression of α2 subunit mRNA. Both allopregnanolone and TBPS had no effect on γ2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.  相似文献   

18.
Cloning and Characterization of a Mouse σ1 Receptor   总被引:1,自引:1,他引:0  
Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ1 cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ1 receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[3H]pentazocine binding with a selectivity profile consistent with σ1 receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[3H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[3H]pentazocine binding site with characteristics of a murine σ1 receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.  相似文献   

19.
Abstract: GABAA receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [3H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [3H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [3H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABAA receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABAA receptors. The presence of α-subunit(s), as revealed by [3H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABAA receptors are extrasynaptic and expressed in astroglia.  相似文献   

20.
Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABAA) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABAA receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.  相似文献   

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