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1.
Abstract: Three novel antisera to the γ2 subunit of the γ-aminobutyric acidA (GABAA) receptor/benzodiazepine receptor (GABAAR/BZDR) complex have been made. Anti-γ2S and anti-γ2L are specific antibodies to synthetic peptides that recognize the γ2S (short) and γ2L (long) forms, respectively, of the γ2 subunit. An antibody (anti-γ2IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ2IL) located between the putative transmembrane segments M3 and M4 of γ2S recognizes both γ2S and γ2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABAAR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-γ2S and anti-γ2L recognize peptides of 45,000 and 47,000 Mr, respectively. Immunoprecipitation experiments indicate that γ2S is more prevalent in hippocampus, whereas γ2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ2S and γ2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ2S and γ2L subunits and 41–48% of the γ2L subunit coexists with γ2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the γ2S and γ2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α6. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain γ2L also contain a γ2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α6, γ2S, and γ2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α6 subunit(s) have neither γ2S nor γ2L subunits. There are also species differences with respect to the relative abundance of γ2S and γ2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABAAR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.  相似文献   

2.
Abstract: Two GABAA receptor subunit-specific antibodies anti-α6 and anti-α1 have been used for elucidating the relationship between the presence of α1 and/or α6 subunits in the cerebellar GABAA receptors and the benzodiazepine-binding specificity. Receptor immunoprecipitation with the subunit-specific antibodies shows that 39% of the cerebellar GABAA receptors have α6, whereas 76% of the receptors have α1 as determined by [3H]muscimol binding. Results show that 42–45% of the receptors having α6 also have α1, whereas 13–15% of the receptors that contain α1 also have α6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both α1 and α6 subunits, a second type has α1 but not α6, and a third type has α6 but not α1 subunits. The results also show that receptors where α1 and α6 subunits coexist have two pharmacologically different benzodiazepine-binding properties, each associated with a different α subunit. The α1 subunit contributes the high-affinity binding of [3H]Ro 15-1788 (flumazenil) and the diazepam-sensitive binding of [3H]Ro 15-4513. The α6 subunit contributes the diazepam-insensitive binding of [3H]Ro 15-4513, but it does not bind [3H]Ro 15-1788 with high affinity. Thus, in the cerebellar α1–α6 GABAA receptors, there is no dominance of the pharmacology of one α subunit over the other.  相似文献   

3.
Abstract: The GABAA receptor is a heterooligomeric protein complex composed of multiple receptor subunits. Developmental changes in the pattern of expression of 11 GABAA receptor subunits in individual rat embryonic hippocampal neurons on days 1–21 in culture and acutely dissociated hippocampal neurons from postnatal day (PND) 5 rat pups were investigated using the technique of single-cell mRNA amplification. We demonstrate that multiple GABAA receptor subunits are expressed within individual hippocampal neurons, with most cells simultaneously expressing α1, α2, α5, β1, and γ2 mRNAs. Further, relative expression of several GABAA receptor subunit mRNAs changes significantly in embryonic hippocampal neurons during in vitro development, with the relative abundance (compared with β-actin) of α1, α5, and γ2 mRNAs increasing 2.3-, 2.7-, and 3.8-fold, respectively, from days 1 to 14, and β1 increasing 5-fold from days 1 to 21. In situ hybridization with antisense digoxigenin-labeled α1, β1, and γ2 RNA probes demonstrates a similar increase in expression of subunit mRNAs as embryonic hippocampal neurons mature in vitro. Relative abundances of α1, β1, and γ2 subunit mRNAs in acutely dissociated PND 5 hippocampal neurons are also significantly greater than in embryonic day 17 neurons on day 1 in vitro and exceed the peak values seen in cultured neurons on days 14–21, suggesting that GABAA receptor subunit mRNA expression within individual hippocampal neurons follows a similar, if somewhat delayed, developmental pattern in vitro compared with in vivo. These findings suggest that embryonic hippocampal neuronal culture provides a useful model in which to study the developmental regulation of GABAA receptor expression and that developmental changes in GABAA receptor subunit expression may underlie some of the differences in functional properties of GABAA receptors in neonatal and mature hippocampal neurons.  相似文献   

4.
Abstract: The pharmacological properties of γ-aminobutyric acidA (GABAA) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABAA receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABAA receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol-dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ~32% in both ethanol-dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol-induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABAA receptor subunit peptide levels are associated with ethanol dependence in rats. GABAA receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABAA receptor gene expression underlie the functional properties of GABAA receptors in ethanol-dependent rats and those undergoing ethanol withdrawal.  相似文献   

5.
Abstract: Previous research has shown that chronic ethanol consumption dramatically alters GABAA receptor α1 and α4 subunit gene expression in the cerebral cortex and GABAA receptor α1 and α6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABAA receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABAA receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABAA receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABAA receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABAA receptor α4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABAA receptor α1, α2, α3, α2/3, or γ2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABAA receptor α4 subunits and significantly decreased the relative expression of cerebral cortical GABAA receptor α1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABAA receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.  相似文献   

6.
Abstract— Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABAA receptor-mediated 36Cl? uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40-50% reduction in GABAA receptor α-subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α1, α2, and α3 subunits of the GABAA receptor in the cerebral cortex and the α1 subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α1-, α2-, and α3-subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABAA receptor α1subunit (51 kDa), 47 ± 8% reduction in level of the α2subunit (53 kDa), and 30 ± 7% reduction in level of the α3subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α1 subunit in the cerebellum. In summary, this ethanol-induced reduction in content of the GABAA receptor α subunits may underlie alterations in the GABAA receptor function and could be related to cellular adaptation to the functional disturbance caused by ethanol.  相似文献   

7.
Abstract: An analogue of colchicine,β-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. β-Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol-stimulated 36Cr-uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopusoocytes expressing human α1β2γ2S, but not α1β2, GABAA receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of β-Lumicolchicine on the GABAA receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, β-Lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.  相似文献   

8.
The GABAA receptor, a multisubunit ligand-gated ion channel, plays a central role in cell–cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the α1, β2/3, and γ2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increase in β2/3 and γ2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABAA receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the α1, β2, β3, and γ2 subunit mRNAs exclusively, suggests that GABAA receptor subunit composition in this cell population does not change during postnatal maturation. 1994 John Wiley & Sons, Inc.  相似文献   

9.
Delta (δ) subunit containing GABAA receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α6 subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABAA receptor pentamers by subunit concatenation. These receptors (composed of α6, β3 and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α6‐β3‐α6/δ receptors showed a substantial response to GABA alone. Three receptors, β3‐α6‐δ/α6‐β3, α6‐β3‐α63‐δ and β3‐δ‐β36‐β3, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α6, β3 and δ subunits.  相似文献   

10.
Abstract: The γ-aminobutyric acidA (GABAA)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (α1–6, β1–4, γ1–4, δ, and ρ1 and ρ2). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABAA/BZ receptor α1, α6, β2, β3, and γ2 subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased α1 subunit mRNA expression in each cell type, whereas the expression of α6 and γ2 subunit mRNA levels increased; no changes were observed in the expression of β2 and β3 subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of α1 and β3 mRNAs, whereas levels of α6, β2, and γ2 mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABAA/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.  相似文献   

11.
To date three β subunits of the GABAA receptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity-purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.  相似文献   

12.
The postnatal development of the gamma-aminobutyric acidA/benzodiazepine receptor (GABAR/BZDR) complex of the rat brain has been investigated using the monoclonal antibody 62-3G1 and the polyclonal rabbit antiserum A, specific for the 57,000 and 51,000 Mr receptor subunits, respectively. Both GABAR and BZDR binding activities co-precipitated during all postnatal ages. Adult rats showed a main 51,000 Mr[3H]flunitrazepam photoaffinity-labeled peptide, whereas newborn rats showed several photolabeled peptides of higher Mr. All the photolabeled peptides could be immunoprecipitated with each antibody regardless of the age of the rats. These results suggest that the physical coupling between the GABAR and the BZDR is already present in newborn animals and it is maintained afterwards during development. Glycosidase and peptidase treatments of the immunoprecipitated GABAR/BZDR complex indicated that all the [3H]flunitrazepam-photolabeled subunits are different peptides, although they seem to conserve a high degree of homology. In addition to the age-dependent heterogeneity, the results also suggest that for each age, there is heterogeneity in the subunit composition of the GABAR/BZDR complex.  相似文献   

13.
Gamma‐aminobutyric acid type A receptors (GABAARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABAARs determine their function and pharmacological profile. GABAARs are heteropentamers of subunits, and (α1)2(β3)2(γ2L)1 is a common subtype. Biochemical and biophysical studies of GABAARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABAAR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)3GK–1D4 GABAAR. These cells achieved expression levels of 70–90 pmol [3H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [3H]flunitrazepam to [3H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABAARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ~30%. Typical purifications yielded 1.0–1.5 nmoles of [3H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [3H]muscimol binding were maintained in the purified state.  相似文献   

14.
Abstract: Antisera were produced in rabbits against synthetic peptides based on subtype-specific regions of the cDNA sequences of the α1, α2, α3, and α4 (also termed α5) subunits of mammalian GABAA receptors. The antigen peptides were chosen from the putative cytoplasmic loop between the proposed third and fourth membrane spanning helices; they were not only subtype-specific sequences, but also their hydrophilicity and predicted secondary structures suggested high potential antigenicity. In all cases, antipeptide antisera recognized on western blots the corresponding α-subunit polypeptide of the GABAA receptors purified from bovine brain by benzodiazepine-affinity chromatography, and were able to immunoprecipitate binding activity from detergent-solubilized purified receptors. The four antisera each recognized a unique polypeptide, and only one, in the purified receptor, with α1, α2, α3, and α4 identified at 51, 52, 56, and 57 kDa, respectively. This represents the first identification of the α4 gene product on a gel. Both the relative amount of staining in immunoblots and the fraction of receptor binding that could be immunoprecipitated by saturating concentrations of each of the four subtypespecific antibodies varied in a consistent manner between receptors purified from different brain regions. Thus, cerebral cortex receptor contained all four α polypeptides on western blots, and significant activity could be precipitated by all four. Hippocampal receptor lacked α3 immunoreactivity on blotting and by immunoprecipitation; α1 was less, whereas both α2 and α4 were more abundant in hippocampus than in cortex by both techniques. Cerebellum receptor contained only α1 of the four α subunits tested, and the anti-α1 antibodies immunoprecipitated >90% of the binding activity. The variable amounts of staining and immunoprecipitation from the three brain areas by the four antisera demonstrate the presence of heterooligomeric receptor complexes with different α-subunit constituents in cortex, hippocampus, and cerebellum. The sum of cortical receptor activity precipitated individually by the four anti-α antisera was > 150%, indicating that some heterooligomers are likely to contain more than one class of α subtype, although most receptor complexes probably contain only one α subtype. These α-subunit subtype-specific antibodies should be useful in analyzing structure, function, and localization of GABAA/benzodiazepine receptors in mammalian brain.  相似文献   

15.
GABAA receptors (GABAAR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABAAR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABAAR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABAAR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function.  相似文献   

16.
The amino acid γ-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotrans-mitter that mediates most of its effects through fast GABA-gated Cl?-channels (GABAAR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes α, β, γ, δ, ε, and ρ, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABAARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABAARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of α, β, and γ subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular α and γ subunit variants. Little is known about the functional properties of the β, δ, and ε subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABAARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.  相似文献   

17.
Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABAA receptor (GABAARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABAAR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABAA sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABAA sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His102 binds at the γ2 subunit interface in proximity to γ2 residues Thr142, Phe77, and Met130; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.  相似文献   

18.
A comparison has been made of the abilities of several neurotoxic and nontoxic phospholipases A2 from snake venoms to inhibit the intake of γ-aminobutyric acid into synaptosomes from rat cerebral cortex. The neurotoxic phospholipases A2 inhibited GABA uptake more than the nontoxic enzymes did. However, there was a poor correlation between the measured specific enzyme activity of a phospholipase A2 and its ability to inhibit the uptake of GABA.  相似文献   

19.
Abstract: The localization of two forms of the γ subunit of G proteins, γ3 and γ12, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ3 tended to decrease with age, whereas that of γ12 in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ3 was abundant in the neuropil, whereas γ12 was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ12-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ12-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ12 is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ12 was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ3 in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ3 and γ12 are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ3 and γ12 in the brain are related to the numbers and/or extent of maturation of these cells.  相似文献   

20.
GABAA receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABAA receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABAA receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn2+ which block α6 containing and incomplete receptors.  相似文献   

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