大鼠眶额叶GABA及其B型受体在应激性抑郁行为发生中的作用及其影响机制

为了探讨眶额叶(orbital frontal cortex,OFC)GABA及其B型受体在应激性抑郁行为发生中的作用及其影响机制,实验采用强迫游泳方法建立急性应激抑郁模型。在OFC区微量注射γ-氨基丁酸(γ-aminobutyric acid,GABA)及其B型受体阻断剂,通过开场实验、强迫游泳方式检测动物行为学表现,用免疫组织化学染色和Western blotting方法检测OFC区Kalirin表达,用高尔基染色法观察锥体细胞树突和树突棘。结果显示:强迫游泳应激引起动物抑郁样行为表现,同时,OFC区Kalirin阳性颗粒数及表达量显著减少,且锥体细胞树突棘密度下降;OFC区微量注射GABA具有抗抑郁效应,使OFC区Kalirin表达显著升高,锥体细胞树突棘密度增加;GABA-B型受体阻断剂CGP35348可以抑制GABA的这种效应。由此可见,通过强迫游泳应激诱发的抑郁样的行为变化与OFC区Kalirin表达减少和神经元树突棘密度降低有关,GABA可能通过GABA-B型受体增加OFC区Kalirin表达,以防止神经元退行性变化而产生抗抑郁作用。     

Allosteric modulation of a human odorant receptor

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Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function

Recent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The gamma-amino-n-butyric acid type B (GABA(B)) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane alpha-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABA(B) ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABA(B) receptor and highlight further the importance of the dimerization process in GPCR activation.     

Developmental regulation of GABA(B) receptor function in rat spinal cord

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.     

Gamma-hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABA B receptor activation in mouse frontal cortex and hippocampus

gamma-Hydroxybutyrate (GHB) naturally occurs in the brain, but its exogenous administration induces profound effects on the central nervous system in animals and humans. The intracellular signaling mechanisms underlying its actions remain unclear. In the present study, the effects of GHB on the activation (phosphorylation) of mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase 1 and 2 (ERK1/2), were investigated. Acute administration of GHB (500 mg/kg, intraperitoneal) induced a fast and long lasting inhibition of MAP kinase phosphorylation in both frontal cortex and hippocampus. The reduced MAP kinase phosphorylation was observed in the CA1 and CA3 areas but not in the dentate gyrus. Pretreatment with the specific gamma-aminobutyric acid, type B (GABAB), receptor antagonist CGP56999A (20 mg/kg, intraperitoneal) prevented the action of GHB, and the effect of GHB was mimicked by baclofen, a selective GABAB receptor agonist, whereas the high affinity GHB receptor antagonist NCS-382 (200 mg/kg, intraperitoneal) had no effect on GHB-inhibited MAP kinase phosphorylation. Moreover, the GHB dehydrogenase inhibitor valproate (500 mg/kg, intraperitoneal), which inhibits the conversion of GHB into GABA, failed to block the effect of GHB on MAP kinase phosphorylation. Altogether, these data suggest that GHB, administered in vivo, reduces MAP kinase phosphorylation via a direct activation of GABAB receptors by GHB. In contrast, GHB (10 mm for 15 min) was found ineffective on MAP kinase phosphorylation in brain slices, indicating important differences in the conditions required for the second messenger activating action of GHB.     

Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.     

GABA(B) receptors function as heterodimers

Our current understanding is that functional GABA(B) receptors exist as heterodimers of two related seven-transmembrane proteins, GABA(B)-R1 and GABA(B)-R2. GABA(B)-R1 requires GABA(B)-R2 to be expressed at the cell surface as a mature glycoprotein. Cloning of the GABA(B) receptor has failed to provide molecular evidence to support the existence of true receptor subtypes. The discovery of the heterodimeric nature of the GABA(B) receptor has already changed the way we think about GPCR function and it is likely that future studies will change our understanding about how receptor subtypes can be formed.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.     

Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.     

NMR structure of an intracellular third loop peptide of human GABA(B) receptor

GABA_B receptor is a G protein-coupled receptor for GABA and drug target for neurological and psychiatric disorders. From the analysis of GTPγS binding assay, we found that a synthesized peptide (GABAb: ETKSVSTEKINDHR) corresponding to the intracellular third loop region of metabotropic GABA_B receptor could activate G_i protein α subunit directly. The three dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H-NMR spectroscopy. GABAb peptide formed an α helical structure and a positive charge cluster at the C-terminal site. These structural features were also found in several other G protein activating peptides. From the comparison among these peptides, we found that peptides with high helical content show the high activity.     

Effects of Ca2+ concentration on GABA(B) receptor function in rat neocortical slices

In rat neocortical slices maintained in Mg2+-free Kreb's medium, the effects of Ca2+ concentration on repetitive spontaneous discharges and on GABA(B) receptor-mediated responses were investigated. Over a concentration range of 0.3-2.4 mM Ca2+, there was a reduction of discharge amplitude, with a 50 +/- 6.5% reduction in 0.3 mM Ca2+, whilst the burst frequency remained unaffected. Baclofen, the GABA(B) receptor agonist, produced a concentration-dependent depression of discharge frequency, reversibly antagonised by the antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch 50911). The EC50 value for baclofen in 2.4 mM Ca2+ was 11 microM, while the EC50 values in 0.3, 0.6, 1.2, and 1.8 mM Ca2+ were 1.3, 2.5, 3.6, and 10 microM, respectively, resulting in 8.5, 4.4, 3.1, and 1.1-fold leftward shifts. This enhanced action of baclofen in low extracellular Ca2+ concentrations in the neocortex may be the result of a lower concentration gradient which reinforces the action of baclofen.     

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

Phenobarbitone modulation of postsynaptic GABA receptor function on cultured mammalian neurons.

The anticonvulsant barbiturate phenobarbitone increases membrane current and conductance responses to gamma-aminobutyric acid (GABA) in cultured mouse spinal neurons. Analyses of GABA current fluctuations under control conditions and in the presence of phenobarbitone show that the principle action is to increase the average time during which GABA-activated channels remain open. The duration of minature synaptic currents with a time constant of decay similar to the mean open-time of GABA-activated channels is prolonged by the drug. The results suggest that (1) the synaptic events are GABA-mediated and (2) the enhancement of these events by barbiturate is due to the postsynaptic action of the drug.     

Identification of GABA(B) receptor in rat testis and sperm

gamma-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABA(A)R, in mammalian sperm. In the present study a second subtype, GABA(B)R, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABA(B)R1 (GABA(B)R1a and GABA(B)R1c) and GABA(B)R2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABA(B)R1a and GABA(B)R1c/lb, respectively, determined by Western blot using polyclonal anti-GABA(B)R1 antibody. By an indirect immunofluorescence technique, GABA(B)R1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABA(B)R.     

Phosphorylation-independent desensitization of GABA(B) receptor by GRK4

Agonist-promoted desensitization of the heterodimeric metabotropic GABA(B) receptor was investigated. Whereas no desensitization was observed in HEK293 cells heterologously expressing the receptor, GABA and the synthetic agonist baclofen induced a robust desensitization in cerebellar granule cells endogenously expressing the receptor. Taking advantage of this cell-specific desensitization phenotype, we identified GRK4 as the kinase involved in the neuronal desensitization. Transfection of small interference RNA directed against GRK4 significantly reduced GRK4 levels in cerebellar granule cells and strongly inhibited the agonist-promoted desensitization. Reciprocally, transfection of GRK4 in HEK293 cells restored agonist-promoted desensitization, confirming that this kinase is sufficient to support desensitization. Surprisingly, this desensitization occurred in the absence of ligand-induced receptor phosphorylation and could be promoted by GRK4 mutants deleted of their kinase domain. Taken together, these results suggest that GRK4 plays a central role in the agonist-promoted desensitization of GABA(B) receptor and that it does so through an atypical mechanism that challenges the generally accepted model linking the kinase activity of GRKs to their role in receptor desensitization.     

Discovery of a novel potent GABA(B) receptor agonist

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.     

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.     

The oligomeric state sets GABA(B) receptor signalling efficacy

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.     

Evolution of GABA(A) receptor diversity in the human genome

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.