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Functional gamma-aminobutyric acid type B (GABA(B)) receptors are normally only observed upon coexpression of GABA(B1) with GABA(B2) subunits. A C-terminal arginine-based endoplasmic reticulum (ER) retention/retrieval signal, RSRR, prevents escape of unassembled GABA(B1) subunits from the ER and restricts surface expression to correctly assembled heteromeric receptors. The RSRR signal in GABA(B1) is proposed to be shielded by C-terminal coiled-coil interaction of the GABA(B1) with the GABA(B2) subunit. Here, we investigated whether the RSRR motif in GABA(B1) remains functional when grafted to ectopic sites. We found that the RSRR signal in GABA(B1) is inactive in any of the three intracellular loops but remains functional when moved within the distal zone of the C-terminal tail. C-terminal deletions that position the RSRR signal closer to the plasma membrane drastically reduce its effectiveness, supporting that proximity to the membrane restricts access to the RSRR motif. Functional ectopic RSRR signals in GABA(B1) are efficiently inactivated by the GABA(B2) subunit in the absence of coiled-coil dimerization, supporting that coiled-coil interaction is not critical for release of the receptor complex from the ER. The data are consistent with a model in which removal of RSRR from its active zone rather than its direct shielding by coiled-coil dimerization triggers forward trafficking. Because arginine-based intracellular retention signals of the type RXR, where X represents any amino acid, are used to regulate assembly and surface transport of several multimeric complexes, such a mechanism may apply to other proteins as well.

In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA(B) receptors (where GABA stands for gamma-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA(B) receptors are assembled from two subunits GABA(B1) (GABA(B) receptor subtype 1) and GABA(B2). For the GABA(B1) subunit, which binds the neurotransmitter GABA, two variants GABA(B1a) (GABA(B) receptor subtype 1 variant a) and GABA(B1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA(B1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA(B2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA(B1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA(B1) can be used for further functional or structural investigations.

Metabotropic GABAB receptors predominantly function as heterodimers of GABAB(1) and GABAB(2) subunits, but GABAB(1) can also form functional receptors in the absence of GABAB(2). Mice lacking the GABAB(1) subunit have altered behavioural responses in tests for anxiety and depression. In these studies, we investigated anxiety and depression in GABAB(2)-deficient mice. We compared the effects directly with that of genetic deletion of the GABAB(1) receptor subunit. Both GABAB(1) and GABAB(2)-deficient mice were found to be more anxious than wild type in the light-dark box paradigm. In contrast, these mice exhibited an antidepressant-like behaviour in the forced swim test. Taken together, these data suggest that heterodimeric GABAB(1,2) receptors are required for the normal regulation of emotional behaviour.

gamma-Aminobutyric acid (GABA) has been implicated in the control of hypophyseal functions. We evaluated whether the constitutive loss of functional GABA(B) receptors in GABA(B1) knockout (GABA(B1)(-/-)) mice alters hormonal levels, under basal and stimulated conditions, and reproductive function. The serum hormone levels were measured by radioimmunoassay, the estrous cyclicity was evaluated by vaginal lavages, and the mating behavior was determined by the presence of vaginal plugs. A moderate hyperprolactinemic condition was observed, in which prolactin increase and thyroid-stimulating hormone decrease were similar between genotypes. Basal luteinizing hormone (LH), follicle-stimulating hormone, thyroid-stimulating hormone, and growth hormone levels were similar between genotypes in each sex. Analysis of the gonadotropin axis revealed no differences in puberty onset between female genotypes. In con trast, the estrous cyclicity was significantly disrupted in GABA(B1)(-/-) female mice, showing significantly extended periods in estrus and shortened periods in proestrus. Reproduction was significantly compromised in GABA(B1)(-/-) females, with a significantly lower proportion of mice (37.5%) getting pregnant during the first 30 days of mating as compared with wild-type controls (87.5%). Moreover, only 14% of vaginal plug positive GABA(B1)(-/-) females had successful pregnancies as compared with 75% in the controls. In addition, the postovariectomy LH rise was significantly advanced in GABA(B1)(-/-) mice, while the response to estradiol feedback was similar in both genotypes. In conclusion, our endocrine analysis of GABA(B1)(-/-) mice reveals that GABA(B) receptors are involved in the regulation of basal prolactin titers. Moreover, the hypothalamic-hypophyseal-ovarian axis is seriously disturbed, with alterations in cyclicity, postcastration LH increase, and fertility indexes. The molecular mechanism underlying these hormonal disturbances remains to be addressed.

Type B gamma-aminobutyric acid receptor (GABABR) is a G protein-coupled receptor that regulates neurotransmitter release and neuronal excitability throughout the brain. In various neurons, GABABRs are concentrated at excitatory synapses. Although these receptors are assumed to respond to GABA spillover from neighboring inhibitory synapses, their function is not fully understood. Here we show a previously undescribed function of GABABR exerted independent of GABA. In cerebellar Purkinje cells, interaction of GABABR with extracellular Ca2+ (Ca(2+)o) leads to a constitutive increase in the glutamate sensitivity of metabotropic glutamate receptor 1 (mGluR1). mGluR1 sensitization is clearly mediated by GABABR because it is absent in GABABR1 subunit-knockout cells. However, the mGluR1 sensitization does not require G(i/o) proteins that mediate the GABABR's classical functions. Moreover, coimmunoprecipitation reveals complex formation between GABABR and mGluR1 in the cerebellum. These findings demonstrate that GABABR can act as Ca(2+)o-dependent cofactors to enhance neuronal metabotropic glutamate signaling.

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function., (Copyright 2004 Wiley-Liss, Inc.)

Balb/c GABA(B(1))(-/-) mice develop complex epileptiform activity, including spontaneous and audiogenic generalized seizures, 6-8 weeks after birth. The neuronal systems involved in these epilepsies have not been identified yet. Because the hippocampus is critically involved in epileptiform activity, we now investigated whether this brain region exhibits seizure-related alterations. Using semi-quantitative immunohistochemistry, we studied the temporal and cellular hippocampal expression pattern of two seizure-sensitive calcium-binding proteins, calbindin-D-28k and calretinin, in GABA(B(1))(-/-) mice. One month after birth, before the onset of overt epileptiform activity, wild-type (WT) and GABA(B(1))(-/-) mice exhibit comparable expression profiles for the two calcium-binding proteins. Three months after birth, once the epileptic phenotype is established, we observe clear alterations in the expression of calcium-binding proteins in the dentate gyrus area. GABA(B(1))(-/-) mice exhibit a 50% decline in the staining intensity of calbindin-D-28k expressing neurons and a 70% increase in the number of calretinin-positive neurons when compared to WT littermates. Six months after birth, the down-regulation of calbindin-D-28k protein is even more pronounced, while the calretinin expression in GABA(B(1))(-/-) mice reverts to the pattern seen in WT littermates. Our data demonstrate that the absence of functional GABA(B) receptors causes epileptiform activity through a mechanism that crucially involves dentate gyrus granule cells, and that this pathological activity is accompanied by adaptive changes.

GABA(B) receptors mediate slow inhibitory GABAergic neurotransmission. They are encoded by two distinct subunits, GABA(B1) (GBR1) and GABA(B2) (GBR2), with two major isoforms of GBR1, GBR1a and GBR1b, arising from differential promoter usage. Heterodimerization of GBR1 and GBR2 is essential for GABA(B) receptor function, as shown in recombinant expression systems and in GBR1(-/-) mice. GABA(B) receptors are highly expressed during ontogeny, prior to synaptogenesis, but their developmental function remains elusive. Here we investigated the postnatal development of GABA(B) receptors in rodent brain, focusing on potential differences in the spatial and temporal expression pattern of GBR1 and GBR2. Immunohistochemistry with subunit-specific antibodies revealed a widespread staining for GBR1a and GBR2 in neonatal rodent brain. During the first 2 weeks, these two subunits exhibited largely overlapping regional distribution, but with profound distinctions in cellular and subcellular localization. The adult-like pattern was established during the third week, with a prominent up-regulation of GBR1b, extensively codistributed with GBR2. Several unexpected features were noted at early stages, notably, a selective GBR2 staining of axonal tracts, such as the corticothalamic projection, and a prominent GBR1 expression in astrocytes. The specificity of the antibody labeling was verified in GBR1- and GBR2-knockout mice. In addition, the analysis of these mutants revealed a partial preservation of GBR2 staining in GBR1(-/-) mice and vice versa. Altogether, the results suggest a functional role for GBR1 and GBR2 proteins in immature brain in addition to their contribution to dimeric GABA(B) receptor complexes.

The role of GABAB receptors in various behavioral processes has been largely defined using the prototypical GABAB receptor agonist baclofen. However, baclofen induces sedation, hypothermia and muscle relaxation, which may interfere with its use in behavioral paradigms. Although there is much evidence for a role of the inhibitory neurotransmitter GABA in the pathophysiology of anxiety, the role of GABAB receptors in these disorders is largely unclear. We recently identified GS39783 (N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine) as a selective allosteric positive modulator at GABAB receptors. The aim of the present study was to broadly characterize the effects of GS39783 in well-validated rodent models for motor activity, cognition, and anxiety. The following tests were included: locomotor activity in rats and mice, rotarod and traction tests (including determinations of core temperature) in mice, passive avoidance in mice and rats, elevated plus maze in rats, elevated zero maze in mice and rats, stress-induced hyperthermia in mice, and pentobarbital- and ethanol-induced sleep in mice. Unlike baclofen and/or the benzodiazepine chlordiazepoxide, GS39783 had no effect in any of the tests for locomotion, cognition, temperature, or narcosis. Most interestingly, GS39783 had anxiolytic-like effects in all the tests used. Overall, the data obtained here suggest that positive modulation of GABAB receptors may serve as a novel therapeutic strategy for the development of anxiolytics, with a superior side effect profile to both baclofen and benzodiazepines.

GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.

GABA(B) receptors are broadly expressed in the nervous system and have been implicated in a wide variety of neurological and psychiatric disorders. The cloning of the first GABA(B) receptor cDNAs in 1997 revived interest in these receptors and their potential as therapeutic targets. With the availability of molecular tools, rapid progress was made in our understanding of the GABA(B) system. This led to the surprising discovery that GABA(B) receptors need to assemble from distinct subunits to function and provided exciting new insights into the structure of G protein-coupled receptors (GPCRs) in general. As a consequence of this discovery, it is now widely accepted that GPCRs can exist as heterodimers. The cloning of GABA(B) receptors allowed some important questions in the field to be answered. It is now clear that molecular studies do not support the existence of pharmacologically distinct GABA(B) receptors, as predicted by work on native receptors. Advances were also made in clarifying the relationship between GABA(B) receptors and the receptors for gamma-hydroxybutyrate, an emerging drug of abuse. There are now the first indications linking GABA(B) receptor polymorphisms to epilepsy. Significantly, the cloning of GABA(B) receptors enabled identification of the first allosteric GABA(B) receptor compounds, which is expected to broaden the spectrum of therapeutic applications. Here we review current concepts on the molecular composition and function of GABA(B) receptors and discuss ongoing drug-discovery efforts.

The mechanism by which the tumor suppressor PTEN slows tumor cell migration is not well characterized. A recent study by Raftopoulou et al. shows that a lack of PTEN protein phosphatase activity accelerates the migration of glioblastoma cells. The protein phosphatase activity of PTEN is directly or indirectly responsible for dephosphorylating a PTEN residue, threonine-383, which is necessary for slowing cell migration. These findings have implications for the design of new therapies against glioblastomas and other highly invasive cancers.

Previous work demonstrated a sexually dimorphic ontogenic expression of gamma-aminobutyric acid receptors (GABA(B)R) in rat pituitary. As sex steroids determine sex-specific expression patterns, we now studied the effect of sex hormones on pituitary GABA(B)R expression. GABA(B)R subunits, measured by Western blot and by semi-quantitative RT-PCR and luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone measured by RIA were determined in two experimental designs: First experimental design: 8- and 15-day-old females (8F, 15F); 8F and 15F treated with 100 mug testosterone propionate (TP) on day 1 of life (8F100TP, 15F100TP), 8- and 15-day-old males (8M, 15M) and 8M and 15M castrated on day 1 (8MC, 15MC). Second experimental design: 8-day-old female and male animals: 8F, 8F100TP, 8F treated with 1 mug/day TP on days 1-4 (8F1TP), 8F treated with the androgen antagonist Flutamide (Flut: 2.5 mg/100 g BW of pregnant mother on days E17-E23) (8F-Flut), 8M, 8MC, 8M treated with Flut as above (8M-Flut) and 8MC-Flut. In these animals, in addition, GABA, glutamate, aspartate and taurine were measured by HPLC in hypothalami and cortex. In the first set of experiments, GABA(B1)R mRNA/protein expression was higher in 8F than in 15F, 8M or 15M. In 8F100TP, GABA(B1)R mRNA/protein decreased to male levels. TP treatment did not alter GABA(B1)R expression in 15F. There was no difference in GABA(B1)R expression between 8M and 15M and neonatal castration did not modify its expression. In the second set of experiments, TP (1 mug) or Flut did not modify GABA(B1)R in 8F, while 100 microg TP continued to decrease GABA(B1)R expression. In 8M, Flut, alone or with castration, increased GABA(B1)R mRNA/protein expression to 8F. Hypothalamic GABA content followed the same pattern as pituitary GABA(B)R expression in 8-day-old animals, suggesting a cross-regulation. With regard to hormonal levels, 100 microg, but not 1 microg TP altered gonadotropins at 8 days, although both treatments effectively androgenized females as evidenced by lack of cycling. We conclude that androgens, acting pre- and postnatally, decrease pituitary GABA(B)R subunit expression.

gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA(B) receptor-mediated activation of K(+) currents in CA1 pyramidal cells, which elevates extracellular K(+) concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA(B) receptors and inhibits GABA(B) receptor-activated K(+) currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA(A) receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA(B) receptor activity.

gamma-Aminobutyric acid-B (GABA(B)) receptors are broadly expressed in the nervous system and have been implicated in a wide variety of neurological and psychiatric disorders. To date the only GABA(B) drug on the market is the agonist baclofen (Lioresal((R))) that is used to treat severe spasticity of cerebral and spinal origin. In addition baclofen is effective in animal models for many central and peripheral disorders, but side-effects and the development of tolerance prohibited a more widespread use of this drug in man. Similarly GABA(B) antagonists show great therapeutic promise but their shortcomings, e.g. the lack of brain penetration or some proconvulsive potential, prevented clinical development. The cloning of GABA(B) receptors in 1997 revived interest in these receptors as drug targets. The long-awaited availability of the tools that were necessary to develop more selective and safer drugs stimulated an impressive activity in the field. The demonstration that GABA(B) receptors needed to heteromerize for function provided new insights into the structure of G-protein coupled receptors in general and enabled to identify allosteric GABA(B) drugs. Gene knockout mice revealed neuronal systems that are under tonic GABA(B) control and therefore best suited for therapeutic intervention. Significant advances were made in clarifying the relationship between GABA(B) receptors and the receptors for gamma-hydroxybutyrate (GHB), a drug of abuse. Here we provide and update on the molecular composition, the physiology and the pharmacology of GABA(B) receptors and discuss to what extent our current knowledge influences ongoing and future drug discovery efforts.

Epilepsy is closely related to an altered transmission of GABA, the major inhibitory transmitter in the brain. GABA acts through two classes of receptors, ionotropic GABA(A) receptors and metabotropic GABA(B) receptors. Using in situ hybridization, receptor autoradiography and immunocytochemistry, we now investigated temporal changes in the expression the GABA(B)-1 and GABA(B)-2 subunits (GABA(B)-1R and GABA(B)-1R, respectively) in the hippocampus following kainic-acid-induced seizures. Significant decreases (by about 40%) in mRNA levels of both splice variants (a and b) of GABA(B)-1R and of GABA(B)-2R were observed in the principal cell layer of the hippocampus 6-12 h after kainic acid injection in the rat. Whereas mRNA levels in the granule cell layer returned to basal after 24 h, the decreases persisted in sectors CA1 and CA3, presumably due to progressing neurodegeneration. In the sector CA3, GABA(B)-R mRNA levels and GABA(B)-R1 immunoreactivity partially recovered 30 days after the initial kainic acid seizures indicating receptor upregulation in surviving neurons.

The scope of the plenary lecture at the occasion of the Xth Meeting on Heterocyclic Structures in Medicinal Chemistry, Palermo 2002, is considerably larger than that of the main lecture at the XVIth International Symposium on Medicinal Chemistry, Bologna 2000, described by Froestl et al. in Farmaco 56 (2001) 101. Additional information is presented, in particular, on the reaction conditions for the 31 step synthesis of the combined affinity chromatography and photoaffinity radioligand [125I]CGP84963 and on the recent developments of the molecular biology of GABA(B) receptors., (Copyright 2003 Editions scienctifiques et médicales Elsevier SAS)

The gamma-aminobutyrate B (GABA(B)) receptor is the first discovered G-protein-coupled receptor (GPCR) that needs two subunits, GB1 and GB2, to form a functional receptor. The GB1 extracellular domain (ECD) binds GABA, and GB2 contains enough molecular determinants for G-protein activation. The precise role of the two subunits in G-protein coupling is investigated. GB1 and GB2 are structurally related to the metabotropic glutamate, Ca(2+)-sensing and other family 3 GPCRs in which the second (i2) as well as the third (i3) intracellular loop play important roles in G-protein coupling. Here, the role of the i2 loops of GB1 and GB2 in the GABA(B) receptor ability to activate G(alpha)-proteins is investigated. To that aim, the i2 loops were swapped between GB1 and GB2 heptahelical domains (HDs), either in the wild-type subunits or in the chimeric subunits GB1/2 that contain the ECD of GB1 and the HD of GB2. The effect of an additional mutation within the i3 loop of GB2 that prevents coupling of the heteromeric receptor was also examined. Combinations of interest were found to be correctly addressed at the cell surface and to assemble into heteromers. Taken together our data revealed the following new information on the G-protein coupling of the heteromeric GABA(B) receptor: 1) the i2 loop of GB2 within the GB2 HD is required for the heteromeric GABA(B) receptor to couple to G-proteins, whereas the i2 loop of GB1 is not; 2) the presence of the i2 loop of GB2 within the GB1 HD is not sufficient to allow coupling of GB1; 3) the GB2 HD activates the Gqi9 protein whether it is associated with the GB2 or GB1 ECD; 4) in the combination with two GB2 HDs, each is able to couple to G-proteins; and finally, 5) the use of mutations in i2, i3, or both within the GB2 HD brings evidence for the absence of domain swapping enabling the exchange of region including i2 and i3 between the subunits.

The actions of the anticonvulsant gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] have been somewhat enigmatic until recently, when it was claimed to be a gamma-aminobutyric acid-B (GABA(B)) receptor agonist acting exclusively at a heterodimeric complex containing the GABA(B(1a)) splice variant (Mol Pharmacol 2001;59:144-152). In this study, we have investigated the effects of gabapentin on recombinant GABA(B(1a)) and GABA(B(1b)) receptors coexpressed with GABA(B(2)) in five different functional recombinant assays, its ability to inhibit [(3)H]GABA binding in a GABA(B) receptor-selective binding assay using rat synaptic membranes, and its ability to inhibit transient lower esophageal sphincter relaxations in Labrador retriever dogs. Up to a concentration of 1 mM, gabapentin displayed no agonistic effects on either the GABA(B(1a,2)) or the GABA(B(1b,2)) heterodimer, when these were expressed in Xenopus laevis oocytes or mammalian cells and assayed by means of electrophysiology, calcium mobilization, inositol phosphate, and fluorometry assays. Gabapentin did not displace [(3)H]GABA from GABA(B) receptor sites in rat synaptic membranes. Finally, in contrast to the classic GABA(B) receptor agonist baclofen, gabapentin was unable to inhibit transient lower esophageal sphincter relaxations in dogs. Because of high levels of GABA(B(1a)) in the canine nodose ganglion, this finding indirectly supports the inactivity of gabapentin on the GABA(B(1a,2)) heterodimer demonstrated in various in vitro assays. In light of these results, we find it highly questionable that gabapentin is a GABA(B) receptor agonist. Hence, the anticonvulsive effects of the compound have to arise from GABA(B) receptor-independent mechanisms. This also implies that the first GABA(B) receptor splice variant-selective ligand remains to be discovered.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152-2159). In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca(2+) channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA(B) receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and G-protein activation, these two functions are assumed by two distinct subunits in the GABA(B) heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype-specific antibodies in pre- and postembedding immunohistochemistry combined with three-dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre- and postsynaptic GABA(B)R1a/b and GABA(B)R2 in the rat cerebellum and ventrobasal thalamus. GABA(B)R1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one-tenth of the synaptic membrane specialization of spine-parallel fibre synapses were labelled at pre- or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABA(B)R1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABA(B)R1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABA(B)Rs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABA(B)Rs differentially regulate activity of different neuronal populations.

The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of gamma-aminobutyric acid(B) (GABA(B)) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate) (GTP gamma[(35)S]) binding to membranes from a GABA(B(1b/2)) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTP gamma[(35)S] binding assay using a native GABA(B) receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTP gamma[(35)S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B(1b/2)) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABA(B(1b)) receptors. In addition to GTP gamma[(35)S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca(2+) signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect of L-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABA(B) receptors may represent a novel means of therapeutic interference with the GABA-ergic system.

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

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.

Although studies in the visual cortex have found gamma-aminobutyric acid B (GABA(B)) receptor-mediated pre- and postsynaptic inhibitory effects on neurons, the subcellular localization of GABA(B) receptors in different types of cortical neurons and synapses has not been shown directly. To provide this information, we have used antibodies against the GABA(B) receptor (R)1a/b and GABA(B)R2 subunits and have studied the localization of immunoreactivities in rat visual cortex. Light microscopic analyses have shown that both subunits are expressed in cell bodies and dendrites of 65-92% of corticocortically projecting pyramidal neurons and in 92-100% of parvalbumin (PV)-, calretinin (CR)-, and somatostatin (SOM)-containing GABAergic neurons. Electron microscopic analyses of immunoperoxidase- and immunogold-labeled tissue revealed staining in the nucleus, cytoplasm and cell surface membranes with both antibodies. Colocalization of both subunits was observed in all of these structures. GABA(B)R1a/b and GABA(B)R2 were concentrated in excitatory and inhibitory synapses and in extrasynaptic membranes. In GABAergic synapses, GABA(B)R1a/b and GABA(B)R2 were more strongly expressed postsynaptically on pyramidal and nonpyramidal cells than presynaptically. In type 1 synapses GABA(B)R1a/b and GABA(B)R2 was found in pre- and postsynaptic membranes. The nuclear localization of GABA(B)R1 and GABA(B)R2 subunits suggests a novel role for neurotransmitter receptors in controlling gene expression. The synaptic colocalization of GABA(B)R1 and GABA(B)R2 indicates that subunits form heteromeric assemblies of the functional receptor in inhibitory and excitatory synapses. Subunit coexpression in GABAergic synapses that include PV-containing and PV-deficient terminals suggests that pre- and postsynaptic GABA(B) receptor activation is provided by several different types of interneurons. The coexpression of both subunits in excitatory synapses suggests a role for GABA(B) receptors in the regulation of glutamate release and raises the question how these receptors are activated in the absence of pre-or postsynaptic GABAergic synaptic inputs to excitatory synapses., (Copyright 2001 Wiley-Liss, Inc.)

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Outlined is the rationale behind the syntheses of radioligands [125I]CGP64213 and [125I]CGP71872, which led to the identification of cloned GABA(B) receptors 1a and 1b 17 years after the first pharmacological characterisation of native GABA(B) receptors by Bowery et al. [Nature 283 (1980) 92-94]. More recently it was shown that the N-terminal extracellular domains of GABA(B) receptors 1a and 1b contain the binding sites for agonists and antagonists [B. Malitschek et al., Mol. Pharmacol. 56 (1999) 448-454]. In order to isolate the extracellular domain(s) of GABA(B) receptors 1a (or 1b) and to purify and crystallise these proteins a third ligand [125I]CGP84963 was designed, which combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as photoaffinity moiety and 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion [W. Froestl et al., Neuropharmacology 38 (1999) 1641-1646].

The gamma-amino-n-butyric acid type B (GABA(B)) receptor is composed of two subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix receptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding proteins that are constituted of two lobes that close upon ligand binding. Here, using molecular modeling and site-directed mutagenesis, we have identified residues in the GABA(B)1 subunit that are critical for agonist binding and activation of the heteromeric receptor. Our data suggest that two residues (Ser(246) and Asp(471)) located within lobe I form H bonds and a salt bridge with carboxylic and amino groups of GABA, respectively, demonstrating the pivotal role of lobe I in agonist binding. Interestingly, our data also suggest that a residue within lobe II (Tyr(366)) interacts with the agonists in a closed form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal role played by the GABA(B)1 subunit in the activation of the heteromeric GABA(B) receptor and are consistent with the idea that a closed state of the binding domain of family 3 receptors is required for their activation.

Kainate receptor activation affects GABAergic inhibition in the hippocampus by mechanisms that are thought to involve the GluR5 subunit. We report that disruption of the GluR5 subunit gene does not cause the loss of functional KARs in CA1 interneurons, nor does it prevent kainate-induced inhibition of evoked GABAergic synaptic transmission onto CA1 pyramidal cells. However, KAR function is abolished in mice lacking both GluR5 and GluR6 subunits, indicating that KARs in CA1 stratum radiatum interneurons are heteromeric receptors composed of both subunits. In addition, we show the presence of presynaptic KARs comprising the GluR6 but not the GluR5 subunit that modulate synaptic transmission between inhibitory interneurons. The existence of two separate populations of KARs in hippocampal interneurons adds to the complexity of KAR localization and function.

gamma-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of gamma-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K(+) (Kir3) conductance. These GABA type B (GABA(B))-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABA(B) agonist baclofen. Recent studies of native GABA(B) receptors (GABA(B)Rs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABA(B)Rs assemble heteromeric complexes from the GABA(B)R1 and GABA(B)R2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABA(B) agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K(+) current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABA(B) antagonists, and prevented by pertussis toxin pretreatment. Both GABA(B)R1 and GABA(B)R2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABA(B)Rs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

The gamma-aminobutyric acid (GABA) receptor type B (GABA(B)R) is constituted of at least two homologous proteins, GABA(B)R1 and GABA(B)R2. These proteins share sequence and structural similarity with metabotropic glutamate and Ca(2+)-sensing receptors, both of which are sensitive to Ca(2+). Using rat brain membranes, we report here that the affinity of GABA and 3-aminopropylphosphinic acid for the GABA(B)R receptor is decreased by a factor >10 in the absence of Ca(2+). Such a large effect of Ca(2+) is not observed with baclofen or the antagonists CGP64213 and CGP56999A. In contrast to baclofen, the potency of GABA in stimulating GTPgammaS binding in rat brain membranes is also decreased by a factor >10 upon Ca(2+) removal. The potency for Ca(2+) in regulating GABA affinity was 37 microM. In cells expressing GABA(B)R1, the potency of GABA, but not of baclofen, in displacing bound (125)I-CGP64213 was similarly decreased in the absence of Ca(2+). To identify residues that are responsible for the Ca(2+) effect, the pharmacological profile and the Ca(2+) sensitivity of a series of GABA(B)R1 mutants were examined. The mutation of Ser269 into Ala was found to decrease the affinity of GABA, but not of baclofen, and the GABA affinity was found not to be affected upon Ca(2+) removal. Finally, the effect of Ca(2+) on the GABA(B) receptor function is no longer observed in cells coexpressing this GABA(B)R1-S269A mutant and the wild-type GABA(B)R2. Taken together, these results show that Ser269, which is conserved in the GABA(B)R1 protein from Caenorhabditis elegans to mammals, is critical for the Ca(2+)-effect on the heteromeric GABA(B) receptor.

Gamma-hydroxybutyrate (GHB) is a neuromodulator with high affinity binding sites in the mammalian brain. However, the receptor for GHB has not yet been identified. There are indications that GHB and gamma-aminobutyric acid (GABA) mediate their effects via the same receptor. We tested this hypothesis using GABA(B)R1/R2 receptors co-expressed with Kir3 channels in Xenopus oocytes. GHB activated these receptors with an EC50 of approximately 5 mM and a maximal stimulation of 69% when compared to the GABA(B) receptor agonist L-baclofen. GHB and L-baclofen did not amplify each others effect nor did they stimulate the GABA(B) receptor in a linearly additive manner. CGP54626A, 2-OH saclofen and CGP35348, three competitive GABA(B) receptor antagonists, inhibited the GHB induced response completely. A concentration of 30 mM GHB displaced [125I]CGP64213 binding at GABA(B)R1 expressed in COS cells by 21%. These results indicate that GHB is a weak partial agonist at the GABA binding site of GABA(B)R1/R2.

Since the discovery that the most abundant inhibitory neurotransmitter in the mammalian brain, GABA (gamma-aminobutyric acid), interacts not only with ionotropic GABA(A) receptors, but also with metabotropic GABA(B) receptors (Bowery et al., 1980) much work has been devoted to the elucidation of the structure of GABA(B) receptors by either affinity chromatography purification or by expression cloning. In 1997 Kaupmann et al. succeeded in cloning two splice variants designated GABA(B) R1a (960 amino acids) and GABA(B) R1b (844 amino acids). Although the amino acid sequences are now known, precise information on the three-dimensional environment of the GABA(B) R1 binding site is still lacking. Recent experiments demonstrated that the amino acids of the seven transmembrane helices are not essential for ligand binding as a soluble GABA(B) receptor fragment is still able to bind antagonists (Malitschek et al., 1999). For the isolation and purification of the soluble N-terminal extracellular domain (NTED) of GABA(B) receptors potent ligands for affinity chromatography were synthesised with the aim of obtaining a crystalline receptor fragment-ligand complex for X-ray structure determination. The most promising ligand [125I]CGP84963 (K(D) = 2 nM) combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as a photoaffinity moiety separated by a spacer consisting of three GABA molecules from 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion.

A gamma-aminobutyric acid (GABA)(B) receptor (named GABA(B)R1) has been recently cloned in the rat and human brain and two variants generated by alternative RNA splicing were identified. In the present study, we addressed the question as to whether these variants contribute to the diversity of GABA(B) receptor-mediated physiological responses and constitute real receptor subtypes with distinct functions. To this aim, we have mapped the GABA(B)R1 (R1a) and GABA(B)R1b (R1b) transcript distribution in the rat brain using in situ hybridization. We have compared the mRNA distribution with the distribution of [(3)H]CGP54626-labeled binding GABA(B)R1 receptor sites as assessed in adjacent cryosections by quantitative autoradiography. We found that GABA(B) receptor transcripts and binding sites are expressed in the brain in almost all neuronal cell populations. Expression in glial cells, if any, is marginal. We observed a good parallelism between GABA(B)R1 mRNA transcripts and binding sites in broad neuroanatomical entities with highest densities in hippocampus, thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit marked differences in their regional and cellular distribution pattern. A typical example is the cerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in the granule, stellate, and basket cells. Data pointing at a pre- versus postsynaptic localization for R1a and R1b, respectively, at some neuronal sites are presented., (Copyright 1999 Wiley-Liss, Inc.)

The novel GABA(B) receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (K(d) = 0.9 nM, B(max) = 760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (K(d) = 0.5 nM, B(max) = 1285 fmol/mg protein). Competition of [3HCGP 35348 = CGP 36742. The GABA(A) ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABA(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA(B)R1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor.

Here we present a novel isoform of the metabotropic G-protein-coupled receptor for gamma-aminobutyric acid (GABA). The isoform, termed GABA(B)R1c (R1c), differs from the recently identified R1a and R1b receptors by an in-frame insertion of 31 amino acids between the second extracellular loop and the fifth transmembrane region. Analysis of the rat GABA(B)R1 gene demonstrates that the insertion is the result of an alternative splicing event within a 567-bp intron between exons 16 and 17. In situ hybridization in the rat brain shows a wide distribution of R1c transcripts and an overlap with the R1a and R1b transcripts. The highest mRNA levels are found in cerebellar Purkinje cells, cerebral cortex, thalamus and hippocampal CA1 and CA3 regions. Western blots and immunodetection of recombinant epitope-tagged receptors as well as [125I]CGP71872 photoaffinity labelling of cell membranes demonstrate that R1c is correctly expressed, although at a lower level than the previously identified isoforms. When coexpressed with the newly characterized GABA(B)R2, R1c functionally couples to G-protein-activated Kir3.1/3.2 channels in Xenopus oocytes and to PLC-activating chimeric G(alpha)qo subunits in HEK-293 cells with a similar EC50 for agonists. These data suggest that the R1c isoform represents a functional GABA(B)R in the rat brain.

The recently identified gamma-aminobutyric acid type B receptors (GABA(B)Rs) share low sequence similarity with the metabotropic glutamate (mGlu) receptors. Like the mGlu receptors, the N-terminal extracellular domain (NTED) of GABA(B)Rs is proposed to be related to bacterial periplasmic binding proteins (PBPs). However, in contrast to the mGlu receptors, the GABA(B)Rs lack a cysteine-rich region that links the PBP-like domain to the first transmembrane domain. This cysteine-rich region is necessary for the PBP-like domain of mGlu receptors to bind glutamate. To delimit the ligand-binding domain of GABA(B)Rs, we constructed a series of chimeric GABA(B)R1/mGluR1 and truncated GABA(B)R1 receptor mutants. We provide evidence that despite the lack of a cysteine-rich region, the NTED of GABA(B)Rs contains all of the structural information that is necessary and sufficient for ligand binding. Moreover, a soluble protein corresponding to the NTED of GABA(B)Rs reproduces the binding pharmacology of wild-type receptors. This demonstrates that the ligand-binding domain of the GABA(B)Rs can correctly fold when dissociated from the transmembrane domains.

B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

gamma-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

Neurophysiological and pharmacological studies suggest a major role of the GABAB receptor in the epileptogenesis of absence seizures. The gene encoding the human GABABR1 receptor (GABABR1) has recently been mapped to human chromosome 6p21.3 by in situ hybridization, a region that harbors a susceptibility locus (EJM1) for idiopathic generalized epilepsy (IGE). We investigated the hypothesis that the GABABR1 gene (GABBR1) represents a candidate gene for EJM1 by: (1) defining the precise localization approximately 130 kilobases telomeric to the HLA-F locus, (2) by characterizing its genomic organization, and (3) by mutation screening of the entire coding region of GABBR1 in 18 German patients with juvenile myoclonic epilepsy (JME) who were derived from families with evidence for linkage to chromosome 6p21.3 (cumulative lod score Z=3.17 at HLA-DQ). The GABAB receptor gene consists of 22 translated exons. The two alternative transcripts, GABABR1a and GABABR1b, are derived from the same locus but they differ in their alternative 5'-exons. Mutation analyses in JME revealed several DNA sequence polymorphisms, two of which result in amino acid changes occurring in all IGE-affected members of two families. However, clinically unaffected relatives did carry the same variations, excluding these amino acid substitutions as the cause for IGE in these families.

The mRNA distribution of the two cloned GABA(B) receptor variants, GABA(B)R1a and -R1b, was analysed in the retina by non-radioactive in situ hybridization. GABA(B)R1a transcripts were found in the inner nuclear and ganglion cell layers, probably in horizontal, amacrine and ganglion cells, whereas GABA(B)R1b transcripts were detected in the ganglion cell layer only. Together with a recent immunohistochemical localization of GABA(B)R1 in the retina, this indicates a differential targeting of the receptor variants to pre- and postsynaptic sites with GABA(B)R1a and -R1b localized to axonal and dendritic compartments, respectively. In this way, inhibition of neurotransmitter release and slow postsynaptic inhibition could be provided by receptor variants derived from the same gene.

Recently, two N-terminal splice variants of the metabotropic receptor for GABA (gamma-amino-butyric acid) were cloned. Here, we describe an antiserum that recognizes the two receptor variants. We demonstrate that these proteins are identical with GABAB receptors that are photoaffinity labeled with [125I]CGP71872 in rat brain. The C-terminal epitopes recognized by the antiserum are conserved in several vertebrate species but not in chicken. No hints for the existence of additional closely related receptor subtypes or variants are found in double-labeling experiments with antibody and photoaffinity ligand. Western blot analysis reveals widespread expression of the GABABR1 receptor proteins in rat brain with the highest level of expression at early postnatal stages. The binding affinity of the GABAB receptor agonist L-baclofen at native R1a and R1b variants is similar. In early postnatal development the affinity at R1a and R1b is 10-fold lower than in adult brain and gradually increases with aging., (Copyright 1998 Academic Press.)

Recently, the long-awaited cloning of the GABAB receptors, the last of the major known neurotransmitter receptors to be identified, has been reported. In addition to an emerging molecular understanding, there have been advances in discerning the specific coupling partners of GABAB receptors in the brain.

L-glutamate, the neurotransmitter of the majority of excitatory synapses in the brain, acts on three classes of ionotropic receptors: NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptors. Little is known about the physiological role of kainate receptors because in many experimental situations it is not possible to distinguish them from AMPA receptors. Mice with disrupted kainate receptor genes enable the study of the specific role of kainate receptors in synaptic transmission as well as in the neurotoxic effects of kainate. We have now generated mutant mice lacking the kainate-receptor subunit GluR6. The hippocampal neurons in the CA3 region of these mutant mice are much less sensitive to kainate. In addition, a postsynaptic kainate current evoked in CA3 neurons by a train of stimulation of the mossy fibre system is absent in the mutant. We find that GluR6-deficient mice are less susceptible to systemic administration of kainate, as judged by onset of seizures and by the activation of immediate early genes in the hippocampus. Our results indicate that kainate receptors containing the GluR6 subunit are important in synaptic transmission as well as in the epileptogenic effects of kainate.

The recently cloned GABA(B) receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (gamma-aminobutyric acid) and GABA(B) receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA(B) receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA(B) receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA(B) receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA(B) receptors. In addition, the extrasynaptic localization of GABA(B) receptors could indicate a paracrine function of GABA in the retina.

In an attempt to gain knowledge of the possible functions of kainate receptors, we have used in situ hybridization to examine the regional and cellular expression patterns of glutamate receptor subunits GluR5-7, KA1 and KA2 in the adult mouse basal ganglia, known to play a pivotal role in the translation of motivation into actions. Kainate receptor subunits were found to be differentially expressed in the circuitry forming the basal ganglia. They differ from each other in expression levels and their spatial localization. GluR6 appeared as the key subunit for the descending gamma-aminobutyric acid (GABA)ergic-glutamatergic pathways, with highest message levels in the caudate putamen, globus pallidus and subthalamic nucleus as well as in the nucleus accumbens and olfactory tubercle. GluR7 exhibited highest expression in the ascending nigrostriatal and mesolimbic dopaminergic neurons. GluR5 had a restricted distribution pattern, with high expression in the ventral pallidum, the islands of Calleja and pars compacta of the substantia nigra. KA2 was usually coexpressed with GluR6, although with a generally lower level of expression. Finally, KA1 mRNA was barely detectable in these neuronal circuits. These data suggest that kainate receptors in general may be involved in the functions associated with the basal ganglia, with a key role in the control of the central dopaminergic transmission. Thus, they might be implicated in the neurodegenerative and psychic disorders associated with an impairment of the basal ganglia.

GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.