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1.
Neuroscience ; 166(2): 377-85, 2010 Mar 17.
Article in English | MEDLINE | ID: mdl-20035841

ABSTRACT

As the pathophysiological mechanism(s) of many neuropsychiatric disorders relate to GABAergic interneuron structure and function, we employed isolation rearing of Wistar rats as a model to correlate developmental emergence of cognitive deficits with the expression of reelin-producing interneurons in the medial prefrontal cortex (PFC). Prepulse inhibition deficits emerged at postnatal day 60 and persisted into adulthood. Paralleling the emergence of these neurobehavioural deficits was an increase in reelin production and reelin-immunopositive cells in layer I of the PFC and this later became significantly reduced at postnatal day 80. Cells expressing reelin immunoreactivity in a horizontal orientation were mainly located to the upper regions of layer I whereas those with a vertical orientation, whose arbors extend into cortical layers II and III, were more numerous in the lower regions of layer I and became significantly dysregulated during postnatal development. No behavioural deficits or altered reelin expression was observed at postnatal days 30 or 40. Developmental emergence of neurobehavioural and reelin deficits in isolation reared animals is proposed to reflect maladaptive wiring within the medial prefrontal cortex during a critical maturation period of this circuitry.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Extracellular Matrix Proteins/metabolism , Interneurons/metabolism , Nerve Tissue Proteins/metabolism , Prefrontal Cortex/metabolism , Sensory Gating/physiology , Serine Endopeptidases/metabolism , Social Isolation , Animals , Behavior, Animal/physiology , Blotting, Western , Cell Count , Housing, Animal , Male , Motor Activity/physiology , Nerve Net/metabolism , Rats , Rats, Wistar , Reelin Protein , Reflex, Startle/physiology
2.
Br J Pharmacol ; 157(2): 307-19, 2009 May.
Article in English | MEDLINE | ID: mdl-19338583

ABSTRACT

BACKGROUND AND PURPOSE: As a combination of 5-HT selective reuptake inhibitor (SSRI) with 5-HT(1A) receptor antagonism may yield a rapidly acting antidepressant, WAY-211612, a compound with both SSRI and 5-HT(1A) receptor antagonist activities, was evaluated in preclinical models. EXPERIMENTAL APPROACH: Occupancy studies confirmed the mechanism of action of WAY-211612, while its in vivo profile was characterized in microdialysis and behavioural models. KEY RESULTS: WAY-211612 inhibited 5-HT reuptake (K(i) = 1.5 nmol.L(-1); K(B) = 17.7 nmol.L(-1)) and exhibited full 5-HT(1A) receptor antagonist activity (K(i) = 1.2 nmol.L(-1); K(B) = 6.3 nmol.L(-1); I(max) 100% in adenyl cyclase assays; K(B) = 19.8 nmol.L(-1); I(max) 100% in GTPgammaS). WAY-211612 (3 and 30 mg.kg(-1), po) occupied 5-HT reuptake sites in rat prefrontal cortex (56.6% and 73.6% respectively) and hippocampus (52.2% and 78.5%), and 5-HT(1A) receptors in the prefrontal cortex (6.7% and 44.7%), hippocampus (8.3% and 48.6%) and dorsal raphe (15% and 83%). Acute or chronic treatment with WAY-211612 (3-30 mg.kg(-1), po) raised levels of cortical 5-HT approximately twofold, as also observed with a combination of an SSRI (fluoxetine; 30 mg.kg(-1), s.c.) and a 5-HT(1A) antagonist (WAY-100635; 0.3 mg.kg(-1), s.c). WAY-211612 (3.3-30 mg.kg(-1), s.c.) decreased aggressive behaviour in the resident-intruder model, while increasing the number of punished crossings (3-30 mg.kg(-1), i.p. and 10-56 mg.kg(-1), po) in the mouse four-plate model and decreased adjunctive drinking behaviour (56 mg.kg(-1), i.p.) in the rat scheduled-induced polydipsia model. CONCLUSIONS AND IMPLICATIONS: These findings suggest that WAY-211612 may represent a novel antidepressant.


Subject(s)
Antidepressive Agents, Second-Generation/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin 5-HT1 Receptor Antagonists , Serotonin Antagonists/pharmacology , Animals , Behavior, Animal/drug effects , Chromatography, High Pressure Liquid , Cyclic AMP/metabolism , Male , Mice , Microdialysis , Rats , Rats, Sprague-Dawley
3.
Biochem Soc Trans ; 33(Pt 4): 553-8, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16042543

ABSTRACT

AD (Alzheimer's disease) is characterized neuropathologically by the presence of amyloid plaques, neurofibrillary tangles and profound grey matter loss. The 'amyloid' hypothesis postulates that the toxic Abeta (amyloid beta) peptide, enzymatically derived from the proteolytic processing of a larger protein called APP (amyloid precursor protein), is one of the principal causative factors of neuronal cell death in the brains of AD patients. As such, methods for lowering Abeta levels in the brain are of significant interest with regard to identifying novel disease modifying therapies for the treatment of AD. In this review, we will review a variety of approaches and mechanisms capable of modulating levels of Abeta.


Subject(s)
Alzheimer Disease/therapy , Amyloid beta-Peptides/metabolism , Alzheimer Disease/immunology , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/drug effects , Aspartic Acid Endopeptidases , Endopeptidases/metabolism , Humans , Immunization , Protein Processing, Post-Translational
4.
J Neurosci ; 25(6): 1459-69, 2005 Feb 09.
Article in English | MEDLINE | ID: mdl-15703400

ABSTRACT

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.


Subject(s)
Cell Surface Extensions/physiology , Lysophospholipids/pharmacology , Nerve Tissue Proteins/physiology , Oligodendroglia/metabolism , Receptors, Lysosphingolipid/physiology , Sphingosine/analogs & derivatives , Amino Acid Sequence , Animals , Ankyrins/analysis , Brain/cytology , Brain/growth & development , Brain Chemistry , Cell Differentiation , Cell Lineage , Cell Shape/drug effects , Cell Surface Extensions/drug effects , Cell Survival/drug effects , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cells, Cultured/ultrastructure , Crosses, Genetic , Female , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Kv1.1 Potassium Channel , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Oligodendroglia/drug effects , Oligodendroglia/ultrastructure , Phosphorylation , Potassium Channels, Voltage-Gated/analysis , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-akt , RNA, Messenger/analysis , RNA, Small Interfering/pharmacology , Rats , Rats, Wistar , Receptors, Lysosphingolipid/deficiency , Receptors, Lysosphingolipid/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Sphingosine/pharmacology , rho-Associated Kinases
5.
Brain Res ; 989(2): 135-46, 2003 Nov 07.
Article in English | MEDLINE | ID: mdl-14556935

ABSTRACT

Using a homology-based bioinformatics approach we have identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) may be a putative novel GABA(B) receptor subunit. We have developed a rabbit polyclonal antisera specific to the GABA(BL) protein and assessed the distribution of GABA(BL) in the rat CNS by immunohistochemistry. Protein expression was particularly dense in regions previously shown to contain known GABA(B) receptor subunits. Dense immunoreactivity was observed in the cortex, major subfields of the hippocampus and the dentate gyrus. GABA(BL) labelling was very conspicuous in the cerebellum, both in the granule cell layer and in Purkinje cells, and was also observed in the substantia gelatinosa and ventral horn motor neurons of the spinal cord. GABA(BL) immunoreactivity was also noted in a subset of parvalbumin positive hippocampal interneurons. Our data suggest a widespread distribution of GABA(BL) throughout the rat CNS.


Subject(s)
Central Nervous System/metabolism , Receptors, GABA-B/metabolism , Animals , Astrocytes/metabolism , Calbindin 2 , Central Nervous System/anatomy & histology , Computational Biology/methods , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Male , Neurons/metabolism , Parvalbumins/metabolism , Peptides/immunology , Rabbits , Rats , Rats, Sprague-Dawley , S100 Calcium Binding Protein G/metabolism , Sequence Homology, Amino Acid
6.
Mol Cell Neurosci ; 24(1): 214-23, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14550781

ABSTRACT

GABA(B) receptor subunits are widely expressed on neurons throughout the CNS, at both pre- and postsynaptic sites, where they mediate the late, slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. The existence of functional GABA(B) receptors on nonneuronal cells has been reported previously, although the molecular composition of these receptors has not yet been described. Here we demonstrate for the first time, using immunohistochemistry the expression of GABA(B1a), GABA(B1b), and GABA(B2) on nonneuronal cells of the rat CNS. All three principle GABA(B) receptor subunits were expressed on these cells irrespective of whether they had been cultured or found within brain tissue sections. At the ultrastructural level GABA(B) receptor subunits were expressed on astrocytic processes surrounding both symmetrical and assymetrical synapses in the CA1 subregion of the hippocampus. In addition, GABA(B1a), GABA(B1b), and GABA(B2) receptor subunits were expressed on activated microglia in culture but were not found on myelin forming oligodendrocytes in the white matter of rat spinal cord. Together these data demonstrate that the obligate subunits of functional GABA(B) receptors are expressed in astrocytes and microglia in the rat CNS.


Subject(s)
Astrocytes/metabolism , Brain/metabolism , Microglia/metabolism , Receptors, GABA-B/biosynthesis , Animals , Astrocytes/ultrastructure , Brain/ultrastructure , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cells, Cultured , Immunohistochemistry , Male , Microglia/ultrastructure , Microscopy, Electron , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , Protein Subunits/biosynthesis , Rats , Rats, Sprague-Dawley , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolism
7.
Brain Res Mol Brain Res ; 110(2): 305-17, 2003 Feb 20.
Article in English | MEDLINE | ID: mdl-12591167

ABSTRACT

Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.


Subject(s)
Brain/metabolism , GTP-Binding Proteins/isolation & purification , Protein Subunits/isolation & purification , Receptors, GABA-B/isolation & purification , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , Cells, Cultured , Chromosome Mapping , Chromosomes, Human, Pair 3/genetics , Cloning, Molecular , DNA, Complementary/analysis , DNA, Complementary/genetics , GTP-Binding Proteins/genetics , Humans , Immunohistochemistry , Male , Mice , Molecular Sequence Data , Molecular Structure , Phylogeny , Protein Structure, Tertiary/genetics , Protein Subunits/genetics , Rats , Receptors, GABA-B/genetics
8.
Auton Autacoid Pharmacol ; 22(3): 147-54, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12452899

ABSTRACT

1. GABA(B1) receptor subunit knockout mice were generated and the effects of the GABA(B) receptor agonist, baclofen, were evaluated within the peripheral nervous system (PNS) of wildtype (+/+), heterozygote (+/-) and knockout (-/-) animals. For this purpose, neuronally-mediated responses were evoked in both the isolated ileum and urinary bladder, using selective electrical field stimulation (EFS). 2. In ileum resected from 4-8-week-old-mice, low frequencies of EFS (0.5 Hz) evoked irregular muscle contractions which were prevented by atropine 1 microM and reduced by baclofen (33.4 +/- 5.6%, 100 microm). The latter effect was antagonized by the GABA(B) receptor antagonist CGP54626 0.2 microm. Baclofen 100 microm did not affect contractions of similar amplitude induced by carbachol, indicating that the ability of baclofen to inhibit cholinergic function in mouse ileum may be due to an action at prejunctional GABA(B) receptors. 3. To avoid the development of grand mal seizure by GABA(B1) (-/-) mice, a behaviour observed when the mice were greater than 3 weeks old, it was necessary to study the effects of this knockout in 1-3-week-old-animals. However, at this age, EFS at 0.5 Hz did not evoke robust muscle contractions. Consequently we used EFS at 5 Hz, which did evoke cholinergically mediated contractions, found to be of similar amplitude in (+/+) and (+/-) mice, of both 1-3 weeks and 4-8 weeks of age. At this frequency of EFS, baclofen reduced the amplitude of the evoked contractions [n = 6 (+/+) and n = 5 (+/-), IC50 19.2 +/- 4.8 microm) and this effect was greatly reduced in the presence of CGP54626 0.2 microm. 4. In urinary bladder from 1-3-week-old-mice, using higher frequencies of EFS to evoke clear, nerve-mediated contractions (10 Hz), baclofen 10-300 microm concentration-dependently inhibited contractions in (+/+) mice (IC50 9.6 +/- 3.8 microm). This effect was inhibited by CGP54626 (0.2 microm, 46.2 +/- 13.6% inhibition, 300 microm baclofen n = 7) a concentration which, by itself, had no effect on the EFS-evoked contractions. 5. The effects of baclofen in both ileum and urinary bladder were absent in the GABA(B1) receptor subunit (-/-) mice; however, responses to EFS were unaffected in (-/-) when compared to the (+/+) mice. 6. Our data suggest that, as in the central nervous system (CNS), the GABA(B1) receptor subunit is an essential requirement for GABA(B) receptor function in the enteric and PNS. As such, these data do not provide a structural explanation for the existence of putative subtypes of GABA(B) receptor, suggested by studies such as those in which different rank-orders of GABA(B) agonist affinity have been reported in different tissues.


Subject(s)
Ileum/physiology , Protein Subunits/deficiency , Receptors, GABA-B/deficiency , Urinary Bladder/physiology , Animals , Dose-Response Relationship, Drug , Female , GABA-B Receptor Agonists , Ileum/drug effects , In Vitro Techniques , Male , Mice , Mice, Knockout , Muscle Contraction/drug effects , Muscle Contraction/physiology , Protein Subunits/agonists , Protein Subunits/genetics , Receptors, GABA-B/genetics , Urinary Bladder/drug effects
9.
Nat Neurosci ; 5(5): 415-24, 2002 May.
Article in English | MEDLINE | ID: mdl-11976702

ABSTRACT

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Receptors, GABA-B/metabolism , Animals , Brain/metabolism , Brain Chemistry , CHO Cells , COS Cells , Cell Membrane/chemistry , Cell Membrane/metabolism , Cells, Cultured , Cricetinae , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , GABA Agonists/pharmacology , GABA-B Receptor Agonists , Humans , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Patch-Clamp Techniques , Phosphoproteins/agonists , Phosphoproteins/analysis , Phosphoproteins/metabolism , Phosphorylation , Potassium Channels/metabolism , Protein Isoforms/agonists , Protein Isoforms/analysis , Protein Isoforms/metabolism , Rats , Receptors, GABA-B/analysis , Recombinant Proteins/metabolism , Signal Transduction/physiology
10.
Neuropharmacology ; 41(8): 965-75, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11747901

ABSTRACT

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.


Subject(s)
Acetates/pharmacology , Amines , Anticonvulsants/pharmacology , Cyclohexanecarboxylic Acids , GABA Agonists/pharmacology , GABA-B Receptor Agonists , gamma-Aminobutyric Acid , Acetates/metabolism , Amino Acid Sequence , Animals , Anticonvulsants/metabolism , Binding, Competitive , Dose-Response Relationship, Drug , GABA Agonists/metabolism , GABA Antagonists/metabolism , GABA Antagonists/pharmacology , GABA-B Receptor Antagonists , Gabapentin , Hippocampus/drug effects , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Rats , Receptors, GABA-B/metabolism
11.
Neuroscience ; 106(3): 447-67, 2001.
Article in English | MEDLINE | ID: mdl-11591450

ABSTRACT

GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.


Subject(s)
Brain/metabolism , Ganglia, Spinal/metabolism , Receptors, GABA-B/metabolism , Receptors, GABA/metabolism , Spinal Cord/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Antibody Specificity/immunology , Brain/cytology , Brain Stem/cytology , Brain Stem/metabolism , Cerebellum/cytology , Cerebellum/metabolism , Diencephalon/cytology , Diencephalon/metabolism , Ganglia, Spinal/cytology , Immune Sera/immunology , Immunohistochemistry , Male , Neurons/cytology , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord/cytology , Telencephalon/cytology , Telencephalon/metabolism
12.
J Neurosci ; 21(20): 8043-52, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11588177

ABSTRACT

GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.


Subject(s)
GTP-Binding Proteins/metabolism , Protein Subunits , Receptors, GABA-B/metabolism , Amino Acids/genetics , Amino Acids/metabolism , Animals , Calcium/metabolism , Cells, Cultured , Dimerization , Humans , Kidney/cytology , Kidney/metabolism , Microinjections , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Protein Binding/physiology , Rats , Receptors, GABA/genetics , Receptors, GABA/metabolism , Receptors, GABA-B/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Analysis, Protein , Signal Transduction/physiology , Structure-Activity Relationship , Superior Cervical Ganglion/cytology , Superior Cervical Ganglion/metabolism , Transfection
13.
Mol Cell Neurosci ; 17(6): 1059-70, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11414794

ABSTRACT

The recent cloning of two GABA(B) receptor subunits, GABA(B1) and GABA(B2), has raised the possibility that differences in GABA(B) receptor subunit composition may give rise to pharmacologically or functionally distinct receptors. If present, such molecular diversity could permit the selective targeting of GABA(B) receptor subtypes specifically involved in pathologies such as drug addiction, spasticity, pain, and epilepsy. To address these issues we have developed a GABA(B1) subunit knockout mouse using gene targeting techniques. In the brains of GABA(B1) null mice, all pre- and postsynaptic GABA(B) receptor function was absent demonstrating that the GABA(B1) subunit is essential for all GABA(B) receptor-mediated mechanisms. Despite this, GABA(B1) null mice appeared normal at birth, although by postnatal week four their growth was retarded and they developed a generalized epilepsy that resulted in premature death. In addition, GABA(B1) heterozygote animals showed enhanced prepulse inhibition responses compared to littermate controls, suggesting that GABA(B1) deficient mice exhibit increased sensorimotor gating mechanisms. These data suggest that GABA(B) receptor antagonists may be of benefit in the treatment of psychiatric and neurological disorders in which attentional processing is impaired.


Subject(s)
Central Nervous System/abnormalities , Epilepsy/congenital , Mice, Knockout/abnormalities , Neural Inhibition/genetics , Neurons/metabolism , Receptors, GABA-B/deficiency , Action Potentials/drug effects , Action Potentials/physiology , Animals , Baclofen/pharmacology , Behavior, Animal/physiology , Central Nervous System/metabolism , Central Nervous System/physiopathology , Down-Regulation/genetics , Epilepsy/genetics , Epilepsy/physiopathology , GABA Agonists/pharmacology , Gene Targeting/methods , Heterozygote , Mice , Mice, Knockout/anatomy & histology , Mice, Knockout/metabolism , Neurons/cytology , Phenotype , RNA, Messenger/metabolism , Radioligand Assay , Receptors, GABA-B/genetics , Receptors, GABA-B/metabolism , Reflex, Startle/drug effects , Reflex, Startle/physiology , Seizures/congenital , Seizures/genetics , Seizures/physiopathology , Synapses/drug effects , Synapses/metabolism , Synapses/ultrastructure , Synaptic Transmission/drug effects , Synaptic Transmission/genetics , gamma-Aminobutyric Acid/metabolism
14.
Neuroreport ; 12(3): 591-5, 2001 Mar 05.
Article in English | MEDLINE | ID: mdl-11234770

ABSTRACT

The distribution of GABA(B) receptor subunits GABA(B(1a)), GABA(B(1b)) and GABA(B2), has been examined in the cerebral cortex and thalamus of adult rats using an immunocytochemical technique. GABA(B(1a)) and GABA(B(1b)) subunits co-localized with GABA(B2) in the cortex, where afferent thalamic GABAergic axons project to pyramidal neurones. The expression patterns of GABA(B(1a)), GABA(B(1b)) and GABA(B2) were similar throughout the thalamus. The data suggest that the GABA(B(1b)) subunit might be the presynaptic isoform in the thalamo-cortical pathway with the GABA(B(1a)) subunit possibly present at postsynaptic sites on cell bodies. This contrasts with our previous data, obtained in cerebellum and spinal cord which indicate opposite locations. Thus, it seems unlikely that functional role along with cellular location can be assigned in a general manner to specific GABA(B) receptor subunit splice variants.


Subject(s)
Cerebral Cortex/chemistry , Receptors, GABA-B/analysis , Thalamus/chemistry , Age Factors , Amino Acid Sequence , Animals , Antibody Specificity , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Receptors, GABA-B/chemistry , Receptors, GABA-B/immunology
15.
Mol Cell Neurosci ; 17(2): 317-28, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11178869

ABSTRACT

Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.


Subject(s)
Receptors, GABA-B/genetics , Receptors, GABA-B/metabolism , Signal Transduction/physiology , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , 14-3-3 Proteins , Animals , Brain Chemistry/physiology , COS Cells , Cell Fractionation , Gene Expression/physiology , Hippocampus/cytology , In Vitro Techniques , Neurons/cytology , Neurons/metabolism , Protein Structure, Tertiary , Rats , Receptors, GABA-B/chemistry , Synapses/metabolism , Transfection , Two-Hybrid System Techniques
16.
Brain Res Mol Brain Res ; 86(1-2): 101-14, 2001 Jan 31.
Article in English | MEDLINE | ID: mdl-11165377

ABSTRACT

Potassium channels are amongst the most heterogeneous class of ion channels known and are responsible for mediating a diverse range of biological functions. The most recently described family of K+ channels, the 'two pore-domain family', contain four membrane spanning domains and two pore-forming domains, suggesting that two channel subunits associate to form a functional K+ pore. Several sub-families of the two pore domain potassium channel family have been described, including the weakly inward rectifying K+ channel (TWIK), the acid-sensitive K+ channel (TASK), the TWIK-related K+ channel (TREK) and the TWIK-related arachidonic acid stimulated K+ channel (TRAAK). However, comparison of the mRNA expression of these channels has been difficult due to the differences in methods used and the species studied. In the present study, we used a single technique, TaqMan semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), to investigate the mRNA distribution of all currently known two pore potassium channels in human central nervous system (CNS) and peripheral tissues. TWIK-1 and the TWIK-1-like channel KCNK7 were predominantly expressed in the CNS, in contrast to TWIK-2 which was preferentially expressed in peripheral tissues such as pancreas, stomach, spleen and uterus. TASK-1 was expressed in the CNS and some peripheral tissues, whereas TASK-2 was exclusively expressed in the periphery except for mRNA expression observed in dorsal root ganglion and spinal cord. In addition, mRNA expression of the recently identified TASK-3, was almost completely exclusive to cerebellum with little or no mRNA detected in any other tissues. TREK-1 and TRAAK mRNA expression was predominantly CNS specific in contrast to the closely related TREK-2, which was expressed in both CNS and peripheral tissues. Studying the mRNA expression profiles of known two pore domain K+ channels will aid in the understanding of the biological roles of these channels. Furthermore, identification of common areas of expression may help identify which channels, if any, associate to form heteromeric K+ channel complexes.


Subject(s)
Central Nervous System/physiology , Ganglia, Spinal/physiology , Nerve Tissue Proteins , Potassium Channels, Tandem Pore Domain , Potassium Channels/chemistry , Potassium Channels/genetics , Central Nervous System/chemistry , Ganglia, Spinal/chemistry , Gene Expression/physiology , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Protein Structure, Tertiary , RNA, Messenger/analysis , Sequence Homology, Amino Acid
17.
J Neurosci ; 21(4): 1203-10, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11160390

ABSTRACT

GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.


Subject(s)
Intracellular Fluid/metabolism , Protein Subunits , Protein Transport/physiology , Receptors, GABA-B/metabolism , Signal Transduction/physiology , Amino Acid Motifs/physiology , Animals , Cell Line , Cricetinae , Dimerization , GTP-Binding Proteins/metabolism , Humans , Mutagenesis, Site-Directed , Protein Structure, Tertiary/physiology , Rats , Receptors, Cell Surface/metabolism , Receptors, GABA-B/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transfection , gamma-Aminobutyric Acid/metabolism
18.
Genomics ; 70(2): 241-52, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11112352

ABSTRACT

Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.


Subject(s)
Membrane Transport Proteins/metabolism , Multienzyme Complexes/genetics , Multigene Family , Neurotransmitter Agents/metabolism , Protein Serine-Threonine Kinases/genetics , Sodium Chloride/metabolism , AMP-Activated Protein Kinases , Amino Acid Sequence , Base Sequence , Cells, Cultured , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Humans , Membrane Transport Proteins/genetics , Molecular Sequence Data , Plasma Membrane Neurotransmitter Transport Proteins , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
20.
Neuroscience ; 100(1): 155-70, 2000.
Article in English | MEDLINE | ID: mdl-10996466

ABSTRACT

GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.


Subject(s)
Central Nervous System/metabolism , Receptors, GABA-B/metabolism , Animals , Brain/metabolism , Female , Humans , Male , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-B/genetics , Spinal Cord/metabolism , Spleen/metabolism , Tissue Distribution , Uterus/metabolism
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