Metabotropic GABA Receptors Research Articles

GABA B receptors: altered coupling to G-proteins in rats sensitized to amphetamine

Modified dopamine and glutamate neurotransmission in discrete brain regions is implicated in stimulant-induced behavioral sensitization. Release of both neurotransmitters is influenced by GABA B metabotropic receptors for the principal inhibitory neurotransmitter GABA. Accordingly, GABA B receptors were examined in rats sensitized to amphetamine by measuring receptor density and coupling to G-proteins indicated as [ 3H]baclofen binding and baclofen-mediated [ 35S]GTPγS binding. Repeated treatment with (+)-amphetamine (5 mg/kg per day, i.p., for five days) sensitized the rats to amphetamine challenge (1 mg/kg) at 14 days, but not one day, later. GABA B receptor density was not altered at either time. Baclofen-mediated [ 35S]GTPγS binding, however, was selectively augmented in the prefrontal cortex and attenuated in the nucleus accumbens at 14 days, but not one day, after amphetamine treatment. Changes in GABA B receptor coupling to G-proteins in rats sensitized to amphetamine, but not in similarly treated but unsensitized rats, lead us to suggest that altered GABA B receptor functioning may contribute to the expression of amphetamine-induced behavioral sensitization.

Synaptic activation of the group I metabotropic glutamate receptor mGlu1 on the thalamocortical neurons of the rat dorsal Lateral Geniculate Nucleus in vitro

Intracellular recordings were made from thalamocortical neurons in slices of rat dorsal lateral geniculate nucleus in vitro, where ionotropic glutamate receptors and ionotropic and metabotropic GABA receptors had been blocked. The activation of specific metabotropic glutamate receptors by exogenous agonists and by the electrical stimulation of the corticothalamic pathway was then assessed using selective antagonists. The specific group I agonist ( S)-3,5-dihydoxyphenylglycine and the non-selective agonist (1 S,3 R)-1-aminocyclo-pentane-1,3-dicarboxylic acid both caused a concentration-dependent depolarization of membrane potential. These effects were associated with an increase in the apparent input resistance, and a more robust expression of both the depolarizing sag of the voltage response and the low-threshold Ca 2+ potential and an increase in thalamocortical neuron excitability. However, group I agonists selective for the mGlu5 receptor and agonists selective for group II and III receptors did not have these effects. Consequently, these data suggested that these actions were mediated specifically by the group I mGlu1 receptor. The activation of cortical fibres, with trains of 50 stimuli at 50 Hz, resulted in a two-component depolarizing response. The first part of this synaptic response and the agonist-induced depolarization of membrane potential were depressed by the novel group I receptor antagonists LY367366 and LY367385, which are active at mGlu1 receptors. However, they were not blocked by 6-methyl-2-(phenylethyl)-pyridine, a highly selective mGlu5 receptor antagonist. Thus, the membrane potential depolarization of thalamocortical neurons caused either by exogenous agonists or by the stimulation of cortical fibres resulted from the specific activation of mGlu1 but not mGlu5 receptors. This result is consistent with the location of this receptor type on the distal dendrites of thalamocortical neurons in the dorsal lateral geniculate nucleus of the thalamus.

Functional effect of metabotropic glutamate and GABA receptors on Insulin Secretion

Functional effect of metabotropic glutamate and GABA receptors on Insulin Secretion

GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia.

The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, approximately 90% was of the GABAB(1a) form and approximately 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.

The metabotropic GABA receptor: molecular insights and their functional consequences.

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.

Intracellular signalling pathways modulate K ATP channels in inspiratory brainstem neurones and their hypoxic activation: involvement of metabotropic receptors, G-proteins and cytoskeleton

K ATP channels regulate the neuronal excitability and their activation during hypoxia/ischemia protect neurons. The activation of K ATP channels during hypoxia is assumed to occur mainly due to the fall in intracellular ATP levels, but other intracellular signalling pathways can be also involved. We measured single K ATP channel currents in inspiratory brainstem neurones of neonatal mice. The activity of K ATP channels was enhanced in hypoosmotic bath solutions, or after applying negative pressure to the recording pipette. Cytochalasin B activated K ATP channels and prevented the effects of osmo-mechanical stress, indicating that cytoskeleton rearrangements, which occur during hypoxia, contribute to the activation of K ATP channels. During hypoxia, extracellular levels of many neurotransmitters increase, leading to activation of corresponding metabotropic receptors that can modulate K ATP channels. K ATP channels were activated by GABA B agonist, baclofen, by mGLUR2/3 agonists and were inhibited by mGLUR1/5 agonists. K ATP channels were activated by phorbol esters and were inhibited by staurosporine. These treatments did not occlude the modulating actions of mGLUR agonists, indicating that they are not mediated by protein kinase C. Activator of α-subunits of G-proteins Mas 7 increased and their inhibitor GPant-2 decreased the activity of K ATP channels. In the presence of either agent, the modulatory actions of baclofen and mGLUR agonists were not observed. We conclude that K ATP channels are modulated by G-proteins that are activated by metabotropic receptors for GABA and glutamate and their release during hypoxia complements activation of channels by osmo-mechanical stress and [ATP] i depletion.

Internal calcium modulates apparent affinity of metabotropic GABA receptors.

The metabotropic GABA receptor (GABA(B)R) regulates calcium influx in neurons. Whole cell voltage-clamp techniques were employed to determine the effects of internal calcium on the activity of GABA(B)Rs. GABA(B)R receptor apparent affinity was maximal when bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) maintained internal calcium below 70 nM. Apparent affinity was reduced as internal calcium increased. EGTA did not produce similar effects, suggesting that localized increases in calcium influenced GABA(B)R apparent affinity. Confocal imaging disclosed relatively high internal calcium just below the plasma membrane of isolated neurons. BAPTA, but not EGTA, reduced this ring of high calcium. Heparin, dantrolene, and ryanodine increased GABA(B)R apparent affinity, effects similar to that of BAPTA. Calmodulin inhibitors also increased receptor apparent affinity. These results suggest that internally released calcium activates calmodulin, which reduces GABA(B)R apparent affinity. This identifies a reciprocal system in which the metabotropic GABA receptor can reduce calcium influx, but internal calcium can suppress this receptor pathway. Metabotropic glutamate receptors linked to inositol 1,4,5 trisphosphate (InsP(3)) raised internal calcium and suppressed the action of GABA(B)Rs. Thus negative feedback systems control the balance between excitatory and inhibitory metabotropic receptor pathways in retinal neurons.

Metabotropic GABA receptors facilitate L-type and inhibit N-type calcium channels in single salamander retinal neurons.

1. Whole-cell voltage clamp experiments were performed on isolated spiking retinal neurons from the salamander retina. Calcium channel currents were studied using barium as the charge carrier while potassium and sodium currents were suppressed with TEA and TTX, respectively. 2. Baclofen, a metabotropic GABA receptor agonist, both enhanced and suppressed high-voltage-activated calcium channel current. Baclofen facilitated an L-type channel current, and this effect was not voltage dependent. As reported previously, baclofen inhibited an N-type channel current and this action was voltage dependent. 3. While the suppressive effect was mediated by a fast-acting, direct G-protein action, the facilitatory effect was slower and was blocked by inhibitors of protein kinase C (PKC), either GF-109203x or the PKC (19-36) sequence fragment. 4. The pharmacology of the inhibitory and facilitatory responses differed. Commonly used antagonists of metabotropic GABA receptors, CGP35348 and CGP55845, were more potent antagonists of the inhibitory response. Similarly, a selective agonist at the metabotropic GABA receptor, APMPA, was also more effective in eliciting the inhibitory response. 5. These observations indicate that there may be two baclofen-sensitive metabotropic GABA receptors with opposing effects on calcium channel current. This is the first description of a facilitatory action of GABAB receptors and indicates that GABA may not function exclusively as an inhibitory transmitter.

Molecular Identification of the Human GABABR2: Cell Surface Expression and Coupling to Adenylyl Cyclase in the Absence of GABABR1

We have identified a gene encoding a GABABreceptor, the human GABABR2, located on chromosome 9q22.1, that is distinct from the recently reported rat GABABR1. GABABR2 structurally resembles GABABR1 (35% identity), having seven transmembrane domains and a large extracellular region, but differs in having a longer carboxy-terminal tail. GABABR2 is localized to the cell surface in transfected COS cells, and negatively couples to adenylyl cyclase in response to GABA, baclofen, and 3-aminopropyl(methyl)phosphinic acid in CHO cells lacking GABABR1. Baclofen action is inhibited by the GABABR antagonist, 2-hydroxysaclofen. The human GABABR2 and GABABR1 genes are differentially expressed in the nervous system, with the greatest difference being detected in the striatum in which GABABR1 but not GABABR2 mRNA transcripts are detected. GABABR2 and GABABR1 mRNAs are also coexpressed in various brain regions such as the Purkinje cell layer of the cerebellum. Identification of a functional homomeric GABABR2 coupled to adenylyl cyclase suggests that the complexity of GABABpharmacological data is at least in part due to the presence of more than one receptor and opens avenues for future research leading to an understanding of metabotropic GABA receptor signal transduction mechanisms.

Demonstration of a tandem pair of complement protein modules in GABA B receptor 1a

We have subcloned and expressed the N-terminal portion of the recently sequenced metabotropic GABA receptor, GABA BR1a. This region of the receptor contains a complement protein-like amino acid sequence. The purified 140-residue recombinant protein fragment was soluble and stable. Mass spectrometry indicated formation of four disulfide bonds, as expected if two complement protein modules (CPs, also known as SCRs, Sushi domains) are formed. The circular dichroism spectrum was unusual and characteristic of CPs. Differential scanning calorimetry demonstrated a melting point (64°C), and total enthalpy commensurate with two fully folded domains. We thus conclude that the 1a subtype of the GABA B receptor, but not the 1b subtype, contains a pair of CPs and we present a three-dimensional model of this region.

Mouse Cerebellar Granule Cell Differentiation: Electrical Activity Regulates the GABAAReceptor α6 Subunit Gene

GABAA receptor alpha6 subunit gene expression marks cerebellar granule cell maturation. To study this process, we used the Deltaalpha6lacZ mouse line, which has a lacZ reporter inserted into the alpha6 gene. At early stages of postnatal cerebellar development, alpha6-lacZ expression is mosaic; expression starts at postnatal day 5 in lobules 9 and 10, and alpha6-lacZ is switched on inside-out, appearing first in the deepest postmigratory granule cells. We looked for factors regulating this expression in cell culture. Membrane depolarization correlates inversely with alpha6-lacZ expression: granule cells grown in 25 mM [K+]o for 11-15 d do not express the alpha6 gene, whereas cultures grown for the same period in 5 mM [K+]o do. This is influenced by a critical early period: culturing for >/=3 d in 25 mM [K+]o curtails the ability to induce the alpha6 gene on transfer to 5 mM [K+]o. If the cells start in 5 mM [K+]o, however, they still express the alpha6-lacZ gene in 25 mM [K+]o. In contrast to granule cells grown in 5 mM [K+]o, cells cultured in 25 mM [K+]o exhibit no action potentials, mEPSCs, or mIPSCs. In chronic 5 mM [K+]o, factors may therefore be released that induce alpha6. Blockade of ionotropic and metabotropic GABA and glutamate receptors or L-, N-, and P/Q-type Ca2+ channels did not prevent alpha6-lacZ expression, but inhibition of action potentials with tetrodotoxin blocked expression in a subpopulation of cells.

Metabotropic GABA receptors regulate acetylcholine responses on snail neurons

1. 1. The A type of acetylcholine response of Helix neurons is downmodulated by low concentrations of GABA that do not elicit any measurable change in membrane potential or conductance. 2. 2. We find that these physiological actions are associated with an increase in both intracellular cyclic AMP levels and 45Ca 2+ influx. 3. 3. The modulation of the acetylcholine response by GABA is blocked when the neurons are injected with EGTA to prevent a rise in intracellular Ca 2+ concentration or when tolbutamide, an inhibitor of protein kinase A, is applied. 4. 4. These results are consistent with the effects of GABA being mediated by a metabotropic GABA receptor that is activated at very low GABA concentrations and mediates modulation of the acetylcholine response via regulation of intracellular Ca 2+ and cyclic AMP levels.

Metabotropic and ionotropic GABA receptors and receptor ligands

Metabotropic and ionotropic GABA receptors and receptor ligands

Characteristics and function of metabotropic receptors for GABA

Characteristics and function of metabotropic receptors for GABA