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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 18, 2003 as doi:10.1096/fj.02-0846fje. |
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-hydroxybutyrate (GHB)1
Institut de Chimie Biologique and INSERM U-575, Faculté de Médecine, 67085 Strasbourg cedex, France
2Correspondence: Institut de Chimie Biologique and INSERM U-575, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg cedex, France. E-mail: maitre{at}neurochem.u-strasbg.fr
SPECIFIC AIMS
-Hydroxybutyrate (GHB) is a putative neuromodulator of the mammalian brain that derives from GABA via transamination and subsequent reduction through a specific succinic semialdehyde reductase (SSR). GHB and SSR are colocalized in some neurons and neuronal endings and GHB is accumulated in synaptic vesicles by an active transport. At micromolar concentrations, GHB exerts a specific influence on some Ca2+ and K+ conductances expressed by certain neuronal cell lines. These effects are thought to be mediated by GHB binding sites, which have specific ontogenesis, pharmacological, and kinetic properties, together with a particular distribution profile in the brain of several species. Peripherally administered GHB potentiates and adapts this endogenous GHB system and is approved in various countries for the treatment of narcolepsy, sleep disturbances, and ethanol withdrawal. GHB is also a drug of abuse for its anxiolytic, euphoric, and sedative properties. In this study, we report the cloning and characterization of the first GHB receptor expressed in the rat brain. This receptor appears to belong to the G-protein-coupled receptor (GPCR) family and can mediate some of the physiological, pharmacological and recreative effects of GHB.
PRINCIPAL FINDINGS
1. The GHB receptor cloned from rat brain has seven putative transmembrane regions and no significant homology with other known GPCR
PCR screening of a rat brain hippocampal cDNA library using primers designed from the sequence of a brain protein with affinity for a GHB-related ligand led to the isolation of a full-length cDNA sequence. The open reading frame encodes for a protein of 512 amino acid residues with a calculated molecular mass of 56,972 Da. Prediction of secondary structure for the protein revealed the presence of seven putative transmembrane regions as is observed for G-protein-coupled receptors. Several putative phosphorylation sites for kinase C and casein kinase II are present along the sequence. No significant sequence homology was found with any known GPCR, but a 65% homology in 175 amino acids exists with a member of the four transmembrane protein superfamily tetraspanin, widely expressed in several tissues, including brain.
2. GHB receptor mRNA is detected only in brain tissue and Southern blot of brain genomic DNA suggests the presence of the receptor in the brain of several species
Northern blot analysis of total RNA extracted from several rat organs (including brain) revealed the existence of specific mRNA only in the brain. Three distinct bands are present, suggesting the existence of a family of GHB receptors in brain.
In situ hybridization studies followed by quantitative image analysis showed that the receptor mRNA is heterogeneously distributed in the rat brain, namely, in the cortex, olfactory bulbs, hippocampus, striatum, thalamus, amygdala, and cerebellar lobules. In all these regions, GHB binding sites have been detected both in human and rat brain; however, the distribution of the mRNA does not reflect the expression of the entire family of GHB receptors.
3. Functional characterization of the GHB receptor in transfected cells
The cloned cDNA was transiently expressed in CHO cells. Using the patch-clamp technique, application of GHB (0.5–20 µM) to transfected cells induced, in contrast to control cells, a reversible response whose amplitude was dose dependent (EC50=4.0±0.3 µM, Fig. 1
). This response was not reproduced by (–) baclofen (10 µM) or sensitive to the GABAB antagonist CGP 55845 (0.1 µM). The current response lasted 1 to 10 min and reversed at a potential (–2.1±0.1 mV), which corresponded under our conditions to the equilibrium potential of monovalent cations (mainly Na+ and K+).
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To ascertain the implication of G-proteins in the mediation of the GHB-induced signal, we dialyzed GTP--S (0.5 or 1 mM) into the recorded cells. Under these conditions, the response of the transfected cells to GHB (1–2 µM) was not reversible during the recording time (1–2 h); no detectable current was seen in nontransfected cells (Fig. 2
).
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4. Pharmacological characterization of the GHB receptor in transfected cells
After transient expression for 48 h in CHO cells, a specific binding for [3H]-GHB was detected on cell membranes (single site, Kd=426±150 nM, Bmax=4.8±1.5 pmol/mg protein). GABA, baclofen, or glutamate (50 or 200 µM) has no capacity to displace radioactive GHB from the binding site. However, several GHB analogs that possess affinity for GHB receptor(s) in ex vivo experiments displace [3H]-GHB from its receptor site (see Table 1
). Analogs and derivatives related to the structural analog of GHB named trans-4-hydroxycrotonate (T-HCA) are the best ligands. However, the GHB receptor antagonist NCS-382, which blocks some of the effects of GHB in vivo, has no affinity for the transfected receptor.
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CONCLUSIONS AND SIGNIFICANCE
The present report is the first on the cloning and expression of a rat brain receptor that is stimulated by and binds the endogenous neuromodulator and drug of abuse GHB. This receptor most probably belongs to the G-protein-coupled receptor. It is only expressed by brain cells and shares some homology with genomic DNA of several species, including humans. Several data argue in favor of a family of GHB receptors in the rat brain. First, several mRNA possess homologies with the isolated receptor. Second, kinetic properties of the cloned receptor expressed in foreign cells are similar to Kd's for GHB binding in some but not all brain regions. Third, the GHB receptor ligand NCS-382, which reduces some in vivo effects of GHB, does not bind to the cloned receptor, indicating the possible existence of several pharmacological classes of GHB receptors. The receptor preference for GHB analogs related to T-HCA supports this hypothesis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0846fje; doi: 10.1096/fj.02-0846fje 
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