α4βδ GABA A receptors are high-affinity targets for γ-hydroxybutyric acid (GHB)
| Autor: | Hans Bräuner-Osborne, Rasmus P. Clausen, Bente Frølund, Nasiara Karim, Gitte M. Knudsen, Mary Chebib, Jesper V. Olsen, Laura F. Eghorn, Petrine Wellendorph, Nathan L. Absalom, Inge S. Villumsen, Tina Bay |
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| Rok vydání: | 2012 |
| Předmět: |
Proteomics
Patch-Clamp Techniques Hydroxybutyrates Photoaffinity Labels Pharmacology Biology Partial agonist Mice Radioligand Assay Xenopus laevis chemistry.chemical_compound Commentaries Radioligand Animals Humans Point Mutation Binding site Receptor Neurotransmitter Electrodes Mice Knockout Binding Sites Multidisciplinary GABAA receptor GHB receptor Brain Receptors GABA-A Recombinant Proteins Rats Pyridazines Benzocycloheptenes Protein Subunits chemistry Biochemistry Protein Binding |
| Zdroj: | Proceedings of the National Academy of Sciences. 109:13404-13409 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABA A receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABA A receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC 50 = 140 nM) over α4β(2/3)δ (EC 50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [ 3 H]( E , RS )-(6,7,8,9-tetrahydro-5-hydroxy-5 H -benzocyclohept-6-ylidene)acetic acid showed a 39% reduction ( P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABA A receptors and postulate a role for extrasynaptic α4δ-containing GABA A receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism. |
| Databáze: | OpenAIRE |
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