α4βδ GABAAreceptors are high-affinity targets for γ-hydroxybutyric acid (GHB)

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 109; no. 33; pp. 13404 - 13409
• Main Authors: Absalom, Nathan, Eghorn, Laura F., Villumsen, Inge S., Karim, Nasiara, Bay, Tina, Olsen, Jesper V., Knudsen, Gitte M., Bräuner-Osborne, Hans, Frølund, Bente, Clausen, Rasmus P., Chebib, Mary, Wellendorph, Petrine
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences 14.08.2012
• Subjects: Agonists; Behavioral neuroscience; Binding sites; Electric potential; Ligands; Messenger RNA; Oocytes; Pharmacology; Proteomics; Receptors
• ISSN: 0027-8424; 1091-6490

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 GABAA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABAA 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 (EC₅₀ = 140 nM) over α4β(2/3)δ (EC₅₀ = 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 [³H](E,RS)-(6,7,8,9-tetra-hydro-5-hydroxy-5H-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 GABAA receptors and postulate a role for extrasynaptic α4δ-containing GABAA 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.