title: α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).
abstract: 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-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 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.
authors: Faculty of Pharmacy A15,  University of Sydney, Sydney, New South Wales 2006,  Australia.
authors: Nathan
authors: N
authors: Absalom
authors: Laura F
authors: LF
authors: Eghorn
authors: Inge S
authors: IS
authors: Villumsen
authors: Nasiara
authors: N
authors: Karim
authors: Tina
authors: T
authors: Bay
authors: Jesper V
authors: JV
authors: Olsen
authors: Gitte M
authors: GM
authors: Knudsen
authors: Hans
authors: H
authors: Bräuner-Osborne
authors: Bente
authors: B
authors: Frølund
authors: Rasmus P
authors: RP
authors: Clausen
authors: Mary
authors: M
authors: Chebib
authors: Petrine
authors: P
authors: Wellendorph
mesh_terms: D000818:Animals; D001567:Benzocycloheptenes / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D004566:Electrodes; D006801:Humans; D006885:Hydroxybutyrates / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018408:Patch-Clamp Techniques; D019920:Photoaffinity Labels; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics; D011724:Pyridazines / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D001567:Benzocycloheptenes; D006885:Hydroxybutyrates; D019920:Photoaffinity Labels; D021122:Protein Subunits; D011724:Pyridazines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C066471:NCS 382; C111420:4-hydroxybutyric acid; C049853:gabazine