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

gamma-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 alpha beta delta -but not alpha beta gamma-receptors, proving that the delta-subunit is essential for potency and efficacy. GHB showed preference for alpha 4 over alpha(1,2,6)-subunits and preferably activated alpha 4 beta 1 delta (EC50 = 140 nM) over alpha 4 beta (2/3)delta (EC50 = 8.41/1.03 mM). Introduction of a mutation, alpha 4F71L, in alpha 4 beta 1(delta)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [H-3](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 alpha 4 KO brain tissue compared with WT controls, corroborating the direct involvement of the alpha 4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with alpha 4-containing GABA(A) receptors and postulate a role for extrasynaptic alpha 4 delta-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.