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.

Fig. 1.

Photoaffinity labeling of high-affinity GHB binding sites from rat brain and isolation of target proteins. SDS/PAGE separations of [¹²⁵I]azido-BnOPh-GHB radiophotoaffinity-labeled partially degraded binding proteins using time-dependent Proteinase K limited proteolysis. Arrows indicate the four protein bands isolated for proteomics analysis (top to bottom: ∼50, ∼28, ∼21, and ∼18 kDa).

Fig. 2.

Pharmacological characterization of GHB at recombinant αβδ-receptors in X. laevis oocytes. Representative GHB current traces at (A) α4β1δ and (B) α4β1 GABA_A receptors. (C) Concentration response curve at α4β1–3δ-receptors normalized to GABA_max (means ± SEM; n = 4–6). (D) GHB (closed circles) and GABA (open circles) I-V relationships at α4β1δ-receptors (n = 7). Reversal potential was not significantly different for GABA and GHB currents (V_rev = −25.2 ± 3.9 and −27.8 ± 4.8, respectively; P = 0.68, Student t test).

Fig. 3.

Abolishment of GHB response by gabazine coapplication and α4F71L point mutation. (A, Left) Representative gabazine inactivation trace of GHB currents at α4β1δ. (A, Right) Summarized data displaying fraction of GHB current at α4β1/δ (means ± SEM; n = 3). (B, Left) Representative traces of GABA and GHB-elicited currents from α4β1, α4(F71L)β1 (Upper), and α4(F71L)β1δ (Lower). (B Right) Summary (means ± SEM) of 30 mM GHB effects at α4β1 vs. α4(F71L)β1 (***P = 6.3 × 10⁻⁵) and 100 μM GHB at α4β1δ vs. α4(F71L)β1δ (*P = 0.011).

Fig. 4.

GHB high-affinity radioligand binding to rat and KO mouse brain preparations. (A) Gabazine and GABA inhibition of [³H]NCS-382 (16 nM) binding to rat brain homogenate (pK_i 4.7 ± 0.11 and 2.7 ± 0.021, respectively; n = 3). (B) Autoradiograms of [¹²⁵I]BnOPh-GHB (100 pM) binding to horizontal brain sections (n = 2). (C and E) Inhibition by GHB and NCS-382 and (D and F) saturation of [³H]NCS-382 binding (16 nM) to membrane preparations from α4- and δ-subunit KO mouse brains, respectively (means ± SEM), showing significantly lower binding in α4 KO vs. WT (P = 0.0056).