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.γ-hydroxybutyric acid receptor | γ-hydroxybutyric acid high-affinity binding sites | α4-subunit knockout | photoaffinity ligand T he GABA metabolite γ-Hydroxybutyric acid (GHB) is present in micromolar concentrations in the mammalian brain, where it has been proposed to act as a neurotransmitter (1). Additionally, GHB is a drug of abuse (Fantasy) and a registered drug for treating narcolepsy (2) and alcoholism (3). GHB binds to at least two distinct populations of low-and high-affinity binding sites in the brain (4). When GHB is ingested in high doses and reaches millimolar concentrations in the brain, it induces behavioral effects such as sedation, motor incoordination and hypothermia (3). These actions are largely mediated by metabotropic GABA B receptors, because effects are prevented by GABA B receptor antagonist pretreatment (5) and completely abolished in GABA B(1)

Extrasynaptic site of action for γ-hydroxybutyrate

“…Among these might be the G protein-coupled GHB site reported by others (18). Nonetheless, the findings of Absalom et al (2) provide direct evidence that at least some of the pharmacological effects of GHB are due to its activation of extrasynaptic GABA A receptors.…”

“…Despite decades of research, significant questions remain about its mechanism of action. In PNAS, Absalom et al (2) identify a distinct population of extrasynaptic GABA A receptors as a target for GHB, providing unique insights into the molecular target for this agent.…”

Systemic Administration of γ-Hydroxybutyric Acid in Adolescent Rat Impairs Contextual Fear Conditioning, But Not Cued Conditioning

“…It acts indirectly through GHB-derived GABA at the high-affinity GABA A receptor and as a partial agonist at the GABA B receptor [1,55]. While many of the behavioral effects of high doses (> 200 mg/kg) of GHB are attributed to its effects on the GABA B receptor [9], it is still unclear which receptor mediates its relatively low dose effects on learning and memory.…”

“…GHB has weak affinity for the GABA B receptor [34]. Recently, it has been shown to bind with high affinity to the α4-containing GABA A receptor [1].…”

Gamma-Hydroxybutyrate (GHB) Content in Hair Samples Correlates Negatively with Age in Succinic Semialdehyde Dehydrogenase Deficiency

“…High levels of GHB and GABA during ontogeny almost certainly offsets the balance of GABA-ergic/glutamatergic neurotransmission (Jansen et al 2008;Vogel et al 2016;Talaei et al 2016). High levels of GHB may impact this balance both through GHB-ergic and GABA B -ergic and GABA Aergic effects (Absalom et al 2012). Fig.…”