Extrasynaptic site of action for γ-hydroxybutyrate

S. J. Enna1 Department of Molecular and Integrative Physiology and Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical School, Kansas City, KS 66160

dvances in pharmacology often lack of concordance between the sensitiv- functional component of the [3H]GHB fi result from identifying the ity of the GABAB receptor to GHB and binding site, con rming earlier suggestions A mechanism of action of drugs the affinity of the [3H]GHB binding sites, that were based on indirect findings found empirically to be bio- with millimolar concentrations of GHB (19–21). The work by Absalom et al. (2) is γ 125 logically active (1). -Hydroxybutyric acid required to activate GABAB receptors in made possible by the synthesis of [ I]4- (GHB), also known as Fantasy, is a cen- vitro (7, 8), whereas the Kd values for the hydroxy-4-[4-(2-iodobenzyloxy)phenyl]bu- trally active agent that has been used high- and low-affinity [3H]GHB binding tanoate ([125I]BnOPh-GHB), a photo- clinically and recreationally for nearly sites are in the nanomolar and micromolar affinity label for the GHB binding site (22– half a century. Because of its history of ranges, respectively (5). These results 24). In a previous study, these investigators abuse, its availability is tightly regulated, suggest that the interaction of GHB with demonstrated that this radioligand labels fi K although it is prescribed for the treatment GABAB receptors mediates responses a single, high-af nity (7-nM d) site in rat of alcoholism and narcolepsy. Depending to high doses but contributes little to the brain membranes that displays a regional 3 on the dose, GHB displays a myriad of effects observed with lower, more clini- distribution identical to [ H]NCS-382 effects on CNS function. Despite decades cally relevant doses. binding (24). Although attachment to the of research, significant questions remain [125I]BnOPh-GHB binding site is inhibited about its mechanism of action. In PNAS, by GHB, GABA is inactive. For the Absalom et al. (2) identify a distinct pop- At least some of the study in PNAS, these investigators sub- jected the [125I]BnOPh-GHB binding ulation of extrasynaptic GABAA receptors pharmacological effects as a target for GHB, providing unique protein to proteolysis. The resultant bands insights into the molecular target for of GHB are due to its represent a variety of GABAA receptor this agent. subunits. A comprehensive functional activation of extrasynaptic screen of human recombinant GABAA GHB and GABA receptor subtypes in Xenopus oocytes re- An involvement of GABAergic systems in vealed that the most GHB-responsive site GABAA receptors. α δ β the actions of GHB has been suspected for contains 4/ - and 1-subunits. They report α δ some time because it is structurally similar that the 4/ -subunit combination is re- fi β to, and a metabolite of, GABA (3). High- Alternatively, these results might sponsible for GHB ef cacy, and the 1- 3 3 and low-affinity [ H]GHB binding sites are indicate that the [ H]GHB binding site is subunit for potency. The EC50 for GHB is present in rat and human brain (4, 5). of no physiological or pharmacological 140 nM. It is concluded that GHB is Because [3H]GHB binding is not inhibited consequence. This appears unlikely, how- a partial agonist at this site because its fi by GABA or various isosteric GABAA ever, because in vivo studies with NCS-382 maximum ef cacy at this recombinant re- receptor ligands, it appears not to attach suggest that it interacts with a functional ceptor is 74% of that displayed by GABA. to the GABA recognition site on this receptor; that is, NCS-382 administration Results from pharmacological and α-sub- pentameric, ligand-gated ion channel re- diminishes the sedative and cataleptic unit mutation experiments demonstrate ceptor. Rather, because [3H]GHB binding effects of GHB and blocks the enhancing that NCS-382 and GHB bind to the in- is influenced by chloride ions and alloste- effects on the spontaneous firing rate of terface of the β-α combination at a site ric GABAA receptor ligands, it has been cortical neurons at low doses of GHB but that overlaps with but is not identical to speculated that its site of action may be not the depressant effects seen at higher the GABAA receptor recognition site. The a component of the GABA receptor doses (14, 15). Because NCS-382 admin- finding of a significant reduction in the A B 3 chloride ion channel (6). istration blocks neither GHB-induced max for [ H]NCS-382 binding in the α δ γ-Hydroxybutyrate–induced neuronal ataxia nor its depressant effects on loco- brains of 4- but not -subunit KO mice α hyperpolarization is blocked by antago- motor activity, learned and unlearned provides further evidence that the 4-sub- nists for metabotropic, heterodimeric behavior, and operant responses (16, 17), unit is a molecular target for GHB. The authors propose that α -containing GABAB receptors (7). Studies with re- theseactionsareprobablymediatedby 4 GABA receptors, in particular α β δ, combinant GABAB receptors indicate that GHB activation of GABAB receptors. A 4 1 represent the primary site of action for GHB is a weak, isosteric GABAB receptor Efforts to characterize the GHB bind- agonist (8), as does the discovery that ing site led to the identification of GHB. The fact that the functional re- the hypolocomotion and hypothermia two parent clones (18). When expressed sponse to GHB requires the presence of a δ-subunit, which is associated with only caused by GHB are absent in GABAB in CHO cells, one of these, C12K32, receptor-deficient mice (9). However, displays NCS-382–sensitive high- and a minor fraction of GABAA receptors, but 3 β α there is no direct association between [3H] low-affinity [ H]GHB binding sites. the binding site is located at the - in- GHB binding or the binding of [3H]NCS- Characterization of the binding compo- terface, which is present in a large number 382, a GHB receptor antagonist (10–12), nent indicates a G protein-coupled site of GABAA receptors, explains why some [3H]NCS-382 binding is present after and GABAB receptors because the at- that is unassociated with either GABAA tachment of these radioligands is unaf- or GABAB receptors. fected in the brains of transgenic mice GHB and Extrasynaptic Receptors Author contributions: S.J.E. wrote the paper. lacking functional GABAB receptors. In addition, [3H]GHB binding is undetect- The study by Absalom et al. (2) provides The author declares no conflict of interest. able in GABAB receptor-transfected HEK evidence directly identifying an extra- See companion article on page 13404. 293 cells (9, 13). Moreover, there is a synaptic GABAA receptor subtype as the E-mail: [email protected].

13142–13143 | PNAS | August 14, 2012 | vol. 109 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1210290109 Downloaded by guest on September 29, 2021 COMMENTARY

deletion of α4-subunits. An alternative sites, and are responsible for tonic in- (THIP) is an example of an extrasynaptic 3 explanation is that this residual [ H]NCS- hibition. Because of their higher affinity GABAA receptor agonist (27). Like GHB, 382 binding represents attachment to for GABA, extrasynaptic receptors are THIP activates αβδ-containing GABAA other molecularly, and possibly function- continuously activated by the small receptors and displays a range of inter- ally, distinct GHB receptors. Among these amounts of endogenous GABA present esting CNS effects. might be the G protein-coupled GHB in the local environment. Whereas the The discovery that the effects of GHB site reported by others (18). Nonetheless, most abundant synaptic receptor is are due to an interaction with extra- the findings of Absalom et al. (2) provide composed of α-, β-, and γ-subunits, the synaptic α4β1δ GABAA receptors opens direct evidence that at least some of extrasynaptic site contains α4-orα6-, β-, the way to designing more selective agents the pharmacological effects of GHB are and δ-subunits (25). Extrasynaptic GA- for this site. The synthesis of such com- due to its activation of extrasynaptic BAA receptors are targets for steroids, pounds could lead to the development GABAA receptors. ethanol, general anesthetics, convul- of a new class of drugs for the treatment Synaptic GABAA receptors mediate sants, hypnotics, and analgesics (26). of anxiety, pain, insomnia, epilepsy, fast phasic inhibition, have a lower af- The rigid GABA analog 4,5,6,7-tetrahy- and other conditions associated with finity for GABA than the extrasynaptic droisoxazolo (5,4-c)pyridine-3-(-ol) GABAergic dysfunction.

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