Extrasynaptic site of action for γ-hydroxybutyrate

Advances in pharmacology often result from identifying the mechanism of action of drugs found empirically to be biologically active (1). γ-Hydroxybutyric acid (GHB), also known as Fantasy, is a centrally active agent that has been used clinically and recreationally for nearly half a century. Because of its history of abuse, its availability is tightly regulated, although it is prescribed for the treatment of alcoholism and narcolepsy. Depending on the dose, GHB displays a myriad of effects on CNS function. 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.

GHB and GABA

An involvement of GABAergic systems in the actions of GHB has been suspected for some time because it is structurally similar to, and a metabolite of, GABA (3). High- and low-affinity [³H]GHB binding sites are present in rat and human brain (4, 5). Because [³H]GHB binding is not inhibited by GABA or various isosteric GABA_A receptor ligands, it appears not to attach to the GABA recognition site on this pentameric, ligand-gated ion channel receptor. Rather, because [³H]GHB binding is influenced by chloride ions and allosteric GABA_A receptor ligands, it has been speculated that its site of action may be a component of the GABA_A receptor chloride ion channel (6).

γ-Hydroxybutyrate–induced neuronal hyperpolarization is blocked by antagonists for metabotropic, heterodimeric GABA_B receptors (7). Studies with recombinant GABA_B receptors indicate that GHB is a weak, isosteric GABA_B receptor agonist (8), as does the discovery that the hypolocomotion and hypothermia caused by GHB are absent in GABA_B receptor-deficient mice (9). However, there is no direct association between [³H]GHB binding or the binding of [³H]NCS-382, a GHB receptor antagonist (10–12), and GABA_B receptors because the attachment of these radioligands is unaffected in the brains of transgenic mice lacking functional GABA_B receptors. In addition, [³H]GHB binding is undetectable in GABA_B receptor-transfected HEK 293 cells (9, 13). Moreover, there is a lack of concordance between the sensitivity of the GABA_B receptor to GHB and the affinity of the [³H]GHB binding sites, with millimolar concentrations of GHB required to activate GABA_B receptors in vitro (7, 8), whereas the K_d values for the high- and low-affinity [³H]GHB binding sites are in the nanomolar and micromolar ranges, respectively (5). These results suggest that the interaction of GHB with GABA_B receptors mediates responses to high doses but contributes little to the effects observed with lower, more clinically relevant doses.

At least some of the pharmacological effects of GHB are due to its activation of extrasynaptic GABA_A receptors.

Alternatively, these results might indicate that the [³H]GHB binding site is of no physiological or pharmacological consequence. This appears unlikely, however, because in vivo studies with NCS-382 suggest that it interacts with a functional receptor; that is, NCS-382 administration diminishes the sedative and cataleptic effects of GHB and blocks the enhancing effects on the spontaneous firing rate of cortical neurons at low doses of GHB but not the depressant effects seen at higher doses (14, 15). Because NCS-382 administration blocks neither GHB-induced ataxia nor its depressant effects on locomotor activity, learned and unlearned behavior, and operant responses (16, 17), these actions are probably mediated by GHB activation of GABA_B receptors.

Efforts to characterize the GHB binding site led to the identification of two parent clones (18). When expressed in CHO cells, one of these, C12K32, displays NCS-382–sensitive high- and low-affinity [³H]GHB binding sites. Characterization of the binding component indicates a G protein-coupled site that is unassociated with either GABA_A or GABA_B receptors.

GHB and Extrasynaptic Receptors

The study by Absalom et al. (2) provides evidence directly identifying an extrasynaptic GABA_A receptor subtype as the functional component of the [³H]GHB binding site, confirming earlier suggestions that were based on indirect findings (19–21). The work by Absalom et al. (2) is made possible by the synthesis of [¹²⁵I]4-hydroxy-4-[4-(2-iodobenzyloxy)phenyl]butanoate ([¹²⁵I]BnOPh-GHB), a photoaffinity label for the GHB binding site (22–24). In a previous study, these investigators demonstrated that this radioligand labels a single, high-affinity (7-nM K_d) site in rat brain membranes that displays a regional distribution identical to [³H]NCS-382 binding (24). Although attachment to the [¹²⁵I]BnOPh-GHB binding site is inhibited by GHB, GABA is inactive. For the study in PNAS, these investigators subjected the [¹²⁵I]BnOPh-GHB binding protein to proteolysis. The resultant bands represent a variety of GABA_A receptor subunits. A comprehensive functional screen of human recombinant GABA_A receptor subtypes in Xenopus oocytes revealed that the most GHB-responsive site contains α₄/δ- and β₁-subunits. They report that the α₄/δ-subunit combination is responsible for GHB efficacy, and the β₁-subunit for potency. The EC₅₀ for GHB is 140 nM. It is concluded that GHB is a partial agonist at this site because its maximum efficacy at this recombinant receptor is 74% of that displayed by GABA. Results from pharmacological and α-subunit mutation experiments demonstrate that NCS-382 and GHB bind to the interface of the β-α combination at a site that overlaps with but is not identical to the GABA_A receptor recognition site. The finding of a significant reduction in the B_max for [³H]NCS-382 binding in the brains of α₄- but not δ-subunit KO mice provides further evidence that the α₄-subunit is a molecular target for GHB. The authors propose that α₄-containing GABA_A receptors, in particular α₄β₁δ, represent the primary site of action for GHB. The fact that the functional response to GHB requires the presence of a δ-subunit, which is associated with only a minor fraction of GABA_A receptors, but the binding site is located at the β-α interface, which is present in a large number of GABA_A receptors, explains why some [³H]NCS-382 binding is present after deletion of α₄-subunits. An alternative explanation is that this residual [³H]NCS-382 binding represents attachment to other molecularly, and possibly functionally, distinct GHB receptors. 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.

Synaptic GABA_A receptors mediate fast phasic inhibition, have a lower affinity for GABA than the extrasynaptic sites, and are responsible for tonic inhibition. Because of their higher affinity for GABA, extrasynaptic receptors are continuously activated by the small amounts of endogenous GABA present in the local environment. Whereas the most abundant synaptic receptor is composed of α-, β-, and γ-subunits, the extrasynaptic site contains α₄- or α₆-, β-, and δ-subunits (25). Extrasynaptic GABA_A receptors are targets for steroids, ethanol, general anesthetics, convulsants, hypnotics, and analgesics (26). The rigid GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridine-3-(-ol) (THIP) is an example of an extrasynaptic GABA_A receptor agonist (27). Like GHB, THIP activates αβδ-containing GABA_A receptors and displays a range of interesting CNS effects.

The discovery that the effects of GHB are due to an interaction with extrasynaptic α₄β₁δ GABA_A receptors opens the way to designing more selective agents for this site. The synthesis of such compounds could lead to the development of a new class of drugs for the treatment of anxiety, pain, insomnia, epilepsy, and other conditions associated with GABAergic dysfunction.