γ-hydroxybutyrate (GHB) is a small molecule with complex pharmacology. Present in low concentrations in the mammalian brain, it acts as a neuromodulator. When taken exogenously, it is used to treat narcolepsy and to ameliorate the withdrawal effects of alcohol, and is used as a recreational drug at higher concentrations, sometimes used as a “date-rape” drug. GHB is known to activate the GABAB receptor at high concentrations, but ligand-binding studies identified “GHB receptors” that bind both GHB and the analogue NCS-382 with high affinity.
In this study, we determined the role of the interaction between GHB, NCS-382, THIP and GABAARs in thermoregulation, and the differences in subtype selectivity that underlies these roles.
Thermoregulation was measured using radiotelemetry in wild-type and knockout mice injected intraperitoneally with GHB, NCS-382 and THIP, a molecule with selectivity for δ-containing GABAARs. The activity of GHB, THIP and NCS-382 at GABAARs was determined by injecting mRNA encoding the sequences of the α4, β1-3 and δ subunits of GABAARs in various ratios and combinations into Xenopus oocytes and measuring currents by two-electrode voltage clamp.
GHB, THIP and NCS-382 all induced hypothermia in wild-type mice, but only THIP-mediated hypothermia was abolished in δ-knockout mice. We then investigated the pharmacology of NCS-382 at α4βδ GABAARs by measuring concentration-response curves of THIP, GHB and NCS-382 on Xenopus oocytes injected with different combinations of α4β1-3 and α4β1-3δ RNA at different ratios. NCS-382 activated β3 homomeric receptors and these currents were inhibited with co-application of GHB. Furthermore, GHB activated α4β1 receptors injected with a 1: 10 ratio significantly more potently than when injected with a 10: 1 ratio.
These data demonstrate that THIP, but not GHB or NCS-382 induce hypothermia via the activation of δ-containing GABAARs. It is likely that NCS-382 and GHB activates GABAA receptors that are expressed in Xenopus oocytes but not readily found on the extracellular surface of native neurons, and these receptors are most likely to contain a β-β interface. While NCS-382 has previously been reported as an antagonist of GHB receptors, the pharmacological profile of NCS-382 is considerably more complicated.