Extrasynaptic δ‐GABAA receptors are high‐affinity muscimol receptors

Muscimol, the major psychoactive ingredient in the mushroom Amanita muscaria, has been regarded as a universal non‐selective GABA‐site agonist. Deletion of the GABAA receptor (GABAAR) δ subunit in mice (δKO) leads to a drastic reduction in high‐affinity muscimol binding in brain sections and to a lower behavioral sensitivity to muscimol than their wild type counterparts. Here, we use forebrain and cerebellar brain homogenates from WT and δKO mice to show that deletion of the δ subunit leads to a > 50% loss of high‐affinity 5 nM [3H]muscimol‐binding sites despite the relatively low abundance of δ‐containing GABAARs (δ‐GABAAR) in the brain. By subtracting residual high‐affinity binding in δKO mice and measuring the slow association and dissociation rates we show that native δ‐GABAARs in WT mice exhibit high‐affinity [3H]muscimol‐binding sites (KD ~1.6 nM on α4βδ receptors in the forebrain and ~1 nM on α6βδ receptors in the cerebellum at 22°C). Co‐expression of the δ subunit with α6 and β2 or β3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [3H]muscimol component. In addition, we compared muscimol currents in recombinant α4β3δ and α4β3 receptors and show that δ subunit co‐expression leads to highly muscimol‐sensitive currents with an estimated EC50 of around 1–2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABAAR muscimol sensitivity. We conclude that biochemical and behavioral low‐dose muscimol selectivity for δ‐subunit‐containing receptors is a result of low nanomolar‐binding affinity on δ‐GABAARs. Muscimol has been regarded as a universal non‐selective GABA‐site agonist. Here, we show that δ subunit incorporation leads to a dramatic increase in GABAA receptor (GABAAR) muscimol sensitivity. The biochemical and behavioral low‐dose muscimol selectivity for δ subunit‐containing receptors was because of low nanomolar‐binding affinity on α4/6βδ GABAARs. This paints a consistent picture in which extrasynaptic δ‐GABAARs are not only exquisitely sensitive to GABA, but also the GABA analogs gaboxadol and muscimol.