Extrasynaptic δ‐ GABA A 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 GABA A receptor ( GABA A R ) δ subunit in mice (δ KO ) 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 δ KO mice to show that deletion of the δ subunit leads to a > 50% loss of high‐affinity 5 nM [ 3 H]muscimol‐binding sites despite the relatively low abundance of δ‐containing GABA A R s (δ‐ GABA A R ) in the brain. By subtracting residual high‐affinity binding in δ KO mice and measuring the slow association and dissociation rates we show that native δ‐ GABA A R s in WT mice exhibit high‐affinity [ 3 H]muscimol‐binding sites ( K D ~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 [ 3 H]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 EC 50 of around 1–2 nM and slow deactivation kinetics. These data indicate that δ subunit incorporation leads to a dramatic increase in GABA A R 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‐GABA A Rs.