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 (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 [³ H]muscimol-binding sites despite the relatively low abundance of δ-containing GABA_A Rs (δ-GABA_A R) 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-GABA_A Rs 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 EC₅₀ 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.