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 GABA(A) receptor (GABA(A)R) delta subunit in mice (delta 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 delta KO mice to show that deletion of the delta subunit leads to a > 50% loss of high-affinity 5 nM [H-3]muscimol-binding sites despite the relatively low abundance of delta-containing GABA(A)Rs (delta-GABA(A)R) in the brain. By subtracting residual high-affinity binding in delta KO mice and measuring the slow association and dissociation rates we show that native delta-GABA(A)Rs in WT mice exhibit high-affinity [H-3]muscimol-binding sites (K-D similar to 1.6 nM on alpha 4 beta delta receptors in the forebrain and similar to 1 nM on alpha 6 beta delta receptors in the cerebellum at 22 degrees C). Co-expression of the delta subunit with alpha 6 and beta 2 or beta 3 in recombinant (HEK 293) expression leads to the appearance of a slowly dissociating [H-3]muscimol component. In addition, we compared muscimol currents in recombinant alpha 4 beta 3 delta and alpha 4 beta 3 receptors and show that delta 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 delta subunit incorporation leads to a dramatic increase in GABA(A)R muscimol sensitivity. We conclude that biochemical and behavioral low-dose muscimol selectivity for delta-subunit-containing receptors is a result of low nanomolar-binding affinity on delta-GABA(A)Rs.