Extrasynaptic δ-GABAA receptors are high-affinity muscimol receptors

Abstract

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.

Keywords: GABAA receptors; affinity; association; binding; dissociation; muscimol.

Figure 1

(a) Structures of the muscimol precursor ibotenic acid, GABA and the GABA_AR agonists muscimol and THIP. The backbone of GABA is shown in bold to illustrate that muscimol and THIP are conformationally restricted GABA analogs. (b) [³H]Muscimol (6 nM) autoradiography in brain sections comparing wild‐type (WT) with α6 knockout (α6KO) and delta knockout (δKO) mouse lines. This shows that high‐affinity muscimol binding in the forebrain is δ subunit dependent, wheras in the cerebellum it is α6 subunit dependent. Figure for δKO and the corresponding WT mice are reproduced with permission of the Proceedings of the National Academy of Sciences, U.S.A. (Mihalek et al. 1999) and that of α6KO and WT mice with permission of the American Society for Pharmacology and Experimental Therapeutics (Mäkelä et al. 1997).

Figure 2

Majority of high‐affinity muscimol binding is δ subunit‐dependent. Association of [³H]muscimol binding to forebrain (n = 8 independent experiments in both mouse lines using individual forebrains in each experiment) and cerebellar (n = 3 independent experiments using pools of 3 individual cerebella from the mouse line in each pool) membranes of WT and δKO mice (mean ± SEM). The experiments were performed in triplicate technical replicates. Forebrain and cerebellar membranes were incubated with 5 nM [³H]muscimol alone and in the presence of 100 μM GABA to determine non‐specific binding. The incubations were terminated at various time points by filtration onto GF/B filters. The values are expressed as fmol/mg protein (left panels) and as % of binding at 15 min (right panels). Binding to δ‐GABA_ARs (WT‐δKO) was calculated by subtracting binding to non‐δ‐GABA_ARS in δKO mice from binding to WT membranes.

Figure 3

The δ subunit leads to very slow muscimol dissociation. Dissociation of 5 nM [³H]muscimol binding from forebrain (n = 4 independent experiments using individual forebrains in each experiment) and cerebellar (n = 3 independent experiments using pools of 3 individual cerebella from the mouse line in each pool) membranes of WT and δKO mice (mean ± SEM). The experiments were performed in triplicate technical replicates. Forebrain and cerebellar membranes of the mouse lines were pre‐incubated for 15 min with 5 nM [³H]muscimol alone and in the presence of 100 μM GABA to determine non‐specific binding. Then 100 μM GABA was added to all tubes to start [³H]muscimol dissociation. The incubations were continued for various durations (30 s to 30 min) and terminated by filtration onto GF/B filters. The values are expressed as fmol/mg protein on the left and as % of control binding at the start of dissociation (0 min) on the right. The values for δ‐GABA_ARs (WT‐δKO) were calculated as described in Materials and methods.

Figure 4

Co‐expression of the δ subunit leads to slow muscimol kinetics, particularly very slow dissociation. Association (a) and dissociation (b–d) of [³H]muscimol binding of recombinant α1β2γ2, α6β2γ2 and α6β2δ receptors expressed in HEK293 cells (mean ± SEM; n = 3–6 independent transfections and independent experiments performed in triplicate technical replicates). HEK293 cell membranes were incubated with 5 nM [³H]muscimol at 22°C (a–c) or on ice (d) in the absence or presence of 100 μM GABA determining the non‐specific binding. Dissociation experiments were performed as described in Materials and Methods. The incubations were terminated at various time points by filtration onto GF/B filters. The values are expressed as % of binding at 15 min (a) or 0 min (b–d).

Figure 5

Subnanomolar concentrations of muscimol evoked currents on recombinant δ subunit‐containing GABA_ARs. Representative concentration‐response data (out of 3 similar recordings made using injections into different batches of oocytes) using muscimol concentrations from 0.1 nM up to 100 μM on (a) α4β3δ‐ or (c) α4β3‐injected oocytes. Muscimol concentrations from 0.1 nM to 30 nM activate currents only in α4β3δ‐injected oocytes, but not in the absence of δ subunits in α4β3 injected oocytes. (b) Slow current activation (association rates are slow at these low muscimol concentrations because association is concentration‐dependent) and also current deactivation at the lowest doses (expanded in b) and the two‐component decay for doses ≥ 10 nM. (d) Superimposed responses to 300 nM muscimol from α4β3δ‐ and α4β3‐injected oocytes. The responses were scaled so that the α4β3 300 nM muscimol current fits the fast current component in α4β3δ‐injected oocytes.