Elsevier

Neuropharmacology

Volume 44, Issue 8, June 2003, Pages 994-1002
Neuropharmacology

The pharmacology of spontaneously open α1β3ε GABAA receptor–ionophores

https://doi.org/10.1016/S0028-3908(03)00116-3Get rights and content

Abstract

Human α1β3ε GABAA receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to α1β3γ2 receptors. The effects of several GABAA agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the ε subunit increased the potency and efficacy of direct activation by partial GABAA agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3α epimers, while chirality at C5 was indifferent. The 3β-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn2+ blocked the spontaneous currents of α1β3ε receptors with different efficacies. Flunitrazepam and 4′-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active α1β3ε GABAA receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

Introduction

The transmitter-gated ion channels comprise different superfamilies of important neurotransmitter receptors (Barnard, 1996). An increasing body of evidence supports the view that it is not just agonist binding that leads to channel opening but unliganded ionophores exist in an equilibrium of open and closed states (Changeux and Edelstein, 1998). The most compelling evidence is the demonstration of spontaneously open channels.

Type A receptors of γ-aminobutyric acid (GABAA) belong to the superfamily of neurotransmitter receptor–ionophores together with glycine receptors, nicotinic acetylcholine receptors and 5-HT3 type serotonin receptors. These receptors have pentameric subunit structures, each subunit having four transmembrane regions (TM1–4) and a cysteine loop in the N-terminal extracellular region (Barnard, 1996). Several GABAA receptor subunits have been cloned, including α1–6, β1–4, γ1–3, δ, ε, π and θ subunits (Whiting et al., 1999). The receptor is subject to allosteric modulation through a number of receptor binding sites, and the precise nature of allosteric modulation is critically dependent on the composition of the receptor.

It is difficult to attribute spontaneous ionophore activity to spontaneously open channels if we cannot exclude the contribution of endogenous agonists. The demonstration of spontaneously open GABAA receptors has been enabled by recombinant receptors. The β1 or β3 subunits can form homomeric GABAA receptors with ionophore function. Rat and murine β1 homomeric receptors are not activated by GABA yet they are open (Sigel et al., 1989, Krishek et al., 1996). Heterooligomeric α6β2γ2 GABAA receptors (Knoflach et al., 1996) also showed some spontaneous ionophore activities, but these activities were so minor that they could not be pharmacologically characterized. Point mutations in the channel-lining TM2 region of α1β2L259Sγ2-, α2S270W- and β1S265W-containing GABAA receptors also led to spontaneous opening, facilitated direct channel opening by agonists, barbiturates, propofol and neurosteroids, and decreased allosteric potentiation (Thompson et al., 1999b, Chang and Weiss, 1999, Findlay et al., 2000). The α1β3ε isoform is the first GABAA receptor with three different endogenous subunits that exhibits substantial spontaneous currents (Neelands et al., 1999). However, its pharmacological characterization has been restricted to openings by GABA and pentobarbital (Thompson et al., 1998) and to blockade by picrotoxin and Zn2+ (Neelands et al., 1999). Spontaneous currents of α1β3ε receptors can be attributed mainly to the presence of the ε subunit, whose structure most closely resembles the γ subunit (Davies et al., 1997). Here, we have studied a number of low efficacy GABA agonists and GABAergic allosteric modulators focusing on the effects of these compounds on the spontaneous activity of α1β3ε GABAA receptors.

Section snippets

Materials

Flunitrazepam, sodium pentobarbital, allopregnanolone, pregnanolone, epipregnanolone, 5β-pregnane-3α,21-diol-20-one (THDOC), dehydroepiandrosterone 3β-sulfate·2H2O (DHEAS), flumazenil (Ro 15-1788), pregnenolone sulfate, piperidine-4-sulphonic acid (P4S), picrotoxin and furosemide were obtained from Sigma (Poole, UK), SR 95531 {2-(3-carboxypropyl)3-amino-6-p-methoxyphenylpyridazinium bromide}, 4′-chlorodiazepam (Ro 5-4864), TBPS (t-butylbicyclo-phosphorothionate) were from Research Biochemicals

Inward currents elicited by GABAergic agents

Concentration–response curves for the inward current elicited by GABA were constructed on α1β3ε and α1β3γ2s receptors (Fig. 1). Similar to receptors composed of α1β1ε (Whiting et al., 1997), the EC50 value of GABA was significantly lower (P<0.0001) for α1β3ε receptors (0.76±0.16 μM, n=9) compared to α1β3γ2s (15.7±1.5 μM, n=12). In addition, the Hill coefficient was 0.75±0.05 (n=9) on α1β3ε compared to 1.54±0.02 (n=12) on α1β3γ2s. Previous reports have identified P4S and thio-4-PIOL as being low

Activation of GABAA ionophores is facilitated by ε versus γ subunits

The EC50 of GABA on α1β3ε GABAA receptors was identical in oocytes here and in mouse fibroblast L929 cells (Neelands et al., 1999). GABA was 20 times more potent when α1β3 subunits were expressed with ε instead of γ2 subunits. The lower Hill coefficients on α1β3ε receptors might be partly attributed to desensitization during activation by high GABA concentrations. The efficacies of the partial agonists P4S and thio-4-PIOL as well as the potency of P4S are higher for α1β3ε receptors here than

Acknowledgments

This study was supported by Grant T 029723 from the Hungarian Science Research Fund OTKA and by the Soros Foundation to Gábor Maksay.

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