The pharmacology of spontaneously open α1β3ε GABAA receptor–ionophores
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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