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.

References (36)

  • A.R. Brooks-Kayal et al.

    Human neuronal γ-aminobutyric acidA receptors: coordinated subunit mRNA expression and functional correlates in individual dentate granule cells

    Journal of Neuroscience

    (1999)
  • P.A. Davies et al.

    Insensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit

    Nature

    (1997)
  • A.J. Delaney et al.

    GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amigdala

    Journal of Neuroscience

    (1999)
  • B. Ebert et al.

    Differences in agonist/antagonist binding affinity and receptor transduction using recombinant γ-aminobutyric acid type A receptors

    Molecular Pharmacology

    (1997)
  • J.L. Fisher et al.

    The role of an α subtype M2-M3 His in regulating inhibition of GABAA, receptor current by zinc and other divalent cations

    Journal of Neuroscience

    (1998)
  • C. Grossman et al.

    Mapping the conformational wave of acetylcholine receptor channel gating

    Nature

    (2000)
  • A.M. Hosie et al.

    Actions of the insecticide fipronil on dieldrin-sensitive and -resistant GABA receptors of Drosophila melanogaster

    British Journal of Pharmacology

    (1995)
  • S. Kasparov et al.

    GABAA receptor ε-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat

    Journal of Physiology

    (2001)
  • Cited by (43)

    • Persistent GABA<inf>A/C</inf> responses to gabazine, taurine and beta-alanine in rat hypoglossal motoneurons

      2016, Neuroscience
      Citation Excerpt :

      Bicuculline-resistant GABA responses showing a high barbiturate-sensitivity were previously described in rat spinal dorsal horn neurons and also likely involve some ρ-containing hybrid hetero-oligomeric receptors (Park et al., 1999). In HMs, the results obtained in the absence of SR did not reveal any constitutive activity that would be both barbiturate-sensitive and bicuculline-resistant, such as the spontaneous activity carried without any agonist by homomeric β GABAA receptors (Krishek et al., 1996; Wooltorton et al., 1997), or by some α1β3ε receptors (Maksay et al., 2003). This is consistent with the previously reported inability of picrotoxin to further decrease the basal current recorded in the presence of bicuculline (Chesnoy-Marchais, 2013).

    • A role for loop F in modulating GABA binding affinity in the GABA <inf>A</inf> receptor

      2012, Journal of Molecular Biology
      Citation Excerpt :

      α1β2γ2S receptors have a GABA EC50 of ~ 59 μM (average of 7 studies). Replacing β2 to form α1β3γ2S receptors decreases GABA EC50 to ~ 34 μM (average of 4 studies), whereas replacing α1 to form α6β2γ2S receptors decreases GABA EC50 to ~ 1.5 μM (average of 2 studies).16–18,60–66 Our simulations show that these small differences in the β-subunit and in the α-subunit loop F enable it to make greater contributions towards ligand binding and modulate affinity.

    • Modulation of neurotransmitter systems by dehydroepiandrosterone and dehydroepiandrosterone sulfate: Mechanism of action and relevance to psychiatric disorders

      2008, Progress in Neuro-Psychopharmacology and Biological Psychiatry
      Citation Excerpt :

      The negatively charged group at C3 is important for this inhibitory activity (Park-Chung et al., 1999). 3α-Steroid sulfates are positive modulators of GABAA receptors (El-Etr et al., 1998; Le Foll et al., 1997; Maksay et al., 2003) while 3β–steroid sulfates are negative modulators (El-Etr et al., 1998) or inactive (Le Foll et al., 1997; Maksay et al., 2003). Other steroids such as pregnenolone sulfate (PREGS) also inhibit GABAA receptors but, in contrast to the effect of DHEAS, PREGS-mediated inhibition is not completely reversible (Le Foll et al., 1997).

    View all citing articles on Scopus
    View full text