Allosteric modulation in spontaneously active mutant γ-aminobutyric acidA receptors in frogs

doi:10.1016/S0304-3940(00)01503-2

Neuroscience Letters

Volume 293, Issue 3, 3 November 2000, Pages 155-158

https://doi.org/10.1016/S0304-3940(00)01503-2 Get rights and content

Abstract

Tryptophan substitutions were made in the second transmembrane domain of the γ-aminobutyric acid_A (GABA_A) receptor α and β subunits and the resulting mutant receptors, containing α₂(S270W) and/or β₁(S265W), were expressed in Xenopus oocytes. Mutation of either or both subunits resulted in receptors that exhibited enhanced sensitivity to agonist and were spontaneously active in the absence of GABA. The spontaneous activity was blocked by picrotoxin or bicuculline. The enhancement of GABA-induced currents by pentobarbital, by the neurosteroid 5α-pregnan-3α-ol-20-one, and by the benzodiazepine flunitrazepam was dramatically reduced in the mutant receptors. These results are consistent with the idea that a mutation that promotes gating behavior in a ligand-gated ion channel will also show reduced effects of all positive allosteric modulators in a generalized manner, even when these modulators act at distinct sites on the receptor.

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γ-aminobutyric acid type A (GABA <inf>A</inf> ) receptor subtype inverse agonists as therapeutic agents in cognition
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The transitions between the various states (R, A, D) have different “energy barriers” and each state can be stabilized by ligand binding (Hogg et al., 2005). Certain types of receptors have been found to be constitutively active (CA or spontaneously active without ligand) and it is likely that, in this case, the open state is energetically relatively more stable (Findlay et al., 2000; Hogg et al., 2005). The existence of constitutive activity for G protein-coupled receptors (GPCRs) was first described by Costa (Costa and Herz, 1989) and is now firmly rooted in receptor pharmacology (Milligan, 2003).
The gabaergic system has been identified as a relevant regulator of cognitive and emotional processing. In fact, the discovery that negative allosteric regulators (or inverse agonists) at GABA_A (γ-aminobutyric acid) α5 subtype receptors improve learning and memory tasks, has further validated this concept. The localization of these extrasynaptic subtype receptors, mainly in the hippocampus, has suggested that they play a key role in the three stages of memory: acquisition, consolidation, and retrieval.
The “α5 inverse agonist” binds to an allosteric site at GABA_A receptor, provoking a reduction of chlorine current, but to elicit this effect, the necessary condition is the binding of agonist neurotransmitter (gamma-amino butyric acid) at its orthosteric site. In this case, the GABA_A receptor is not a “constitutively active receptor” and, however, the presence of spontaneous opening channels for native GABA_A receptors is rare.
Here, we present various classes of nonselective and α5 selective GABA_A receptor ligands, and the in vitro and in vivo tests to elucidate their affinity and activity.
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Transmembrane residues define the action of isoflurane at the GABA <inf>A</inf> receptor alpha-3 subunit
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These receptors displayed ∼100-fold increase in sensitivity to GABA (EC50 = 0.22 μM, n = 12), while the Hill coefficient (nH = 1.17 ± 0.08) and maximal currents (IMAX = −634 ± 134 pA) were unchanged. Previous reports have demonstrated that mutation of specific residues within the TM domains of GABAA subunits associated with increased agonist potency produces spontaneously open channels [6,13]. To test this possibility in the α3 subunit, we applied 100 μM of the channel blocker picrotoxin (PTX) in the absence of GABA to HEK293 cells expressing wild-type and mutant α3 receptors.
The γ-aminobutyric acid type A (GABA_A) receptor is the target of a structurally diverse group of sedative, hypnotic, and anesthetic drugs, including the volatile anesthetic isoflurane. Previous studies on the GABA_A receptor have suggested the existence of a cavity located between transmembrane (TM) segments 2 and 3 in both alpha-1 and alpha-2 subunits, within which volatile anesthetics might bind. In this study, we have used site-directed mutagenesis to investigate the involvement of homologous residues of the GABA_A alpha-3 subunit in allosteric modulation by isoflurane. Mutation of serine residue 294 within the TM2 to histidine or tyrosine increased the potency of GABA and decreased positive modulation by isoflurane. Mutation of alanine residue 315 within the TM3 to tryptophan increased the potency of GABA and abolished isoflurane modulation. The activity of the intravenous anesthetic propofol was unaltered from wild-type at these mutant receptors. These findings are consistent with the action of isoflurane on a critical site within the transmembrane domains of the receptor and suggest a degree of functional homology between the GABA_A alpha-1, -2, and -3 subunits.
The pharmacology of spontaneously open α<inf>1</inf>β<inf>3</inf>ε GABA<inf>A</inf> receptor-ionophores
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Human α₁β₃ε GABA_A 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 GABA_A 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 GABA_A 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 GABA_A receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.
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Both possibilities would, however, suggest that, in this case, the mutations we studied affected transduction of a nonspecific effect of SDS on interfacial properties. This is also consistent with mutation of amino acid 270 of the GABAA receptor to other residues, which have been shown to affect signal transduction which causes mutation of the serine in position 270 of the GABAA receptor to tryptophan affects channel gating, but also reduces the effect of other positive modulators.27 The prediction that GABA would positively modulate Gly receptor function, and that Gly would modulate GABAA receptor function, was based on a theory of anesthesia that proposes that anesthetics modulate bilayer properties that are coupled to receptor function.17
No theory of inhaled anesthetic action requires volatility of the anesthetic to accomplish the biophysical interaction of anesthetic with biological target. The identification of mutations that attenuate the effect of inhaled anesthetics on various receptors raises the possibility that nonvolatile compounds with anesthetic effects can be identified with the aid of these receptors. In previous studies, we identified compounds that were either charged or had an exceptionally low vapor pressure and which modulated anesthetic-sensitive receptors in a manner similar to inhaled anesthetics. We tested whether these, and another charged compound, shared a common mechanism with volatile anesthetics, by comparing their effect on wild-type γ-aminobutyric acid type A (GABA_A) or glycine receptors and mutant receptors that were engineered to be relatively resistant to inhaled anesthetics.
The effect of β-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA were tested on homomeric α₁ and mutant α₁ (S267I) glycine receptors. The effect of sodium dodecyl sulfate and glycine were tested on α₁b₂γ_2s and mutant α₁(S270I)β₂γ_2s GABA_A receptors. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. For both GABA_A and glycine receptors, isoflurane and ethanol were used as positive controls and propofol as a negative control (i.e., unaffected by the mutation).
β-hydroxybutyric acid, ammonium chloride, diethylhexyl phthalate, and GABA all enhanced glycine receptor function. This effect was reduced by the S267I mutations. Sodium dodecyl sulfate and glycine enhanced GABA_A receptor function, and the S270I mutation attenuated this effect.
These findings support the hypothesis that the compounds studied modulate GABA_A or glycine receptors by a mechanism similar to that of isoflurane and ethanol. Comparing the effect of drugs on anesthetic-sensitive wild-type receptors with relatively less sensitive mutant receptors may help identify compounds with anesthetic effects.

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Allosteric modulation in spontaneously active mutant γ-aminobutyric acidA receptors in frogs

Abstract

Trends Pharmacol. Sci.

Biophys. J.

Trends Pharmacol. Sci.

Use of constitutive G protein-coupled receptor activity for drug discovery

Mol. Pharmacol.

Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes

Alcohol Clin. Exp. Res.

Agonist gating and isoflurane potentiation in the human gamma-aminobutyric acid type A receptor determined by the volume of a second transmembrane domain residue

Mol. Pharmacol.

Propofol and other intravenous anesthetics have sites of action on the γ-aminobutyric acid type A receptor distinct from that for isoflurane

Mol. Pharmacol.

Allosteric modulation in spontaneously active mutant γ-aminobutyric acid_A receptors in frogs

Ethanol, flunitrazepam, and pentobarbital modulation of GABA_A receptors expressed in mammalian cells and Xenopus oocytes