Low nanomolar GABA effects at extrasynaptic α4β1/β3δ GABA(A) receptor subtypes indicate a different binding mode for GABA at these receptors

Ionotropic GABA_A receptors are a highly heterogenous population of receptors assembled from a combination of multiple subunits. The aims of this study were to characterize the potency of GABA at human recombinant δ-containing extrasynaptic GABA_A receptors expressed in Xenopus oocytes using the two-electrode voltage clamp technique, and to investigate, using site-directed mutagenesis, the molecular determinants for GABA potency at α4β3δ GABA_A receptors. α4/δ-Containing GABA_A receptors displayed high sensitivity to GABA, with mid-nanomolar concentrations activating α4β1δ (EC₅₀ = 24 nM) and α4β3δ (EC₅₀ = 12 nM) receptors. In the majority of oocytes expressing α4β3δ subtypes, GABA produced a biphasic concentration-response curve, and activated the receptor with low and high concentrations (EC₅₀(1) = 16 nM; EC₅₀(2) = 1.2 μM). At α4β2δ, GABA had low micromolar activity (EC₅₀ = 1 μM). An analysis of 10 N-terminal singly mutated α4β3δ receptors shows that GABA interacts with amino acids different to those reported for α1β2γ2 GABA_A receptors. Residues Y205 and R207 of the β3-subunit significantly affected GABA potency, while the residue F71 of the α4- and the residue Y97 of the β3-subunit did not significantly affect GABA potency. Mutating the residue R218 of the δ-subunit, equivalent to the GABA binding residue R207 of the β2-subunit, reduced the potency of GABA by 670-fold, suggesting a novel GABA binding site at the δ-subunit interface. Taken together, GABA may have different binding modes for extrasynaptic δ-containing GABA_A receptors compared to their synaptic counterparts.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

3-Hydroxy-2'-methoxy-6-methylflavone: a potent anxiolytic with a unique selectivity profile at GABA(A) receptor subtypes

Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA_A receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA_A receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC₅₀ values between 38 and 106 μM) and occurred in a flumazenil-insensitive manner. 3-OH-2′MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4–11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2′MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors (EC₅₀ values between 1.4 and 2.5 μM) was 10-fold higher than at α4β1δ GABA_A receptors. 3-OH-2′MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2′MeO6MF (1–100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2′MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA_A receptor subtypes.     

Development of a Robust Mammalian Cell‐based Assay for Studying Recombinant α4β1/3δ GABAA Receptor Subtypes

δ‐Containing GABA_A receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ‐containing GABA_A receptors. Inconveniently, the recombinant expression of especially α₄β_1/3δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96‐well format cell‐based assay for extrasynaptic α₄β_1/3δ receptors, we have engineered and validated a HEK293 Flp‐In? cell line stably expressing the human GABA_A δ‐subunit. Upon co‐transfection of α₄ and β_1/3 subunits, at optimized ratios, we have established a well‐defined system for expressing α₄β_1/3δ receptors and used the fluorescence‐based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α₄β_1/3δ and binary α₄β_1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ‐containing receptors, whereas Zn²⁺ had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ‐selective compounds, AA29504 and 3‐OH‐2'MeO6MF, were non‐selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set‐up, based on transient expression of human α₄ and β_1/3 subunits into a δ‐subunit stable HEK293 Flp‐In? cell line, portrays a simple 96‐well format assay as a useful supplement to electrophysiological recordings on δ‐containing GABA_A receptors.     

Novel compounds selectively enhance δ subunit containing GABAA receptors and increase tonic currents in thalamus

Inhibition in the brain is dominated by the neurotransmitter γ-aminobutyric acid (GABA); operating through GABA_A receptors. This form of neural inhibition was presumed to be mediated by synaptic receptors, however recent evidence has highlighted a previously unappreciated role for extrasynaptic GABA_A receptors in controlling neuronal activity. Synaptic and extrasynaptic GABA_A receptors exhibit distinct pharmacological and biophysical properties that differentially influence brain physiology and behavior. Here we used a fluorescence-based assay and cell lines expressing recombinant GABA_A receptors to identify a novel series of benzamide compounds that selectively enhance, or activate α4β3δ GABA_A receptors (cf. α4β3γ2 and α1β3γ2). Utilising electrophysiological methods, we illustrate that one of these compounds, 4-chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS1) potently (low nM) enhances GABA-evoked currents mediated by α4β3δ receptors. At similar concentrations DS1 directly activates this receptor and is the most potent known agonist of α4β3δ receptors. 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide (DS2) selectively potentiated GABA responses mediated by α4β3δ receptors, but was not an agonist.Recent studies have revealed a tonic form of inhibition in thalamus mediated by the α4β2δ extrasynaptic GABA_A receptors that may contribute to the regulation of thalamocortical rhythmic activity associated with sleep, wakefulness, vigilance and seizure disorders. In mouse thalamic relay cells DS2 enhanced the tonic current mediated by α4β2δ receptors with no effect on their synaptic GABA_A receptors. Similarly, in mouse cerebellar granule cells DS2 potentiated the tonic current mediated by α6βδ receptors. DS2 is the first selective positive allosteric modulator of δ-GABA_A receptors and such compounds potentially offer novel therapeutic opportunities as analgesics and in the treatment of sleep disorders. Furthermore, these drugs may be valuable in elucidating the physiological and pathophysiological roles played by these extrasynaptic GABA_A receptors.     

3-Hydroxy-2′-methoxy-6-methylflavone: A potent anxiolytic with a unique selectivity profile at GABAA receptor subtypes

Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA_A receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA_A receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC₅₀ values between 38 and 106 μM) and occurred in a flumazenil-insensitive manner. 3-OH-2′MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4–11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2′MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors (EC₅₀ values between 1.4 and 2.5 μM) was 10-fold higher than at α4β1δ GABA_A receptors. 3-OH-2′MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2′MeO6MF (1–100 mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2′MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA_A receptor subtypes.     

The protective effect of M40401, a superoxide dismutase mimetic, on post-ischemic brain damage in Mongolian gerbils

Background

Amino acids in the β subunit contribute to the action of general anaesthetics on GABA_A receptors. We have now characterized the phenotypic effect of two β subunit mutations in the most abundant GABA_A receptor subtype, α1β2γ2.

Results

The β2(N265M) mutation in M2 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, while the direct actions of propofol, etomidate and alphaxalone were impaired. The β2(M286W) mutation in M3 decreased the modulatory actions of propofol, etomidate and enflurane, but not of alphaxalone, whereas the direct action of propofol and etomidate, but not of alphaxalone, was impaired.

Conclusions

We found that the actions of general anaesthetics at α1β2(N265M)γ2 and α1β2(M286W)γ2 GABA_A receptors are similar to those previously observed at α2β3(N265M)γ2 and α2β3(M286W)γ2 GABA_A recpetors, respectively, with the notable exceptions that the direct action of propofol was decreased in α1β2(M286W)γ2 receptors but indistinguishable form wild type in α2β3(M286W)γ2 receptors and that the direct action of alphaxalone was decreased in α1β2(N265M)γ2 but not α2β3(N265M)γ2 receptors and indistinguishable form wild type in α1β2(M286W)γ2 receptors but increased in α2β3(M286W)γ2 receptors. Thus, selected phenotypic consequences of these two mutations are GABA_A receptor subtype-specific.     

Distinct actions of etomidate and propofol at β3-containing γ-aminobutyric acid type A receptors

Etomidate and propofol have clearly distinguishable effects on the central nervous system. However, studies in knock-in mice provided evidence that these agents produce anesthesia via largely overlapping molecular targets, namely GABA_A receptors containing β3 subunits. Here the authors address the question as to whether etomidate and propofol are targeting different subpopulations of β3 subunit containing GABA_A receptors.The effects of etomidate and propofol (0.5 μM and 1.0 μM) on spontaneous activity of neocortical neurons were investigated in organotypic slice cultures from wild-type and β3(N265M) knock-in mice. Firing patterns were characterized by mean burst length and number of action potentials per burst. Additionally, etomidate and propofol actions on GABA_A receptor-mediated currents were investigated by whole-cell voltage clamp recordings.On the network level, the duration of spontaneously occurring bursts of action potentials was decreased by etomidate but increased by propofol in the wild-type. The effects of etomidate were abolished in β3(N265M) mutant slices while those of propofol were qualitatively inverted. On the receptor level, GABA_A receptor-mediated inhibition of cortical neurons was modulated by etomidate and propofol in different ways. Again, drug-specific actions of etomidate and propofol were largely attenuated by the β3(N265M) mutation.Etomidate and propofol alter the firing patterns and GABA_A receptor-mediated inhibition of neocortical neurons in different ways. This suggests that etomidate and propofol act via non-uniform molecular targets. Because the major effects induced by these anesthetics were attenuated by the β3(N265M) mutation, different subpopulations of β3-containing GABA_A receptors are likely to be involved.     

Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

Zolpidem is a widely prescribed sleep aid with relative selectivity for GABA_A receptors containing α1–3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABA_A receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABA_A receptor-mediated tonic current (I_tonic) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that α1–3 subunits are expressed in synaptic GABA_A receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABA_A receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABA_A receptors containing α1–3 subunits contribute to the I_tonic.     

Volatile anesthetic effects on isolated GABA synapses and extrasynaptic receptors

The volatile anesthetics enhance GABAergic inhibitory transmission at synaptic and extrasynaptic sites at central neurons. In the present study, we investigated the effects of three volatile anesthetics (isoflurane, enflurane and sevoflurane) on synaptic and extrasynaptic GABA_A receptor responses using mechanically dissociated rat hippocampal CA1 neurons in which functional native nerve endings (boutons) were retained. The extrasynaptic GABA_A receptors were activated by exogenous GABA application while synaptic ones were assessed by miniature and evoked inhibitory postsynaptic currents (mIPSCs and eIPSCs, respectively). All volatile anesthetics concentration-dependently enhanced the exogenous GABA-induced postsynaptic responses. The structural isomers, isoflurane and enflurane, increased mIPSC frequency while sevoflurane had no effect. None of these anesthetics altered mIPSC amplitudes at their clinically relevant concentrations. Sevoflurane prolonged event kinetics by increasing decay time of mIPSCs and eIPSCs at clinically relevant concentration. On the other hand, both isoflurane and enflurane only prolonged the kinetics of these events at 1 mM of high concentration. For GABAergic eIPSCs, both isoflurane and enflurane decreased the evoked response amplitude and increased the failure rate (Rf), while sevoflurane decreased the amplitude without affecting Rf. These results suggest that isoflurane and enflurane at the clinically relevant concentrations predominantly act on GABAergic presynaptic nerve endings to decrease action potential dependent GABA release. It was concluded that these anesthetics have heterogeneous effects on mIPSCs and eIPSCs with different modulation of synaptic and extrasynaptic GABA_A receptors.     

Characterization of neurosteroid effects on hyperpolarizing current at α4β2δ GABAA receptors

Rationale

The neurosteroid 3α,5β-THP (3α-OH-5β-pregnan-20-one, pregnanolone) is a modulator of the GABA_A receptor (GABAR), with α4β2δ GABARs the most sensitive. However, the effects of 3α,5β-THP at α4β2δ are polarity-dependent: 3α,5β-THP potentiates depolarizing current, as has been widely reported, but decreases hyperpolarizing current by accelerating desensitization.

Objectives

The present study further characterized 3α,5β-THP inhibition of hyperpolarizing current at this receptor and compared effects of other related steroids at α4β2δ GABARs.

Methods

α4β2δ GABARs were expressed in HEK-293 cells, and agonist-gated current recorded with whole cell voltage-clamp techniques using a theta tube to rapidly apply agonist before and after application of neurosteroids.

Results

The GABA-modulatory steroids (30 nM) 3α,5α-THP (3α-OH-5α-pregnan-20-one, allopregnanolone) and THDOC (3α,21-dihydroxy-5α-pregnan-20-one) inhibited hyperpolarizing GABA (10 μM)-gated current at α4β2δ GABARs similar to 3α,5β-THP, while the inactive 3β,5β-THP isomer had no effect. Greater inhibition was seen for current gated by the high efficacy agonist gaboxadol (THIP, 100 μM) than for GABA (0.1–1000 μM), consistent with an effect of 3α,5β-THP on desensitization. Inhibitory effects of the steroid were not seen under low [Cl^?] conditions or in the presence of calphostin C (500 nM), an inhibitor of protein kinase C. Chimeras swapping the IL (intracellular loop) of α4 with α1, when expressed with β2 and δ, produced receptors (α[414]β2δ) which were not inhibited by 3α,5β-THP when GABA-gated current was hyperpolarizing, while α[141]β2δ exhibited steroid-induced polarity-dependent modulation.

Conclusions

These findings suggest that numerous neurosteroids exhibit polarity-dependent effects at α4β2δ GABARs, which are dependent upon protein kinase C and the IL of α4.     

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

Fragile X syndrome is the most common heritable form of mental retardation. It is caused by silencing of the Fmr1 gene and the absence of the encoded protein. The purpose of this study was to examine global protein expression levels of GABA_A and GABA_B receptors, and GABAergic enzymes and trafficking proteins in fragile X knockout mice during brain maturation. Quantitative western blotting of homogenates of forebrain revealed that the levels of GABA_A β1 and β3, GABA_B-R1, NKCC1, KCC2, gephyrin and ubiquilin were not significantly different from wild-type mice at any of the postnatal time points examined.In contrast, the GABA_A receptor α1, β2, and δ subunits, and the GABA enzymes GABA transaminase and succinic semialdehyde dehydrogenase were down-regulated during postnatal development, while GAD65 was up-regulated in the adult knockout mouse brain. The GABA_A receptor α1 and β2 subunits displayed a divergent pattern of developmental expression whereby α1 was reduced in the immature brain but regained a level of expression similar to wild-type mice by adulthood, while the expression of β2 was similar to wild-types at postnatal day 5 but reduced at day 12 and in the adult brain. The GABA_A receptor δ subunit and the GABA catabolic enzymes GABA transaminase and succinic semialdehyde dehydrogenase were simultaneously but transiently decreased only at postnatal day 12. Our results demonstrate that GABA_A receptor subunits and GABA enzymes display complex patterns of changes during brain development suggesting that dynamic interactions may occur between GABA transmitter levels and GABA receptors in fragile X syndrome.     

GABAA receptor open-state conformation determines non-competitive antagonist binding

The γ-aminobutyric acid (GABA) type A receptor (GABA_AR) is one of the most important targets for insecticide action. The human recombinant β3 homomer is the best available model for this binding site and 4-n-[³H]propyl-4′-ethynylbicycloorthobenzoate ([³H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the β3 homomer relative to the much-less-active but structurally very-similar β1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The β1 and β3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera β3/β1 with the β3 subunit extracellular domain and the β1 subunit transmembrane helices retained the high [³H]EBOB binding level of the β3 homomer while chimera β1/β3 with the β1 subunit extracellular domain and the β3 subunit transmembrane helices had low binding activity similar to the β1 homomer. GABA at 3 μM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive β1/β3 chimera close to wildtype α1β1 activity. EBOB binding was significantly altered by mutations β1S15′N and β3N15′S compared with wildtype β1 and β3, respectively. However, the binding activity of α1β1S15′N was insensitive to GABA and α1β3N15′S was stimulated much less than wildtype α1β3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation β3N15′S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABA_A receptor sensitivity.     

Methionine 286 in transmembrane domain 3 of the GABAA receptor β subunit controls a binding cavity for propofol and other alkylphenol general anesthetics

γ-Aminobutyric acid type A (GABA_A) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA_A receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA_A receptor β₂ subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the β₂ subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the β₂(M286W) mutation resulted in a decrease in the ‘cut-off’ volume for propofol analog molecules to enhance GABA responses at GABA_A α₁β₂γ_2s receptors. This suggests that mutation of M286 in the GABA_A β₂ subunit alters the dimensions of a ‘binding pocket’ for propofol and related alkylphenol general anesthetics.     

The effects of a point mutation of the beta2 subunit of GABA(A) receptor on direct and modulatory actions of general anesthetics

The gamma-aminobutyric acid type A receptor (GABA(A) receptor) sites involved in the direct and modulatory actions of general anesthetics remain to be elucidated. The mutation of tyrosine at position 157 in the beta2 GABA(A) receptor subunit was reported to reduce sensitivity to activation by GABA, but not pentobarbital. We examined whether this mutation of the beta2 subunit (Tyr157-->Phe) affects the direct and modulatory actions of other general anesthetics such as propofol and etomidate. Using the two-electrode voltage clamp method, we recorded Cl- current in Xenopus oocytes expressing alpha1beta2gamma2s and alpha1-mutated beta2gamma2s subunits. The mutation of the beta2 subunit reduced the apparent affinity for propofol. However, the mutation had no effect on both the direct actions of pentobarbital and etomidate or on the modulatory actions of pentobarbital, propofol and etomidate. These results suggest that unique loci may exist for the direct action of propofol and that the GABA binding site may not mediate the modulatory actions of general anesthetics at GABA(A) receptors.     

Triazolam is more efficacious than diazepam in a broad spectrum of recombinant GABAA receptors

Benzodiazepine-induced modifications of GABA (γ-aminobutyric acid) activated Cl⁻ currents were studied in native GABA_A receptors expressed in neonatal rat brain cortical neurons in primary cultures and in recombinant GABA_A receptors expressed in transformed human embryonic kidney cells (293) after a transient transfection with cDNAs encoding for different molecular forms of α, β, and γ subunits of GABA_A receptors. The efficacy of triazolam in cortical neurons was higher than that of diazepam. In transfected cells, triazolam showed a greater efficacy as a positive modulator of GABA-elicited Cl⁻ currents in α1β1γ1, α1β1γ2, α1β1γ3, α6β1γ2 and α1β3γ2 receptors than diazepam, except in α3β1γ2 receptors where diazepam was more efficacious. When triazolam and diazepam were applied together to GABA_A receptors assembled by transfecting cDNAs encoding for α1β1γ1 subunits, the action of triazolam was curtailed in a manner related to the dose of diazepam. In recombinant receptors assembled with α1β1γ1 receptors, maximally active doses of triazolam were more efficacious than those of clonazepam, alpidem, zolpidem, diazepam or bretazenil.     

Flavan-3-ol esters: new agents for exploring modulatory sites on GABA(A) receptors

BACKGROUND AND PURPOSE

Enhancement of GABAergic function is the primary mechanism of important therapeutic agents such as benzodiazepines, barbiturates, neurosteroids, general anaesthetics and some anticonvulsants. Despite their chemical diversity, many studies have postulated that these agents may bind at a common or overlapping binding site, or share an activation domain. Similarly, we found that flavan-3-ol esters act as positive modulators of GABA_A receptors, and noted that this action resembled the in vitro profile of general anaesthetics. In this study we further investigated the interactions between these agents.

EXPERIMENTAL APPROACH

Using two-electrode voltage clamp electrophysiological recordings on receptors of known subunit composition expressed in Xenopus oocytes, we evaluated positive modulation by etomidate, loreclezole, diazepam, thiopentone, 5α-pregnan-3α-ol-20-one (THP) and the flavan-3-ol ester 2S,3R-trans 3-acetoxy-4′-methoxyflavan (Fa131) on wild-type and mutated GABA_A receptors.

KEY RESULTS

The newly identified flavan, 2S,3S-cis 3-acetoxy-3′,4′-dimethoxyflavan (Fa173), antagonized the potentiating actions of Fa131, etomidate and loreclezole at α1β2 and α1β2γ2L GABA_A receptors. Furthermore, Fa173 blocked the potentiation of GABA responses by high, but not low, concentrations of diazepam, but did not block the potentiation induced by propofol, the neurosteroid THP or the barbiturate thiopental. Mutational studies on ‘anaesthetic-influencing’ residues showed that, compared with wild-type GABA_A receptors, α1M236Wβ2γ2L and α1β2N265Sγ2L receptors are resistant to potentiation by etomidate, loreclezole and Fa131.

CONCLUSIONS AND IMPLICATIONS

Fa173 is a selective antagonist that can be used for allosteric modulation of GABA_A receptors. Flavan-3-ol derivatives are potential ligands for etomidate/loreclezole-related binding sites at GABA_A receptors and the low-affinity effects of diazepam are mediated via the same site.     

The wake-promoting transmitter histamine preferentially enhances α-4 subunit-containing GABAA receptors

Histamine is an important wake-promoting neurotransmitter that activates seven-transmembrane G-protein coupled histamine receptors. However, histamine demonstrates target promiscuity, including direct interaction with the structurally unrelated glutamate (NMDA) and GABA_A receptor channels. Previous work showed that histamine enhances the activity of recombinant GABA_A receptor isoforms typically found in synaptic locations, although co-release of histamine and GABA is not known to occur in vivo. Here we used patch clamp recordings of various recombinant GABA_A receptor isoforms (α1-6, β1-3, γ1-3, δ) to test the hypothesis that histamine might show subunit preference under low GABA concentration (extrasynaptic) conditions. We found that histamine potentiated the whole-cell responses to GABA for all tested subunit combinations. However, the magnitude of enhancement was largest (∼400% of EC₁₀ GABA-evoked currents) with α4β3 and α4β3X isoforms, where X could be γ or δ. In contrast, histamine (1 mM) had small effects on prolonging deactivation of α4β3γ2 receptors following brief (5 ms) pulses of 1 mM GABA. These findings suggest GABA-histamine cross-talk may occur preferentially at low GABA concentrations, which could theoretically be inhibitory (via enhancing tonic inhibition), directly excitatory (via enhancing presynaptic GABAergic signaling), or indirectly excitatory (via inhibiting GABAergic interneurons).     

Role of the alpha subunit in the modulation of GABA(A) receptors by anabolic androgenic steroids

Neural transmission mediated by circuits expressing α₂ subunit-containing γ-aminobutyric acid type A (GABA_A) receptors is critical for the expression of behaviors known to be altered by anabolic androgenic steroids (AAS). Here we show that micromolar concentrations of AAS, which reflect levels found in steroid abusers, induce positive modulation of currents from α₂β₃γ_2L recombinant receptors elicited by pulses of GABA that mimic synaptic conditions in a manner that is mechanistically distinct from modulation induced at α₁β₃γ_2L receptors. Specifically, at α₂-containing receptors, the AAS, 17α-methyltestosterone (17α-MeT) enhanced peak current, slowed deactivation, diminished desensitization, and promoted entry of receptors into more distal states along the activation pathway. Analysis of GABA_A receptor-mediated synaptic currents in primary cortical neurons followed by single cell real-time RT-PCR demonstrated that 17α-MeT enhancement of synaptic currents is proportional to the ratio of α₂ to α₁ subunit mRNA. Finally, we show that the modulation elicited by AAS is not comparable to that produced by micromolar concentrations of other positive allosteric modulators at α₂-containing receptors. In sum, these data indicate that AAS elicit effects on GABA_A receptor function that depend significantly on α subunit composition and that the mechanism of AAS modulation of GABA_A receptors is distinct from that of other positive allosteric modulators.