Negative modulation of the GABAAρ1 receptor function by histamine.

Besides its function as a local mediator of the immune response, histamine can play a role as a neurotransmitter and neuromodulator. Histamine actions are classically mediated through four different G protein-coupled receptor subtypes but non-classical actions were also described, including effects on many ligand-gated ion channels. Previous evidence indicated that histamine acts as a positive modulator on diverse GABAA receptor subtypes, such as GABAAα1ß2γ2, GABAAα2ß3γ2, GABAAα3ß3γ2, GABAAα4ß3γ2 and GABAAα5ß3γ2. Meanwhile, its effects on GABAAρ1 receptors, known to stand for tonic currents in retinal neurons, had not been examined before. The effects of histamine on the function of human homomeric GABAAρ1 receptors were studied here, using heterologous expression in Xenopus laevis oocytes followed by the electrophysiological recording of GABA-evoked Cl- currents. Histamine inhibited GABAAρ1 receptor-mediated responses. Effects were reversible, independent of the membrane potential, and strongly dependent on both histamine and GABA concentration. A rightward parallel shift in the concentration-response curve for GABA was observed in the presence of histamine, without substantial change in the maximal response or the Hill coefficient. Results were compatible with a competitive antagonism of histamine on the GABAAρ1 receptors. This is the first report of inhibitory actions exerted by histamine on an ionotropic GABA receptor.

Reactive Oxygen Species in the Regulation of the GABA Mediated Inhibitory Neurotransmission.

Reactive oxygen species (ROS) are best known for being involved in cellular metabolism and oxidative stress, but also play important roles in cell communication. ROS signaling has become increasingly recognized as a mechanism implicated in the regulation of synaptic neurotransmission, under both physiological and pathological conditions. Hydrogen peroxide (H2O2) and superoxide anion are the main biologically relevant endogenous ROS in the nervous system. They are predominantly produced in the mitochondria of neurons and glial cells and their levels are tightly regulated by the antioxidant cell machinery, which allows for dynamic signaling through these agents. Physicochemical and biological properties of H2O2 enable it to effectively play an important role in signaling. This review brings up some or the most significant evidence supporting ROS as signaling agents in the nervous system and summarizes data showing that ROS modulate γ-aminobutyric acid (GABA)-mediated neurotransmission by pre- and postsynaptic mechanisms. ROS induce changes on both, the activity of phasic and tonic GABAA receptors and GABA release from presynaptic terminals. Based on these facts, ROS signaling is discussed as a possible selective mechanism linking cellular metabolism to inhibitory neurotransmission through the direct or indirect modulation of the GABAA receptor function. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABAA ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABAA ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABAA ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABAA ρ1 receptors.

Organization of Circadian Behavior Relies on Glycinergic Transmission.

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.

Dynamic Regulation of the GABAA Receptor Function by Redox Mechanisms.

Oxidizing and reducing agents, which are currently involved in cell metabolism and signaling pathways, can regulate fast inhibitory neurotransmission mediated by GABA receptors in the nervous system. A number of in vitro studies have shown that diverse redox compounds, including redox metabolites and reactive oxygen and nitrogen species, modulate phasic and tonic responses mediated by neuronal GABAA receptors through both presynaptic and postsynaptic mechanisms. We review experimental data showing that many redox agents, which are normally present in neurons and glia or are endogenously generated in these cells under physiologic states or during oxidative stress (e.g., hydrogen peroxide, superoxide and hydroxyl radicals, nitric oxide, ascorbic acid, and glutathione), induce potentiating or inhibiting actions on different native and recombinant GABAA receptor subtypes. Based on these results, it is thought that redox signaling might represent a homeostatic mechanism that regulates the function of synaptic and extrasynaptic GABAA receptors in physiologic and pathologic conditions.

Enhancement of tonic and phasic GABAergic currents following nitric oxide synthase inhibition in hippocampal CA1 pyramidal neurons.

Nitric oxide (NO) is involved in synaptic plasticity in the hippocampus through different presynaptic and postsynaptic mechanisms that include the modulation of the GABAergic neurotransmission. Inhibitory synapses on hippocampal pyramidal neurons are known to possess the molecular machinery for retrograde NO-signaling, but the modulation of GABAARs function by NO in these neurons and the mechanisms of action involved have not been fully characterized. Here we show that suppression of the endogenous NO generation by the nitric oxide synthase (NOS) inhibitor L-NAME produces significant and reversible increases in the magnitude of both tonic and phasic GABAergic currents in CA1 hippocampal pyramidal neurons. GABA-evoked chloride currents were measured in the presence or absence of L-NAME using whole-cell patch-clamp recordings in acute hippocampal slices from young adult mice. Enhancement of the tonic GABA responses induced by L-NAME was insensitive to TTX and decreased by co-incubation with the NO donor DEA/NO. Applications of DEA/NO alone did not produce significant effects on tonic GABA responses. L-NAME treatment also increased the amplitude of phasic GABAergic currents evoked by GABA-puffs. Our results indicate that the extent of tonic and phasic inhibition mediated by GABAA receptors in CA1 hippocampal pyramidal neurons is affected by endogenous NO production.

Benzodiazepine modulation of homomeric GABAAρ1 receptors: differential effects of diazepam and 4'-chlorodiazepam.

GABA(A) receptors (GABA(A)Rs) are ligand-gated ion channels that mediate inhibitory neurotransmission in the central nervous system (CNS). They are members of the Cys-loop receptor family and display marked structural and functional heterogeneity. Many GABA(A)Rs receptor subtypes are allosterically modulated by benzodiazepines (BDZs), which are drugs extensively used as anxiolytics, sedative-hypnotics and anticonvulsants. One high-affinity site and at least three additional low-affinity sites for BDZ recognition have been identified in several heteromeric and homomeric variants of the GABA(A)Rs (e.g.: α1ß2γ2, α1ß2/3, ß3, etc.). However, the modulation of homomeric GABA(A)ρRs by BDZs was not previously revealed, and these receptors, for a long a time, were assumed to be fully insensitive to the actions of these drugs. In the present study, human homomeric GABA(A)ρ1 receptors were expressed in Xenopus oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of BDZs. GABA(A)ρ1 receptor-mediated responses were modulated by diazepam and 4'-chlorodiazepam in the micromolar range, in a concentration-dependent, voltage-independent and reversible manner. Diazepam produced potentiating effects on GABA-evoked Cl(-) currents and 4'-Cl diazepam induced biphasic effects depending on the GABA concentration, whereas Ro15-4513 and alprazolam were negative modulators. BDZ actions were insensitive to flumazenil. Other BDZs showed negligible activity at equivalent experimental conditions. Our results suggest that GABA(A)ρ1 receptor function can be selectively and differentially modulated by BDZs.

An intracellular redox sensor for reactive oxygen species at the M3-M4 linker of GABAA ρ1 receptors.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) are normally involved in cell oxidative stress but also play a role as cellular messengers in redox signalling; for example, modulating the activity of neurotransmitter receptors and ion channels. However, the direct actions of ROS on GABAA receptors were not previously demonstrated. In the present work, we studied the effects of ROS on GABAA ρ1 receptor function. EXPERIMENTAL APPROACH: GABAA ρ1 receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of ROS. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis studies were used to identify protein residues involved in ROS actions. KEY RESULTS: GABAA ρ1 receptor-mediated responses were significantly enhanced in a concentration-dependent and reversible manner by H2O2. Potentiating effects were attenuated by a free radical scavenger, lipoic acid or an inhibitor of the Fenton reaction, deferoxamine. Each ρ1 subunit contains only three cysteine residues, two extracellular at the Cys-loop (C¹77 and C¹9¹) and one intracellular (C³64) at the M3-M4 linker. Mutant GABAA ρ1 receptors in which C³64 was exchanged by alanine were completely insensitive to modulation, implying that this site, rather than a cysteine in the Cys-loop, is essential for ROS modulation. CONCLUSION AND IMPLICATIONS: Our results show that the function of GABAA ρ1 receptors is enhanced by ROS and that the intracellular C³64 is the sensor for ROS actions.

Quercetin antagonism of GABAAρ1 receptors is prevented by ascorbic acid through a redox-independent mechanism.

Quercetin is a natural flavonoid widely distributed in plants that acts as a neuroprotective agent and modulates the activity of different synaptic receptors and ion channels, including the ionotropic GABA receptors. GABA(Aρ1) receptors were shown to be antagonized by quercetin, but the mechanisms underlying these antagonistic actions are still unknown. We have analyzed here if the antagonistic action produced by quercetin on GABA(Aρ1) receptors was related to its redox activity or due to alternative mechanism/s. Homomeric GABA(Aρ1) receptors were expressed in frog oocytes and GABA-evoked responses electrophysiologically recorded. Quercetin effects on GABA(Aρ1) receptors were examined in the absence or presence of ascorbic acid. Chemical protection of cysteines by selective sulfhydryl reagents and site directed mutagenesis experiments were also used to determine ρ1 subunit residues involved in quercetin actions. Quercetin antagonized GABA(Aρ1) receptor responses in a dose-dependent, fast and reversible manner. Quercetin inhibition was prevented in the presence of ascorbic acid, but not by thiol reagents that modify the extracellular Cys-loop of these receptors. H141, an aminoacidic residue located near to the ρ1 subunit GABA binding site, was involved in the allosteric modulation of GABA(Aρ1) receptors by several agents including ascorbic acid. Quercetin similarly antagonized GABA-evoked responses mediated by mutant (H141D)GABA(Aρ1) and wild-type receptors, but prevention exerted by ascorbic acid on quercetin effects was impaired in mutant receptors. Taken together the present results suggest that quercetin antagonistic actions on GABA(Aρ1) receptors are mediated through a redox-independent allosteric mechanism.

Allosteric modulation of retinal GABA receptors by ascorbic acid.

Ionotropic GABA receptors (GABA(A) and GABA(C)) belong to the Cys-loop receptor family of ligand-gated ion channels. GABA(C) receptors are highly expressed in the retina, mainly localized at the axon terminals of bipolar cells. Ascorbic acid, an endogenous redox agent, modulates the function of diverse proteins, and basal levels of ascorbic acid in the retina are very high. However, the effect of ascorbic acid on retinal GABA receptors has not been studied. Here we show that the function of GABA(C) and GABA(A) receptors is regulated by ascorbic acid. Patch-clamp recordings from bipolar cell terminals in goldfish retinal slices revealed that GABA(C) receptor-mediated currents activated by tonic background levels of extracellular GABA, and GABA(C) currents elicited by local GABA puffs, are both significantly enhanced by ascorbic acid. In addition, a significant rundown of GABA puff-evoked currents was observed in the absence of ascorbic acid. GABA-evoked Cl(-) currents mediated by homomeric ρ(1) GABA(C) receptors expressed in Xenopus laevis oocytes were also potentiated by ascorbic acid in a concentration-dependent, stereo-specific, reversible, and voltage-independent manner. Studies involving the chemical modification of sulfhydryl groups showed that the two Cys-loop cysteines and histidine 141, all located in the ρ(1) subunit extracellular domain, each play a key role in the modulation of GABA(C) receptors by ascorbic acid. Additionally, we show that retinal GABA(A) IPSCs and heterologously expressed GABA(A) receptor currents are similarly augmented by ascorbic acid. Our results suggest that ascorbic acid may act as an endogenous agent capable of potentiating GABAergic neurotransmission in the CNS.

Redox modulation of homomeric rho1 GABA receptors.

The activity of many receptors and ion channels in the nervous system can be regulated by redox-dependent mechanisms. Native and recombinant GABA(A) receptors are modulated by endogenous and pharmacological redox agents. However, the sensitivity of GABA(C) receptors to redox modulation has not been demonstrated. We studied the actions of different reducing and oxidizing agents on human homomeric GABArho(1) receptors expressed in Xenopus laevis oocytes. The reducing agents dithiothreitol (2 mM) and N-acetyl-L-cysteine (1 mM) potentiated GABA-evoked Cl(-) currents recorded by two-electrode voltage-clamp, while the oxidants 5-5'-dithiobis-2-nitrobenzoic acid (500 microM) and oxidized dithiothreitol (2 mM) caused inhibition. The endogenous antioxidant glutathione (5 mM) also enhanced GABArho(1) receptor-mediated currents while its oxidized form GSSG (3 mM) had inhibitory effects. All the effects were rapid and easily reversible. Redox modulation of GABArho(1) receptors was strongly dependent on the GABA concentration; dose-response curves for GABA were shifted to the left in the presence of reducing agents, whereas oxidizing agents produced the opposite effect, without changes in the maximal response to GABA and in the Hill coefficient. Our results demonstrate that, similarly to GABA(A) receptors and other members of the cys-loop receptor superfamily, GABA(C) receptors are subjected to redox modulation.

Functional activation by central monoamines of human dopamine D(4) receptor polymorphic variants coupled to GIRK channels in Xenopus oocytes.

We studied the functional activation of different polymorphic variants of the human dopamine D(4) receptors by the three major central monoamines, dopamine, noradrenaline and serotonin. Dopamine D(4) receptors carrying two (D4.2), four (D4.4) or seven (D4.7) repeats within the third intracellular domain were co-expressed with G protein-regulated inwardly rectifying potassium channels (GIRK1) in frog oocytes. All the dopamine D(4) receptor variants coupled to oocyte G(i/o) proteins and modulated co-expressed GIRK1 channels. Monoamine-induced responses were detected as increases in voltage-clamp recorded GIRK1 currents. Dopamine, noradrenaline as well as serotonin stimulated dopamine D(4) receptors. Dose-response analysis showed that dopamine and noradrenaline are full agonists whereas serotonin acted as partial agonist. Dopamine was 5-fold more potent on D4.2 and D4.7 (EC(50)=1 nM) than on D4.4 (EC(50)=5 nM) suggesting that the actions of dopamine and therapeutic drugs on dopamine D(4) receptors might vary among individuals depending on their repertoire of expressed alleles. In contrast, noradrenaline and serotonin did not discriminate among dopamine D(4) receptor variants (EC(50 NA)=50 nM, EC(50 5-HT)=1.5 microM). All monoamine effects were blocked by the specific dopaminergic D(4) antagonist (S)-(-)-4-[4-[2-(Isochroman-1-yl)ethyl]piperazin-1-yl]benzenesulfonamide (PNU101387). Sequence analyses of dopamine D(4) receptors and related monoamine receptors revealed that dopamine D(4) receptors have most aminoacidic residues necessary for binding of dopamine, noradrenaline and serotonin. Our data indicate that dopamine D(4) receptors can be pharmacologically stimulated by any the three major central monoamines.

Analysis of macroscopic ionic currents mediated by GABArho1 receptors during lanthanide modulation predicts novel states controlling channel gating.

Lanthanide-induced modulation of GABA(C) receptors expressed in Xenopus oocytes was studied. We obtained two-electrode voltage-clamp recordings of ionic currents mediated by recombinant homomeric GABArho(1) receptors and performed numerical simulations of kinetic models of the macroscopic ionic currents.GABA-evoked chloride currents were potentiated by La(3+), Lu(3+) and Gd(3+) in the micromolar range. Lanthanide effects were rapid, reversible and voltage independent. The degree of potentiation was reduced by increasing GABA concentration.Lu(3+) also induced receptor desensitization and decreased the deactivation rate of GABArho(1) currents. In the presence of 300 microM Lu(3+), dose-response curves for GABA-evoked currents showed a significant enhancement of the maximum amplitude and an increase of the apparent affinity. The rate of onset of TPMPA and picrotoxin antagonism of GABArho(1) receptors was modulated by Lu(3+). These results suggest that the potentiation of the anionic current was the result of a direct lanthanide-receptor interaction at a site capable of allosterically modulating channel properties. Based on kinetic schemes, which included a second open state and a nonconducting desensitized state that closely reproduced the experimental results, two nonexclusive probable models of GABArho(1) channels gating are proposed.

Studies on the mechanisms of action of picrotoxin, quercetin and pregnanolone at the GABA rho 1 receptor.

1. The mechanisms of action of antagonists of the gamma-aminobutyric acid C (GABA(C)) receptor picrotoxin, quercetin and pregnanolone were studied. 2. Ionic currents (chloride), mediated through human homomeric GABA rho(1) receptors expressed in Xenopus oocytes, were recorded by two-electrode voltage clamp. 3. Dose-response (D-R) curves and kinetic measurements of GABA rho(1) currents were carried out in the presence or absence of antagonists. Use-dependent actions were also evaluated. 4. Picrotoxin, quercetin and pregnanolone exerted noncompetitive actions. 5. IC(50) values measured at the EC(50) for GABA (1 microM) were as follows: picrotoxin 0.6+/-0.1 microM (Hill coefficient n=1.0+/-0.2); quercetin 4.4+/-0.4 microM (n=1.5+/-0.2); pregnanolone 2.1+/-0.5 microM (n=0.8+/-0.1). 6. These antagonists produced changes only in the slope of the linear current-voltage relationships, which was indicative of voltage-independent effects. 7. The effect of picrotoxin on GABA rho(1) currents was use-dependent, strongly relied on agonist concentration and showed a slow onset and offset. The mechanism was compatible with an allosteric inhibition and receptor activation was a prerequisite for antagonism. 8. The effect of quercetin was use-independent, showed relatively fast onset and offset, and resulted in a slowed time course of the GABA-evoked currents. 9. The effect of pregnanolone was use-independent, presented fast onset and a very slow washout, and did not affect current activation. 10. All the antagonists accelerated the time course of deactivation of the GABA rho(1) currents.

Flavonoid modulation of ionic currents mediated by GABA(A) and GABA(C) receptors.

The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.