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.

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.