GABA_AR Properties

GABA_ARs are heteropentameric GABA-gated chloride channels that belong to the Cys-loop ligand-gated ion channel superfamily (Fig. 1A) (Smart and Paoletti, 2012). Diversity in subunit composition underlies the variation in the physiologic and pharmacological properties of GABA_ARs. To date, 19 GABA_AR subunits have been cloned in the mammalian central nervous system (CNS) and classified into classes based on the following sequence identity: α(1–6), β(1–3), γ(1, 2S, 2L, 3), δ, ε, π, θ, and ρ(1–3) (Alexander et al., 2013b). Each individual subunit is composed of a large extracellular N terminus, four transmembrane domains (termed M1-M4, with M2 contributing to the ionic channel), a short intracellular loop (M1-M2 linker), a small extracellular loop (M2-M3 linker), a bulky intracellular loop (M3-M4 linker), and an extracellular C-terminus (Fig. 1). Two cysteine residues located at the external N-terminus of the GABA_AR subunits form a disulfide bridge that delimitate the Cys-loop, a distinct domain conserved in all the members of the receptor superfamily that is a critical target for redox agents within the GABA_AR (Fig. 1A) (Pan et al., 1995; Amato et al., 1999; Calero and Calvo, 2008). Most of the GABA_AR subunits contain extra cysteines (1–11) that can significantly contribute to redox modulation (Fig. 1, A and B, and Supplemental Table 1) (Beltrán González et al., 2014), but the sensitivity of the diverse GABA_AR subtypes to endogenous redox agents is not well characterized, and the functional role of most of these other cysteine residues is presently unknown.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

GABA_AR structure, subunit topology, and identification of cysteine residues located in the different receptor domains. (A, left) Schematic representation showing the transmembrane topology of a single GABA_AR subunit. (A, middle) Crystal structure of the human GABA_Aβ3R homopentamer (Protein Data Bank ID, 4COF.pdb). Cysteine residues at the Cys-loop are marked in red. Note that crystallized GABA_Aβ3Rs have truncated M3-M4 linkers (Miller and Aricescu, 2014). (A, right) Model of γ2 subunit generated with Phyre². Cysteine residues at the Cys-loop, the transmembrane domains, and the intracellular M3-M4 linker are marked in red. Molecular graphics and analyses performed with Chimera, package developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (https://www.cgl.ucsf.edu/chimera/docs/credits.html). (B) Comparison of the primary sequences in the M1, M3, and M3-M4 GABA_AR domains showing the conservation of several cysteine residues at specific positions. Alignments were performed with Clustal Omega.

Phasic and Tonic GABA_AR-Mediated Neurotransmission

Activation of GABA_ARs, after vesicular GABA release from presynaptic terminals, increases the permeability of the postsynaptic membrane to chloride and bicarbonate ions, leading to a net inward flow of anions (Miller and Smart, 2010). GABAergic neurotransmission can take place in two modalities, each one mediated by different GABA_AR subtypes (Farrant and Nusser, 2005). Phasic GABAergic synaptic transmission is mediated by the most abundant heteromeric GABA_AR subtype expressed in brain containing the γ2 subunit (e.g., α1β2γ2). This modality operates point to point, fast, and transiently at high GABA concentrations to build hyperpolarizing postsynaptic responses called inhibitory postsynaptic potentials. In addition, the activation of GABA_ARs by ambient GABA during spillover can induce slower, persistent, and less spatially and temporally restricted “tonic” responses. Tonic GABA responses are commonly considered to be evoked by low concentrations of GABA acting on extrasynaptic receptors located beyond the synaptic cleft, on presynaptic terminals, or at neighboring synapses on the same or adjacent neurons. Tonic GABA responses are usually mediated by GABA_ARs of defined subunit compositions (e.g., α5βγ2 and α4βδ in the hippocampus; α6βδ in the cerebellum; homomeric ρ1 in the retina) (Jones and Palmer, 2009; Brickley and Mody, 2012). The contrasting features of phasic and tonic inhibition, mediated by one or the other GABA_AR subtypes, add extra complexity to the integrative mechanisms of dynamic signaling in neurons. Thus, redox-based regulation of phasic and tonic GABA_A responses could represent new elaborate forms of synaptic modulation, with a differential impact on the excitability of different neuronal types and the activity of discrete CNS circuits.

Redox Agents and Their Role in the Nervous System

A wide variety of redox agents are found in the nervous system, including antioxidant/reducing agents (e.g., ascorbic acid, GSH, lipoic acid, l-cysteine, l-carnitine, l-carnosine, homocysteine, retinol, α-tocopherol, ubiquinol, flavonoids, and carotenoids); enzymes (e.g., superoxide dismutase, glutathione peroxidases, catalase); oxidizing agents such as reactive oxygen species (ROS) [e.g., H₂O₂, the free radicals hydroxyl (OH^•), nitrosium, nitroxyl, and superoxide (O₂^−•)]; and reactive nitrogen species (RNS) (e.g., NO, which reacts with O₂^−• to form the highly toxic peroxynitrite). The brain is particularly vulnerable to oxidative damage due to its high oxygen utilization, a high content of oxidizable polyunsaturated fatty acids, and the presence of redox-active metals (Cu and Fe). Redox homeostasis is maintained in the cells by the balance between the generation and elimination of oxidants by the antioxidant systems. However, redox status can generally undergo fluctuations during both normal and pathologic conditions. Redox signaling is mediated by small and ubiquitous molecules that show high reactivity toward specific redox-sensitive thiols, such as H₂O₂, O₂^−•, OH^•, and NO (Thomas et al., 2008; Rice, 2011; Schieber and Chandel, 2014). These local diffusible messengers commonly produce neurotoxic effects during oxidative and nitrosative stress, and are involved in normal aging and neurodegenerative disorders (Thomas et al., 2008).

ROS are primarily generated as byproducts of mitochondrial oxidative metabolism (Schieber and Chandel, 2014). The damaging effects of ROS can be counteracted by the specific detoxifying enzymes and high levels of antioxidant agents in a sophisticated way such that their local concentration and distribution can be rapidly and precisely controlled to allow dynamic cell signaling (Rhee, 2006; Bao et al., 2009; Woo et al., 2010; Rice, 2011; Finkel, 2011). ROS effects in the nervous system can include transient changes in neuronal activity and synaptic plasticity (Klann, 1998; Bao et al., 2009), as well as diverse actions on both excitatory and inhibitory neurotransmission (Frantseva et al., 1998; Sah and Schwartz-Bloom, 1999; Kamsler and Segal, 2003), including regulatory changes on neurotransmitter receptors (Aizenman et al., 1990; Chu et al., 2006; Coddou et al., 2009; Accardi et al., 2014, 2015; Beltrán González et al., 2014; Penna et al., 2014).

RNS are also involved in the regulation of neuronal excitability and induce several forms of synaptic plasticity (Garthwaite, 2008; Steinert et al., 2010). NO is an unstable free radical gas, produced from l-arginine by the NO synthase (NOS) that acts as a short-lived cell-signaling molecule. However, at high concentrations NO can be a neurotoxic agent producing neuronal injury and apoptotic cell death (Garthwaite, 2008; Hardingham et al., 2013). NO effects are mainly mediated by the activation of a soluble guanylyl cyclase that leads to increased cGMP levels, which in turn activates the cGMP-dependent protein kinase (Bradley and Steinert, 2016). However, NO actions can also take place through cGMP/cGMP-dependent protein kinase–independent pathways, including cysteine S-nitrosylation and S-glutathionylation, which are reversible post-translational modifications that convey redox-based cellular signals (Hess et al., 2005; Okamoto and Lipton, 2015).

Besides the role of the reactive species, the intrinsic antioxidant mechanisms can also contribute to redox signaling (Papadia et al., 2008; Harrison and May, 2009; Aquilano et al., 2014; Covarrubias-Pinto et al., 2015). Neurons and glial cells accumulate ascorbate at millimolar concentrations by specific transporters (Hediger, 2002; Harrison and May, 2009). The extracellular concentrations of ascorbate are also substantial and can transiently undergo large increases during neuronal firing, mainly due to extensive extrusion through the neuronal sodium-dependent vitamin C transporter (Portugal et al., 2009). However, the effects of ascorbate on neurotransmission, and particularly on the function of excitatory and inhibitory receptors, have not been extensively studied. Ascorbate exerts diverse neuromodulatory actions, including redox modulation of synaptic receptors (Majewska et al., 1990; Calero et al., 2011), either directly by reducing amino acidic residues or indirectly by scavenging ROS capable of modifying redox-sensitive residues (Covarrubias Pinto et al., 2015). Meanwhile, GSH, the major thiol antioxidant and redox buffer of the cell, is found at very high concentrations (millimolar) in all the neuronal compartments and in the extracellular milieu (Do et al., 2009). GSH exerts a protective role against oxidative stress, for example, in the detoxification of H₂O₂ and lipid peroxides (as cofactor of the GSH peroxidase), scavenging of OH^• and the regeneration of the most important antioxidants, vitamins C and E. GSH was also involved in the regulation of many synaptic functions and in neuronal plasticity (Do et al., 2009; Robillard et al., 2011; Aquilano et al., 2014).

Phasic and tonic GABA_ARs are targets for the actions of all the above-mentioned redox agents. Thus, the study of the redox-dependent modulation of GABA_ARs is fundamental for understanding the physiologic and pharmacological mechanisms that control neuronal inhibition.

Redox Modulation of the GABA_AR Function

Redox modulation of GABA_AR function was established in several regions of the nervous system through the use of several different in vitro experimental models, including freshly dissociated neurons, acute slices, and whole-mount preparations (Fig. 2) (Pan et al., 1995; Amato et al., 1999; Sah et al., 2002; Calero et al., 2011; Penna et al., 2014; Accardi et al., 2014, 2015). Previous receptor-binding studies indicated that sulfhydryl and disulfide groups may play a role in the responses to GABA and glycine (referenced in Pan et al., 1995); however, the functional modulation of GABA_ARs by redox agents was revealed by studying the effects of endogenous and exogenous thiol reagents on inhibitory responses in retinal neurons (Pan et al., 1995) (Fig. 2). Whole-cell patch-clamp recordings in retinal ganglion cells showed that GABA_A responses were reversibly potentiated in the presence of the reducing agents DTT and GSH, and were inhibited by thiol oxidant agents such as DTNB and oxidized GSH (GSSG) (Fig. 2). Effects were surmounted by pretreatment with the irreversible thiol alkylating agent N-ethylmaleimide, which suggests that, as in other neurotransmitter receptors and ion channels, cysteine residues at the GABA_AR are likely targets for the actions of redox reagents. Thiol reagents elicited opposite effects on glycine-evoked responses evoked in these cells, indicating that other Cys-loop receptors could also be sensitive to redox modulation (Pan et al., 1995). Redox susceptibility of GABA_ARs to diverse endogenous agents (e.g., ascorbic acid, GSH, ROS, RNS) was later corroborated in several neuronal types, including cerebellar granule cells (Amato et al., 1999; Accardi et al., 2015), superior cervical ganglionic neurons (Amato et al., 1999), retinal bipolar neurons (Calero et al., 2011), hippocampal CA1 pyramidal neurons (Penna et al., 2014), and cerebellar stellate cells (Accardi et al., 2014) (Fig. 2). Redox modulation of GABA_AR responses typically depended on GABA concentration; receptors were more sensitive to redox reagents at low GABA levels, and differences in redox sensitivity were associated with different GABA_AR subtypes. For example, GABA_AR-mediated responses in cerebellar granule neurons showed much less susceptibility to redox modulation by thiol reagents than in retinal ganglion neurons (Fig. 2) (Pan et al., 1995, Amato et al., 1999). These results were consistent with the fact that diversity in subunit composition causes variation in the functional properties of GABA_ARs. Additional studies showed that diverse recombinant GABA_AR subtypes, expressed in heterologous systems, also displayed differential susceptibilities to redox agents (Fig. 2 and Supplemental Table 2). As a rule, GABA_AαβRs or GABA_Aρ1Rs were more vulnerable to regulation by redox agents than GABA_Aαβγ2Rs (Amato et al., 1999; Pan et al., 2000; Calero and Calvo, 2008).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Redox modulation of GABA_AR-mediated responses. Retina: (A) Effects of thiol reagents on phasic GABA_AR currents in ganglion neurons (GABA 5 μM; DTT, GSH and GSSG 5 mM; DTNB 500 μM) (Pan et al., 1995). (B) Effects of ascorbate (3 mM) on phasic currents (miniature synaptic currents, left) and tonic currents (induced by bath application of GABA, right) (Calero et al., 2011). GABA_AR currents recorded in synaptic terminals of bipolar neurons. In (A) and (B) and following records, the membrane potential is −60 mV. Hippocampus: (A) H₂O₂ (200 μM) produced no changes in spontaneous synaptic GABA_A currents (left), but enhanced tonic GABA_A currents [right, GABA 0.5 μM; without bicuculline (BIC) 100 μM] in pyramidal neurons (Penna et al., 2014). (B) NOS inhibition by l-NAME (100 μM) potentiated both phasic currents (left, induced by GABA puffs) and tonic currents (right, GABA 5 μM, without bicuculline 100 μM) GABA_AR responses (Gasulla and Calvo, 2015). Cerebellum: (A) GABA_A responses (GABA 10 μM) in acutely dissociated granule cells showed very low sensitivity to DTT (5 mM) and DTNB (500 μM) (Amato et al., 1999). (B) After an increase in ROS in granule cells, induced by mitochondrial uncoupling (bottom trace) (Accardi et al., 2015), a higher frequency of low-amplitude postsynaptic responses was detected. Expression in oocytes: (A) Effects of DTT (2 mM) on responses mediated by human GABA_AR variants (GABA 10 μM) (Pan et al., 2000). (B) Effects of GSH (5 mM), GSSG (3 mM), ascorbate (3 mM), H₂O₂ (500 μM), and the NO donor 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine sodium (DEA) (100 μM) on responses mediated by human GABA_Aρ1Rs (GABA 0.3 μM) (Calero and Calvo, 2008; Calero et al., 2011; Beltrán González et al., 2014; Gasulla et al., 2012). Expression in human embryonic kidney (HEK) cells: (A) Effects of GSH (5 mM) and DTT (2 mM) on responses mediated by murine GABA_ARs (GABA 10 μM). GABA_Aα1β2γ2Rs exhibited lower redox sensitivity than GABA_Aα1β2Rs (Amato et al., 1999). (B) Effects of ascorbate (1 mM) on responses mediated by human GABA_Aα1β2Rs and GABA_Aα1β2γ2sRs (recorded at the corresponding EC₅₀, and GABA 1 and 5 μM respectively) (Calero et al., 2011). The γ2 subunit containing GABA_ARs showed a decreased sensitivity to ascorbate.

It is important to mention that basal contaminating levels of Zn²⁺ can exert a standing inhibition of the responses mediated by GABA_AαβRs by acting on specific histidine residues (Pan et al., 2000; Wilkins and Smart, 2002). Zn²⁺ inhibition is diminished in the presence of redox agents that chelate this cation, such as DTT, and this results in a potentiating effect on GABA_AR currents that is not related to the actual redox mechanism that normally involves sulfhydryl residues. Thus, selective Zn²⁺ chelating agents such as tricine and N,N,N9,N9-tetrakis(2-pyridylmethyl)ethane1,2-diamine, which do not interact with amino acidic residues at the GABA_AR and do not interfere with redox modulation, were needed to avoid overestimation of redox modulation (Pan et al., 2000; Wilkins and Smart, 2002). Other receptor subtypes, such as the GABA_Aα1β2γ2R and GABA_Aρ1R, are less sensitive to ambient Zn²⁺, and redox modulation induced by DTT was not affected by tricine (Pan et al., 2000; Wilkins and Smart, 2002; Calero and Calvo, 2008). It remains unknown whether endogenous Zn²⁺ can also mask the modulation exerted on GABA_ARs by endogenous redox agents.

Modulation of GABA_AR-Mediated Responses by Physiologically Relevant Redox Agents

Possible Sites of Action of Endogenous Redox Agents at the GABA_ARs

Many of the specific structural components of the GABA_AR that may contribute to redox sensitivity have not been determined yet. Mutational analysis of the diverse GABA_AR subunits is still incipient, but a number of cysteine residues were already identified as targets for redox actions (Amato et al., 1999; Pan et al., 2000; Calero and Calvo, 2008; Calero et al., 2011; Beltrán González et al., 2014). Oxidation/reduction of these cysteine residues may induce allosteric transitions in the receptors that modify channel gating, causing reversible potentiation or inhibition of the GABA responses (Miller and Smart, 2010; Nemecz et al., 2016).

The contribution of extracellular cysteines at the N-terminal domain was suggested using chemical modification of GABA_AR with thiol reagents. The conserved cysteine residues at the Cys-loop participate in GSH potentiation of GABA_AR-mediated responses in retinal ganglion neurons (Fig. 2) (Pan et al., 1995), recombinant GABA_Aα1β2R expressed in human embryonic kidney cells (Fig. 2) (Amato et al., 1999), and GABA_Aρ1Rs expressed in oocytes (Fig. 2) (Calero and Calvo, 2008). Cys-loop cysteines were also essential for the potentiating effects of ascorbate and NO on GABA_Aρ1R expressed in oocytes (Fig. 2) (Calero et al., 2011; Gasulla et al., 2012). As site-directed mutagenesis affecting the disulphide bond disturbed the capacity of the GABA_AR subunits to assemble (Amin et al., 1994), this approach was avoided during heterologous expression of GABA_ARs (Amato et al., 1999; Pan et al., 2000; Calero and Calvo, 2008; Calero et al., 2011; Gasulla et al., 2012). An extracellular oxidative reaction at the GABA_AR was also proposed to mediate the increase in tonic GABA_AR responses produced by H₂O₂ in hippocampal CA1 pyramidal neurons (Penna et al., 2014), and the corresponding amino acidic residues acting as ROS targets were not identified in this case.

Redox sensitivity was even observed in other members of the Cys-loop receptor superfamily (Pan et al., 1995; Thio and Zhang, 2006), but GABA_AR variants insensitive to redox modulation were also found (Amato et al., 1999; Pan et al., 2000), indicating that simply the presence of the Cys-loop is insufficient to confer redox susceptibility. GABA_AR subunits can have additional cysteines at the N-terminal domain aside from those involved in the Cys-loop, including α4 (C⁹), β3 (C³⁷), ε (C¹¹⁰), π (C⁴), and ρ3 (C¹¹ and C¹⁹) (Supplemental Table 1). The potential role of these residues to act as targets for redox actions was not explored.

Transmembrane domains can contain cysteines whose positions show a relatively high degree of conservation. For example, a single cysteine at the M3 domain is present in all GABA_AR subunit subtypes, except in ρ1-3 (Fig. 1B). The role of this residue has only been studied in homomeric GABA_Aβ3Rs, which yield spontaneous currents when expressed in oocytes (Pan et al., 2000). Cysteine replacement by alanine in the mutant GABA_Aβ3^C288ARs produced a significant change in redox sensitivity, but the heterologous expression of heteromeric GABA_Aα1β3^C288AR or GABA_Aβ3^C288Aγ2SR restored the original sensitivity. These results suggested that other cysteine residues in the α- and γ-subunits may be conveniently redox modulated (Pan et al., 2000) and that folding can also be a critical factor in determining redox sensitivity. A single cysteine is also conserved at the M1 domain of α (1–6) and γ (1–3) subunits (Fig. 1B), whereas M2 and M4 domains rarely contain cysteines (with a few exceptions, π (C²⁶⁰ in M2), γ2 (C⁴¹⁵ in M4), and ε (C⁴⁸¹ in M4) (Supplemental Table 1). The possible role of these cysteine residues in redox modulation requires analysis.

A single study (Beltrán González et al., 2014) indicated that intracellular cysteines can also contribute to redox modulation of GABA_ARs. A cysteine located at the M3-M4 linker (Fig. 1, A and B) of each ρ1 subunit (C³⁶⁴) acts as an intracellular sensor for the actions of ROS on homomeric GABA_Aρ1Rs expressed in oocytes. This cysteine residue is also present in ρ2 subunits, but not in ρ3 subunits. Meanwhile, γ-subunits have a group of five cysteines whose relative positions are conserved at this domain, with two of them contiguous, in sharp contrast with those exhibited by the ρ-subunits that show a different arrangement (Fig. 1B). The M3-M4 linker of the Cys-loop receptors is a large intracellular loop (∼100-200 amino acids) whose structure can be predicted as a disorganized region (Fig. 1A). This loop contains motifs required for pentameric assembly; diverse protein-binding domains important for receptor clustering, sorting, targeting, and trafficking; phosphorylation sites for protein kinase A, protein kinase C, and protein tyrosine kinase; and structural determinants that are critical for single-channel conductance, desensitization properties, and interactions with other neurotransmitter receptors and the cytoskeleton (Macdonald and Botzolokis, 2009; Papke and Grosman, 2014). As the M3-M4 linker supports a variety of functions, redox modulation at this level could possibly have an important impact in many functions mediated by GABA_ARs.

Variation in the number and location of cysteines in the diverse GABA_AR subunit subtypes, could account for differential redox sensitivities observed between receptor variants. But the potential contribution of the remaining cysteines to redox modulation needs to be further investigated. Other amino acidic residues at the GABA_AR subunits, such as methionine, tyrosine, phenylalanine, histidine, or lysine, could also undergo redox-dependent side chain modification. However, it is possible that the chemical modification of such residues requires highly extreme conditions. In addition, redox modulation of the GABA_AR function can also be mediated indirectly, compromising additional targets for endogenous agents, such as receptor associated-proteins (Wang et al., 1999). As in the S-nitrosylation–induced, gephyrin-mediated regulation of the synaptic GABA_AR density in hippocampal neurons (Dejanovic and Schwarz, 2014), these pathways need to be more closely analyzed using different neuronal types.

Physiologic Significance

Endogenous redox agents are generated and/or accumulated in neural tissue under physiologic conditions and oxidative stress. Therefore, effects such as those described here for H₂O₂, NO, ascorbate, and GSH on GABA_ARs could be relevant for neuronal excitability. These redox agents exert a dynamic modulation on the function of diverse GABA_AR subtypes in various areas of the CNS, including the hippocampus, cerebellum, and retina (Pan et al., 1995; Amato et al., 1999; Calero et al., 2011; Accardi et al., 2014, 2015; Penna et al., 2014). However, the significance of these modulatory actions is not yet well understood.

In the hippocampus, the selective potentiation of tonic GABA_A currents in CA1 pyramidal cells induced during H₂O₂ application was equivalent to that observed by using an oxygen-glucose deprivation protocol, which produces high endogenous levels of H₂O₂ and other ROS (Penna et al., 2014). These results suggested a novel link between cellular metabolism and GABA_AR activity in hippocampal neurons. In the cerebellum, ROS were also proposed as homeostatic signaling molecules coupling cellular metabolism to the strength of inhibitory transmission, by inducing synaptic plasticity through the recruitment of nonresident GABA_ARs in granule and stellate cells (Accardi et al., 2014, 2015). In the retina, there is considerable production of free radicals, and ascorbate is accumulated at high concentrations (Harrison and May, 2009). Hence, given the fact that GABA_ARs mediate several modes of inhibitory actions in retinal neurons (Jones and Palmer, 2009), the modulatory effects observed for free radicals and antioxidants on tonic and phasic GABA_AR responses in retinal bipolar cells might be physiologically relevant (Calero et al., 2011; Gasulla et al., 2012; Beltrán González et al., 2014). Changes in GABA_AR function were also observed in retinal neurons during experimental diabetes (Ramsey et al., 2007) and in anoxia (Katchman et al., 1994); GABA_ARs were also involved in mechanisms of cell death during ROS-induced oxidative stress (Okumichi et al., 2008), but the underlying mechanisms and the particular roles of free radicals and ascorbate in this process were not determined.

The effects of ROS and RNS on GABA_ARs depended on many factors, including the CNS region and the neuronal type involved (Penna et al., 2014; Dejanovic and Schwarz, 2014; Accardi et al., 2014, 2015), in addition to structural determinants of the GABA_ARs and their functional relations with the cell machinery (e.g., redox-sensitive residues, receptor folding, and associated proteins) (Amato et al., 1999; Pan et al., 2000; Calero et al., 2011; Gasulla et al., 2012; Dejanovic and Schwarz, 2014). Other critical aspects include the interaction of the generated redox agents, or reactive species, with cellular antioxidant systems and the metabolic state of the cells in the experimental preparations (e.g., levels of ascorbate, glutathione, and antioxidant enzymes, oxidative agents such as CO₂, O₂, and light). All concomitant factors must be considered in future experiments to determine whether endogenous redox agents are actually acting as signaling molecules, as well as to characterize the specific pathways and mechanisms involved.

Concluding Remarks

Modulation of tonic and phasic GABA_AR responses by endogenous redox agents has been demonstrated in different CNS regions using a diverse range of in vitro models. Future studies are needed to evaluate the interplay between these endogenous redox agents, the cellular antioxidant systems, as well as the possible role of multiple cysteines at the different GABA_AR subunits in redox modulation. In vivo studies will also be required to more precisely define the relevance that this redox modulation might have in normal physiologic conditions and during oxidative stress.