Mechanisms of GABAA receptor assembly and trafficking: implications for the modulation of inhibitory neurotransmission

Fast synaptic inhibition in the brain is largely mediated by ionotropic GABA receptors, which can be subdivided into GABAA and GABAC receptors based on pharmacological and molecular criteria. GABAA receptors are important therapeutic targets for a range of sedative, anxiolytic, and hypnotic agents and are implicated in several diseases including epilepsy, anxiety, depression, and substance abuse. In addition, modulating the efficacy of GABAergic neurotransmission may play a key role in neuronal plasticity. Recent studies have begun to reveal that the accumulation of ionotropic GABAA receptors at synapses is a highly regulated process that is facilitated by receptor-associated proteins and other cell-signaling molecules. This review focuses on recent experimental evidence detailing the mechanisms that control the assembly and transport of functional ionotropic GABAA receptors to cell surface sites, in addition to their stability at synaptic sites. These regulatory processes will be discussed within the context of the dynamic modulation of synaptic inhibition in the central nervous system (CNS).     

GABAA receptors: properties and trafficking

Fast synaptic inhibition in the brain and spinal cord is mediated largely by ionotropic gamma-aminobutyric acid (GABA) receptors. GABAA receptors play a key role in controlling neuronal activity; thus modulating their function will have important consequences for neuronal excitation. GABAA receptors are important therapeutic targets for a range of sedative, anxiolytic, and hypnotic agents and are involved in a number of CNS diseases, including sleep disturbances, anxiety, premenstrual syndrome, alcoholism, muscle spasms, Alzheimer's disease, chronic pain, schizophrenia, bipolar affective disorders, and epilepsy. This review focuses on the functional and pharmacological properties of GABAA receptors and trafficking as an essential mechanism underlying the dynamic regulation of synaptic strength.     

Fragrances in oolong tea that enhance the response of GABAA receptors

We electrophysiologically investigated the effect of some fragrant compounds in oolong tea on the response of ionotropic gamma-aminobutyric acid (GABA) receptors (GABAA receptors) which were expressed in Xenopus oocytes. Of the tested fragrances in oolong tea, cis-jasmone, jasmine lactone, linalool oxide and methyl jasmonate significantly potentiated the response. Among these, cis-jasmone and methyl jasmonate potently potentiated the response, having a respective dissociation constant of the compound (Kp) and maximum potentiation (Vm) of 0.49 mM and 322% for cis-jasmone, and 0.84 mM and 450% for methyl jasmonate. Inhalation of 0.1% cis-jasmone or methyl jasmonate significantly increased the sleeping time of mice induced by pentobarbital, suggesting that these fragrant compounds were absorbed by the brain and thereby potentiated the GABAA receptor response. Both of these compounds may therefore have a tranquillizing effect on the brain.     

Receptors for gamma-aminobutyric acid and voltage-dependent chloride channels as targets for drugs and toxicants

The function of chloride (Cl-) channel proteins is to regulate the transport of Cl- across membranes. There are two major kinds of Cl- channels: 1) those activated by binding of a transmitter such as gamma-aminobutyric acid (GABA), glycine, or glutamate, and thus are receptors; and 2) those activated by membrane depolarization or by calcium. There are two kinds of GABA receptors: GABAA is the major inhibitory receptor of vertebrate brain and the one that operates a Cl- channel, and the GABAB receptor, which is proposed to regulate cAMP production that is stimulated by other receptors. Except for binding of GABA, these two GABA receptors differ completely in their drug specificities. However, there are many similarities among the GABAA receptor, the glycine receptor, and the voltage-dependent Cl- channel. The two receptors and Cl- channels bind avermectin, whereas bicuculline binds only to mammalian GABAA and glycine receptors, not to the insect brain GABAA receptor. Barbiturates bind to GABAA and voltage-dependent Cl- channels, possibly directly activating them. Benzodiazepines potentiate both the glycine and GABAA receptors. Several insecticides act on the GABAA receptor and voltage-dependent Cl- channel. It is suggested that the GABAA receptor is the primary target for the action of toxaphene and cyclodiene insecticides but a secondary target for lindane and type II pyrethroids. On the other hand, the Cl- channel may be a primary target for avermectin and lindane but a secondary one for cyclodienes. The similarity in certain drug specificities and the operation of Cl- channels suggest a degree of homology between the subunits of GABAA and glycine receptors and the voltage-dependent Cl- channels.     

Extrasynaptic GABAA receptors in the crosshairs of hormones and ethanol

Gamma-aminobutyric acid (GABA) is the main chemical inhibitory neurotransmitter in the brain. In the central nervous system (CNS) it acts on two distinct types of receptor: an ion channel, i.e., an "ionotropic" receptor permeable to Cl- and HCO3- (GABAA receptors) and a G-protein coupled "metabotropic" receptor that is linked to various effector mechanisms (GABAB receptors). This review will summarize novel developments in the physiology and pharmacology of GABAA receptors (GABAARs), specifically those found outside synapses. The focus will be on a particular combination of GABAAR subunits sensitive to ovarian and adrenal cortical steroid hormone metabolites that are synthesized in the brain (neurosteroids) and to sobriety impairing concentrations of ethanol. These receptors may be the final common pathway for interactions between ethanol and ovarian and stress-related neurosteroids.     

gamma-Aminobutyric acid (GABA) induces a receptor-mediated reduction in GABAA receptor alpha subunit messenger RNAs in embryonic chick neurons in culture.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, is known to interact with a subclass of receptors that activate a ligand-gated chloride ion channel. Exposure of cultured embryonic chick neurons to physiological concentrations of GABA results in a time-dependent down-regulation of these GABAA receptors. To delineate the cellular mechanism(s) responsible for agonist-induced down-regulation of GABAA receptors we quantified the levels of GABAA receptor alpha subunit messenger RNAs, which encode the subunit(s) containing agonist recognition site(s), and observed a marked reduction in alpha subunit mRNAs following exposure of embryonic chick neurons to GABA. Both the down-regulation of GABAA receptors and the reduction in alpha subunit mRNAs induced by GABA were completely antagonized by the specific GABAA receptor antagonist SR-95531. These data demonstrate the presence of an agonist-induced receptor-mediated mechanism for regulating the expression of receptor subunit-encoding mRNAs that may be involved in the development of tolerance to the pharmacological actions of drugs known to act via GABAA receptors.     

Asymmetric cross-inhibition between GABAA and glycine receptors in rat spinal dorsal horn neurons

Presynaptic nerve terminals of inhibitory synapses in the dorsal horn of the spinal cord and brain stem can release both GABA and glycine, leading to coactivation of postsynaptic GABAA and glycine receptors. In the present study we have analyzed functional interactions between GABAA and glycine receptors in acutely dissociated neurons from rat sacral dorsal commissural nucleus. Although the application of GABA and glycine activates pharmacologically distinct receptors, the current induced by a simultaneous application of these two transmitters was less than the sum of currents induced by applying two transmitters separately. Sequential application of glycine and GABA revealed that the GABA-evoked current is more affected by glycine than glycine-evoked responses by GABA. Activation of glycine receptors decreased the amplitude and accelerated the rate of desensitization of GABA-induced currents. This asymmetric cross-inhibition is reversible, dependent on the agonist concentration applied, but independent of both membrane potential and intracellular calcium concentration or changes in the chloride equilibrium potential. During sequential applications, the asymmetric cross-inhibition was prevented by selective GABAA or glycine receptor antagonists, suggesting that occupation of binding sites did not suffice to induce glycine and GABAA receptors functional interaction, and receptor channel activation is required. Furthermore, inhibition of phosphatase 2B, but not phosphatase 1 or 2A, prevented GABAA receptor inhibition by glycine receptor activation, whereas inhibition of phosphorylation pathways rendered cross-talk irreversible. Taken together, our results demonstrated that there is an asymmetric cross-inhibition between glycine and GABAA receptors and that a selective modulation of the state of phosphorylation of GABAA receptor and/or mediator proteins underlies the asymmetry in the cross-inhibition.     

Differential effects of baclofen andγ-aminobutyric acid (GABA) on rat piriform cortex pyramidal neuronsin vitro

1. The effects of baclofen and GABA on rat piriform cortex neurons were investigated electrophysiologically using a brain slice preparation. 2. At resting potential GABA depolarized and baclofen hyperpolarized the cell, probably through activation of Cl and K conductances acting at GABAA and GABAB receptors, respectively. 3. The GABAA receptors were concentrated on the apical and basal dendrites near the cell body, while the baclofen-sensitive GABA receptors were concentrated particularly on the basal dendrites. 4. The different distributions of receptor localization must have functional consequences which remain to be elucidated.     

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.     

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.     

Role of GABA receptors in fetal lung development in rats

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl-) fluxes. Since fetal lung actively secretes Cl--rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl- efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion.     

Gamma-aminobutyric acid inhibits T cell autoimmunity and the development of inflammatory responses in a mouse type 1 diabetes model

Gamma-aminobutyric acid (GABA) is both a major inhibitory neurotransmitter in the CNS and a product of beta cells of the peripheral islets. Our previous studies, and those of others, have shown that T cells express functional GABAA receptors. However, their subunit composition and physiological relevance are unknown. In this study, we show that a subset of GABAA receptor subunits are expressed by CD4+ T cells, including the delta subunit that confers high affinity for GABA and sensitivity to alcohol. GABA at relatively low concentrations down-regulated effector T cell responses to beta cell Ags ex vivo, and administration of GABA retarded the adoptive transfer of type 1 diabetes (T1D) in NOD/scid mice. Furthermore, treatment with low dose of GABA (600 microg daily) dramatically inhibited the development of proinflammatory T cell responses and disease progression in T1D-prone NOD mice that already had established autoimmunity. Finally, GABA inhibited TCR-mediated T cell cycle progression in vitro, which may underlie GABA's therapeutic effects. The immunoinhibitory effects of GABA on T cells may contribute to the long prodomal period preceding the development of T1D, the immunological privilege of the CNS, and the regulatory effects of alcohol on immune responses. Potentially, pharmacological modulation of GABAA receptors on T cells may provide a new class of therapies for human T1D as well as other inflammatory diseases.     

Identification and immunohistochemical mapping of GABAA receptor subtypes containing the delta-subunit in rat brain.

Synaptic inhibition in brain is mainly mediated via GABAA receptors which display a striking structural heterogeneity. A novel type of GABAA receptor subunit, the delta-subunit, has recently been described based on molecular cloning of its cDNA. To identify the prevalence and distribution of GABAA receptors which contain the delta-subunit protein in situ, polyclonal site-directed antisera were developed against three synthetic peptides derived form the rat delta-subunit cDNA-sequence. All antisera specifically recognized a 54 kDa protein in GABAA receptor preparations. Nearly 30% of the GABAA receptors contained the delta-subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the delta-subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABAA receptors with a delta-subunit have now been visualized and made accessible for a functional analysis of this GABAA receptor subtype in situ.     

Effect of delta-opioid receptor over-expression on cortical expression of GABAA receptor alpha1-subunit in hypoxia

Recent studies show that both delta-opioid receptors (DOR) and GABA receptors play a neuroprotective role in the mature cortex. Since we have observed that DOR over-expression renders the cortex more tolerant to hypoxic stress, we asked whether DOR over-expression affects GABA receptors expression in the cortex under hypoxia. As the first step, we investigated the expression of GABAA receptor alpha1-subunit (GABAA Ralpha1, the most abundant alpha-subunit of GABA receptors in the adult brain) in the mouse cortex with transgenic DOR over-expression after hypoxia. The results showed that GABAA Ralpha1 expression was lower in the transgenic than wild-type cortex, suggesting that DOR overexpression induces an inhibitory effect on GABAA receptor expression. Hypoxia for 1-3 days significantly increased GABAA Ralpha1 expression in the wild-type cortex, which may be an adaptive strategy for protecting the cortex against hypoxic stress. Interestingly, such increase was not found in the transgenic cortex with DOR over-expression. This may represent an interactive regulation in the transgenic cortex to efficiently balance energy production and consumption for better adaptation to hypoxic environment. Since DOR over-expression increases cortical tolerance to hypoxia, an increase in GABA receptors expression (an energy-costing process) may not be necessary in the cortex with DOR over-expression.     

Myoclonus: analysis of monoamine, GABA, and other systems

Myoclonus is a clinical term meaning a quick involuntary jerk, seen in normal subjects under certain circumstances, including sleep, and in certain disease states. It is important as a symptom that may impair function and as an indicator of neurological dysfunction. Not until patients with myoclonus and major functional disability were reported in the 1960s was attention given to understanding its basis and pharmacotherapy. Reports of myoclonus developing after anoxic brain injury, and its response to treatment with the serotonin precursor 5-hydroxytryptophan (5-HTP), drew special attention. Further experience showed that only a few patients with myoclonus benefit from 5-HTP therapy. Benzodiazepines (BDZs) are often helpful in the treatment of myoclonus. Their beneficial effects decline with chronic administration because of drug tolerance, and the theoretical basis for BDZ responses remains unclear. The relationships between myoclonus, clonus, and epilepsy are discussed, as is the possible contribution of slow signaling transmembrane receptors to synchronization of motoneuron firing, which is suggested as a hallmark of myoclonus. Myoclonus may originate in many CNS sites, but the brain-stem reticular formation is especially relevant to myoclonus. Brain-stem serotonin neurons have special influence on spinal motoneurons, on startle responses, and on myoclonus. Among 5-HT receptors, 5-HT1A receptors are related to some forms of myoclonus, although 5-HT2 receptors are also implicated. GABAA receptors are related to some forms of myoclonus. Blockade of GABAA receptors or GABA synthesis regularly evokes convulsive seizures, but administration of many GABA agonists and some GABA uptake blockers paradoxically may evoke myoclonus. Injection of GABA receptor blockers into some brain areas has anticonvulsant effects. Stimulation of GABAA receptors may therefore promote or antagonize myoclonus depending on which GABA receptors are involved, the state of the system, etc. The role of glycine receptors is well established in some animal models, but has yet to be clearly established for human myoclonus. Opiates may produce myoclonus when given intrathecally or in high dosage. The concept of excitant anesthetics and special function of certain GABA receptors is discussed.     

The molecular aspects of the functional heterogeneity of the GABA receptors

It is generally accepted that gamma-aminobutyric acid (GABA) is one of the main inhibitory transmitter in the mammalian brain. There are three types of GABA receptors in the vertebrata central nervous system: the GABAA, GABAB and GABAC receptors. The GABAA receptor is a GABA-gated Cl- channel and is the tetramer ore the pentamer made of some classes of subunit (alpha, beta, gamma, delta). GABAB receptors are not affiliated with Cl(-) ionophore. GABAB receptors appear to be coupled to Ca2+ and K+ channels of presynaptic membranes. It seems they regulate the release of neurotransmitters release. The structural and functional properties of GABA receptors are discussed.     

Characteristics of GABA receptors on the ocellar L-neurons of American cockroach Periplaneta americana

The ocellar L-neurons of cockroach Periplaneta americana were used in the present study as model systems to investigate the pharmacological properties of the GABA receptors. To do so, a glass microelectrode was impaled into the axon of the L-neurons to record the membrane potential intracellularly and to monitor membrane response to GABA treatment and cercal stimulation by air puff. The traditional GABA and their receptor agonists were introduced through perfusion and/or iontophoresis to monitor their effects on the L-neurons. The GABA receptor antagonists were administered by perfusion to examine if the response of the L-neurons to GABA and/or cercal stimulation was changed. The results revealed that administration of GABA, muscimol and imidazole acetic acid, two GABAA agonists, produced depolarization on the L-neurons. However, treatment of 3-APS and guanidine acetic acid, another two GABAA agonists, evoked hyperpolarization on the L-neurons. Among those tested antagonists, only picrotoxin, GABAA antagonist, antagonize the depolarization induced by GABA and/or cercal stimulation. More interestingly, administration of strychnine, glycine receptor antagonist, largely attenuated the depolarization response of the L-neurons to cercal stimulation. This attenuation caused by strychnine was even stronger than that initiated by varied GABA antagonists. In addition, phaclofen, a GABAB receptor antagonist, showed no antagonistic effect. These results strongly suggest that the characteristics of GABA receptors of the ocellar L-neurons may differ from those in vertebrates. It may be more likely to be a novel GABA receptor.     

Zn2+对爪蟾卵母细胞表达鲫鱼脑GABA受体的调制作用

爪蟾卵母细胞注射鲫鱼脑ｍＲＮＡ后表达的ＧＡＢＡ受体中约８５％为ＧＡＢＡＡ受体。约１５％的成分为ＧＡＮＡＣ受体。本文利用双电极电压箝方法结合药物灌流研究了Ｚｎ６２＋对这两型受体的作用。我们观察到了Ｚｎ＾２＋对它们的调制都是可抑制性的,可逆的。     

Pyritinol inhibition of the GABA and benzodiazepine receptor

In 100 and 200 mumol/l concentration, pyritinol inhibited GABA binding to the GABA receptors of brain synaptosomal membranes. GABA receptors from the cerebral cortex, diencephalon and striatum were inhibited to approximately the same degree; those from the cerebellum and spinal cord were inhibited more. Both high and low affinity receptors were inhibited. Pyritinol did not greatly affect the number of binding sites (Bmax), but reduced the affinity (raised the dissociation constant KD) of both receptors. The benzodiazepine receptor, which is connected with the postsynaptic GABAA receptor, was also inhibited by pyritinol. The character of inhibition was the same as for GABA receptors, i.e. there was no change in the number of binding sites, but there was a decrease in their affinity. It is assumed that the similarity of the effect on GABA and benzodiazepine receptors is associated with their occurrence on one, or on two relatively firmly interconnected, protein molecules. Depression of the affinities of GABA and the associated benzodiazepine receptor, together with inhibition of GABA synthesis, in the presence of pyritinol indicate that diminished activity of the GABA system in the brain might be related to the activating effect of pyritinol.     

Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABAA receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABAA receptor gamma2 subunit (gamma2+/-) were studied using ligand autoradiographic assays on brain cryostat sections. The [35S]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant alpha1/6beta3 than in alpha1/2beta3gamma2 or beta2 subunit-containing GABAA receptors expressed in HEK 293 cells. Increased GABA-insensitive [35 S]TBPS binding in the gamma2+/- mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of gamma2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [3H]Ro 15-4513 autoradiography. As the gamma2 subunit protein is needed for synaptic clustering of GABAA receptors, these results indicate that the extrasynaptic alphabeta3 receptors can be visualized in vitro as atypical GABA-insensitive [35S]TBPS binding sites. The results suggest that GABAAergic synaptic inhibition is widely decreased in the brains of anxiety-prone gamma2+/- mice, while extrasynaptic GABAA receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABAA receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.