Neurexins and neuroligins: new partners for GABAA receptors at synapses

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. As one of several types of endogenous receptors, GABA_A receptors have been shown to be essential in most, if not all, aspects of brain functioning, including neural development and information processing. Mutations in genes encoding GABA_A receptors and alterations in the function of GABA_A receptors are associated with many neurologic diseases, and GABA_A receptors have been clinically targeted by many drugs, such as benzodiazepines and general anesthetics. Extensive studies have revealed a number of intracellular chaperons/interactions for GABA_A receptors, providing a protein-protein network in regulating the trafficking and location of GABA_A receptors in the brain. Recently, neurexins and neuroligins, two families of transmembrane proteins present at neurological synapses, are implicated as new partners to GABA_A receptors. These works shed new light on the synaptic regulation of GABA_A receptor activity. Here, we summarized the proteins that were implicated in the function of GABA_A receptors, including neurexins and neuroligins.     

The diversity of GABAA receptors

The amino acid γ-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotrans-mitter that mediates most of its effects through fast GABA-gated Cl^?-channels (GABA_AR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes α, β, γ, δ, ε, and ρ, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABA_ARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and ethanol. To understand the physiology and diversity of GABA_ARs, the native isoforms have to be identified by their localization in the brain and by their pharmacology. In heterologous expression systems, channels require the presence of α, β, and γ subunits in order to mimic the full repertoire of native receptor responses to drugs, with the BZ pharmacology being determined by the particular α and γ subunit variants. Little is known about the functional properties of the β, δ, and ε subunit classes and only a few receptor subtype-specific substances like loreclezole and furosemide are known that enable the identification of defined receptor subtypes. We will summarize the pharmacology of putative receptor isoforms and emphasize the characteristics of functional channels. Knowledge of the complex pharmacology of GABA_ARs might eventually enable site-directed drug design to further our understanding of GABA-related disorders and of the complex interaction of excitatory and inhibitory mechanisms in neuronal processing.     

Structure,Function, and Modulation of GABAA Receptors

The GABA_A receptors are the major inhibitory neurotransmitter receptors in mammalian brain. Each isoform consists of five homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA. How many isoforms of the receptor exist is far from clear. GABA_A receptors located in the postsynaptic membrane mediate neuronal inhibition that occurs in the millisecond time range; those located in the extrasynaptic membrane respond to ambient GABA and confer long-term inhibition. GABA_A receptors are responsive to a wide variety of drugs, e.g. benzodiazepines, which are often used for their sedative/hypnotic and anxiolytic effects.     

Assembly of GABAA receptors (Review)

GABA_A receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA_A receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA_A receptor subtypes in the brain. The majority of GABA_A receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABA_A receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA_A receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA_A receptors are discussed in the light of recent publications.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

Regional variations in the effects of chronic ethanol administration on GABAA receptor expression: potential mechanisms

Gamma-aminobutyric acid type A (GABA_A) receptors in brain adapt to chronic ethanol exposure via changes in receptor function and subunit expression. The present review summarizes currently available data regarding changes in GABA_A receptor subunit mRNA and peptide expression. Data are presented from various different brain regions and the variations between specific brain regions used to draw conclusions about mechanisms that may underlie GABA_A receptor adaptations during chronic ethanol exposure. In the whole cerebral cortex, chronic ethanol exposure leads to a reduction of GABA_A receptor α1 subunit mRNA and peptide levels and a near equivalent increase in α4 subunit mRNA and peptide levels. This observation is the primary support for the hypothesis that altered receptor composition is a mechanism for GABA_A receptor adaptation produced by chronic ethanol exposure. However, other brain regions do not display similar patterns of subunit changes. Moreover, subregions within cortex (prefrontal, cingulate, parietal, motor, and piriform) exhibit patterns of changes in subunit expression that differ from whole cortex. Therefore, regional differences in GABA_A receptor subunit expression are evident following chronic ethanol administration, thus suggesting that multiple mechanisms contribute to the regulation of GABA_A receptor expression. These mechanisms may include the involvement of other neurotransmitter systems, endogenous steroids and second or third messenger cross-talk.     

GABAA receptor diversity and pharmacology

Because of its control of spike-timing and oscillatory network activity, γ-aminobutyric acid (GABA)-ergic inhibition is a key element in the central regulation of somatic and mental functions. The recognition of GABA_A receptor diversity has provided molecular tags for the analysis of distinct neuronal networks in the control of specific pharmacological and physiological brain functions. Neurons expressing α₁GABA_A receptors have been found to mediate sedation, whereas those expressing α₂GABA_A receptors mediate anxiolysis. Furthermore, associative temporal and spatial memory can be regulated by modulating the activity of hippocampal pyramidal cells via extrasynaptic α₅GABA_A receptors. In addition, neurons expressing α₃GABA_A receptors are instrumental in the processing of sensory motor information related to a schizophrenia endophenotype. Finally, during the postnatal development of the brain, the maturation of GABAergic interneurons seems to provide the trigger for the experience-dependent plasticity of neurons in the visual cortex, with α₁GABA_A receptors setting the time of onset of a critical period of plasticity. Thus, particular neuronal networks defined by respective GABA_A receptor subtypes can now be linked to the regulation of various clearly defined behavioural patterns. These achievements are of obvious relevance for the pharmacotherapy of certain brain disorders, in particular sleep dysfunctions, anxiety disorders, schizophrenia and diseases associated with memory deficits.     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Bicuculline-insensitive GABA binding to catfish neuronal membranes

GABA receptor binding to mammalian neuronal membranes has been classified into at least 2 subtypes—GABA_A and GABA_B binding sites. In catfish brain GABA_A receptor sites have previously been demonstrated. Evidence is now presented that under appropriate conditions which rule out GABA_A receptor binding, [³H]GABA binds to membranes prepared from catfish brain. This binding is bicuculline-insensitive but differs enough from mammalian GABA_B binding to cast some doubt on the idea that GABA_B receptors exist in catfish brain. Specific binding was detected that was saturable and exhibited a dissociation constant of 4μM. (±)Baclofen, a potent inhibitor in rat brain, was a weak inhibitor, producing a maximum of 43% inhibition. This inhibitory effect could be enhanced, however, in the presence of 320 μM isoguvacine. [³H]GABA binding was unaffected by bicuculline. Thus bicuculline-insensitive GABA binding sites exist in catfish brain but they differ in a number of ways from the GABA_B receptor site found in mammals. Furthermore, a third [³H]GABA binding site appears to exist that is both baclofen- and bicuculline-insensitive, yet is inhibited by high concentrations of isoguvacine, a known GABA_A agonist.     

Downregulation of GABA<Subscript>A</Subscript> Receptor Recycling Mediated by HAP1 Contributes to Neuronal Death in In Vitro Brain Ischemia

Downregulation of GABAergic synaptic transmission contributes to the increase in overall excitatory activity in the ischemic brain. A reduction of GABA_A receptor (GABA_AR) surface expression partly accounts for this decrease in inhibitory activity, but the mechanisms involved are not fully elucidated. In this work, we investigated the alterations in GABA_AR trafficking in cultured rat hippocampal neurons subjected to oxygen/glucose deprivation (OGD), an in vitro model of global brain ischemia, and their impact in neuronal death. The traffic of GABA_AR was evaluated after transfection of hippocampal neurons with myc-tagged GABA_AR β3 subunits. OGD decreased the rate of GABA_AR β3 subunit recycling and reduced the interaction of the receptors with HAP1, a protein involved in the recycling of the receptors. Furthermore, OGD induced a calpain-mediated cleavage of HAP1. Transfection of hippocampal neurons with HAP1A or HAP1B isoforms reduced the OGD-induced decrease in surface expression of GABA_AR β3 subunits, and HAP1A maintained the rate of receptor recycling. Furthermore, transfection of hippocampal neurons with HAP1 significantly decreased OGD-induced cell death. These results show a key role for HAP1 protein in the downmodulation of GABAergic neurotransmission during cerebral ischemia, which contributes to neuronal demise.     

γ-Hydroxybutyric Acid (GHB) Is Not an Agonist of Extrasynaptic GABAA Receptors

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABA_B receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABA_A receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABA_A receptors, where GHB acts as an agonist with an EC₅₀ of 140 nM. We investigated three neuronal cell types that express a tonic GABA_A receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABA_A receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABA_B receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.     

Revisiting the TRAK Family of Proteins as Mediators of GABAA Receptor Trafficking

γ-Aminobutyric acid type A (GABA_A) receptor interacting factor-1 (GRIF-1) was originally discovered as a result of studies aiming to find the elusive GABA_A receptor clustering protein. It was identified as a GABA_A receptor associated protein by virtue of its specific interaction with the GABA_A receptor β2 subunit intracellular loop in a yeast two-hybrid screen of a rat brain cDNA library. Further work however, established that GRIF-1, now known as trafficking kinesin protein 2 (TRAK2), is a member of the TRAK family of kinesin adaptor proteins. A pivotal role for TRAK1 and TRAK2 in the transport of mitochondria is well recognized. Notwithstanding this progress, there is a body of evidence that still supports a role for TRAKs in the intracellular transport of GABA_A receptors. This is critically reviewed in this article.     

Inhibitory effects of insulin on GABAA currents modulated by the GABAA alpha subunit

Abstract

Insulin, when co-applied with GABA, can cause an inhibition of the induced current at GABA_A receptors. This study investigated that inhibitory effect of insulin at a variety of receptor isoforms, concentrating on α1, α2 and α4 containing receptors. Various isoforms were expressed in Xenopus oocytes and currents determined using two-electrode voltage clamp. Submaximal GABA currents at all isoforms studied were inhibited by nanomolar concentrations of insulin. At α2 and α4 containing forms, insulin could inhibit maximal GABA currents. The ability to inhibit maximal currents, and the general potency and effects at submaximal currents paralleled the number of potential MAPK sites on the α subunits. The differences in insulin inhibition of GABA currents at different α containing GABA_A receptors could be important in autocrine and paracrine control of hormone secretion in the pancreas, and in control of reward and food intake circuits of the brain.     

DBI (Diazepam Binding Inhibitor): The precursor of a family of endogenous modulators of GABA A receptor function. History,perspectives, and clinical implications

Biochemical, electrophysiological, and lately, molecular biological techniques have shown that GABA_A receptors are heterogeneous supramolecular complexes and can be divided into at least three major subgroups: GABA_A1, GABA_A2, and GABA_A3. They differ mainly in the structural and functional properties of the allosteric modulatory center associated with each one of them. This paper will review the present state of research based on the evidence that DBI (diazepam binding inhibitor) and its natural processing products can selectively modulate GABAergic transmission at different GABA_A receptor subtypes. Furthermore, the possibility that the DBI family of peptides represents a novel and meaningful neurochemical correlate for neuropsychiatric pathology, sustained by an alteration of GABAergic transmission, will be discussed.Special issue dedicated to Dr. Erminio Costa     

Differential Drug Responses on Native GABAA Receptors Revealing Heterogeneity in Extrasynaptic Populations in Cultured Hippocampal Neurons

Hippocampal pyramidal neurons potentially express multiple subtypes of GABA_A receptors at extrasynaptic locations that could therefore respond to different drugs. We activated extrasynaptic GABA_A receptors in cultured rat hippocampal pyramidal neurons and measured single-channel currents in order to compare the actions of two drugs that potentially target different GABA_A receptor subtypes. Despite the possible difference in receptor targets of etomidate and diazepam, the two drugs were similar in their actions on native extrasynaptic GABA_A receptors. Each drug produced three distinct responses that differed significantly in current magnitude, implying heterogeneous GABA_A receptor populations. In the majority of patches, drug application increased both the single-channel conductance (>40 pS) and the open probability of the channels. By contrast, in the minority of patches, drug application caused an increase in open probability only. In the third group high-conductance channels were observed upon GABA activation and drug application increased their open probability only. The currents potentiated by etomidate or diazepam were substantially larger in patches displaying high-conductance GABA channels compared to those displaying only low-conductance channels. Factors contributing to the large magnitude of these currents were the long mean open time of high-conductance channels and the presence of multiple channels in these patches. In conclusion, we suggest that the local density of extrasynaptic GABA_A receptors may influence their single-channel properties and may be an additional regulating factor for tonic inhibition and, importantly, differential drug modulation. This work is dedicated to the memory of Professor P. W. Gage.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

Autoradiographic Analysis of GABAA Receptors in μ-Opioid Receptor Knockout Mice

Anatomical evidence indicates that γ-aminobutyric acid (GABA)-ergic and opioidergic systems are closely linked and act on the same neurons. However, the regulatory mechanisms between GABAergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are changes in GABA_A receptors in mice lacking μ-opioid receptor gene. The GABA_A receptor binding was carried out by autoradiography using [³H]-muscimol (GABA_A), [³H]-flunitrazepam (FNZ, native type 1 benzodiazepine) and [³⁵S]-t-butylbicyclophosphorothionate (TBPS, binding to GABA_A-gated chloride channels) in brain slices of wild type and μ-opioid receptor knockout mice. The binding of [³H]-FNZ in μ-opioid receptor knockout mice was significantly higher than that of the wild type controls in most of the cortex and hippocampal CA1 and CA2 formations. μ-Opioid receptor knockout mice show significantly lower binding of [³⁵S]-TBPS than that of the wild type mice in few of the cortical areas including ectorhinal cortex layers I, III, and V, but not in the hippocampus. There was no significant difference in binding of [³H]-muscimol between μ-opioid receptor knockout and wild type mice in the cortex and hippocampus. These data indicate that there are specific regional changes in GABA_A receptor binding sites in μ-opioid receptor knockout mice. These data also suggest that there are compensatory up-regulation of benzodiazepine binding site of GABA_A receptors in the cortex and hippocampus and down-regulation of GABA-gated chloride channel binding site of GABA_A receptors in the cortex of the μ-opioid receptor knockout mice.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.