The GABA-benzodiazepine interaction fifteen years later

The main steps are presented that led to our current understanding of the interaction between benzodiazepine receptor ligands and the GABA_A receptor. The benzodiazepine receptor is a modulatory site located on the GABA_A receptor-chloride channel complex that has the unique property of being able to mediate positive as well as negative modulation of the chloride channel gating by the GABA_A receptor. Some critical issues concerning the structure of the receptor-channel complex remain to be clarified. Research on the benzodiazepine-GABA interaction has led to novel concepts of drug action and receptor function and provides the basis for a whole spectrum of potential drugs with therapeutic utility.Special issue dedicated to Dr. Erminio Costa     

Endogenous GABAergic modulators in the pathogenesis of hepatic encephalopathy

Theories on the neurochemical etiology for hepatic encephalopathy have recently focussed on activation of inhibitory neurotransmitter GABA systems. Modulators of the GABA_A receptor complex, including diazepam binding inhibitor, are significantly and selectively altered in hepatic encephalopathy. In animals and humans, benzodiazepine receptor antagonists rapidly ameliorate this syndrome suggesting the possible existence of an endogenous benzodiazepine-like substance. Endogenous GABAergic modulators may contribute to the neurochemical pathogenesis of hepatic encephalopathy.Special issue dedicated to Dr. Erminio Costa     

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     

From binding studies to the molecular biology of GABA receptors

Conclusions The GABA_A-complex remains enigmatic despite the substantial amount of information that has been gathered. The work of Barnard's group on the structure of the complex and the relationship of the recognition sites for GABA and benzodiazepines to its protein subunits has raised as many questions as have been answered. The existence of multiple forms of the subunit and the ability of various combinations of the subunits to mediate GABA-gated choloride conductance suggest that there may be many forms of the GABA_A-complex. Thus, the identification of previously unsuspected drug interactions, and evidence for the existence of additional endogenous ligands beside GABA (e.g. benzodiazepine-binding inhibitor (68) and certain steroids (69), all suggest that the characterization of the GABA_A-complex has only begun.Special issue dedicated to Dr. Erminio Costa     

GABAergic mechanisms in the electrophysiological actions of ethanol on cerebellar neurons

We have found that the partial inverse benzodiazepine agonists Ro 15-4513 and FG 7142 antagonize the depressant electrophysiologial effects of locally applied ethanol in the cerebellum. Although absolute tissue concentrations are not known, dose-response curves constructed using pressure-ejection doses as previously described (31, 25) we found that FG 7142 was more efficacious, but less potent than Ro 15-4513. Our observation that ethanol and inverse benzodiazepine agonists have interactions which are not competitive might suggest that these two drugs act through separate, but interactive mechanisms in order to produce the observed ethanol antagonism. If such independent interactions were mediated at different sites on a given macromolecular complex, such as the GABA_a/Cl^– channel, then one might expect to find allosteric interactions between those sites as well as with the functional response of the complex to GABA activation. Indeed, this hypothesis is consistent with the recent finding of Harris and collaborators that ethanol potentiates the inverse agonist actions of Ro 15-4513 and FG 7142. On the other hand, we were unable to find large ethanol-induced potentiations of GABA effects on all neurons which showed depressant responses to ethanol administration in rat cerebellum. However we did find that the GABA_a antagonist, bicuculline, blocks the depressant effects of ethanol on the same neurons. We conclude that the interaction between ethanol and GABA probably does not occur directly at the GABA_a receptor site, but that the GABA_a mechanism does play a permissive role in the ethanol-induced depressions of cerebellar Purkinje neurons. Thus, although a postsynaptic GABA_a mechanism may not be the primary locus of action at which ethanol causes depressant electrophysiological responses of neurons, activation of the GABA_a receptor may be required to make cerebellar Purkinje neurons responsive to the depressant actions of ethanol. Further investigations will be required to determine the pre vs postsynaptic nature of this interaction of ethanol with the GABA mechanism of action.Special issue dedicated to Dr. Erminio Costa     

A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Plasticity of GABA<Subscript>A</Subscript> Receptors in Brains of Rats Treated with Chronic Intermittent Ethanol

The study of alcohol dependence mechanisms has been aided by work in rodents, where regimens of intermittent chronic administration with repeated episodes of intoxication and withdrawal can be coupled with controlled timing of in vitro studies and the possibility of relating them to behavior. The chronic intermittent ethanol (CIE) model in the rat has been found to be a good model of human alcohol dependence, showing persistent signs of withdrawal and self-administration. Studies in CIE rats suggest that plastic changes in GABA-mediated inhibition involving the GABA_A receptor system may be responsible for the behavioral alterations. Here we summarize a combination of evidence that the alcoholic rat CIE model demonstrates changes in GABA_A receptor subunit levels, in receptor localization, and in physiology and pharmacology, leading to alterations in behavior that contribute to the hyperexcitable alcohol withdrawal state (anxiety, insomnia, seizure susceptibility) and alcohol dependence. Special Issue dedicated to Dr. Simo S. Oja     

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.     

Taurine Interaction with Neurotransmitter Receptors in the CNS: An Update

Taurine appears to have multiple functions in the brain participating both in volume regulation and neurotransmission. In the latter context it may exert its actions by serving as an agonist at receptors of the GABAergic and glycinergic neurotransmitter systems. Its interaction with GABA_A and GABA_B receptors as well as with glycine receptors is reviewed and the physiological relevance of such interactions is evaluated. The question as to whether local extracellular concentrations of taurine are likely to reach the threshold level for the pertinent receptor populations cannot presently be answered satisfactorily. Hence more sophisticated analytical methods are warranted in order to obtain a definite answer to this important question. Special issue dedicated to Dr. Simo S. Oja     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

GABA_A-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. At the light microscope level specific staining of GABA_A-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling of GABA_A-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations to be performed. Quantification of the labeling intensity revealed a higher concentration of GABA_A-receptors per m plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABA_A-receptors was observed. No specific labeling of GABA_A-receptors was observed in dissociated primary cultures of cerebellar astrocytes.Special issue dedicated to Dr. Eugene Roberts.     

Heterogeneity of GABAA-receptors: cell-specific expression,pharmacology, and regulation

Vigilance, anxiety, memory, epileptogenic activity and muscle tension can be regulated by a modulation of GABA_A-receptor function. A multitude of different GABA_A-receptors exist in the brain due to the combinatorial assembly of various subunits encoded by at least 15 genes. The clarification of the physiological and pharmacological significance of GABA_A-receptor subtypes, in combination with their cellular localization, will make it possible to identify the neuronal circuits regulating the respective CNS states and to provide strategies for the development of subtype-specific drugs for selective therapies.Special issue dedicated to Robert Balázs     

Brain GABAA receptors studied with subunit-specific antibodies

Brain GABA_A/benzodiazepine receptors are highly heterogeneous. This heterogeneity is largely derived from the existence of many pentameric combinations of at least 16 different subunits that are differentially expressed in various brain regions and cell types. This molecular heterogeneity leads to binding differences for various ligands, such as GABA agonists and antagonists, benzodiazepine agonists, antagonists, and inverse agonists, steroids, barbiturates, ethanol, and Cl⁻ channel blockers. Different subunit composition also leads to heterogeneity in the properties of the Cl⁻ channel (such as conductance and open time); the allosteric interactions among subunits; and signal transduction efficacy between ligand binding and Cl⁻ channel opening. The study of recombinant receptors expressed in heterologous systems has been very useful for understanding the functional roles of the different GABA_A receptor subunits and the relationships between subunit composition, ligand binding, and Cl⁻ channel properties. Nevertheless, little is known about the complete subunit composition of the native GABA_A receptors expressed in various brain regions and cell types. Several laboratories, including ours, are using subunit-specific antibodies for dissecting the heterogeneity and subunit composition of native (not reconstituted) brain GABA_A receptors and for revealing the cellular and subcellular distribution of these subunits in the nervous system. These studies are also aimed at understanding the ligand-binding, transduction mechanisms, and channel properties of the various brain GABA_A receptors in relation to synaptic mechanisms and brain function. These studies could be relevant for the discovery and design of new drugs that are selective for some GABA_A receptors and that have fewer side effects.     

Electrophysiology of GABA-mediated synaptic transmission and possible roles in epilepsy

Epileptogenic conditions come about from a disequilibrium between excitatory and inhibitory mechanisms, creating a state of neuronal hypersynchrony. From experimental studies in animal models of epilepsy it appears that several mechanisms, alone or in combination, could be responsible for this imbalance. An alteration of GABA-mediated inhibition has long been considered to be one of the most likely candidates. We review recent data on the synaptic physiology of GABA-mediated inhibition, with emphasis on GABA_A and GABA_B receptors and their conductances. We describe the integrative role of GABAergic local-circuit neurons in the normal control of recurrent excitation. We then discuss possible alterations in GABA_A-mediated inhibition in two chronic animal models of epilepsy, the kindled rat and the kainate-treated rat. Finally, we review studies on GABA inhibition in human epileptic cortex resected for the treatment of intractable epilepsy.Special issue dedicated to Dr. Eugene Roberts.     

Cerebral GABAB receptor: Proposed mechanisms of action and purification procedures

The GABA_B receptor in brain is one of the GABA receptor subtypes, and has been found to be negatively coupled to adenylate cyclase and phosphatidylinositide turnover. This receptor easily solubilizes from cerebral synaptic membrane preparations by 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. GABA_B receptor solubilized from bovine cerebral cortex was purified using baclofen-coupled affinity beads (baclofen-coupled Toyopearl beads). Using these procedures, almost pure GABA_B receptor (80 KDa protein) was obtained in the affinity eluate. A monoclonal antibody has been also raised against the purified GABA_B receptor. The antibody recognized a protein of about 80 KDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove more than 90% of the baclofen suppressive GABA binding activity in the solubilized synaptic membrane, and this system was found to be useful for the immunoaffinity column chromatographic separation of GABA_B receptor. Preliminary studies of immunohistochemical visualization of GABA_B receptor in the rat cerebellum suggested that this receptor may be exclusively localized at the presynaptic site of GABAergic neurons.Special issue dedicated to Dr. Claude Baxter.     

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.     

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor

A novel series of agonists at the benzodiazepine binding site of the GABA_A receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA_A receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.     

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.