Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

Levels of Benzodiazepine Receptor Subtypes and GABAA Receptor α-Subunit mRNA Do Not Correlate During Development

Abstract: Developmental changes in the pharmacological properties of the GABA_A receptor have been suggested to result from changes in the subunit composition of the receptor complex. The nicotinic acetylcholine receptor is structurally related to the GABA_A receptor and undergoes a developmental subunit switch at the neuromuscular synapse. To examine the mechanistic similarities between these systems we sought to find whether the changes in GABA_A receptor subunits are controlled by changes in messenger RNA levels, as they are for the nicotinic acetylcholine receptor. We found a 10-fold increase in the level of α1-subunit mRNA, and a small increase in levels of GABA_A/benzodiazepine receptors from day 1 to day 24 of rat cerebellar development. We also found that the levels of α1-subunit mRNA were higher than the levels of mRNA encoding other α subunits at all developmental time points. The low levels of messenger RNA for α2, α3, and α5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these α subunits have been shown to form GABA_A receptors with type II benzodiazepine binding. These findings are inconsistent with simple models that would explain the developmental differences in GABA_A receptor pharmacology simply as a result of changes in α-subunit gene expression.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

Polyclonal Antibodies Directed Against an Epitope Specific for the α4-Subunit of GABAA Receptors Identify a 67-kDa Protein in Rat Brain Membranes

Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α4-subunit. These anti-peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti-peptide α4 (517–523) antibodies and could then be identified by the anti-α4-antibodies as well as by the GABA_A receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABA_A receptors. Intact GABA_A receptors appeared to be retained by the immunoaffinity column because other GABA_A receptor subunit proteins like the β2/β3-subunits and the γ2-subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67-kDa protein, a 51-kDa protein could be detected by the antibody bd-28 and the anti-peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1-subunit-specific anti-peptide α1 (1–9) antibody. In contrast to the α1-subunit, the 51-kDa protein identified by the anti-α4 antibody could not be deglycosylated by N -Glycanase. The identity of the 51-kDa protein identified by the anti-α4-antibodies thus must be further investigated.     

GABAA Receptors Containing the α4-Subunit: Prevalence, Distribution, Pharmacology, and Subunit Architecture In Situ

Abstract: Recombinant GABA_A receptors, expressed from α-, β-, and γ2-subunits, are diazepam-insensitive when the α-subunit is either α4 or α6. In situ, diazepam-insensitive receptors containing the α6-subunit are almost exclusively expressed in the granule cell layer of the cerebellum. However, diazepam-insensitive receptors are also expressed in forebrain areas. Here, we report on the presence of diazepam-insensitive GABA_A receptors in various brain areas containing the α4-subunit. GABA_A receptors immunoprecipitated with a newly developed α4-subunit-specific antiserum displayed a drug binding profile that was indistinguishable from those of α4β2γ2-recombinant receptors and diazepam-insensitive [³H]Ro 15-4513 binding sites in rat brain membranes. In addition, α4-subunit containing receptors and forebrain diazepam-insensitive receptors are present at comparably low abundance in rat brain and exhibit virtually identical patterns of distribution. Analysis of the subunit architecture of α4-subunit containing receptors revealed that the α4-subunit contributes to several receptor subtypes. Depending on the brain region, the α4-subunit can be coassembled with a second type of α4-subunit variant being α1, α2, or α3. The data demonstrate that native receptors containing the α4-subunit are structurally heterogeneous, expressed at very low abundance in the brain, and display the drug binding profile of diazepam-insensitive [³H]Ro 15-4513 binding sites. Pharmacologically, these receptors may contribute to the actions of nonclassical ligands such as Ro 15-4513 and bretazenil.     

The α5(H105R) mutation impairs α5 selective binding properties by altered positioning of the α5 subunit in GABAA receptors containing two distinct types of α subunits

GABA_A receptors are pentameric ligand-gated ion channels that are major mediators of fast inhibitory neurotransmission. Clinically relevant GABA_A receptor subtypes are assembled from α5(1-3, 5), β1-3 and the γ2 subunit. They exhibit a stoichiometry of two α, two β and one γ subunit, with two GABA binding sites located at the α/β and one benzodiazepine binding site located at the α/γ subunit interface. Introduction of the H105R point mutation into the α5 subunit, to render α5 subunit-containing receptors insensitive to the clinically important benzodiazepine site agonist diazepam, unexpectedly resulted in a reduced level of α5 subunit protein in α5(H105R) mice. In this study, we show that the α5(H105R) mutation did not affect cell surface expression and targeting of the receptors or their assembly into macromolecular receptor complexes but resulted in a severe reduction of α5-selective ligand binding. Immunoprecipitation studies suggest that the diminished α5-selective binding is presumably due to a repositioning of the α5(H105R) subunit in GABA_A receptor complexes containing two different α subunits. These findings imply an important role of histidine 105 in determining the position of the α5 subunit within the receptor complex by determining the affinity for assembly with the γ2 subunit.     

Chronic Ethanol Administration Regulates the Expression of GABAA Receptor α1 and α5 Subunits in the Ventral Tegmental Area and Hippocampus

Abstract: Ethanol dependence and tolerance involve perturbation of GABAergic neurotransmission. Previous studies have demonstrated that ethanol treatment regulates the function and expression of GABA_A receptors throughout the CNS. Conceivably, changes in receptor function may be associated with alterations of subunit composition. In the present study, a comprehensive (1–12 weeks) ethanol treatment paradigm was used to evaluate changes in GABA_A receptor subunit expression in several brain regions including the cerebellum, cerebral cortex, ventral tegmental area (VTA) (a region implicated in drug reward/dependence), and the hippocampus (a region involved in memory/cognition). Expression of α₁ and α₅ subunits was regulated by ethanol in a region-specific and time-dependent manner. Following 2–4 weeks of administration, cortical and cerebellar α₁ and α₅ subunit immunoreactivity was reduced. In the VTA, levels of α₁ subunit immunoreactivity were significantly decreased after 12 weeks but not 1–4 weeks of treatment. Hippocampal α₁ subunit immunoreactivity and mRNA content were also significantly reduced after 12 but not after 4 weeks of treatment. In contrast, α₅ mRNA content was increased in this brain region. These data indicate that chronic ethanol administration alters GABA_A receptor subunit expression in the VTA and hippocampus, effects that may play a role in the abuse potential and detrimental cognitive effects of alcohol.     

Modulation of GABAA Receptor tert-[35S]Butylbicyclophosphorothionate Binding by Antagonists: Relationship to Patterns of Subunit Expression

Abstract: The multisubunit γ-aminobutyric acid type A (GABA_A) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABA_A-receptor ionophore ligand tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) and its modulation by GABA and the GABA_A antagonists bicuculline and 2'-(3'-carboxy-2',3'-propyl)-3-amino-6- p -methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABA_A subunits in brain as reported previously. In some brain regions SR 95531 reduced [³⁵S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [³⁵S]TBPS binding. This ratio correlated positively with α2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit-containing GABA_A receptors. The data aid in developing GABA_A-receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABA_A antagonists.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Distinct Loci Mediate the Direct and Indirect Actions of the Anesthetic Etomidate at GABAA Receptors

Abstract: Most general anesthetics produce two distinct actions at GABA_A receptors. Thus, these drugs augment GABA-gated chloride currents (referred to as an indirect action) and, at higher concentrations, elicit chloride currents in the absence of GABA (referred to as a direct action). Because a β subunit appears to be required for the direct action of intravenous anesthetics in recombinant GABA_A receptors, site-directed mutagenesis of the β3 subunit was performed to identify amino acid residues that are critical for this action. In HEK293 cells expressing a prototypical GABA_A receptor composed of α1β3γ2 subunits, mutation of amino acid 290 from Asn to Ser dramatically reduced both etomidate-induced chloride currents and its ability to stimulate [³H]flunitrazepam binding. By contrast, the ability of etomidate to augment GABA-gated chloride currents and GABA-enhanced [³H]flunitrazepam binding was retained. The demonstration that the direct, but not the indirect, actions of etomidate are dependent on β3(Asn²⁹⁰) indicates that the dual actions of this intravenous anesthetic at GABA_A receptors are mediated via distinct loci.     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Microtubule Depolymerization Inhibits Ethanol-Induced Enhancement of GABAA Responses in Stably Transfected Cells

Abstract: We studied whether microtubule organization is important for actions of ethanol on GABA_A ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk⁻) cells stably transfected with GABA_A receptor α₁β₁γ_2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. β-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicine did not alter the potentiating actions of flunitrazepam or pentobarbital on muscimol-stimulated chloride uptake. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABA_A responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.     

Chronic Dizocilpine (MK-801) Reversibly Delays GABAA Receptor Maturation in Cerebellar Granule Neurons In Vitro

Abstract: We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABA_A receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK-801 during the first week in vitro, a decrease in the α1, γ2S, and γ2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the α1 and γ2L mRNAs. Finally, if MK-801 was present during the first week and removed during the second week, these changes reversed. Whole-cell voltage-clamp recordings showed that treatment with MK-801 during either the first or second week increased the EC₅₀ of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK-801 was removed after the first week in vitro. Our results suggest that MK-801 reversibly inhibits GABA_A receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the γ2 receptor subunit.     

Benzodiazepine Treatment Causes Uncoupling of Recombinant GABAA Receptors Expressed in Stably Transfected Cells

Abstract: GABA_A and benzodiazepine receptors are allosterically coupled, and occupation of either receptor site increases the affinity of the other. Chronic exposure of primary neuronal cultures to benzodiazepine agonists reduces these allosteric interactions. Neurons express multiple GABA_A receptor subunits, and it has been suggested that uncoupling is due to changes in the subunit composition of the receptor. To determine if uncoupling could be observed with expression of defined subunits, mouse Ltk⁻ cells stably transfected with GABA_A receptors (bovine α1, β1, and γ2L subunits) were treated with flunitrazepam (Flu) or clonazepam. The increase in [³H]Flu binding affinity caused by GABA (GABA shift or coupling) was significantly reduced in cells treated chronically with the benzodiazepines, whereas the K _D and B _max of [³H]Flu binding were unaffected. The uncoupling caused by clonazepam treatment occurred rapidly with a t _1/2 of ∼30 min. The EC₅₀ for clonazepam treatment was ∼0.3 µ M , and cotreatment with the benzodiazepine antagonist Ro 15-1788 (5.6 µ M ) prevented the effect of clonazepam. The uncoupling observed in this system was not accompanied by receptor internalization, is unlikely to be due to changes in receptor subunit composition, and probably represents posttranslational changes. The rapid regulation of allosteric coupling by benzodiazepine treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABA_A and benzodiazepine receptors as well as benzodiazepine tolerance.     

Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABAA receptors

GABA_A receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of α₁S205C and α₁T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (–NCS). We also provide new data that identify α₁H101C and α₁N102C as exclusive sites of the reaction of a diazepam derivative where the –Cl atom is replaced by a –NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of α₁β₂γ₂ receptors.     

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABA_A) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABA_A receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [³H]muscimol and [³H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.     

Differential pharmacological properties of GABAA receptors in axon terminals and soma of dentate gyrus granule cells

Although it has been well established that GABA_A receptors are molecular targets of a variety of allosteric modulators, such as benzodiazepines, the pharmacological properties of presynaptic GABA_A receptors are poorly understood. In this study, the effects of diazepam and Zn²⁺ on presynaptic GABA_A receptors have been investigated by measuring the GABA_A receptor-mediated facilitation of spontaneous glutamate release in mechanically dissociated rat CA3 pyramidal neurons. Diazepam significantly enhanced the muscimol-induced facilitation (particularly at submicromolar concentrations) of spontaneous glutamate release and shifted the concentration–response relationship for muscimol toward the left, whereas Zn²⁺ (≤ 100 μM) had little effect on the muscimol-induced facilitation of spontaneous glutamate release. In contrast, Zn²⁺ significantly suppressed the muscimol-induced currents mediated by GABA_A receptors expressed on dentate gyrus granule cells, which are parent neurons of mossy fibers, whereas the effect of diazepam on GABA_A receptors expressed on dentate gyrus granule cells was lesser than that on presynaptic GABA_A receptors. The results suggest that the pharmacological properties of GABA_A receptors differ considerably between presynaptic (axon terminals) and somatic regions in the same granule cell and that presynaptic GABA_A receptors should be considered as one of the important pharmacological targets of many drugs affecting GABA_A receptors.