Ectopic expression of the GABA(A) receptor alpha6 subunit in hippocampal pyramidal neurons produces extrasynaptic receptors and an increased tonic inhibition.

We generated transgenic (Thy1alpha6) mice in which the GABA(A) receptor alpha6 subunit, whose expression is usually confined to granule cells of cerebellum and cochlear nuclei, is ectopically expressed under the control of the pan-neuronal Thy-1.2 promoter. Strong Thy1alpha6 subunit expression occurs, for example, in deep cerebellar nuclei, layer V iscocortical and hippocampal pyramidal cells and dentate granule cells. Ligand binding and protein biochemistry show that most forebrain alpha6 subunits assemble as alpha6betagamma2-type receptors, and some as alpha1alpha6betagamma2 and alpha3alpha6betagamma2 receptors. Electron microscopic immunogold labeling shows that most Thy1-derived alpha6 immunoreactivity is in the extrasynaptic plasma membrane of dendrites and spines in both layer V isocortical and CA1pyramidal cells. Synaptic immunolabeling is rare. Consistent with the alpha6 subunits' extrasynaptic localization, Thy1alpha6 CA1 pyramidal neurons have a five-fold increased tonic GABA(A) receptor-mediated current compared with wild-type cells; however, the spontaneous IPSC frequency and the mIPSC amplitude in Thy1alpha6 mice decrease 37 and 30%, respectively compared with wild-type. Our results strengthen the idea that GABA(A) receptors containing alpha6 subunits can function as extrasynaptic receptors responsible for tonic inhibition and further suggest that a homeostatic mechanism might operate, whereby increased tonic inhibition causes a compensatory decrease in synaptic GABA(A) receptor responses.

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells.

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site.

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.

A significant part of native gamma-aminobutyric AcidA receptors containing alpha4 subunits do not contain gamma or delta subunits.

Using a novel antibody directed against the alpha4 subunit of gamma-aminobutyric acidA (GABAA) receptors, 5% of all [3H]muscimol but only about 2% of all [3H]Ro15-4513 binding sites present in brain membrane extracts could be precipitated. This indicated that part of the alpha4 receptors containing [3H]muscimol binding sites did not contain [3H]Ro15-4513 binding sites. Immunoaffinity purification and Western blot analysis of alpha4 receptors demonstrated that not only alpha1, alpha2, alpha3, beta1, beta2, and beta3 subunits but also gamma1, gamma2, gamma3, and delta subunits can be colocalized with alpha4 subunits in native GABAA receptors. Quantification experiments, however, indicated that only 7, 33, 4, or 7% of all alpha4 receptors contained gamma1, gamma2, gamma3, or delta subunits, respectively. These data not only explain the low percentage of [3H]Ro15-4513 binding sites precipitated by the anti-alpha4 antibody but also indicate that approximately 50% of the alpha4 receptors did not contain gamma1, gamma2, gamma3, or delta subunits. These receptors, thus, either are composed of alpha4 and beta1-3 subunits only, or additionally contain epsilon, pi, or so far unidentified GABAA receptor subunits.

Alterations in the expression of GABAA receptor subunits in cerebellar granule cells after the disruption of the alpha6 subunit gene.

Any given subunit of the heteromultimeric type-A gamma-aminobutyric acid (GABA) GABAA receptor may be present in several receptor subtypes expressed by individual neurons. Changes in the expression of a subunit may result in differential changes in the expression of other subunits depending on the subunit composition of the receptor subtype, leading to alterations in neuronal responsiveness to GABA. We used the targeted disruption of the alpha6 subunit gene to test for changes in the expression of other GABAA receptor subunits. Immunoprecipitation and ligand binding experiments indicated that GABAA receptors were reduced by approximately 50% in the cerebellum of alpha6 -/- mice. Western blot experiments indicated that the alpha6 subunit protein completely disappeared from the cerebellum of alpha6 -/- mice, which resulted in the disappearance of the delta subunit from the plasma membrane of granule cells. The amount of beta2, beta3 and gamma2 subunits was reduced by approximately 50%, 20% and 40%, respectively, in the cerebella of alpha6 -/- mice. A comparison of the reduction in the level of alpha1, beta2, beta3, gamma2, or delta-subunit-containing receptors in alpha6 -/- cerebellum with those observed after removal of alpha6-subunit-containing receptors from the cerebella of alpha6 +/+ mice by immuno-affinity chromatography demonstrated the presence of a significantly higher than expected proportion of receptors containing beta3 subunits in alpha6 -/- mice. The receptors containing alpha1, beta2, beta3 and gamma2 subunits were present in the plasma membrane of granule cells of alpha6 -/- mice at both synaptic and extrasynaptic sites, as shown by electron microscopic immunocytochemistry. Despite the changes, the alpha1 subunit content of Golgi-cell-to-granule-cell synapses in alpha6 -/- animals remained unaltered, as did the frequency of alpha1 immunopositive synapses in the glomeruli. Furthermore, no change was apparent in the expression of the alpha1, beta2 and gamma2 subunits in Purkinje cells and interneurons of the molecular layer. These results demonstrate that in alpha6 -/- mice, the cerebellum expresses only half of the number of GABAA receptors present in wild-type animals. Since these animals have no gross motor deficits, synaptic integration in granule cells is apparently maintained by alpha1-subunit-containing receptors with an altered overall subunit composition, and/or by changes in the expression of other ligand and voltage gated channels.

Subunit composition and quantitative importance of hetero-oligomeric receptors: GABAA receptors containing alpha6 subunits.

In cerebellum, GABAA receptors containing alpha6 subunits are expressed exclusively in granule cells. The number of alpha6 receptor subtypes formed in these cells and their subunit composition presently are not known. Immunoaffinity chromatography on alpha6 subunit-specific antibodies indicated that 45% of GABAA receptors in cerebellar extracts contained alpha6 subunits. Western blot analysis demonstrated that alpha1, beta1, beta2, beta3, gamma2, and delta subunits co-purified with alpha6 subunits, suggesting the existence of multiple alpha6 receptor subtypes. These subtypes were identified using a new method based on the one-by-one immunochromatographic elimination of receptors containing the co-purifying subunits in parallel or subsequent experiments. By quantification and Western blot analysis of alpha6 receptors remaining in the extract, the proportion of alpha6 receptors containing the eliminated subunit could be calculated and the subunit composition of the remaining receptors could be determined. Results obtained indicated that alpha6 receptors in cerebellum are composed predominantly of alpha6betaxgamma2 (32%), alpha1alpha6betaxgamma2 (37%), alpha6betaxdelta (14%), or alpha1alpha6betaxdelta (15%) subunits. Other experiments indicated that 10%, 51%, or 21% of alpha6 receptors contained homogeneous beta1, beta2, or beta3 subunits, respectively, whereas two different beta subunits were present in 18% of all alpha6 receptors. The method presented can be used to resolve the total number, subunit composition, and abundancy of GABAA receptor subtypes in the brain and can also be applied to the investigation of other hetero-oligomeric receptors.

Stoichiometry and assembly of a recombinant GABAA receptor subtype.

GABAA receptors are ligand-gated chloride ion channels that are presumed to be pentamers composed of alpha, beta, and gamma subunits. The subunit stoichiometry, however, is controversial, and the subunit arrangement presently is not known. In this study the ratio of subunits in recombinant alpha1beta3gamma2 receptors was determined in Western blots from the relative signal intensities of antibodies directed against the N terminus or the cytoplasmic loop of different subunits after the relative reactivity of these antibodies had been determined with GABAA receptor subunit chimeras composed of the N-terminal domain of one and the remaining part of the other subunit. Via this method a subunit stoichiometry of two alpha subunits, two beta subunits, and one gamma subunit was derived. Similar experiments investigating the composition of alpha1beta3 receptors expressed on the surface of human embryonic kidney (HEK) 293 cells cotransfected with alpha1 and beta3 subunits resulted in a stoichiometry of two alpha and three beta subunits. Density gradient centrifugation studies indicated that combinations of alpha1beta3gamma2 or alpha1beta3 subunits expressed in HEK 293 cells are able to form pentamers, whereas combinations of alpha1gamma2 or beta3gamma2 subunits predominantly form heterodimers. These results provide valuable information on the mechanism of GABAA receptor assembly and support the conclusion that GABAA receptors are pentamers in which a total of four alternating alpha and beta subunits are connected by a gamma subunit.

Rat beta 3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of gamma-aminobutyric acid type A receptors.

Human embryonic kidney 293 cells transiently transfected with beta 3 subunits of gamma-aminobutyric acid type A receptors from the rat exhibited a specific high affinity binding for [35S]t-butylbicyclophosphorothionate (TBPS) that could be inhibited by pentobarbital, etazolate, (+)-etomidate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864. The potency of these compounds for inhibition of [35S]TBPS binding was similar in membranes from beta 3 subunit-transfected human embryonic kidney 293 cells and in cerebellar membranes. In contrast to maximally inhibiting concentrations of unlabeled TBPS or picrotoxin, which caused a monophasic and rather slow dissociation of [35S]TBPS, maximally inhibiting concentrations of pentobarbital, etazolate, alphaxalone, propofol, chlormethiazole, and Ro 5-4864 accelerated the dissociation of [35S]TBPS from beta 3 subunit-containing membranes. Immunoaffinity chromatography and Western blot analysis with subunit-specific antibodies indicated that other endogenous subunits possibly present in these cells were not associated with beta 3 subunits. These results appear to indicate that most of the allosteric binding sites present on gamma-aminobutyric acid type A receptors can be formed by the beta subunit of these receptors. Homo-oligomeric beta 3 receptors therefore are an excellent model system for the structural investigation of gamma-aminobutyric acid type A receptors.

Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A2.

The N-linked carbohydrate chains of phospholipase A2 from honeybee (Apis mellifera) were released from glycopeptides with peptide-N-glycanase A and reductively aminated with 2-aminopyridine. The fluorescent derivatives were separated by size-fractionation and reverse-phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500-MHz 1H-NMR spectroscopy. Ten oligosaccharides consisted of mannose, N-acetylglucosamine and fucose alpha 1-6 and/or alpha 1-3 linked to the innermost N-acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with N-acetylgalactosamine. The structures of the 14 N-glycans from honeybee phospholipase A2 can be arranged into the following three series: [formula: see text]

Fucose alpha 1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals.

The reactivity of sera from honeybee venom allergic patients with the N-glycan of phospholipase A2 was investigated using neoglycoproteins with an enzyme-linked immunosorbent assay. Of 122 sera with appreciable levels of IgE antibodies directed against bee venom as measured by radioallergosorbent test, 34 sera exhibited significant amounts of glycan-reactive IgE. These sera cross-reacted with the N-glycan from the plant glycoprotein bromelain. The interaction of IgE with the N-glycan from phospholipase could be inhibited with glycopeptides from bromelain which shares the alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine with bee venom phospholipase. Since defucosylated bromelain glycopeptides or glycopeptides containing a Man3GlcNAc2 oligosaccharide were not recognized by most of these sera, we conclude that alpha 1,3-fucosylation of the innermost N-acetylglucosamine residue of N-glycoproteins forms an IgE-reactive determinant. This structural element is frequent in glycoproteins from plants, and it occurs also in insects. It is suspected to be one of the major causes of the broad allergenic cross-reactivity among various allergens from insects and plants.

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue.

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.