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D Benke

Publications and source records attributed to D Benke.

53 records · Page 3Linked to original sources
PubMed1993

Molecular distinction of three N-methyl-D-aspartate-receptor subtypes in situ and developmental receptor maturation demonstrated with the photoaffinity ligand 125I-labeled CGP 55802A.

Activation of N-methyl-D-aspartate (NMDA) receptors is essential for synaptic plasticity in the central nervous system and contributes to neuronal death under various pathological conditions. Although several subunits have been cloned, the structure of NMDA receptors in situ is unresolved. By using a photoreactive antagonist with nanomolar affinity to the NMDA-binding site, three types of receptors were differentiated by their pattern of photoaffinity-labeled subunits. In adult brain, a protein of 175-kDa was photoreactive that displayed a profile of ligand binding and autoradiographical distribution corresponding to NMDA receptors. In contrast, in early postnatal brain, proteins of both 175 kDa and 115 kDa were photolabeled. This labeling pattern is switched to that of adult brain around postnatal day 10, pointing to a structural maturation of NMDA receptors. A third type of receptor could be identified in cerebellar granule cell cultures, where NMDA receptors mediate trophic effects and photolabeling was exclusively targeted to a 115-kDa protein. To identify the proteins labeled in situ, recombinant receptors were subjected to photolabeling. When the NR1 subunit was coexpressed with either the NR2A, NR2B, or NR2C subunit, only the combination of NR1/NR2A was photoreactive. Both the NR1 and NR2A subunits were photolabeled, corresponding in size to the proteins labeled in situ. However, the lack of subunit-selectivity in photolabeling the NR1/NR2A combination suggests the presence of additional receptor components in situ to explain the subunit-selective photoreactivity in adult brain (175 kDa) and in cerebellar granule cells (115 kDa). The subunit combination NR1/NR2A by itself appears insufficient to describe a major population of NMDA receptors, in particular, in adult brain.

Affinity Labels↗
PubMed1993

Photoaffinity labeling of the NMDA receptor.

The structure of NMDA receptors in situ has been probed with the novel photoaffinity ligand 125I-CGP 55802A. By covalently linking the radioactive high-affinity photolabel to NMDA receptors in bovine brain we have identified a protein of 175 kDa associated with the binding site for NMDA receptor agonists and competitive antagonists. Based on its molecular size the photolabeled protein is likely to correspond to the NR2A and/or NR2B subunit. The photoaffinity ligand will permit the assessment of regulatory changes in NMDA receptor subunit expression.

Affinity Labels↗
PubMed1993

GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by alpha 5- and delta-subunit-specific immunopurification.

The pharmacological significance and structural basis of the gamma-aminobutyrate (GABAA) receptor heterogeneity was investigated in situ by an immunobiochemical analysis of receptor populations characterized by the delta- and alpha 5-subunit. Using an antiserum specific for the delta-subunit, a population of GABAA receptors (21 +/- 2% of solubilized receptors) was immunoprecipitated from rat brain extracts which contained high affinity benzodiazepine binding sites. They were distinguished from those immunoprecipitated by the alpha 1- and alpha 3-subunit antisera by a 4-5-fold and 5-10-fold higher affinity for diazepam and beta CCM, respectively. Using the delta-antiserum in immunoaffinity chromatography the delta-subunit was found to be associated with the alpha 1-, alpha 3-, beta 2/3-, and gamma 2-subunits, suggesting that the latter conveys benzodiazepine receptor sensitivity also to GABAA receptors containing the delta-subunit. The receptor population immunoprecipitated by the alpha 5-subunit antiserum (10 +/- 2% of receptors solubilized from whole brain extracts) was characterized by affinities for zolpidem, beta CCM, and CL 218872 which distinguished it from all other known receptor populations. The alpha 5-subunit was associated with the alpha 1-, alpha 3-, beta 2/3- and gamma 2-subunits pointing to differential subunit combinations. Indeed, when receptor populations were immunoprecipitated by the alpha 5-subunit antiserum from different brain regions, zolpidem displayed striking differences in affinity pointing to the role of subunits other than alpha 5 in determining receptor affinity.

Animals↗
PubMed1993

Neuron-specific expression of GABAA-receptor subtypes: differential association of the alpha 1- and alpha 3-subunits with serotonergic and GABAergic neurons.

GABAA-receptors in the brain display a striking structural heterogeneity, which is based on a multiplicity of diverse subunits. The allocation of GABAA-receptor subtypes to identified neurons is essential for an analysis of the functional significance of receptor heterogeneity. Among GABA-receptive neurons, well-characterized examples include the serotonergic and GABAergic neurons in the raphe nuclei. The GABAA-receptor subtypes expressed in these two types of neurons were analysed using antisera which recognize selectively the alpha 1- and alpha 3-subunits, and their co-localization with serotonin and glutamate decarboxylase was assessed by confocal laser microscopy in double and triple immunofluorescence staining in the rat. The vast majority of serotonergic neurons express strong alpha 3-subunit-immunoreactivity, but are devoid of alpha 1-subunit staining. In contrast, both the alpha 1- and alpha 3-subunit-immunoreactivities are present in glutamate decarboxylase-positive neurons. Thus, serotonergic and GABAergic neurons selectively express distinct patterns of alpha subunits, suggesting that they possess distinct subtypes of GABAA-receptors. The occurrence of neuron-specific GABAA-receptor subtypes may open new possibilities for the targeting of drugs with selective therapeutic actions.

Animals↗
PubMed1993

GABAA-receptors: drug binding profile and distribution of receptors containing the alpha 2-subunit in situ.

The highest structural diversity of GABAA-receptor subunits is observed among members of the alpha-subunit class. Using subunit-specific antisera, the receptors containing the alpha 2-subunit were characterized. Western blots revealed an apparent molecular size of 52 kDa for the alpha 2-subunit. Immunohistochemically, the alpha 2-subunit was most preponderant in areas which lack the alpha 1-subunit, e.g. striatum and olfactory bulb granule cell layer, suggesting that these two subunits represent largely distinct receptor subtypes. Pharmacologically, the receptor population which was immunoprecipitated by the alpha 2-subunit-specific antisera displayed a drug binding profile characterized by a low affinity for CL 218872, beta CCM and zolpidem. This is in striking contrast to the high affinities of these ligands displayed by receptors immunoprecipitated by the alpha 1-subunit-specific antiserum. Thus, the alpha 1- and the alpha 2-subunit characterize two GABAA-receptor populations which greatly differ in brain distribution and pharmacological profile.

Amino Acid Sequence↗
PubMed1992

Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies.

The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABAA) receptors in the brain is expected to give rise to different modes of GABAergic synaptic inhibition and different profiles of modulatory drugs effective in anxiolytic, hypnotic, and antiepileptic therapy. To identify receptor subtypes in situ, the most prevalent subunits were visualized by double and triple immunofluorescence staining in rat brain, using polyclonal antibodies to the alpha 1, alpha 3, and gamma 2 subunits and a monoclonal antibody to locate both the beta 2 and the beta 3 subunit. At both cellular and subcellular levels five distinct patterns of subunit colocalization were identified: I, alpha 1 beta 2,3 gamma 2; II, alpha 3 beta 2,3 gamma 2; III, alpha 1 alpha 3 beta 2,3 gamma 2; IV, alpha 3 gamma 2; and V, alpha 1 alpha 3 gamma 2. As analyzed by confocal laser microscopy, different subunits displayed the same local variations of staining intensity ("hot spots") along the plasma membrane. The covisualized subunits appear therefore to be coassembled in receptor subtypes. Most neurons expressed only a single major receptor subtype with no apparent distinction between synaptic and extrasynaptic sites. However, in some neurons, most notably in Purkinje cells, the subunit composition varied between the soma and the dendrites, pointing to the existence of receptor heterogeneity within single neurons. Furthermore, different populations of neurons may be characterized by particular receptor subtypes. Cells displaying alpha 1-subunit immunoreactivity were mostly identified as GABAergic, whereas monoaminergic neurons displayed intense alpha 3-subunit immunoreactivity but virtually no alpha 1-subunit immunoreactivity. The allocation of defined GABAA receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.

Animals↗
PubMed1992

GABAA-receptor subtypes differing in alpha-subunit composition display unique pharmacological properties.

GABAA-receptor subtypes in rat brain were characterized using anti-peptide antisera specific for the alpha 1-, alpha 3- and alpha 5-subunits. While a high abundance of alpha 1-containing receptors was demonstrated by immunoprecipitation (80-90% of receptors), the receptors precipitated with the alpha 3- and the alpha 5-antiserum were less frequent (18-25% and 10-23%, respectively). The three receptor populations displayed unique pharmacological properties as shown by radioligand binding. Diazepam, flumazenil and flunitrazepam displayed similar displacing potencies in [3H]-flumazenil binding. However, the affinities of CL 218872, beta CCM and zolpidem were up to 10-fold lower in the alpha 3- than the alpha 1-receptor population while intermediate values were found for the alpha 5-receptor population. In many brain areas, a neuron-specific expression of receptors containing either alpha 1- or alpha 3-subunits could be visualized immunohistochemically. It will be of major interest to determine, whether ligands with differential affinities for receptor subtypes in situ will provide novel therapeutic profiles.

Animals↗
PubMed1991

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

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

Amino Acid Sequence↗
PubMed1991

GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits.

Recombinant GABAA (gamma-aminobutyrate-Type A) receptors that are sensitive to benzodiazepine receptor ligands can be generated by coexpression of alpha-, beta-, and gamma 2-subunit cDNAs (Pritchett, D. B., Sontheimer, H., Shivers, B. D., Ymer S., Kettenmann, H., Schofield, P. R., and Seeburg, P. H. (1989) Nature 338, 582-585; Pritchett, D. B., Lüddens, H., and Seeburg, P. H. (1989) Science 245, 1389-1392; Malherbe, P., Sigel, E., Baur, R., Perssohn, E., Richards, J. G., and Mohler, H. (1990) J. Neurosci. 10, 2330-2337). However, in brain tissue, only alpha- and beta-subunit proteins have so far been detected. To identify the size and distribution of the gamma 2-subunit protein in brain tissue, polyclonal antibodies were prepared against two synthetic peptides corresponding to amino acids 1-15 and 336-350 of the cDNA-derived rat gamma 2-subunit sequence. On Western blots, both anti-gamma 2-subunit antisera selectively labeled a 43-kDa protein. gamma 2-Subunit immunoreactivity was detected immunohistochemically in various brain regions, e.g. in the olfactory bulb, cerebral cortex, islands of Calleja, hippocampus, substantia nigra, and cerebellum. Immunoprecipitation with both antisera identified the gamma 2-subunit immunoreactivity in 40 and 50% of the native GABAA receptors purified from bovine and rat brains, respectively. Monoclonal antibody bd24 selectively recognizes the alpha 1-subunit, whereas bd17 recognizes both the beta 2- and beta 3-subunits (Ewert, M., Shivers, B. D., Lüddens, H., Mohler, H., and Seeburg, P. H. (1990) J. Cell Biol. 110, 2043-2048). Since either of these monoclonal antibodies (bd17 and bd24) precipitated approximately 90% of the GABAA receptors, the gamma 2-subunit is frequently associated with the alpha 1-subunit and the beta 2- and/or beta 3-subunit in vivo.

Amino Acid Sequence↗
PubMed1991

A computer controlled device to facilitate studies of the kinetics of ligand-binding: binding of diazepam to bovine brain membranes.

A device to facilitate kinetic receptor filtration assays is described. The receptor containing membranes and the labeled ligand are placed in two separate syringes and are rapidly mixed into a collecting syringe using a pneumatic ram. Shortly after the start of mixing, a pneumatically controlled valve switches the collecting syringe containing the receptor-ligand mixture to the filtration unit. Filtration is performed on glass/microfiber filters or equivalent by pushing the plunger of the collecting syringe by a stepper motor. A valve positioner controlling several valves allows the filtered membranes to be washed and dried by pressure in any user programmable sequence. Further filtration of the receptor-ligand mixture can be programmed at selected time points. The entire system is controlled by an IBM-PC. With this system, the association and dissociation of diazepam from crude bovine-brain membranes has been studied at 4 degrees C. The dissociation shows a biphasic pattern with half lifes of 1.3 and more than 23 minutes respectively. Association appears to be into a single compartment.

Animals↗
PubMed1991

Immunochemical identification of the alpha 1- and alpha 3-subunits of the GABAA-receptor in rat brain.

To identify subunit variants of the GABAA-receptor antisera were developed against specific cDNA-derived peptide sequences of the alpha 1- and alpha 3-subunits of rat brain. The alpha 1-subunit antiserum selectively recognized a protein of 50 +/- 1 kDa in rat and bovine GABAA-receptor preparations, while the alpha 3-subunit antiserum interacted with a protein doublet of 59 +/- 2 kDa and 61 +/- 3 kDa. The alpha 1-subunit immunoreactivity resides in a large population of GABAA-receptors as shown by immunoprecipitation of 63 +/- 6% of [3H]flumazenil binding sites with the alpha 1-subunit antiserum. In contrast, only 24 +/- 3% of receptor binding sites were precipitated with the alpha 3-subunit antiserum. Co-precipitation studies suggest that the alpha 1- and alpha 3-subunit immunoreactivities do not share the same receptor population while the gamma 2-subunit immunoreactivity is associated with the alpha 1-subunit immunoreactivity.

Amino Acid Sequence↗
PubMed1990

Identification of the gamma 2-subunit protein in native GABAA receptors in brain.

GABAA receptors with functional benzodiazepine receptors have been reconstituted by coexpression of alpha-, beta- and gamma-subunit cDNAs. In brain, proteins of the alpha- and beta-subunits, but not the gamma 2-subunit have been identified. Using an antipeptide antiserum, we now demonstrate that the gamma 2-subunit is a protein of 43 kDa. It is present in at least 50% of GABAA receptors, as shown by immunoprecipitation.

Amino Acid Sequence↗
PubMed1989

Comparison of acetylcholine and alpha-bungarotoxin binding sites in insects and vertebrates.

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology. 2. Hill plot analysis indicated a non-cooperative binding of acetylcholine. 3. In membrane preparations from locust ganglia and mouse brain the number of binding sites for ACh was about ten fold lower than for BGTX, whereas in membranes from electric tissue both sites occurred in similar concentrations. 4. Drug binding studies suggest that the high affinity binding sites for ACh and BGTX in preparations from insect and mouse are different; whereas in electric tissue both sites are very similar. 5. Precipitation experiments using immobilized BGTX and specific antibodies indicated that in insect nervous tissue as in electric tissue the ACh and BGTX binding sites are located on the same receptor molecule and occupy distinct partially overlapping binding sites, whereas in the vertebrate brain both sites are located on distinct binding proteins.

Animals↗
PubMed1987

Expression of neuronal acetylcholine receptor polypeptides in vitro.

1. Translation of poly(A) RNA extracted from the nervous tissue of locusts in a reticulocyte lysate system led to polypeptides with a broad spectrum of molecular weights. 2. Using anti-locust acetylcholine receptor (AChR) antisera, polypeptides with a molecular weight of about 50,000 were immunoprecipitated. These peptides comprised about 0.3% of the total translation products. 3. Cotranslational incubation with pancreatic rough microsomes resulted in a glycosylated 60,000-dalton immunoprecipitate. 4. Density-gradient analysis of in vitro synthesized and glycosylated receptor polypeptides indicated that no assembly of subunits had taken the place under the in vitro conditions.

Animals↗
PubMed1986

Messenger RNA from insect nervous tissue induces expression of neuronal acetylcholine receptors in Xenopus oocytes.

mRNA isolated from the nervous tissue of young insects microinjected in Xenopus oocytes induced the expression of alpha-toxin binding sites which were inserted into the surface membrane. Immunoprecipitation experiments revealed that the coded receptor polypeptides were processed to the authentic size, and ion flux studies demonstrated that functional nicotinic acetylcholine receptors were produced and inserted into the oocyte membrane.

Animals↗
PubMedDate not supplied

Immunobiochemical characterization of the NMDA-receptor subunit NR1 in the developing and adult rat brain.

To investigate the developmental and regional expression of the NR1-subunit of the NMDA-receptor on the protein level, two polyclonal antisera [NR1(N) and NR1(C)] were raised against fusion proteins derived from the N- and C-terminal domain of the NR1-subunit, respectively. In Western blots of rat brain membranes, both antisera specifically recognized a single protein band with an apparent molecular size of 115 kDa. The regional distribution of the NR1-subunit immunoreactivity was analyzed in the developing and adult rat brain using sections blotted onto nitrocellulose membranes for immunostaining. With the NR1(N)-antiserum, strongest signals were detected in hippocampus, followed by cortex, striatum and thalamus, and weaker staining was observed in tectum, brainstem and cerebellum of adult brain. The NR1(C)-immunoreactivity exhibited a similar distribution, except that the staining in thalamus, tectum, brainstem and cerebellum was faint or virtually absent. The distinct pattern of NR1(N)- and NR1(C)-immunoreactivity arose during postnatal development. At birth, moderate staining with both NR1-subunit antisera was observed throughout the brain increasing strongly in most brain regions until postnatal day 21. In some brain areas, however, the NR1(C)-, in contrast to the NR1(N)-staining, decreased postnatally e.g. in thalamus, tectum and brainstem. The restricted staining intensity of the NR1(C)-antiserum in particular areas of adult and developing brain appears to reflect the emergence of C-terminal splice variants of the NR1-subunit which are not recognized by the NR1(C)-antiserum.

Animals↗