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B Laube

Publications and source records attributed to B Laube.

At least 37 records · Page 2Linked to original sources
PubMed1998

The human glycine receptor subunit alpha3. Glra3 gene structure, chromosomal localization, and functional characterization of alternative transcripts.

The neuronal glycine receptor is a ligand-gated chloride channel composed of ligand binding alpha and structural beta polypeptides. Homology screening of a human fetal brain cDNA library resulted in the identification of two alternative splice variants of the glycine receptor alpha3 subunit. The amino acid sequence predicted for the alpha3L variant was largely identical to the corresponding rat subunit. In contrast, the novel splice variant alpha3K lacked the coding sequence for 15 amino acids located within the cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and TM4. Using P1 artificial chromosome (PAC) clones, the structure of the GLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situ hybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to alpha3L and alpha3K, respectively, were identified and found to be widely distributed throughout the human central nervous system. Structural analysis of the GLRA3 gene revealed that the alpha3K transcript resulted from a complex splice event where excision of the novel exon 8A comprising the alternative sequence of 45 base pairs coincides with the persistence of a large intronic sequence in the 3'-untranslated region. Functional expression in HEK 293 cells of alpha3L and alpha3K subunits resulted in the formation of glycine-gated chloride channels that differed significantly in desensitization behavior, thus defining the cytoplasmic loop as an important determinant of channel inactivation kinetics.

Alternative Splicing↗
PubMed1998

Differentiation of glycine antagonist sites of N-methyl-D-aspartate receptor subtypes. Preferential interaction of CGP 61594 with NR1/2B receptors.

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.

Allosteric Regulation↗
PubMed1998

Evidence for a tetrameric structure of recombinant NMDA receptors.

The amino acids L-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1(Q387K), and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopus oocytes. In both cases, analysis of the resulting dose-response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1(R505K) and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.

Animals↗
PubMed1997

Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit.

NMDA receptors require both L-glutamate and the coagonist glycine for efficient channel activation. The glycine binding site of these heteromeric receptor proteins is formed by regions of the NMDAR1 (NR1) subunit that display sequence similarity to bacterial amino acid binding proteins. Here, we demonstrate that the glutamate binding site is located on the homologous regions of the NR2B subunit. Mutation of residues within the N-terminal domain and the loop region between membrane segments M3 and M4 significantly reduced the efficacy of glutamate, but not glycine, in channel gating. Some of the mutations also decreased inhibition by the glutamate antagonists, D-AP5 and R-CPP. Homology-based molecular modeling of the glutamate and glycine binding domains indicates that the NR2 and NR1 subunits use similar residues to ligate the agonists' alpha-aminocarboxylic acid groups, whereas differences in side chain interactions and size of aromatic residues determine ligand selectivity.

2-Amino-5-phosphonovalerate↗
PubMed1996

Molecular cloning and functional expression of a novel rat heart P2X purinoceptor.

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full-length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40-47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK-293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP-activated current reveals little agonist desensitization, is not activated by alpha,beta-meATP and is completely blocked by suramin and PPADS.

Amino Acid Sequence↗
PubMed1996

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

Amino Acid Sequence↗
PubMed1996

Migration of human glioma cells on myelin.

Histoanatomically invading astrocytoma cells appear to migrate along distinct structures within the brain. Astrocytoma invasion may occur along extracellular matrix (ECM) protein-containing structures, such as blood vessels, but most frequently occurs along tracts of myelinated fibers. This behavior most likely is a consequence of the use of constitutive extracellular ligands expressed along the pathways of preferred dissemination. Enzymatic modification of the extracellular space or deposition of ECM by the tumor cells may also create a more permissive environment. Established human glioma cell lines and two preparations of primary cells isolated from glioblastoma biopsies were studied with the use of cell adhesion and monolayer migration assays to investigate whether crude human central nervous system myelin extracts present specific cell adhesion ligands that promote glioma attachment and cell migration. Two cell lines showed high levels of adhesion and migration on central nervous system myelin similar to levels of migration on the ECM protein merosin, which has previously been shown to be a highly permissive substrate for cultured astrocytoma cells. Two other cell lines showed lower but specific migratory response; one cell line did not attach or specifically migrate on crude myelin extracts. For both glioblastoma primary cell preparations, myelin and merosin were the most permissive substrates for attachment and migration. Other ECM proteins (collagen type IV, fibronectin, and vitronectin) were moderate or nonpermissive substrates. Our findings indicated that astrocytoma cells may be able to use oligodendrocyte membrane-associated ligands as well as ECM proteins of the basement membranes for invasion of normal brain.

Astrocytoma↗
PubMed1995

Hyperekplexia mutations of the glycine receptor unmask the inhibitory subsite for beta-amino-acids.

beta-Alanine and taurine are agonists of the glycine receptor (GlyR) which, at low concentrations, antagonize the action of the principal agonist glycine. We analysed the potency of these ligands on alpha 1 subunits mutated at residue R271. GlyRs formed from alpha 1R271K subunits showed a reduction of beta-alanine and taurine affinities and maximal inducible currents; the mutants alpha 1R271Q and alpha 1R271L associated with human hyperekplexia gave no responses to these ligands. Inhibition of glycine-evoked currents by beta-alanine and taurine, however, was similar for all mutant GlyRs. These data are consistent with the existence of two subdomains within the ligand binding region of the GlyR, an agonistic one, which depends on arginine 271, and an antagonistic subsite, which is not connected to this residue.

Animals↗
PubMed1995

Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors.

1. The effect of the divalent cation Zn2+ on inhibitory glycine receptor (GlyR) currents was investigated in rat embryonic spinal cord neurons and Xenopus oocytes expressing recombinant GlyRs. 2. In cultured spinal neurons, Zn2+ potentiated glycine-induced whole-cell currents about 3-fold when applied extracellularly at concentrations of 0.5-10 microM. In contrast, higher concentrations (> 100 microM) of Zn2+ decreased the glycine response. 3. A similar biphasic modulation of glycine-induced currents by Zn2+ was also found with recombinant homo- and hetero-oligomeric GlyRs generated in Xenopus oocytes. Dose-response analysis showed that both the potentiating and inhibitory effects of Zn2+ result from changes in apparent agonist affinity. 4. Analysis of chimeric constructs of the GlyR alpha 1- and beta-subunits revealed that the positive and negative modulatory effects of Zn2+ are mediated by different regions of the alpha 1-subunit. 5. Our data indicate the existence of distinct high- and low-affinity Zn2+ binding sites on the ligand-binding alpha-subunits of the GlyR. These sites may be implicated in the regulation of synaptic efficacy within glycinergic pathways.

Amino Acid Sequence↗
PubMed1994

Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia.

Hereditary hyperekplexia is a dominant neurological disorder associated with point mutations at the channel-forming segment M2 of the glycine receptor alpha 1 subunit. Voltage-clamp recordings from the heterologously expressed mutants (alpha 1R271L or alpha 1R271Q) revealed 146- to 183-fold decreased potencies of glycine to activate the chloride channel, and significantly reduced maximal whole-cell currents as compared with wild-type receptors. In contrast, the ability of the competitive antagonist strychnine to block glycine-induced currents was similar in all cases. Radioligand binding assays showed a 90- to 1365-fold reduction in the ability of glycine to displace [3H]strychnine from its binding site on the mutant receptors. Paralleling the reductions in whole-cell current, the elementary main-state conductances of the mutants (alpha 1R271L, 64 pS; alpha 1R271Q, 14 pS) were lower than that of the wild-type receptor (86 pS). The decreased agonist affinities and chloride conductances of the mutants are likely to cause neural hyperexcitability of affected patients by impairing glycinergic inhibition. In addition, our data reveal that structural modifications of the ion-channel region can affect agonist binding to the glycine receptor.

Animals↗
PubMed1994

Mutational analysis of the glycine-binding site of the NMDA receptor: structural similarity with bacterial amino acid-binding proteins.

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

Amino Acid Sequence↗
PubMed1994

Structure, diversity and synaptic localization of inhibitory glycine receptors.

The inhibitory glycine receptor (GlyR) mediates postsynaptic inhibition in spinal cord, brain stem and other regions of the vertebrate central nervous system. Biochemical and molecular approaches have identified different developmentally and regionally regulated GlyR isoforms that result from the differential expression of at least four genes coding for different variants of the ligand-binding alpha subunit. Molecular studies have allowed identification of GlyR subunit domains implicated in ligand binding, channel formation and receptor assembly. At the postsynaptic membrane, the GlyR colocalizes with a 93-kDa tubulin-binding peripheral membrane protein, gephyrin. Antisense inhibition of gephyrin expression prevents GlyR accumulation at postsynaptic membrane specialization. Thus, gephyrin is essential for postsynaptic receptor topology.

Animals↗
PubMed1993

Glycine-glutamate interactions at the NMDA receptor: role of cysteine residues.

The NMDA subtype of ionotropic glutamate receptors occupation by both L-glutamate and the co-agonist glycine for efficient channel opening. To elucidate the role of disulfide bridges for the allosteric interaction of these agonists we mutated the cysteine residues in the ligand-binding NMDAR1 (NR1 or zeta) subunit of the rodent NMDA receptor and co-expressed the resulting mutants with the NR2B (epsilon 2) subunit in Xenopus oocytes. Most of the cysteine substitutions had no effect on agonist responses. However, replacement of cysteines 402 and 418 by alanine largely abolished the potentiation of glutamate currents by glycine. These cysteine residues in the putative extracellular domain of the NR1 subunit may form a disulfide bridge important for agonist interaction.

Animals↗
PubMed1993

Glutamate receptors of Drosophila melanogaster. Primary structure of a putative NMDA receptor protein expressed in the head of the adult fly.

The NMDA subtype of ionotropic glutamate receptors has been implicated in the activity-dependent modification of synaptic efficacy in the mammalian brain. Here we describe a cDNA isolated from Drosophila melanogaster which encodes a putative invertebrate NMDA receptor protein (DNMDAR-I). The deduced amino acid sequence of DNMDAR-I displays 46% amino acid identity to the rat NMDAR1 polypeptide and shows significant homology (16-23%) to other vertebrate and invertebrate glutamate receptor proteins. The DNMDAR-I gene maps to position 83AB of chromosome 3R and is highly expressed in the head of adult flies. Our data indicate that the NMDA subtype of glutamate receptors evolved early during phylogeny and suggest the existence of activity-dependent synaptic plasticity in the insect brain.

Amino Acid Sequence↗
PubMed1993

Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry.

The inhibitory glycine receptor (GlyR) is a pentameric protein composed of two types (alpha and beta) of membrane-spanning subunits. Coexpression in Xenopus oocytes of a low affinity mutant of the alpha 2 subunit with the alpha 1 and beta subunits indicated that GlyRs assembled from alpha 1 and alpha 2 polypeptides contain variable subunit ratios, whereas alpha/beta hetero-oligomers have an invariant (3:2) stoichiometry. Analysis of different alpha/beta chimeric constructs revealed that this difference in assembly behavior is mediated by the N-terminal extracellular regions of the receptor subunits. Substitution of residues diverging between the alpha and beta subunits identified combinations of sequence motifs determining subunit stoichiometry.

Amino Acid Sequence↗
PubMed1992

Glutamate receptors of Drosophila melanogaster: cloning of a kainate-selective subunit expressed in the central nervous system.

We report the isolation and functional characterization of cDNAs encoding a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits significant homology to mammalian glutamate receptor subunits. Injection of DGluR-I cRNA into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and philanthotoxin. DGluR-I transcripts are differentially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1992

Structure and function of the cochlea in the African mole rat (Cryptomys hottentotus): evidence for a low frequency acoustic fovea.

The cochlea of the mole rat Cryptomys hottentotus was investigated with physiological and anatomical methods. In order to reveal the place-frequency map of the cochlea, iontophoretic HRP-applications were made in the cochlear nucleus at physiologically characterized locations. Subsequent HRP-transport in auditory nerve fibres and labeling patterns of spiral ganglion cells within the cochlea were evaluated. A cochlear place-frequency map was constructed from 17 HRP-applications in the cochlear nucleus at positions where neurons had characteristic frequencies between 0.1 and 12.6 kHz. As in other mammals, high frequencies were found to be represented at the cochlear base, low frequencies at the cochlear apex. The place-frequency map had three distinct parts which were characterized by their different slopes. A clear over-representation of the frequencies between 0.6 and 1 kHz was revealed, in this frequency range the slope of the place-frequency map amounted to 5.3 mm/octave. As calculated from the regression analysis, below 0.6 kHz the slope of the cochlear place-frequency map amounted to 0.24 mm/octave, above 1 kHz to 0.9 mm/octave. As in other mammals width of the basilar membrane (BM) increased from the cochlear base towards the cochlear apex. Also in concordance with the findings in other mammals, BM-thickness decreased from the cochlear base to the apex. However, it was remarkable to find that there was no or little change in BM-width and thickness between 40 and 85% BM-length. It was also revealed that scala tympani was only 1/10th the size found in the rat or other mammals of similar body size.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗