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V I Teichberg

Publications and source records attributed to V I Teichberg.

At least 19 recordsLinked to original sources
PubMed2001

Identification of domains and amino acids involved in GLuR7 ion channel function.

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed in Xenopus oocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured in Xenopus oocytes. In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7. Our data show that EC(50) values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC(50) values in GluR6. The high EC(50) for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain. Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.

Amino Acids↗
PubMed2000

Mutant cycle analysis of the active and desensitized states of an AMPA receptor induced by willardiines.

The halogenated willardiines are agonists at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors. Although they differ only by the nature of the halogen substituent, they display marked differences in their efficacy to activate the receptor channel opening and in causing desensitization. We have studied the origin of the different agonist properties of the willardiines and in particular the nature of the structural element within the receptor binding domain that is able to distinguish between willardiines at a subatomic resolution of 0.6 A (the difference in radius between F and Br) and allow (S)-5-fluorowillardiine to cause receptor desensitization much more than (S)-5-bromowillardiine. For this purpose, we analyzed, with the thermodynamic mutant cycle method, the active and desensitized states induced by the willardiines in the GluR1 subtype of AMPA receptors and GluR1 mutants in which residues E398, Y446, L646, and S650, within the agonist binding domain, were mutated. The results were used to generate a 3D model of the willardiine docking mode. We suggest that the active and desensitized states of the AMPA-R correspond, respectively, to the open-lobe and closed-lobe conformations of the agonist binding domain.

Alanine↗
PubMed2000

How well can molecular modelling predict the crystal structure: the case of the ligand-binding domain of glutamate receptors.

The concept that the ligand-binding domain of vertebrate glutamate receptor channels and bacterial periplasmic substrate-binding proteins (PBPs) share similar three-dimensional (3D) structures has gained increasing support in recent years. On the basis of a dual approach that included computer-assisted molecular modelling and functional studies of site-specific mutants, theoretical 3D models of this domain have been proposed. This article reviews to what extent these models could predict the crystal structure of the ligand-binding domain of an ionotropic glutamate receptor subunit recently determined at high resolution by X-ray diffraction studies.

Amino Acid Sequence↗
PubMed1999

Lectin-induced inhibition of desensitization of the kainate receptor GluR6 depends on the activation state and can be mediated by a single native or ectopic N-linked carbohydrate side chain.

The ionotropic glutamate receptor GluR6 exhibits strongly and rapidly desensitizing current responses. Treatment of heterologically expressed GluR6 with the lectin concanavalin A (ConA) in Xenopus oocytes as well as in human embryonic kidney-293 cells results in a considerable increase of the steady-state current, presumably by inhibiting receptor desensitization. In the present study, we investigated the molecular basis of this effect using a systematic mutagenesis approach. We found that although N-glycosylation is an absolute prerequisite for the lectin-mediated inhibition of desensitization, no single one of the nine extracellular consensus sites for N-glycosylation of GluR6 is required. Rather, each of the nine N-linked carbohydrate side chains is independently capable of modulatory interaction with the lectin. Moreover, even artificially introduced N-glycosylation sites can substitute for native sites. Thus, the specific site of the lectin binding does not appear to be important for its desensitization-inhibiting action. Furthermore, we show that the extent of the receptor's ConA sensitivity depends on its state of activation, because the desensitized GluR6 exhibits significantly lower lectin sensitivity than the nondesensitized receptor. We conclude that binding of ConA "locks" the receptor in the activatable state, thereby inhibiting conformational changes required to shift the receptor to the desensitized state.

Animals↗
PubMed1998

Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin.

The N-methyl-D-aspartate receptor (NMDA-R) and brain spectrin, a protein that links membrane proteins to the actin cytoskeleton, are major components of post-synaptic densities (PSDs). Since the activity of the NMDA-R channel is dependent on the integrity of actin and leads to calpain-mediated spectrin breakdown, we have investigated whether the actin-binding spectrin may interact directly with NMDA-Rs. Spectrin is reported here to interact selectively in vitro with the C-terminal cytoplasmic domains of the NR1a, NR2A and NR2B subunits of the NMDA-R but not with that of the AMPA receptor GluR1. Spectrin binds at NR2B sites distinct from those of alpha-actinin-2 and members of the PSD95/SAP90 family. The spectrin-NR2B interactions are antagonized by Ca2+ and fyn-mediated NR2B phosphorylation, but not by Ca2+/calmodulin (CaM) or by Ca2+/CaM-dependent protein kinase II-mediated NR2B phosphorylation. The spectrin-NR1 interactions are unaffected by Ca2+ but inhibited by CaM and by protein kinase A- and C-mediated phosphorylations of NR1. Finally, in rat synaptosomes, both spectrin and NR2B are loosened from membranes upon addition of physiological concentrations of calcium ions. The highly regulated linkage of the NMDA-R to spectrin may underlie the morphological changes that occur in neuronal dendrites concurrently with synaptic activity and plasticity.

Animals↗
PubMed1998

A tetrameric subunit stoichiometry for a glutamate receptor-channel complex.

The structure of glutamate receptor-channel (GluR) subunits has recently been shown to differ from that of other ligand-gated channels and to contain a voltage-gated channel-like pore-forming motif. The view that the structure of GluR complexes is similar to the pentameric structure of other ligand-gated channels was questioned here. Studies of the response properties of the GluR1 subunit of the AMPA subtype of GluRs, co-expressed in Xenopus oocytes with its L646A mutant, which differs only by a greatly reduced sensitivity to quisqualate, provide new evidence suggesting that the GluR1 homomeric receptor channel has a tetrameric structure.

Animals↗
PubMed1996

A venus flytrap mechanism for activation and desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.

Desensitization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of glutamate receptor channels is an important process shaping the time course of synaptic excitation. Upon desensitization, the receptor channel closes and the agonist affinity increases. So far, the nature of the structural rearrangements leading to these events was unknown. On the basis of the structural homology of the ligand binding domains of AMPA receptors and of the bilobated bacterial periplasmic proteins, we now show that agonist interaction with one lobe of the GluR1 subunit of homomeric AMPA receptors controls channel activation while additional interactions with the other lobe cause channel desensitization. Accordingly, we suggest that the transition of the AMPA receptor channel to the desensitized state involves the agonist-mediated stabilization of the closed lobe conformation of its binding domain and is a process akin to that used by the venus flytrap.

Animals↗
PubMed1996

Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor.

The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine-rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive-like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha-32P]GTP. The reaction was blocked by KA, glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a peptide containing the glycine-rich motif. Site-directed mutagenesis of residues K72 and Y73 within the glycine-rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine-rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine-rich motif is the binding site at which guanine nucleotides inhibit the glutamate-mediated responses of various members of the subfamily of glutamate ionotropic receptors.

Affinity Labels↗
PubMed1996

CDNA cloning of chick brain alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors reveals conservation of structure, function and post-transcriptional processes with mammalian receptors.

Several types of functional ionotropic glutamate receptor have been cloned in the recent years from the mammalian central nervous system, but till now, none from other vertebrate species. Here, we report the cloning and functional analysis of four chick brain cDNAs, coding for members of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptors. These receptors are highly homologous to the mammalian GluR1-4 (A-D) receptors ( > 90%), and conserve their post-transcriptional modifications. The flip/flop exons are conserved not only at the amino acid level but also at the nucleotide level, and the intron of GluR4 involved in the RNA editing of the R/G site displays a rat-chick sequence conservation of 95%. Significant sequence differences are found only in the region containing the immunogenic epitope of neuroactive anti-GluR3 antibodies. Chick AMPA receptors are expressed in both the cerebrum and cerebellum. The ion channel activities of chick GluR1-4 were analyzed in Xenopus oocytes and found to be similar to those of mammalian AMPA receptors. Though their contribution to kainate binding activity in the cerebellum is minor, the profile of channel activity of the chick GluR1-4 suggests that they account for the kainatergic channel activity expressed by total chick cerebellar mRNAs.

Amino Acid Sequence↗
PubMed1996

Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor.

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

A ligand binding study of the interactions of guanine nucleotides with non-NMDA receptors.

The interactions of guanine nucleotides, and particularly GTP, with the [3H]-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and [3H]-kainate (KA) binding sites present on brain membranes was studied, using the ligand binding methodology and Scatchard analysis, in order to establish the competitive/non competitive nature of the interaction and determine whether guanine nucleotides, KA and AMPA share common binding sites. GTP was found to block [3H]-AMPA and [3H]-KA binding to rat cortical membranes with IC50 values of 0.4 mM and 1 mM respectively and the [3H] KA-binding to chick cerebellar membranes with a IC50 value of 20 microM. Scatchard analysis of [3H]-KA binding performed in the absence or presence of 1 mM GTP or 0.25 mM AMPA reveals that the high affinity [3H]-KA binding component is not affected by GTP but blocked in a non competitive fashion by AMPA while the low affinity [3H]-KA binding component is not affected by AMPA but blocked by GTP. Scatchard analysis of [3H]-KA binding to chick cerebellar membranes performed in the absence or presence of 33 microM GTP reveals a single binding site blocked in a competitive fashion by GTP. Scatchard analysis of [3H]-AMPA binding performed in the absence or presence of 0.5 mM GTP or 30 microM KA reveals that the high affinity [3H]-AMPA binding component is affected in a non competitive fashion by both GTP and KA while the low affinity [3H] AMPA binding component is affected in a competitive fashion by both GTP and KA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Organization and expression of the gene encoding chick kainate binding protein, a member of the glutamate receptor family.

The gene encoding chick cerebellar Bergmann glia-specific kainate binding protein (chKBP), has been isolated, characterized and expressed in heterologous systems. The structural gene spans 11.2 kb and contains 11 exons and 10 introns. Several of the exons encode specific receptor domains, including each of the predicted transmembrane regions. Exon/intron boundaries flanking the second, putative channel-forming transmembrane domain are conserved between chKBP and other glutamate/kainate receptor subunits. The putative promoter region 5' to the first exon displays high GC content and TATA, CAAT and AP1 consensus sequences. Transcription of the chKBP gene is evident prior to full cerebellar cortical maturation. Transcripts are abundant in cells consistent with Bergmann glia, as revealed by in situ hybridization. Transfection of 293 kidney cell cultures with chKBP cDNA or chKBP gene expression constructs confers CNQX-sensitive kainate binding with the pharmacological specificity displayed by both chKBP and kainate receptors. However, expression of the same constructs in Xenopus oocytes fails to yield detectable agonist-activated currents.

Amino Acid Sequence↗
PubMed1991

Properties of kainate receptor/channels on cultured Bergmann glia.

Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

Glial glutamate receptors: likely actors in brain signaling.

It has become clear that the neurotransmitter glutamate does not confine its excitatory effects to central nervous system neurons but interacts also with glial cells. Neurons and glia share the same types of ionotropic and metabotropic glutamate receptors except for the N-methyl-D-aspartate receptor, which is not found on glia. Applied on cultured glial cells, glutamate regulates the opening of receptor channels, activates second messengers, and causes the release of neuroactive compounds. Although glutamate and glutamate receptors confer on cultured glia the ability to receive and emit signals, it remains to be established whether glial signaling takes place in vivo. The chick Bergmann glial cells provide a unique experimental system with which to test the contribution of glial glutamate receptors to neuronal electrical activity. These cells are the exclusive carriers in the cerebellum of functional kainate receptors. The synaptic location of these receptors, their ion channel properties, and their regulation by phosphorylation reactions suggest that glial kainate receptors play a role in regulating synaptic efficacy and plasticity. If proved, this concept may require a modification of the anatomical and functional definition of a synapse to include a glial component as well.

Animals↗
PubMed1990

Phosphorylation of the 49-kDa putative subunit of the chick cerebellar kainate receptor and its regulation by kainatergic ligands.

The traffic of ions through the kainic acid (KA) receptor/channels present on chick cerebellar glia is modulated by intracellular events likely to involve phosphorylation reactions. The protein carrying the KA binding sites has been isolated from chick cerebellum and shown to be composed of a 49-kDa polypeptide. Its primary structure, established via cDNA cloning, shows the presence of two putative phosphorylation sites. We report here that this 49-kDa polypeptide is a substrate of the cAMP-dependent protein kinase which catalyzes the incorporation of up to 2 mol of phosphate/mol of KA binding site. KA prevents this phosphorylation reaction in a concentration range similar to that needed to activate the KA receptor/channels but higher by 3 orders of magnitude than that needed to saturate the KA binding sites. Kainatergic ligands produce similar effects to those of KA, but 1 mM N-methyl-D-aspartic acid and 1 mM quisqualic acid have no effect. However, 0.01 mM quisqualic acid prevents the inhibitory action of KA. These results raise the possibility that the phosphorylation of KA receptor/channels in their cellular environment is negatively regulated by KA.

Animals↗
PubMed1990

Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellar cortex.

A monoclonal antibody, IX-50, that was raised against a kainate binding protein (Mr = 49,000) from chicken cerebellum, was used in light and electron microscopic immunocytochemical studies to localize putative kainate receptors. Pre- and postembedding immunoperoxidase and immunogold methods were used in the cerebellar cortices of one to 26-day old chickens and adult rainbow trout. Immunoreactivity was detected only in association with Golgi epithelial/Bergmann glial cells. Intracellular immunoreactivity was present in the granular and agranular endoplasmic reticulum, Golgi apparatus and in lysosomes, representing the sites of synthesis, glycosylation and degradation of the protein. In the fish the granular endoplasmic reticulum was not immunoreactive. Extracellular immunoreactivity was associated with the plasma membrane. In the fish it was established that the epitope is on the outer surface of the membrane. The protein seems to be uniformly distributed along the membrane including the somata, the radial stem processes and the leafy lamellae surrounding Purkinje cell dendrites. Areas of the glial membrane in contact with other glial cells were also immunopositive. High-resolution light microscopy demonstrated all the Bergmann glial plasma membrane in the cortex, providing a "negative" image of Purkinje cell dendrites. It is apparent that Bergmann glial processes selectively outline the dendrites of the Purkinje cells by surrounding the parallel fibre terminal/Purkinje cell spine synaptic complexes. The parallel fiber terminals were highly immunoreactive for glutamate, as shown by an immunogold procedure. The association of Bergmann glial processes, carrying the Mr = 49,000 kainate binding protein, with the Purkinje cell dendrites and spine synapses could provide a basis for neuronal signalling to the Bergmann glia, possibly by glutamate.

Animals↗
PubMed1989

Molecular structure of the chick cerebellar kainate-binding subunit of a putative glutamate receptor.

Kainate receptors mediate some of the excitatory transactions carried out in the central nervous system by the neurotransmitter glutamate. They are involved in neurotoxicity, possibly in neurodegenerative disorders and it has been suggested that they have a role in long-term potentiation. Kainate receptors are present both on neuronal and glial cell membranes where they regulate the gating of a voltage-independent ion channel. Nothing is known about their molecular structure. Taking advantage of the unusually high abundance of 3H-kainate binding sites in the chick cerebellum, we have isolated an oligomeric protein that displays a pharmacological profile similar to that of a kainate receptor, and have demonstrated, using the monoclonal antibody IX-50, that this protein is composed of a single polypeptide of Mr 49,000 which harbours the specific kainate recognition site. The structure of this kainate binding protein (KBP) is also of interest because of its exclusive cerebellar localization on Bergmann glial membrane in close proximity to established glutamatergic synapses. We now report the isolation of the complementary DNA containing the complete coding region of the kainate binding protein. The predicted structure of the mature protein has four putative transmembrane domains with a topology analogous to that found in the superfamily of ligand-gated ion channels. This raises the possibility, that kainate binding protein may form part of an ion channel and may be a subunit of a kainate subtype of glutamate receptor.

Amino Acid Sequence↗