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H Betz

Publications and source records attributed to H Betz.

At least 91 records · Page 5Linked to original sources
PubMed1996

The membrane-bound rat serotonin transporter, SERT1, is an oligomeric protein.

The synaptic actions of the neurotransmitter serotonin are terminated by a selective re-uptake system located in the axonal membrane. To gain information about the quaternary structure of this membrane protein, we transiently expressed the recombinant rat serotonin transporter, SERT1, in human embryonic kidney 293 cells. Treatment with sulfhydryl oxidizing agents and the homobifunctional cross-linker dimethyl suberimidate (DMS) generated adducts of 130-180 kDa and 220-270 kDa, respectively. These data indicate an oligomeric structure of SERT1.

Animals↗
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

Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice.

Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses.

Animals↗
PubMed1996

The sensory innervation of the mouse spinal cord may be patterned by differential expression of and differential responsiveness to semaphorins.

To better understand the regulatory processes underlying axonal pathfinding we analyzed the embryonic expression of seven murine semaphorin genes by in situ hybridization In the spinal cord, transcripts of all seven semaphorin genes were detected from Embryonic Day 11.5 (E11.5) onward and restricted to distinct regions at E15.5. Interestingly, semE, F, and G mRNAs were in addition differentially expressed in the ventricular zone of the telencephalon. In order to correlate these expression patterns to the behavior of different types of sensory afferents, we tested their response to recombinant semaphorin proteins. Specific subpopulations of dorsal root ganglion sensory neurons displayed a developmentally regulated differential response to Sem D. Whereas extension of both NGF- and NT-3-dependent neurites was inhibited by Sem D at E12.5, only neurites formed in the presence of NGF responded at E14.5. This suggests that Sem D may be involved in preventing an early penetration of the spinal cord by sensory afferents and subsequently shaping their lamina-specific termination.

Animals↗
PubMed1996

Immunocytochemical detection of the serotonin transporter in rat brain.

The regional distribution of the serotonin uptake system was studied in rat brain using a specific polyclonal antibody raised against the putative extracellular loop between transmembrane domains 7 and 8 of the cloned rat serotonin transporter. Light microscope analysis with fluorescence and avidin-biotin-peroxidase techniques revealed a punctate staining as well as numerous labelled thin fibres, which exhibited accumulation of reaction end-product deposit over varicosities. These immunopositive processes were widely and heterogeneously distributed in the rat brain. High densities of immunoreactivity were seen within the caudate-putamen, amygdaloid complex, cortical areas, substantia nigra, ventral pallidum, Islands of Calleja, septal nuclei, interpeduncular nucleus, trigeminal motor nucleus and olfactory nuclei. We also found strong expression of serotonin transporter in the stratum oriens of area CA3 and, to a lesser extent, in the stratum oriens of CA1 and the stratum lacunosum molecular of CA1-CA3 regions of the hippocampus. Within the raphe nuclei, a moderate to high incidence of stained processes was observed, and immunopositive cell bodies were detected in the dorsal raphe nucleus. In addition, some immunoreactive fibres were present in the molecular and granular layers of the cerebellum as well as in the cochlear and olivary nuclei. In none of the regions analysed was evidence for glial staining obtained. The present immunocytochemical data reveal a widespread and heterogeneous distribution of the serotonin transporter in rat brain and suggest that serotoni transporter is preferentially sorted into axons, where it appears concentrated at varicosities and terminal boutons.

Animals↗
PubMed1996

A novel class of murine semaphorins with homology to thrombospondin is differentially expressed during early embryogenesis.

The semaphorins are a family of proteins thought to be involved in axonal guidance. Most of the known semaphorins have a similar primary structure characterized by the semaphorin domain and a carboxy-terminal Ig motif. Here we report the cloning of two members (semF and G) of a novel class of membrane-bound semaphorins which contain seven carboxy-terminal thrombospondin repeats, a motif known to promote neurite outgrowth. SemF and G transcripts are expressed, together with semD and E, in specific regions of young mouse embryos, demarcating distinct compartments of the developing somites or the undifferentiated neuroepithelium. The identification of semF and G increases the number of vertebrate semaphorins to at least 20 and suggests that some semaphorins might act as positive axonal guidance cues.

Age Factors↗
PubMed1996

Expression of glycine receptor alpha subunits and gephyrin in cultured spinal neurons.

The inhibitory glycine receptor is a pentameric membrane protein composed of alpha and beta subunits. In the postsynaptic membrane, the glycine receptor and the copurifying peripheral membrane protein gephyrin are clustered underneath glycine-releasing nerve terminals. Here, we describe the expression of gephyrin and the neonatal and adult glycine receptor alpha subunit isoforms alpha1 and alpha2 during in vitro differentiation of rat spinal neurons. Analysis by immunoassays and the reverse transcriptase-polymerase chain reaction showed that gephyrin and alpha subunit mRNA and protein levels exhibited a marked increase from 1 to 5 days in vitro, i.e. prior to the formation of functional synaptic contacts. Using confocal and standard immunofluorescence, we determined the number of immunoreactive cells and the cellular localization of the alpha subunits and gephyrin. At 3 days in vitro, glycine receptor immunoreactivity revealed by the monoclonal antibody mAb4a was found in < 10% of cells and was mainly localized intracellularly; in contrast, gephyrin was detected in in vitro, gephyrin was essentially localized at the neuronal surface. At this stage, the number of glycine receptor-positive cells approached that of gephyrin-containing neurons (50%), and glycine receptor antigen was found both intracellularly and at the periphery of the cells. The antibody mAb2b, which binds exclusively to the alpha1 subunit, revealed aggregates at the surface of a few neurons. At 10 days in vitro, glycine receptor and gephyrin staining was localized in clusters at the periphery of the soma and the neurites. This quantitative analysis corroborates temporal differences in the cellular distribution of gephyrin and glycine receptor alpha subunits, the former being accumulated first at the neuronal surface.

Animals↗
PubMed1996

The plasma membrane protein SNAP-25, but not syntaxin, is present at photoreceptor and bipolar cell synapses in the rat retina.

The two neuronal plasma membrane proteins synaptosomal associated protein 25 kDa (SNAP-25) and syntaxin form, in current models of synaptic vesicle exocytosis, a docking complex on the presynaptic plasma membrane. This docking complex interacts with the integral synaptic vesicle protein, synaptobrevin. We have examined, in the rat retina, the distribution of SNAP-25 and syntaxin using light and electron microscopic immunocytochemistry. SNAP-25 immunoreactivity was present in the outer and inner nuclear layers at the membranes of photoreceptor and amacrine cell somata, in the outer plexiform layer in the terminals of photoreceptor cells and throughout the inner plexiform layer in the terminals of bipolar and amacrine cells. In contrast, syntaxin immunoreactivity was not found in the terminals of photoreceptor and bipolar cells, but only in the terminals of amacrine cells. Because of the absence of detectable syntaxin from the terminals of photoreceptor and bipolar cells, we propose that either a novel syntaxin isoform or a syntaxin-like protein exists at the ribbon synapses formed by these neurons.

Animals↗
PubMed1996

Localization of the serotonin transporter in rat spinal cord.

The spinal cord is richly innervated by serotoninergic fibres originating from the raphe nuclei. The localization of the terminating component of serotoninergic neurotransmission, the serotonin transporter SERT1, was found in both the dorsal and ventral horns, especially at the level of the cervical and lumbar segments. Within the thoracic region, we observed a heavily labelled bundle in the intermediolateral nucleus of lamina VII. A low density of stained fibres was encountered in the sacral spinal cord. In contrast to homogeneous staining of motor nuclei, a differential labelling of laminae was seen in the dorsal horn, with laminae I, III and IV exhibiting a higher density of immunopositive terminals than the medial part of lamina II. High magnification revealed a preferential accumulation of serotonin transporter staining within nerve endings and varicosities of thin fibres. Double immunofluorescence staining demonstrated a co-localization of serotonin and its uptake system within these varicosities. These results show that the serotonin transporter is highly expressed in the rat spinal cord and that its distribution parallels the serotoninergic innervation. They also reinforce the view that varicosities are important neuronal structures, which modulate the function of dorsal and ventral horn neurons by releasing serotonin.

Animals↗
PubMed1996

Novel antagonists of the inhibitory glycine receptor derived from quinolinic acid compounds.

Binding of the coagonist glycine to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is potently antagonized by 2-carboxy-4-hydroxyquinolines. We show that closely related derivatives, 4-hydroxy-quinolines and 4-hydroxquinoline-3-carboxylic acids, antagonize the agonist response of the recombinant inhibitory glycine receptor (GlyR). In Xenopus laevis oocytes expressing the GlyR alpha 1 subunit, the chloride-substituted derivatives 5,7-dichloro-4-hydroxyquinoline-3-carboxylic acid and 7-chloro-4-hydroxyquinoline inhibited glycine currents in a mixed high affinity competitive and low-affinity noncompetitive fashion, whereas the related compounds 7-trifluoromethyl-4-hydroxyquinoline-3-carboxylic acid and 7-trifluoromethyl-4-hydroxyquinoline showed purely competitive antagonism. Our data suggest a model of the pharmacophore of the GlyR that displays significant similarity to that proposed for the glycine binding site of the N-methyl-D-aspartate receptor.

Animals↗
PubMed1995

The recombinant GABA transporter GAT1 is downregulated upon activation of protein kinase C.

Treatment of human embryonic kidney 293 cells expressing the rat gamma-aminobutyric acid (GABA) transporter 1 (GAT1) with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to decrease the velocity of specific [3H]GABA uptake. This downregulation varied with extracellular GABA concentration and was blocked by the PKC inhibitors 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) and staurosporine. An about 50% reduction of uptake velocity by PMA treatment was observed at GABA concentrations > 1 microM, whereas only a minor effect was seen at low substrate concentrations. These data indicate that GAT1 activity is downregulated by PKC activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Heterogeneity of antidepressant binding sites on the recombinant rat serotonin transporter SERT1.

Antidepressant drugs block the uptake of serotonin into serotonergic nerve terminals and blood platelets. Here, binding of the tricyclic antidepressant [3H]imipramine to the recombinant rat serotonin transporter SERT1 expressed in human embryonic kidney cells was found to be nonhomogeneous. Scatchard analysis and competition experiments revealed the existence of two distinct antidepressant binding sites. At site 1, [3H]imipramine binding was strictly sodium-dependent with an apparent KD of approximately 10 nM. In contrast, [3H]imipramine binding to site 2 occurred also in the absence of sodium and exhibited a lower affinity. Binding of the nontricyclic antidepressant [3H]citalopram was observed only at site 2. The natural substrate of this carrier, serotonin, competitively inhibited antidepressant binding at both sites; however, its affinity to site 2 was approximately 5-fold lower. These data provide a molecular explanation for the distinct pharmacological actions of different antidepressants.

Animals↗
PubMed1995

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.

Adenosine Triphosphate↗
PubMed1995

Baculovirus-driven expression and purification of glycine receptor alpha 1 homo-oligomers.

The glycine receptor is a ligand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human alpha 1 subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4- to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the alpha 1 receptor was found to exist in a predominantly monodisperse state and could be affinity-purified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.

Animals↗
PubMed1995

Colocalization of gephyrin and GABAA-receptor subunits in the rat retina.

Gephyrin is a protein that copurifies with the glycine receptor (GlyR) and is required for the clustering of GlyRs at postsynaptic sites. Previously, it was thought that antibody mAb 7a, directed against gephyrin, was a specific marker for GlyR. However, there is evidence that gephyrin can also be found at nonglycinergic synapses. Here, immunocytochemistry was applied to show this directly for the rat retina. Both gephyrin and different subunits of the gamma-aminobutyric acid (GABA)A receptor were localized to discrete puncta in the inner plexiform layer, and these puncta were shown by electron microscopy to represent synaptic sites. Double immunocytochemistry revealed that GABAA receptors and GlyRs are not colocalized. However, gephyrin and different subunits of GABAA receptors were found to occur at the same synapses. The amount of colocalization varied with the GABAA receptor subunit composition and was most extensive for the alpha 2 subunit, less for the alpha 3 subunit, and minimal for the alpha 1 subunit. The gephyrin present at GABAergic synapses of the retina might also be involved with clustering of receptors at the postsynaptic sites. Hence, localization of gephyrin can no longer be considered as a unique marker of glycinergic synapses.

Animals↗
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↗