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H Betz

Publications and source records attributed to H Betz.

At least 145 records · Page 8Linked to original sources
PubMed1993

Widespread expression of gephyrin, a putative glycine receptor-tubulin linker protein, in rat brain.

The peripheral membrane protein gephyrin co-purifies with the inhibitory postsynaptic glycine receptor (GlyR) of mammalian spinal cord. By immunoelectron microscopy, gephyrin has been localized at the cytoplasmic face of glycinergic postsynaptic membrane specializations. Here, we used specific monoclonal antibodies to demonstrate the presence of gephyrin in all regions of rat brain known to contain synapses and compared its histochemical distribution with that of GlyR antigens. In most brain structures, gephyrin is expressed independently of the GlyR alpha 1 subunit, but its distribution is very similar to the pattern obtained with mAb 4a, a monoclonal antibody recognizing the known GlyR alpha and beta subunits. Our data suggest a much wider distribution of gephyrin and GlyR proteins in the mammalian CNS than anticipated previously.

Animals↗
PubMed1993

Distribution of gephyrin transcripts in the adult and developing rat brain.

The peripheral membrane protein gephyrin copurifies with the inhibitory glycine receptor of mammalian spinal cord. It binds with high affinity to polymerized tubulin and has been implicated in the anchoring of the glycine receptor to cytoskeletal elements. Recently, cDNA cloning has identified variants of the gephyrin mRNA, which originate from alternative splicing of four exonic regions (cassettes 1-4). In this study, the expression patterns of gephyrin splice variants were determined in the adult and developing rat brain by in situ hybridization with synthetic oligonucleotide probes. Gephyrin transcripts were detected throughout the brain and spinal cord, with mRNAs containing cassette 2 (C2 transcripts) being predominant in adult animals. C3 and C4 transcripts were seen in cerebellar granule cells and in the dentate gyrus, whereas a C1 probe did not produce detectable hybridization signals. During development, C2 and C3 mRNAs were found in most brain regions. Generally, the spatial and temporal distribution of gephyrin transcripts is similar to that of the glycine receptor beta subunit mRNA reported previously.

Animals↗
PubMed1993

Synaptophysin and synaptoporin expression in the developing rat olfactory system.

The expressions of two closely related synaptic vesicle antigens synaptophysin and synaptoporin were examined in the olfactory system of the adult rat and during pre- and postnatal development. In the adult, immunocytochemistry showed that the continuously regenerating olfactory receptor neurons (primary neurons) produce both synaptophysin and synaptoporin which were localized in the cell bodies of the receptor neurons in the olfactory epithelium, their dendrites, axonal processes in the olfactory nerve and their terminals in the olfactory bulb glomeruli. Furthermore, ultrastructural analysis revealed synaptophysin- and synaptoporin-immunoreactivities associated with synaptic vesicles in most olfactory receptor axonal terminals impinging on dendrites of the mitral and tufted neurons (secondary neurons in the olfactory bulb circuitry) in the olfactory glomeruli. In like manner, tufted neurons, granule and periglomerular neurons (interneurons in the olfactory bulb circuitry) express both synaptophysin and synaptoporin. In contrast, mitral neurons expressed only the synaptophysin antigen which was likewise associated with mitral axonal terminals in their target the olfactory cortex. The patterns of synaptophysin and synaptoporin expressions in mitral neurons (synaptophysin only) and tufted neurons (synaptophysin and synaptoporin) were similar in prenatal, postnatal and adult rats as revealed by immunocytochemistry and in situ hybridization. However, the biosynthesis of synaptophysin and synaptoporin by granule and periglomerular neurons, olfactory bulb interneurons, occurred mainly postnatally.

Animals↗
PubMed1993

On the structure of the 'synaptosecretosome'. Evidence for a neurexin/synaptotagmin/syntaxin/Ca2+ channel complex.

Recent experiments have identified interactions between presynaptic and synaptic vesicle membrane proteins, that might be important in organizing the components of the fast neurotransmitter release mechanism to ensure that the process follows a rapid time course. Here we extend previous investigations to show that in addition to the alpha-latrotoxin receptor (neurexin) and synaptotagmin another presynaptic protein, syntaxin, co-purifies on a alpha-latrotoxin affinity column. This implies that syntaxin is associated with these two molecules in a complex; a conclusion supported by the immunoprecipitation of [125I]latrotoxin binding by syntaxin antibodies. In addition, antibodies against syntaxin and the alpha-latrotoxin receptor immunoprecipitate [125I]omega-conotoxin binding sites, indicating that calcium channels are associated with this complex. Thus, neurexin, synaptotagmin, syntaxin, and calcium channels can be found in a structure we propose to call the 'synaptosecretosome'. The components of the synaptosecretosome, in association with additional proteins, are postulated to organize the process of neurotransmitter release.

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

Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis.

Neurotransmitter release is triggered by Ca2+ ions binding to an unknown Ca2+ receptor within presynaptic terminals. Synaptotagmin, a Ca2(+)-binding protein of synaptic and other secretory vesicles, has been proposed to mediate vesicle-plasma membrane interactions during neurotransmitter release. Here we test this hypothesis using the giant synapse of the squid Loligo pealei, which because of its unusually large size and well established physiology is uniquely suited for dissecting presynaptic events. We find that injection of peptides from the C2 domains of synaptotagmin into squid giant presynaptic terminals rapidly and reversibly inhibits neurotransmitter release. Our data are consistent with these peptides competitively blocking release after synaptic vesicle docking and indicate that Ca2+ probably initiates neurotransmitter release by regulating the interaction of synaptotagmin with an acceptor protein.

Amino Acid Sequence↗
PubMed1993

Ontogeny of synaptophysin and synaptoporin in the central nervous system: differential expression in striatal neurons and their afferents during development.

The expression of the synaptic vesicle antigens synaptophysin (SY) and synaptoporin (SO) was studied in the rat striatum, which contains a nearly homogeneous population of GABAergic neurons. In situ hybridization revealed high levels of SY transcripts in the striatal anlage from embryonic day (E) 14 until birth. In contrast, SO hydridization signals were low, and no immunoreactive cell bodies were detected at these stages of development. At E 14, SY-immunoreactivity was restricted to perikarya. In later prenatal stages of development SY-immunoreactivity appeared in puncta (identified as terminals containing immunostained synaptic vesicles), fibers, thick fiber bundles and 'patches'. In postnatal and adult animals, perikarya of striatal neurons exhibited immunoreaction for SO; ultrastructurally SO antigen was found in the Golgi apparatus and in multivesicular bodies. SO-positive boutons were rare in the striatum. In the neuropil, numerous presynaptic terminals positive for SY were observed. Our data indicate that the expression of synaptic vesicle proteins in GABAergic neurons of the striatum is developmentally regulated. Whereas SY is prevalent during embryonic development, SO is the major synaptic vesicle antigen expressed postnatally by striatal neurons which project to the globus pallidus and the substantia nigra. In contrast synapses of striatal afferents (predominantly from cortex, thalamus and substantia nigra) contain SY.

Animals↗
PubMed1993

Expression of inhibitory glycine receptors in postnatal rat cerebral cortex.

The developmental expression of inhibitory glycine receptors was analyzed in postnatal rat cerebral cortex using the specific monoclonal antibody, MAb 4a. This antibody defines an epitope common to all known glycine receptor alpha-subunits. At birth, high levels of immunoreactivity were found, which transiently increased during the second postnatal week, but subsequently declined to low adult levels. Biochemical analysis of the MAb 4a antigen from parietal areas indicates that cortical glycine receptors correspond to the neonatal receptor isoform previously identified in spinal cord of newborn animals. Immunocytochemistry showed that, within 2 weeks after birth, MAb 4a-reactive glycine receptors changed their distribution. In the occipital cortex of the neonatal rat, a dense labeling of closely packed neurons was found in the superficial layers while, in the adult rat, glycine receptors were predominantly localized on perikarya and processes of pyramidal neurons in layers II/III and V of the cortex.

Animals↗
PubMed1993

Residues within transmembrane segment M2 determine chloride conductance of glycine receptor homo- and hetero-oligomers.

We have expressed glycine receptor (GlyR) alpha and beta subunit cDNAs in HEK-293 cells to study the functional properties of homo- versus hetero-oligomeric GlyR channels. Dose-response curves of whole-cell currents in cells expressing alpha 1 subunits revealed an average Hill coefficient of h = 4.2. Co-expression with the beta subunit markedly increased glycine-gated whole-cell currents, which now exhibited a mean Hill coefficient of only h = 2.5. For alpha 1, alpha 2 and alpha 3 homo-oligomers, the main-state single-channel conductances were 86, 111 and 105 pS, respectively, recorded at symmetrical Cl- concentrations of 145 mM. The mutant alpha 1 G221A gave rise to a main-state of 107 pS. This indicates that the main-state of alpha homo-oligomers depends on residue 221 which is located within transmembrane segment M2. Importantly, the main-state conductances of alpha 1/beta, alpha 2/beta and alpha 3/beta hetero-oligomers were only 44, 54 and 48 pS, respectively. The latter values are similar to those found in spinal neurons, suggesting that native GlyRs are predominantly alpha/beta hetero-oligomers. Co-expression of alpha 1 with mutant beta subunits revealed that residues within and close to segment M2 of the beta subunit determine the conductance differences between homo- and hetero-oligomers.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of Drosophila glutamate receptors.

Insects and other invertebrates use L-glutamate as a neurotransmitter in the central nervous system and at the neuromuscular junction. In contrast to the well-studied effects of L-glutamate on invertebrate muscle cells, relatively little is known about the physiological role of glutamate receptors (GluRs) in the invertebrate central nervous system. We have applied a molecular cloning approach to elucidate the molecular structure of neuronal and muscle-specific Drosophila glutamate receptor subunits (DGluRs). Several domains conserved between rat GluR subunits and DGluRs indicate regions of high functional significance. Drosophila genetics may now be used as a valuable experimental tool to gain further insight into the role of DGluRs in development, synaptic plasticity and control of gene expression.

Animals↗
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

Cross-linking of 125I-alpha-bungarotoxin to Drosophila head membranes identifies a 42 kDa toxin binding polypeptide.

The nicotinic acetylcholine receptor (nAChR) antagonist alpha-bungarotoxin (alpha-Btx) binds to two different classes of high affinity binding sites from the Drosophila central nervous system. We have used the bivalent reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to cross-link 125I-alpha-Btx (M(r) = 8 kDa) to Drosophila head membranes. Upon sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), one major adduct of M(r) approximately 50 kDa was identified, suggesting that a 42 kDa polypeptide binds the toxin. Adduct formation was inhibited by other cholinergic ligands. Detergent-solubilized receptor complexes containing the cross-linked products were immunoprecipitated by antisera against two nAChR subunits previously identified by molecular cloning, the ALS and ARD proteins, suggesting that the 42 kDa toxin binding polypeptide constitutes a component of the previously described class 1 alpha-Btx binding site.

Animals↗
PubMed1992

Neurotransmitter transporters. A novel family of integral plasma membrane proteins.

The re-uptake of neurotransmitters into the nerve terminal terminates synaptic transmission at most central synapses and constitutes a key step in the modulation of synaptic efficacy. Recently, the cloning of several Na(+)-driven neurotransmitter transporters has resulted in the description of a novel family of homologous membrane proteins, each with 12 transmembrane segments. These transporters constitute major targets of widely used drugs, and modulation of transporter gene expression and/or activity may represent an important substrate for plasticity in the nervous system.

Amino Acid Sequence↗
PubMed1992

Primary structure and functional expression of a choline transporter expressed in the rat nervous system.

Synthesis of the neurotransmitter acetylcholine in cholinergic nerve terminals is regulated by a sodium-driven high-affinity choline uptake system in the plasma membrane. We have isolated cDNAs from rat spinal cord and brainstem which encode a choline transporter (CHOT1). The predicted protein shares considerable amino acid identity and several structural features including twelve putative transmembrane regions with other neurotransmitter transporters. Expression of in vitro transcribed CHOT1 RNA in Xenopus oocytes generated Na(+)-dependent choline uptake, which was not seen in control oocytes. Amplification by polymerase chain reaction (PCR) revealed significant amounts of CHOT1 mRNA in brain, cerebellum, spinal cord and, to a lesser extent, heart, but only very low expression in lung, kidney and muscle.

Amino Acid Sequence↗
PubMed1992

Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein, whose ligand binding alpha subunit occurs in several isoforms in the mammalian central nervous system. Here we show that coexpression of the GlyR-associated protein gephyrin changes the agonist and antagonist binding affinities of GlyRs generated by alpha 2 subunit expression in 293 kidney cells. Thus, a receptor-associated protein modifies the functional properties of a neurotransmitter receptor. This may contribute to an optimization of the postsynaptic neurotransmitter response.

Animals↗
PubMed1992

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.

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