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H Betz

Publications and source records attributed to H Betz.

At least 127 records · Page 7Linked to original sources
PubMed1994

Na(+)-dependent high-affinity uptake of choline into cultured fibroblasts.

The acetylcholine precursor choline is transported into cholinergic neurons by a high-affinity, sodium-dependent mechanism that is selectively localized to the cholinergic nerve terminal. In addition, a low-affinity, sodium-independent choline uptake system is present in cholinergic and non-cholinergic cells which deliver choline for cell membrane anabolism. Here, we show that uptake of [3H]choline into cultured fibroblast cell lines exhibits high affinity (Km < or = 10 microM), is sodium-dependent, and is blocked by hemicholinium, a classical inhibitor of neuronal high-affinity choline uptake. Our data indicate that sodium-dependent high-affinity choline transport systems are also present in non-cholinergic cells.

Biological Transport, Active↗
PubMed1994

Expression of the human glycine receptor alpha 1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.

Animals↗
PubMed1994

The putative rat choline transporter CHOT1 transports creatine and is highly expressed in neural and muscle-rich tissues.

A rabbit homolog of the putative rat choline transporter CHOT1 has recently been shown to mediate creatine transport (Guimbal and Kilimann, J. Biol. Chem., 1993). Here, the functional properties of the CHOT1 cDNA were studied by heterologous expression in HEK-293 cells. After transfection, the cells displayed a 7-8fold stimulation of [14C]creatine uptake with a Km of 46.2 microM. This transport was inhibited by the creatine uptake inhibitor beta-guanidinopropionate (Ki of 23 microM). Northern blot analysis revealed a major transcript of 4.8 kb in brain, heart, skeletal muscle and kidney. In situ hybridization showed high transcript levels in the cerebellum, hippocampus and other brain regions. High expression was also seen in the rat embryo along the entire neuraxis and in some non-neuronal tissues. These data establish CHOT1 as a widely expressed rat creatine transporter.

Animals↗
PubMed1994

Structural analysis of mouse glycine receptor alpha subunit genes. Identification and chromosomal localization of a novel variant.

The inhibitory glycine receptor is a ligand-gated ion channel protein that occurs in different developmentally regulated isoforms in the mammalian central nervous system. Here, we have analyzed genomic clones covering the coding regions of the murine glycine receptor alpha 1 and alpha 2 subunit genes. Both genes contain eight intronic regions with precisely conserved boundaries. The same structure was also found for seven exons of a third homologous gene, alpha 4, identified during screening. The predicted alpha 4 polypeptide displays very high homology to the alpha 2 subunit. Like the alpha 2 gene, the alpha 4 gene maps to the mouse X chromosome. Our data indicate that the genomic organization of glycine receptor alpha subunit genes is conserved during evolution.

Amino Acid Sequence↗
PubMed1994

The spastic mouse: aberrant splicing of glycine receptor beta subunit mRNA caused by intronic insertion of L1 element.

Mice homozygous for the spastic mutation (spa) suffer from a complex motor disorder resulting from reduced CNS levels of the adult glycine receptor isoform GlyRA, which is composed of ligand-binding alpha 1 and structural beta polypeptides. The beta subunit-encoding gene (Glyrb) was mapped near the spa locus on mouse chromosome 3. In spa/spa mice, aberrant splicing of the beta subunit pre-mRNA strikingly diminishes the CNS contents of full-length transcripts, whereas truncated beta subunit mRNAs accumulate. This is a result of exon skipping, which causes translational frameshifts and premature stop codons. Intron 5 of the spa Glyrb gene contains an L1 transposable element that apparently is causal for the aberrant splicing of beta subunit transcripts.

Amino Acid Sequence↗
PubMed1994

A post-docking role for synaptobrevin in synaptic vesicle fusion.

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%-68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

Amino Acid Sequence↗
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↗
PubMed1994

Neurotransmitter transporters: new members of known families.

The identification of two gene families encoding neurotransmitter transporters was a major step towards a better understanding of these proteins and their function in neurotransmission. The recent isolation of additional members of these families underscores their high molecular diversity and implies a delicate regulation of transmitter uptake.

Animals↗
PubMed1994

Molecular pathways for presynaptic calcium signaling.

The results presented in this article describe two distinct, Ca-regulated molecular pathways in presynaptic terminals and implicate these two pathways in differentially mediating neurotransmitter secretion and PTP. Our current view of the Ca-dependent triggering of secretion and PTP is shown in Fig. 9. According to this scheme, differential activation of these two pathways is achieved by a combination of diffusion-based dilution of Ca that enters the terminal through voltage-gated Ca channels and by coupling these pathways to Ca receptors with different affinities for Ca ions. A simple way to achieve these conditions is to position these two receptors at different distances from the Ca channels, as shown in Fig. 2. Given that Ca ions are involved in activating many different presynaptic processes (Fig. 1), we propose that closer scrutiny of the molecular physiology of nerve terminals will reveal a wide variety of Ca-activated pathways responsible for producing these diverse processes.

Amino Acid Sequence↗
PubMed1994

Sequence of a Drosophila ligand-gated ion-channel polypeptide with an unusual amino-terminal extracellular domain.

We report the isolation of a full-length clone from a Drosophila melanogaster head cDNA library that encodes a 614-residue polypeptide that exhibits all of the features of a ligand-gated chloride-channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor-like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32-37% identity to the GABAA receptor-like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino-terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane-spanning domain, of 75 amino acids that is not found in any other ligand-gated chloride-channel subunit. Analysis of cDNA and genomic DNA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.

Amino Acid Sequence↗
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

Mutation of glycine receptor subunit creates beta-alanine receptor responsive to GABA.

The amino acid at position 160 of the ligand-binding subunit, alpha 1, is an important determinant of agonist and antagonist binding to the glycine receptor. Exchange of the neighboring residues, phenylalanine at position 159 and tyrosine at position 161, increased the efficacy of amino acid agonists. Whereas wild-type alpha 1 channels expressed in Xenopus oocytes required 0.7 millimolar beta-alanine for a half-maximal response, the doubly mutated (F159Y,Y161F) alpha 1 subunit had an affinity for beta-alanine (which was more potent than glycine) that was 110-fold that of the wild type. Also, gamma-aminobutyric acid and D-serine, amino acids that do not activate wild-type alpha 1 receptors, efficiently gated the mutant channel. Thus, aromatic hydroxyl groups are crucial for ligand discrimination at inhibitory amino acid receptors.

Amino Acid Sequence↗