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Biomedical subjects

J Kuhse

Publications and source records attributed to J Kuhse.

At least 37 records · Page 2Linked to original sources
PubMed1995

The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex.

Significant progress has been made towards the identification of functional domains of the inhibitory glycine receptor. Several residues crucial for ligand binding, ion-channel properties and stoichiometric subunit assembly have been identified. A major recent advance has been the finding that the biogenesis of postsynaptic glycine receptor clusters requires the tubulin-binding protein, gephyrin. Another area of exciting research has focused on mutations of glycine receptor alpha and beta subunit genes, which have been found to be causal for different hereditary motor disorders.

Animals↗
PubMed1994

Point mutation of glycine receptor alpha 1 subunit in the spasmodic mouse affects agonist responses.

Homozygotic spasmodic (spd/spd) mice suffer from a motor disorder resembling poisoning by the glycine receptor antagonist strychnine. Here, a point mutation was identified in the glycine receptor alpha 1 subunit gene of the spasmodic mouse which predicts an alanine-to-serine exchange at position 52 of the mature polypeptide. Upon expression in Xenopus laevis oocytes, alpha 1A52S receptor channels displayed reduced responses to glycine, beta-alanine and taurine when compared to recombinant alpha 1 glycine receptors. As glycine receptor content in spinal cord and native molecular weight appeared unaltered, this suggests that the spasmodic phenotype results from an altered neurotransmitter sensitivity of the mutant alpha 1A52S subunit.

Alanine↗
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

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

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↗
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

Agonist pharmacology of neonatal and adult glycine receptor alpha subunits: identification of amino acid residues involved in taurine activation.

The inhibitory glycine receptor (GlyR) is a pentameric chloride channel protein which mediates postsynaptic inhibition in the mammalian central nervous system. In spinal cord, different GlyR isoforms originate from the sequential expression of developmentally regulated variants of the ligand binding alpha subunit. Here, neonatal alpha 2 and adult alpha 1 subunits are shown to generate GlyRs with distinct agonist activation profiles upon heterologous expression in Xenopus oocytes. Whereas alpha 1 receptors are efficiently gated by beta-alanine and taurine, alpha 2 GlyRs show only a low relative response to these agonists, which also display a reduced sensitivity to inhibition by the glycinergic antagonist strychnine. Construction of an alpha 2/alpha 1 subunit chimera and site-directed mutagenesis of the extracellular region of the alpha 1 sequence identified amino acid positions 111 and 212 as important determinants of taurine activation. Our results indicate the existence of distinct subsites for agonists on alpha 1 and alpha 2 GlyRs and suggest that the ligand binding pocket of these receptor proteins is formed from discontinuous domains of their extracellular region.

Adult↗
PubMed1991

Alternative splicing generates two isoforms of the alpha 2 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein which displays developmental heterogeneity in the mammalian central nervous system. Here we describe 2 novel cDNA variants of the rat GlyR alpha 2 subunit and demonstrate that alternative splicing generates these 2 isoforms. The deduced protein sequences (alpha 2A and alpha 2B) exhibit 99% identity with the previously characterized human alpha 2 subunit. In situ hybridization revealed expression of both alpha 2A and alpha 2B mRNAs in the prenatal rat brain, suggesting that these variant proteins may have a role in synaptogenesis. Heterologous expression in Xenopus oocytes showed that the more abundantly expressed alpha 2A subunit forms strychnine-sensitive ion channels which resemble human alpha 2 subunit GlyRs in their electrophysiological properties.

Amino Acid Sequence↗
PubMed1991

Alternative splicing generates two variants of the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) in mammalian spinal cord displays pharmacological and molecular heterogeneity of its strychnine binding alpha subunit. Here, cDNAs were isolated which encode a variant (alpha ins 1) of the rat GlyR alpha 1 subunit that contains eight additional amino acids in its putative cytoplasmic domain. Analysis of the corresponding genomic sequence showed that alpha ins 1 transcripts result from alternative splice acceptor site selection. S1 nuclease protection experiments, Northern blot analysis, and RNA amplification by polymerase chain reaction revealed alpha 1 and alpha ins 1 mRNA in postnatal spinal cord, but not in other brain regions. Expression of synthetic alpha ins 1 RNA in Xenopus oocytes generated glycine-gated strychnine-sensitive chloride channels. These data indicate that alternative splicing contributes to GlyR alpha subunit heterogeneity in the mammalian central nervous system.

Amino Acid Sequence↗
PubMed1991

Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain.

The inhibitory glycine receptor (GlyR) is a ligand-gated ion channel which mediates post-synaptic inhibition in spinal cord and other regions of the vertebrate central nervous system. Previous biochemical and molecular cloning studies have indicated heterogeneity of GlyRs during development. Here, the distribution of GlyR subunit transcripts in rat brain and spinal cord was investigated by in situ hybridization using sequence-specific oligonucleotide probes. In adult animals, GlyR alpha 1 subunit mRNA was abundant in spinal cord, but was also seen in a few brain areas, e.g. superior and inferior colliculi, whereas alpha 2 transcripts were found in several brain regions including layer VI of the cerebral cortex and hippocampus. GlyR alpha 3 subunit mRNA was expressed at low levels in cerebellum, olfactory bulb and hippocampus, while high amounts of beta subunit transcripts were widely distributed throughout spinal cord and brain. During development, alpha 2 mRNA accumulated already prenatally and decreased after birth, whereas alpha 1 and alpha 3 subunit transcripts appeared only in postnatal brain structures. Hybridization signals of beta subunit mRNA were seen already at early embryonic stages and continuously increased to high levels in adult rats. These data reveal unexpected differences in the regional and developmental expression of GlyR subunit mRNAs and point to novel functions of GlyR proteins in the mammalian central nervous system.

Animals↗
PubMed1991

How to build a glycinergic postsynaptic membrane.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein found at many synapses of the mammalian central nervous system. During development, distinct isoforms of the GlyR are generated by the sequential expression of different alpha subunit variants. The appearance of adult-type GlyRs in spinal cord is accompanied by the accumulation of a 93 x 10(3) Mr receptor-associated peripheral membrane protein. The latter has been localized at the cytoplasmic face of glycinergic postsynaptic membranes and is thought to anchor GlyRs beneath glycinergic nerve terminals. The 93 x 10(3) Mr protein binds with high affinity to polymerized tubulin, suggesting that it functions as a receptor-microtubule linking component. Our data suggest that the interaction of developmentally regulated receptor isoforms with specialized microtubule-associated proteins represents a crucial step in the assembly of postsynaptic receptor matrices.

Animals↗
PubMed1990

Identification and functional expression of a novel ligand binding subunit of the inhibitory glycine receptor.

By homology screening of a rat brain cDNA library we have identified a novel alpha subunit variant of the inhibitory glycine receptor, alpha 3. The deduced protein exhibits 82-83% amino acid identity to the previously characterized rat and human alpha 1 and alpha 2 subunit sequences. Upon heterologous expression in Xenopus laevis oocytes, the alpha 3 subunit formed functional agonist-gated chloride channels which displayed a low affinity for glycine and only small responses to taurine. Amplification of alpha 3 transcripts by polymerase chain reaction revealed high levels in spinal cord at later postnatal stages of development. These data indicate alpha subunit heterogeneity of the inhibitory glycine receptor in adult rats that may result in pharmacologically distinct receptor subtypes.

Amino Acid Sequence↗