Heinrich Betz

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.

GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization

Prostaglandin E2 (PGE2) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PGE2-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy.

Evidence for a Tetrameric Structure of Recombinant NMDA Receptors

The amino acids l-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1Q387K, and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopusoocytes. In both cases, analysis of the resulting dose–response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1R505K and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.

Identification of a gephyrin binding motif on the glycine receptor β subunit

Abstract The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GIyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GIyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the β subunit. GIyR α subunits and GABA A receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GIyR β subunit into the GABA A receptor β1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that β subunit expression is essential for the formation of a postsynaptic GIyR matrix.

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.

Murine semaphorin D/collapsin is a member of a diverse gene family and creates domains inhibitory for axonal extension

Members of the collapsin/semaphorin gene family have been proposed to act as growth cone guidance signals in vertebrates and invertebrates. To identify candidate molecules involved in axonal pathfinding during mouse embryogenesis, we isolated cDNAs encoding five new members of the semaphorin family (Sem A-Sem E). The murine semaphorin genes are differentially expressed in mesoderm and neuroectoderm before and during the time when axons select their pathways in the embryo. In explant cultures, recombinant Sem D/collapsin converts a matrix permissive for axonal growth into one that is inhibitory for neurites of peripheral ganglia. Our data demonstrate that semaphorins are a diverse family of molecules that may provide local signals to specify territories nonaccessible for growing axons.

Glycine receptor heterogeneity in rat spinal cord during postnatal development.

Two different isoforms of the inhibitory glycine receptor were identified during postnatal development of rat spinal cord. A neonatal form characterized by low strychnine binding affinity, altered antigenicity, and a ligand binding subunit differing in mol. wt (49 kd) from that of the adult receptor (48 kd) predominates at birth (70% of the total receptor protein). Separation from the adult form could be achieved by either use of a selective antibody or glycine gradient elution of 2‐aminostrychnine affinity columns. Both isoforms co‐purify with the mol. wt 93 kd peripheral membrane protein of the postsynaptic glycine receptor complex.

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand‐gated ion channel superfamily. Here, a beta subunit‐specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co‐transfected with alpha and beta cDNAs. Further, inhibition of alpha homo‐oligomeric GlyRs by picrotoxinin, a non‐competitive blocker of ion flow, was reduced 50‐ to 200‐fold for alpha/beta hetero‐oligomeric receptors generated by cotransfection. Site‐directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

The β Subunit Determines the Ligand Binding Properties of Synaptic Glycine Receptors

Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.