Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor.

Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyl-dimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABAA receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA(A) receptors expressed in Xenopus oocytes.

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site.

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.

Developmental expression of the neurotransmitter transporter GAT3.

Neurotransmitter transporters are involved in termination of the synaptic neurotransmission and they also play a key role in neuroregulation and brain development. In this report, we describe the developmental distribution of the y-aminobutyric acid transporter GAT3 which transports gamma-aminobutyric acid (GABA) and beta-alanine in a sodium chloride-dependent manner. GAT3 was localized to the meninges in developmental stages where two other GABA transporters, GAT1 and GAT4, were adjacently expressed. In later developmental stages, only GAT3 remained in this area. The expression of GAT3 in the peripheral embryonic tissues was confined to the liver, to a layer of cells under the skin, to the mouse kidney, and to hipoccampal blood vessels only in late developmental stages. The developmental distribution of GAT3 suggests involvement in central nervous system (CNS) maturation.

Developmental expression of the neurotransmitter transporter NTT4.

Neurotransmitter transporters are involved in termination of the synaptic neurotransmission and are implicated as the sites of action of antidepressant medicines and illicit drugs. In addition to their function in neurotransmission, neurotransmitter transporters play a key role in neuroregulation and brain development. In this report, the developmental distribution of the "orphan" transporter NTT4, whose substrate has not yet been shown, is described. Immunohistochemical studies have previously shown NTT4 to be specifically and widely localized to the central nervous system. In this report, the distribution of NTT4 in brain areas enriched in glutamatergic and gamma-aminobutyric acid-ergic innervations is further substantiated. NTT4 is detected beginning at E18 in various parts of the rat brain, including the cerebral cortex, fimbria hippocampi, fornix, lateral lemniscus, anterior commissure, and spinal cord. At E18, strong immunoreactivity of NTT4 is observed in the cortical subplate and marginal layers that later develops into the fimbria hippocampi, and at P22, the expression of NTT4 in the hippocampal formation reaches the mature form. The expression of NTT4 in the spinal cord begins at E18 in the ventral white matter. Heavy staining for NTT4 is observed in the substantia nigra since birth and through all time points examined. Transient immunoreactivity is observed in the inferior colliculus, reaching maximal expression at P10, whereas the superior colliculus commences to express NTT4 only after this time point. The globus pallidus is highly stained after birth, and the caudate putamen shows strong staining for NTT4 only at P22. In the adult rat brain, NTT4 is strongly expressed in the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamus, substantia nigra, pontine nucleus, cerebellum, and spinal cord. The developmental distribution of NTT4 suggests involvement in central nervous system maturation.

Developmental expression of GABA transporters GAT1 and GAT4 suggests involvement in brain maturation.

cDNA clones representing four pharmacologically distinct GABA transporters (GAT1-GAT4) were previously identified in mouse brain. Two of these, GAT1 and GAT4, were found to be brain specific. We studied GAT1 and GAT4 in the developing rat brain using polygonal antibodies against recombinant fusion proteins. Patterns of immunoreactivity were very similar in the embryonic and early postnatal stages for both transporters. However, whereas GAT1 immunoreactivity was detected in distinct patterns in gray matter and growing axons, GAT4 immunoreactivity was found in a subset of radial glial cell fascicles. These patterns usually oriented perpendicularly to the axons expressing GAT1. Our results suggest a transient relationship between GAT4-expressing radial glial elements and GAT1-expressing axons. The presence of GAT1 in the cortical marginal zone and the numerous GAT4-positive fascicles observed in the fetal anterior commissure indicate that both transporters may play a role in processes of brain maturation. Because the beginning of expression for both GAT1 and GAT4 correlates with the expression of the alpha1 subunit of the GABA receptor, the transporters may be connected with the maturation of adult-type GABAergic inhibitory system in the brain.

Developmental expression of the glycine transporters GLYT1 and GLYT2 in mouse brain.

Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

Distribution and sites of synthesis of NTT4, an orphan member of the Na+/Cl(-)-dependent neurotransmitter transporter family, in the rat CNS.

The distribution and sites of synthesis in rat CNS of NTT4, a novel orphan member of the Na+/Cl(-)-dependent neurotransmitter transporter family, were determined by immunohistochemistry and hybridization histochemistry. Antibodies raised against recombinant fusion proteins, corresponding to residues of NTT4, and 35S-labelled oligodeoxyribonucleotide probes, were used to delineate the cellular distribution of the transporter at the protein and mRNA levels. High levels of immunoreactivity (mainly in the neuropil) were found in the olfactory bulb, cerebral cortex, striatum, hippocampus, thalamus, substantia nigra, pontine nuclei, cerebellum and spinal cord. The lowest levels were associated with the lateral hypothalamic area and deep mesencephalic nuclei. In situ hybridization signals correlated well with the immunoreactivity, and demonstrated a widespread distribution of NTT4 transcripts exclusively in neurons. NTT4 transcripts appeared widely codistributed with the N-methyl-D-aspartate receptor subunit 1 (1-4b), i.e. spliced variants characterized by a common 5' 63 bp insertion. These results indicate that the transporter was associated with neuronal processes in specific glutamate innervated CNS regions. Although the substrate transported by NTT4 remains unknown, our findings suggest a possible role for this carrier protein in glutamate/glycine neurotransmission.

Localization of glycine neurotransmitter transporter (GLYT2) reveals correlation with the distribution of glycine receptor.

We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N-terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal-rostral gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunoreactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.

Structure, function and brain localization of neurotransmitter transporters.

We studied four different cDNAs encoding GABA transporters and three different cDNAs encoding glycine transporters in mouse and rat brains. A genomic clone of two of the glycine transporters (GLYT1a and GLYT1b) revealed that they derive from differential splicing of a single gene. The third glycine transporter (GLYT2) is encoded by a separate gene. Antibodies were raised against seven of these neurotransmitter transporters and their cytochemical localization in the mouse brain was studied. In general, we observed a deviation from the classical separation of neuronal and glial transporters. It seems that each of the neurotransmitter transporters is present in specific places in the brain and is expressed in a different way in very specific areas. For example, the GABA transporter GAT4, which also transports beta-alanine, was localized to neurons. However, GAT1, which is specific for GABA, was localized not only to neurons but also to glial cells. The recently discovered glycine transporter GLYT2 was of particular interest because of its deviation from the general structure by a very extended N terminus containing multiple potential phosphorylation sites. Western analysis and immunocytochemistry in frozen sections of mouse brain demonstrated a clear caudal-rostral gradient of GLYT2 distribution, with massive accumulation in the spinal cord and brainstem and less in the cerebellum. Its distribution is typically neuronal and it is present in processes with varicosities. A correlation as observed between the pattern we obtained and that observed previously from strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission at the classical inhibitory system in the hindbrain. The availability of four different GABA transporters made it possible to look for specific binding sites upon the neurotransmitter transporters. An extensive program of site-directed mutagenesis led us to identify a potential neurotransmitter binding site on the GABA transporters.

DNA sequences from Saccharomycopsis fibuligera capable of autonomous replication in Saccharomyces cerevisiae.

A Sau3AI digest of total DNA from Saccharomycopsis fibuligera was cloned with the yeast-integrating plasmid YEp24 delta EcoRI and the capacity for autonomous replication (ARS) was assayed in yeast. From eighty clones, five mitoticaly unstable yeast transformants were picked up, recombinant plasmids from these clones were recovered with Escherichia coli, mapped, hybridized with total DNA of S. fibuligera and tested to mitotical stability in Saccharomyces cerevisiae. Experiments suggested the existence of DNA sequences from dimorphic Saccharomycopsis fibuligera with ARS activity in Saccharomyces cerevisiae.