Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel ( GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats, cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.

The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [H-3]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degreesC until assay. Both Con-GA7 and ifenprodil inhibited [H-3]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC50 changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC50 with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [H-3]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC50, while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

High throughput analysis of endogenous glutamate release using a fluorescence plate reader

Recent discoveries of different modes of exocytosis and a plethora of molecules involved in neurotransmitter release has resulted in demand for more rapid and efficient methods for monitoring endogenous glutamate release from various tissue sources. In this article, we describe a high throughput microplate version of the enzyme-linked fluorescence detection method for the measurement of released glutamate, which utilises glutamate dehydrogenase, and the reduction of NADP to NADPH. Previous versions of this method rely upon cuvette-based fluorimeters for detection that are limited by large sample volumes and small numbers of samples that can be measured simultaneously. Comparison between the two methods shows that the microplate assay has comparable performance to the cuvette-based assay but has the capacity to analyse many times more samples in a given run. This increased capacity provides improved experimental design opportunities, higher experimental throughput and better comparison between experimental conditions. (c) 2005 Elsevier B.V. All rights reserved.

Differential expression of the NMDA NR2B receptor subunit in motoneuron populations susceptible and resistant to amyotrophic lateral sclerosis

We have compared the expression pattern of NMDA receptor subunits (NR1 and NR2A-D)and NRI splice variants (NR1-1a/1b,-2a/2b,-3a/3b,4a/4b) in motor neuron populations from adult Wistar rats that are vulnerable (hypoglossal, XII) or resistant (oculomotor, III) to death in amyotrophic lateral sclerosis (ALS). The major finding was higher levels of expression of the NR2B subunit in the hypoglossal nucleus. Quantitative real-time PCR showed that NR1 was expressed at a greater level than any of the NR2 subunits (> 15 fold greater, P

Chronic smoking and alcoholism change expression of selective genes in the human prefrontal cortex

Background: Alcoholism is commonly associated with chronic smoking. A number of gene expression profiles of regions within the human mesocorticolimbic system have identified potential alcohol-sensitive genes; however, the influence of smoking on these changes was not taken into account. This study addressed the impact of alcohol and smoking on the expression of 4 genes, previously identified as alcoholism-sensitive. in the human prefrontal cortex (PFC). Methods: mRNA expression of apolipoprotein D, tissue inhibitor of the metalloproteinase 3, high-affinity glial glutamate transporter and midkine, was measured in the PFC of alcoholic Subjects and controls with and without smoking comorbidity using real-time polymerase chain reaction. Results: The results show that alcohol affects transcription of some of these genes. Additionally, smoking has a marked influence on gene expression. Conclusion: This study emphasizes the need for careful case selection in future gene expression studies to delineate the adaptive molecular process associated with smoking and alcohol.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group l etabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Genes and gene expression in the brains of human alcoholics

Chronic alcohol misuse by human subjects leads to neuronal loss in regions such as the superior frontal cortex (SFC). Propensity to alcoholism is associated with several genes. γ-Aminobutyric acid (GABA)A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the regional presentation of GABAA and glutamate-NMDA (N-methyl-d-aspartate) receptors in SFC. Autopsy tissue was obtained from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and matched controls. ADH1C, DRD2B, EAAT2, and APOE genotypes modulated GABAA-β subunit protein expression in SFC toward a less-effective form of the receptor. Most genotypes did not divide alcoholics and controls on glutamate-NMDA receptor pharmacology, although gender and cirrhosis did. Genotype may affect amino acid transmission locally to influence neuronal vulnerability.

Comorbidity and genotype modify receptor expression in human alcoholics

Chronic alcohol misuse leads to both widespread and localized damage in human cerebral cortex. The latter, as neuronal loss, is marked in superior frontal cortex (SFC) but milder in primary motor cortex (PMC) and elsewhere. Quantitative morphometry by Harper et al showed that neuronal loss is greater in alcoholics with comorbidity (Wernicke Korsakoff syndrome, liver cirrhosis). Previous work revealed a paradox: the marked differences in GABAA receptor density, pharmacology, and expression between alcoholics without cormorbidity and controls are muted or absent in cirrhotic alcoholics. This concurs with work by the Butterworth group on hepatic encephalopathy cases — most of whom had an alcoholic ætiology — who show only minor differences from controls. Glutamate receptor differences are muted in many autopsy studies, though we have evidence that NMDA site pharmacology may vary in cirrhotic alcoholics. Here we used Real-Time PCR normalized to GAPDH deltaCT to quantify NMDA NR1, NR2A and NR2B subunit expression in SFC and PMC samples obtained at autopsy from alcoholics with and without comorbid cirrhosis and matched controls. Overall subunit transcript expression was signifi cantly lower in alcoholic cirrhotics than in either of the other groups (F2,42 = 12.942, P < 0.001). The effect was most marked for the NR1 subunit; males differed from females, particularly in SFC. The data suggest that if excitotoxicity mediates neuronal loss in SFC, it may be implemented differently: passively in uncomplicated alcoholics, by altered GABAergic transmission; actively in cirrhotic alcoholics, by altered glutamatergic transmission. We also subdivided cases on a panel of genetic markers. Different genotypes interacted with NMDA and GABAA pharmacology and expression. Cirrhotic and uncomplicated alcoholics may differ pathogenically because of inherent characteristics in addition to possible neurotoxic sequelæ to the liver damage.

Genes and gene expression in human alcoholic brain

Chronic alcoholism leads to localized brain damage, which is prominent in superior frontal cortex but mild in motor cortex. The likelihood of developing alcohol dependence is associated with genetic markers. GABA-A receptor expression differs between alcoholics and controls, whereas glutamate receptor differences are muted. We determined whether genotype differentiated the localized expression of glutamate N-methyl-D-aspartate (NMDA) and GABA-A receptors to influence the severity of alcohol-induced brain damage. Cerebral cortex tissue was obtained at autopsy from alcoholics without disease comorbid with alcoholics, alcoholics with cirrhosis, and matched controls. DRD2A, DRD2B, GABRB2, SLC1A2, and 5HTT genotypes did not divide alcoholic cases and controls on NMDA receptor parameters. In contrast, a specific alcohol dehydrogenase (ADHIC) genotype interacted significantly with NMDA efficacy and affinity in a region-specific manner SLC1A2 (glutamate transporter-2) genotype interacted significantly with local GABAA receptor b subunit mRNA expression, and ADHIC, DRD2B, SLC1A2, and APOE genotypes with b subunit isoform protein expression. In the latter instance, possession of the alcoholism- associated allele altered b isoform protein expression patterns toward a less-efficacious form of the GABA-A receptor in the pathologically vulnerable region. GABRB2 and GRIN2B (NMDA receptor 2B subunit} Genotypes were associated with significant regional difference in the pattern of b subunit protein isoform expression, but this was not influenced by alcoholism status. Genotype may modulate amino acid transmission locally so as to mediate neuronal vulnerability. This has implications for the effectiveness of pharmacological interventions aimed at ameliorating brain damage and, possibly, dependence.

Interactions between serotonergic genotype and amino acid transmitter receptor expression in human alcoholics

Serotonin can modulate the activity of neural reward pathways that are strongly implicated in mediating the effects of chronic alcohol misuse, and its treatment, in human subjects. In previous work and as discussed elsewhere at this meeting, we and others have found consistent differences in the parameters of GABA and glutamate receptors, and the expression of their component subunit transcripts and proteins, in areas of the alcoholic brain that are altered by alcoholism. We did not fi nd clear changes in GABA and glutamate transport function in such samples, but a series of microarray analyses showed consistent upregulation of the presynaptic GABA/betaine transporter SLC6A12. Microarray studies showed no signifi cant differences in the expression of transcripts associated with 5HT transmission; however, only a small number of such elements were present on the arrays. Here we partitioned GABAA and NMDA pharmacology, and subunit mRNA and protein expression, measured in samples of frontal and motor cortex obtained at autopsy from alcoholics without comorbid disease, alcoholics with liver cirrhosis, and controls, according to 5HTTLPR (SLC6A4) and 5HT1B (HTR1B) polymorphisms. We found no effect of these genotypes on the expression of GABAA receptor gene products, but there was a signifi cant mRNA Transcript X Area X Group X 5HTTLPR Interaction with NMDA subunit isoform expression measured by Real Time PCR with GAPDH normalization. Further analysis showed the effect to be selective for alcoholics with cirrhosis, to be most marked in the pathologically vulnerable frontal cortex, and to vary with subunit transcript (F2,76 = 6.545, P = 0.002). NR1 expression was most affected, followed by NR2A, with NR2B expression least altered. Pilot data suggest 5HT1B genotype may also modulate NMDA subunit expression. Interactions between amino acid and serotonin transmission may infl uence susceptibility to alcohol dependence or pathogenesis