Novel dicarboxylate selectivity in an insect glutamate transporter homolog.

Mammals express seven transporters from the SLC1 (solute carrier 1) gene family, including five acidic amino acid transporters (EAAT1-5) and two neutral amino acid transporters (ASCT1-2). In contrast, insects of the order Diptera possess only two SLC1 genes. In this work we show that in the mosquito Culex quinquefasciatus, a carrier of West Nile virus, one of its two SLC1 EAAT-like genes encodes a transporter that displays an unusual selectivity for dicarboxylic acids over acidic amino acids. In eukaryotes, dicarboxylic acid uptake has been previously thought to be mediated exclusively by transporters outside the SLC1 family. The dicarboxylate selectivity was found to be associated with two residues in transmembrane domain 8, near the presumed substrate binding site. These residues appear to be conserved in all eukaryotic SLC1 transporters (Asp444 and Thr448, human EAAT3 numbering) with the exception of this novel C. quinquefasciatus transporter and an ortholog from the yellow fever mosquito Aedes aegypti, in which they are changed to Asn and Ala. In the prokaryotic EAAT-like SLC1 transporter DctA, a dicarboxylate transporter which was lost in the lineage leading to eukaryotes, the corresponding TMD8 residues are Ser and Ala. Functional analysis of engineered mutant mosquito and human transporters expressed in Xenopus laevis oocytes provide support for a model defining interactions of charged and polar transporter residues in TMD8 with α-amino acids and ions. Together with the phylogenetic evidence, the functional data suggest that a novel route of dicarboxylic acid uptake evolved in these mosquitos by mutations in an ancestral glutamate transporter gene.

Connexin-deficiency affects expression levels of glial glutamate transporters within the cerebrum.

The glial glutamate transporter subtypes, GLT-1/EAAT-2 and GLAST/EAAT-1 clear the bulk of extracellular glutamate and are severely dysregulated in various acute and chronic brain diseases. Despite the previous identification of several extracellular factors modulating glial glutamate transporter expression, our knowledge of the regulatory network controlling glial glutamate transport in health and disease still remains incomplete. In studies with cultured cortical astrocytes, we previously obtained evidence that glial glutamate transporter expression is also affected by gap junctions/connexins. To assess whether gap junctions would likewise control the in vivo expression of glial glutamate transporters, we have now assessed their expression levels in brains of conditional Cx43 knockout mice, total Cx30 knockouts, as well as Cx43/Cx30 double knockouts. We found that either knocking out Cx30, Cx43, or both increases GLT-1/EAAT-2 protein levels in the cerebral cortex to a similar extent. By contrast, GLAST/EAAT-1 protein levels maximally increased in cerebral cortices of Cx30/Cx43 double knockouts, implying that gap junctions differentially affect the expression of GLT-1/EAAT-2 and GLAST/EAAT-1. Quantitative PCR analysis further revealed that increases in glial glutamate transporter expression are brought about by transcriptional and translational/posttranslational processes. Moreover, GLT-1/EAAT-2- and GLAST/EAAT-1 protein levels remained unchanged in the hippocampi of Cx43/Cx30 double knockouts when compared to Cx43fl/fl controls, indicating brain region-specific effects of gap junctions on glial glutamate transport. Since astrocytic gap junction coupling is affected in various forms of brain injuries, our findings point to gap junctions/connexins as important regulators of glial glutamate turnover in the diseased cerebral cortex.

l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation.

l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using (14)C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (K(m)=69.4 µM). Also, l(2)01810 uptakes glutamate in a Na(+)-independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na(+)-independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein's localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg(146) in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.

Region- and age-specific changes in glutamate transport in the AßPP23 mouse model for Alzheimer's disease.

Using 8- and 18-month-old AßPP23 mice, we investigated the involvement of high-affinity glutamate transporters (GLAST, GLT-1, EAAC1), vesicular glutamate transporters (VGLUT1-3) and xCT, the specific subunit of system x(c)⁻, in Alzheimer's disease (AD) pathogenesis. Transporter expression was studied in cortical and hippocampal tissue and linked to extracellular glutamate and glutamate reuptake activity as measured using in vivo microdialysis. In 8-month-old animals, we could not observe plaque formation or gliosis. Yet, in hippocampus as well as cortex GLAST and GLT-1 expression was decreased. Whereas in cortex this was accompanied by upregulated VGLUT1 expression, extracellular glutamate concentrations were decreased. Surprisingly, inhibiting glutamate reuptake with TBOA revealed increased glutamate reuptake activity in cortex of AßPP23 mice, despite decreased GLAST and GLT-1 expression, and resulted in status epilepticus in all AßPP23 mice, contrary to wildtype littermates. In hippocampus of 8-month-old AßPP23 mice, we observed increased EAAC1 expression besides the decrease in GLAST and GLT-1. Yet, glutamate reuptake activity was drastically decreased according to the decreased GLAST and GLT-1 expression. In 18-month-old AßPP23 mice, plaque formation and gliosis in cortex and hippocampus were accompanied by decreased GLT-1 expression. We also showed, for the first time, increased cortical expression of VGLUT3 and xCT together with a strong tendency towards increased cortical extracellular glutamate levels. VGLUT2 expression remained unaltered in all conditions. The present findings support the hypothesis that alterations in transport of glutamate, and more particular via GLT-1, may be involved in AD pathogenesis.

Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement.

L-Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high-affinity Glu transporters (EAAT1-5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post-translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.

Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons.

Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ.

Pharmacological manipulation of glutamate transport.

L-Glutamic acid acts as the major excitatory neurotransmitter and, at the same time, represents a potential neurotoxin for the mammalian central nervous system (CNS). The termination of excitatory transmission and the maintenance of physiologic levels of extracellular glutamate, which is necessary to prevent excitotoxicity, are prominently mediated by a family of high-affinity sodium-dependent excitatory amino acid transporters (EAATs). Five subtypes of EAATs have been cloned, possessing distinct pharmacology, localization, sensitivity to transport inhibitors and modulatory mechanisms. Expression and activity of EAATs have been shown to be amenable to fine endogenous and, potentially, pharmacological regulation by substrate itself, growth factors, second messengers, hormones, biological oxidants, inflammatory mediators and pathological conditions. The present review describes basic pharmacological studies, mostly performed on animal models or cell preparations, in order to obtain an updated picture of the known regulatory mechanisms of single EAAT expression and activity. New insight into molecular pathways involved in EAAT regulation will allow pharmacological manipulation of excitatory CNS activity, possibly avoiding adverse effects of glutamate receptor blockade.

Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight.

After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-associated protein termed excitatory amino acid transporter 3 (EAAT3). Following entry into the nerve terminal, glutamate is pumped into synaptic vesicles by a vesicular transport system. Three different vesicular glutamate transporter proteins (VGLUT1-3) representing unique markers for glutamatergic neurons were recently characterized. The presence of EAAT3, glutaminase and VGLUT1-3 was examined in mouse, rat and rabbit species at mRNA and protein levels in hypothalamic neurons which are involved in the regulation of body weight using in situ hybridization and immunohistochemistry. EAAT3 and glutaminase mRNAs were demonstrated in all parts of the arcuate nucleus in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamic area. VGLUT1 mRNA was present in the magnocellular lateral hypothalamic nucleus. VGLUT2 mRNA was demonstrated in a subpopulation of neurons in the arcuate nucleus and in the ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamic area. Few VGLUT3 mRNA expressing neurons were scattered throughout the medial and lateral hypothalamus. EAAT3-like immunoreactivity (-li) was demonstrated in glutamate, neuropeptide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanin-concentrating hormone and orexin-immunoreactive (-ir) neurons. VGLUT2-li could only be demonstrated in POMC- and CART-ir neurons of the ventrolateral arcuate nucleus. The results show that key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NPY/AGRP neurons situated in the ventromedial part of the arcuate nucleus are mainly GABAergic, it is shown that several anorexigenic POMC/CART neurons of the ventromedial arcuate nucleus are most likely glutamatergic [corrected].

Roles and regulation of glutamate transporters in the central nervous system.

1. Glutamate transporters (also known as excitatory amino acid transporters or EAAT) are solely responsible for the removal of the excitatory neurotransmitter l-glutamate (Glu) from the extracellular space and, thus, permit normal transmission, as well as preventing cell death due to the excessive activation of Glu receptors. 2. Five subtypes of glutamate transporter (EAAT1-5) exist, possessing distinct pharmacology, cellular localization and modulatory mechanisms. 3. Experimental inhibition of EAAT activity in vitro and in vivo results in increased extracellular concentrations of Glu and in neuronal death via excitotoxicity, highlighting the importance of EAAT in normal excitatory neurotransmission. 4. Dysfunction of EAAT may contribute to the pathology of both acute neuronal injury and chronic neurodegenerative conditions, so correction of EAAT function under these conditions may provide a valuable therapeutic strategy. 5. The present review describes basic pharmacological studies that allow new insights into EAAT function and suggest possible strategies for the therapeutic modulation of EAAT.