Identification of truncated human glutamate transporter.
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Excitatory amino-acid carrier 1 (EAAC1) is a high affinity Na+-dependent L-glutamate/D, L-Aspartate transporter protein. A truncated form of EAAC1 (tEAAC1) was identified by reverse transcription and polymerase chain reaction in the human cell line, ACHN, in which EAAC1 mRNA was highly expressed. The deduced amino acid sequence of tEAAC1 lacks 31-77 amino acids including the first extracellular domain. The mRNA encoding tEAAC1 was detected in various cells of human origin but not in cells of rat or mouse origin. The expression of tEAAC1 mRNA was proportional to that of full-length EAAC1 (fEAAC1) mRNA, suggesting common transcriptional regulation between tEAAC1 and fEAAC1. In addition, the expression of EAAC1 mRNA was relatively low or non-existent in non-adherent cells. (+info)
Differential expressions of glycine transporter 1 and three glutamate transporter mRNA in the hippocampus of gerbils with transient forebrain ischemia.
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The extracellular concentrations of glutamate and its co-agonist for the N-methyl-d-aspartate (NMDA) receptor, glycine, may be under the control of amino acid transporters in the ischemic brain. However, there is little information on changes in glycine and glutamate transporters in the hippocampal CA1 field of gerbils with transient forebrain ischemia. This study investigated the spatial and temporal expressions of glycine transporter 1 (GLYT1) and three glutamate transporter (excitatory amino acid carrier 1, EAAC1; glutamate/aspartate transporter, GLAST; glutamate transporter 1, GLT1) mRNA in the gerbil hippocampus after 3 minutes of ischemia. The GLYT1 mRNA was transiently upregulated by the second day after ischemia in astrocytelike cells in close vicinity to hippocampal CA1 pyramidal neurons, possibly to reduce glycine concentration in the local extracellular spaces. The EAAC1 mRNA was abundantly expressed in almost all pyramidal neurons and dentate granule cells in the control gerbil hippocampus, whereas the expression level in CA1 pyramidal neurons started to decrease by the fourth day after ischemia in synchrony with degeneration of the CA1 neurons. The GLAST and GLT1 mRNA were rather intensely expressed in the dentate gyrus and CA3 field of the control hippocampus, respectively, but they were weakly expressed in the CA1 field before and after ischemia. As GLAST and GLT1 play a major role in the control of extracellular glutamate concentration, the paucity of these transporters in the CA1 field may account for the vulnerability of CA1 neurons to ischemia, provided that the functional GLAST and GLT1 proteins are also less in the CA1 field than in the CA3 field. This study suggests that the amino acid transporters play pivotal roles in the process of delayed neuronal death in the hippocampal CA1 field. (+info)
Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+-dependent glutamate transport.
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Spinal cord injury is a devastating condition, with much of the clinical disability resulting from disruption of white matter tracts. Recent reports suggest a component of glutamate excitotoxicity in spinal cord injury. In this study, the role of glutamate and mechanism of release of this excitotoxin were investigated in rat dorsal column slices subjected to 60 min of anoxia or 15 sec of mechanical compression at a force of 2 gm in vitro. The broad-spectrum glutamate antagonist kynurenic acid (1 mm) and the selective AMPA antagonist GYKI52466 (30 microm) were protective against anoxia (compound action potential amplitude recovered to 56 vs 27% without drug). GYKI52466 was also effective against trauma (65 vs 35%). Inhibition of Na(+)-dependent glutamate transport with dihydrokainate or l-trans-pyrrolidine-2,4-dicarboxylic acid (1 mm each) protected against anoxia (65-75 vs 25%) and trauma (70 vs 35%). The depletion of cytosolic glutamate in axon cylinders and oligodendrocytes by anoxia was completely prevented by glutamate transport inhibition. Immunohistochemistry revealed that a large component of injury occurred in the myelin sheath and was prevented by AMPA receptor blockade or glutamate transport inhibitors. We conclude that release of glutamate by reversal of Na(+)-dependent glutamate transport with subsequent activation of AMPA receptors is an important mechanism in spinal cord white matter anoxic and traumatic injury. (+info)
Functional diversity of excitatory amino acid transporters: ion channel and transport modes.
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Recent studies of glutamate transporters in the central nervous system indicate that in addition to their fundamental role in mediating neurotransmitter uptake, these proteins may contribute to the modulation of a variety of cellular processes. Activation of the excitatory amino acid (EAA) carriers generates an electrogenic current attibutable to ion-coupled cotransport. In addition to this transport-associated current, a substrate-gated thermodynamically uncoupled anion flux has been identified that has been proposed to dampen neuronal excitability. Arachidonic acid has been reported to modulate a variety of membrane proteins involved in cellular signaling. Here we discuss recent findings that indicate arachidonic acid stimulates a previously uncharacterized proton-selective conductance in the Purkinje cell-specific subtype, EAAT4. The unique channel-like porperties of the EAATs, their unexpected localization, and physiological evidence propose a modulatory role for the EAATs in neuronal signaling and suggest a broader role for glutamate transporters than simply the clearance of synaptically released glutamate. Thus, the identification of this arachidonate-stimulated proton conductance extends the complexity of mechanisms through which glutamate transporters modulate neuronal excitability. (+info)
Glutamate transporters in kidney and brain.
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Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells with glutamate for metabolic purposes. In the kidney, glutamate transporters are involved in reabsorption of filtered acidic amino acids, regulation of ammonia and bicarbonate production, and protection of cells against osmotic stress. (+info)
Glutamate transport and renal function.
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Brush border gamma-glutamyltransferase-glutaminase activity and the high-affinity glutamate transporter EAAC1 function as a unit in generating and transporting extracellular glutamate into proximal tubules as a signal that modulates intracellular glutamine/glutamate metabolism, paracellular permeability, and urinary acidification. The reported presence of a second glutamate transporter, GLT1, on the antiluminal tubule surface points to specific functional roles for each subtype in physiological and pathophysiological processes. (+info)
Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.
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Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid. (+info)
Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters.
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Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells. (+info)