Molecular cloning,gene structure,expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.     

GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation

Glutamate transporter associated protein 3–18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.     

Methyl-beta-cyclodextrin influences glutamate transport in the rat brain nerve terminals by depletion of membrane cholesterol

Role of membrane cholesterol in direct and reversed function of Na+ -dependent glutamate transporters and exocytosis was investigated. The depletion of membrane cholesterol by methyl-beta-cyclodextrin (MebetaCD) resulted in a dose-dependent significant reduction of the L-[14C]glutamate uptake by synaptosomes. Treatment of synaptosomes with 15 mM MebetaCD caused a decrease in the velocity of L-[14C]glutamate uptake by 49 +/- 4% (P < or = 0.05). The depolarization stimulated Ca2+ -dependent glutamate release that occurred via reverse functioning of glutamate transporters decreased insignificantly for 1 min from 8.0 +/- 0.4% to 6.7 +/- 0.4% of total accumulated synaptosomal label after MebetaCD treatment. The depletion of membrane cholesterol resulted in a reduction of the depolarization evoked exocytotic release from 8.0 +/- 1.0% to 4.2 +/- 1.0% of total synaptosomal label. Thus, cholesterol depletion was found to decrease significantly the Na+ -dependent uptake and exocytotic release of glutamate.     

Modulation of neuronal glutathione synthesis by EAAC1 and its interacting protein GTRAP3-18

Glutathione (GSH) plays essential roles in different processes such as antioxidant defenses, cell signaling, cell proliferation, and apoptosis in the central nervous system. GSH is a tripeptide composed of glutamate, cysteine, and glycine. The concentration of cysteine in neurons is much lower than that of glutamate or glycine, so that cysteine is the rate-limiting substrate for neuronal GSH synthesis. Most neuronal cysteine uptake is mediated through the neuronal sodium-dependent glutamate transporter, known as excitatory amino acid carrier 1 (EAAC1). Glutamate transporters are vulnerable to oxidative stress and EAAC1 dysfunction impairs neuronal GSH synthesis by reducing cysteine uptake. This may start a vicious circle leading to neurodegeneration. Intracellular signaling molecules functionally regulate EAAC1. Glutamate transporter-associated protein 3-18 (GTRAP3-18) activation down-regulates EAAC1 function. Here, we focused on the interaction between EAAC1 and GTRAP3-18 at the plasma membrane to investigate their effects on neuronal GSH synthesis. Increased level of GTRAP3-18 protein induced a decrease in GSH level and, thereby, increased the vulnerability to oxidative stress, while decreased level of GTRAP3-18 protein induced an increase in GSH level in vitro. We also confirmed these results in vivo. Our studies demonstrate that GTRAP3-18 regulates neuronal GSH level by controlling the EAAC1-mediated uptake of cysteine.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.     

Modulation of the neural glutamate transporter EAAC1 by the addicsin-interacting protein ARL6IP1

Addicsin (Arl6ip5) is a murine homologue of rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a putative negative modulator of Na+-dependent neural glutamate transporter-excitatory amino acid carrier 1 (EAAC1). Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6ip1) is a novel addicsin-associated partner that indirectly promotes EAAC1-mediated glutamate transport activity in a protein kinase C activity-dependent manner. Like addicsin, Arl6ip1 is expressed in numerous tissues and proved likely to be co-localized with addicsin in certain neurons in the matured brain. Arl6ip1 was not translocated from the subcellular compartments under any of the test conditions and had no association with any molecules on the plasma membrane. Immunoprecipitation assay demonstrated that Arl6ip1 bound directly to addicsin and that the hydrophobic region located at amino acids 103-117 of addicsin was crucial to the formation of the Arl6ip1-addicsin heterodimer and addicsin homodimer. Glutamate transport assay revealed that increasing the expression of Arl6ip1 in C6BU-1 cells markedly enhanced Na+-dependent EAAC1-mediated glutamate transport activity in the presence of 100 nm phorbol 12-myristate 13-acetate. Under these conditions, kinetic analyses demonstrated that EAAC1 altered glutamate transport activity by increasing its glutamate affinity but not its maximal velocity. Meanwhile, increasing expression of addicsin Y110A/L112A mutant lacking binding ability for Arl6ip1 showed no enhancement of EAAC1-mediated glutamate transport activity, regardless of phorbol 12-myristate 13-acetate activation, suggesting that association between addicsin and Arl6ip1 causes altered EAAC1-mediated glutamate transport activity. Our findings suggest that Arl6ip1 is a novel addicsin-associated partner that promotes EAAC1-mediated glutamate transport activity by decreasing the number of addicsin molecules available for interaction with EAAC1.     

Caspase-3 cleaves and inactivates the glutamate transporter EAAT2

EAAT2 is a high affinity, Na+-dependent glutamate transporter with predominant astroglial localization. It accounts for the clearance of the bulk of glutamate released at central nervous system synapses and therefore has a crucial role in shaping glutamatergic neurotransmission and limiting excitotoxicity. Caspase-3 activation and impairment in expression and activity of EAAT2 are two distinct molecular mechanisms occurring in human amyotrophic lateral sclerosis (ALS) and in the transgenic rodent model of the disease. Excitotoxicity caused by down-regulation of EAAT2 is thought to be a contributing factor to motor neuron death in ALS. In this study, we report the novel evidence that caspase-3 cleaves EAAT2 at a unique site located in the cytosolic C-terminal domain of the transporter, a finding that links excitotoxicity and activation of caspase-3 as converging mechanisms in the pathogenesis of ALS. Caspase-3 cleavage of EAAT2 leads to a drastic and selective inhibition of this transporter. Heterologous expression of mutant SOD1 proteins linked to the familial form of ALS leads to inhibition of EAAT2 through a mechanism that largely involves activation of caspase-3 and cleavage of the transporter. In addition, we found evidence in spinal cord homogenates of mutant SOD1 ALS mice of a truncated form of EAAT2, likely deriving from caspase-3-mediated proteolytic cleavage, which appeared concurrently to the loss of EAAT2 immunoreactivity and to increased expression of activated caspase-3. Taken together, our findings suggest that caspase-3 cleavage of EAAT2 is one mechanism responsible for the impairment of glutamate uptake in mutant SOD1-linked ALS.     

Elevated ammonium levels: differential acute effects on three glutamate transporter isoforms

Increased ammonium (NH(4)(+)/NH(3)) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [(3)H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na(+) were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.     

Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains: importance for excitatory amino acid transporter localization and function

In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced Na(+)-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-beta-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na(+)-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function.     

Thrombin-stimulated glutamate uptake in human platelets is predominantly mediated by the glial glutamate transporter EAAT2

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.     

Activation of P2X(7) receptors decreases glutamate uptake and glutamine synthetase activity in RBA-2 astrocytes via distinct mechanisms

Glutamate clearance by astrocytes is critical for controlling excitatory neurotransmission and ATP is an important mediator for neuron-astrocyte interaction. However, the effect of ATP on glutamate clearance has never been examined. Here we report that treatment of RBA-2 cells, a type-2-like astrocyte cell line, with ATP and the P2X(7) receptor selective agonist 3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) decreased the Na+-dependent [3H]glutamate uptake within minutes. Mechanistic studies revealed that the decreases were augmented by removal of extracellular Mg2+ or Ca2+, and was restored by P2X7 selective antagonist , periodate-oxidized 2',3'-dialdehyde ATP (oATP), indicating that the decreases were mediated through P2X(7) receptors. Furthermore, stimulation of P2X7 receptors for 2 h inhibited both activity and protein expression of glutamine synthetase (GS), and oATP abolished the inhibition. In addition, removal of extracellular Ca(2+) and inhibition of protein kinase C (PKC) restored the ATP-decreased GS expression but failed to restore the P2X(7)-decreased [3H]glutamate uptake. Therefore, P2X7-mediated intracellular signals play a role in the down-regulation of GS activity/expression. Activation of P2X7 receptors stimulated increases in intracellular Na+ concentration ([Na+](i)) suggesting that the P2X(7)-induced increases in [Na+](i) may affect the local Na+ gradient and decrease the Na+-dependent [3H]glutamate uptake. These findings demonstrate that the P2X7-mediated decreases in glutamate uptake and glutamine synthesis were mediated through distinct mechanisms in these cells.     

The endoplasmic reticulum exit of glutamate transporter is regulated by the inducible mammalian Yip6b/GTRAP3-18 protein

GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na(+)/K(+) glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.     

A novel alternative splicing form of excitatory amino acid transporter 1 is a negative regulator of glutamate uptake

Abstract EAAT1 is a major glutamate transporter in the CNS and is required for normal neurotransmission and neuroprotection from excitotoxicity. In the present study, we have identified a novel form of the human EAAT1, named here as EAAT1ex9skip, which lacks the entire exon 9. Quantitative PCR analysis indicates that this variant is expressed throughout the CNS, both in grey matter and axonal tracts, at levels ranging between 10% and 20% of the full-length EAAT1 form. When expressed in HEK293 cells, EAAT1ex9skip mRNA is translated into a truncated protein localized in the endoplasmic reticulum. EAAT1ex9skip has no functional glutamate uptake activity but instead, exerts a dominant negative effect over full-length EAAT1 function. In turn, co-expression of full-length EAAT1 and EAAT1ex9skip variants reduces the insertion of the former into the plasma membrane. Together, these results indicate that the EAAT1ex9skip splice variant is a negative regulator of full-length EAAT1 function in the human brain.     

Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.

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.     

Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.     

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.     

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.     

Hetero-oligomerization of neuronal glutamate transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

Steroid hormone-inducible expression of the GLT-1 subtype of high-affinity l-glutamate transporter in human embryonic kidney cells.

The cDNA encoding the predominant rat brain high-affinity l-glutamate transporter GLT-1 was isolated and subcloned into the pIND expression vector for the establishment of steroid hormone inducible expression in vitro using the ecdysone-inducible mammalian expression system. Steroid hormone-inducible expression was demonstrable in a stable cell line designated HEK/GLT-1. Treatment of HEK/GLT-1 cells with 10 microM ponasterone A for 24 hincreased the maximum velocity (V(max)) of Na(+)-dependent l-glutamate uptake by greater than 10-fold, as compared with the uninduced cells. Equivalent levels of l-glutamate transport capacity were observed in the uninduced GLT-1 cell line and the host cell line indicating that the expression of GLT-1 was tightly regulated. To confirm that the increased l-glutamate uptake observed in HEK/GLT-1 cells following induction was attributable to the expression of GLT-1, rather than the up-regulation of the endogenously expressed EAAT3 subtype present in the host cells, we evaluated the effects of the selective GLT-1 inhibitors dihydrokainate (DHK) and kainate. Both DHK and kainate produced concentration-dependent inhibition of the l-glutamate uptake into HEK/GLT-1 cells, and the estimated IC(50) values were consistent with those described for the cloned GLT-1. These results demonstrate that the expression of GLT-1 can be tightly regulated in vitro using the ecdysone system.