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Originally published In Press as doi:10.1074/jbc.M412440200 on December 17, 2004

J. Biol. Chem., Vol. 280, Issue 8, 6621-6626, February 25, 2005
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High Affinity Transport of Taurine by the Drosophila Aspartate Transporter dEAAT2*

Marie Thérèse Besson{ddagger}, Diane B. Ré§, Matthieu Moulin{ddagger}, and Serge Birman{ddagger}

From the {ddagger}Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université de la Méditerranée, Developmental Biology Institute of Marseille, Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France and the §Laboratoire Interactions Cellulaires, Neurodégénérescence et Neuroplasticité, UMR 6186 CNRS-Université de la Méditerranée, Campus du GLM, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France

Received for publication, November 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na+/K+-dependent uptake of the amino acids L-glutamate and DL-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar Km and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na+ ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The vertebrate glutamate/neutral amino acid transporter family is composed of five glutamate/aspartate transporters, also designated the XAG system or EAATs,1 and two neutral amino acid transporters known as alanine-serine-cysteine transporters 1 and 2 (ASCT1 and ASCT2) (for reviews, see Refs. 1 and 2). The EAATs are credited with many aspects of brain functioning in normal and disease states, including synaptic signal termination, transmitter recycling, prevention of excitotoxicity, and homeostatic regulation of excitatory amino acid levels. In Drosophila, two EAATs have been identified, namely dEAAT1, which ensures glutamate buffering in the brain (3), and dEAAT2, which has an unknown function. Both transporters are specifically expressed in the nervous system (4, 5). All of the EAAT proteins share nearly identical hydropathy profiles and predicted membrane topologies. However, transport mechanisms can differ considerably in this family (2). For instance, we reported previously that dEAAT2 exhibits a unique preference for D- and L-aspartate as substrates over L-glutamate (6), whereas all of the other known EAATs transport L-glutamate and D- or L-aspartate with similar affinities (7).

Taurine is the second most abundant amino acid after glutamate in the mammalian and insect brains (8–10). This sulfur-containing amino acid is widely, although not evenly, distributed in neural and non-neural tissues. In the mammalian brain, extracellular levels of taurine are regulated by two specific, high affinity transporters with significant sequence homology to GABA transporters (11, 12), which are not related to EAATs. The physiological functions of taurine and its transporters in the nervous system remain poorly understood. Taurine appears to act as an osmolyte in cell volume regulation (13–15) and a neuromodulator (16), especially in developing nervous systems (17). In the hippocampus, taurine protects neural cells from excitotoxic damage induced by massive release of EAAs under ischemic or hypoxic conditions (18) and from free radical injury (19). It has been suggested that taurine inhibits neuronal discharges by acting as an agonist at GABAA (20) and glycine receptors (21). Taurine also plays a role in long lasting changes in synaptic activity (22–24). Thus, taurine appears to be linked to various physiological functions and could interact at different levels with neurotransmitter systems (25).

Here we tested a number of neuroactive compounds for their ability to interfere with dEAAT2-mediated D-aspartate transport. We found that three insect neurotransmitters, GABA, histamine, and octopamine, were inefficient at competing and that only high concentrations of L-cysteine and L-cystine somewhat decreased D-aspartate uptake. In contrast and surprisingly, we observed that taurine markedly inhibited D-aspartate uptake by dEAAT2 and that this transporter mediates high affinity Na+-dependent uptake of taurine. Specific antagonists of the mammalian taurine transporters have no inhibitory effect on dEAAT2. High affinity taurine transport may be a specific property of dEAAT2 in this family, because it was not shared by two other EAATs we tested, Drosophila dEAAT1 and the human transporter hEAAT2. Additionally, we observed that taurine and D-aspartate acted as mutual competitive inhibitors for dEAAT2 transport and that this transporter can operate in an exchange mode with a preferred orientation, namely D-aspartate outwards and taurine inwards.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—D-[2,3-3H]Aspartic acid and [1,2-3H]taurine were purchased from Amersham Biosciences. L-Glutamine and L-cysteine were purchased from Fluka, guanidinoethyl sulfonate was from Toronto Research Chemicals, and DL-threo-{beta}-benzyloxyaspartate (TBOA) came from Tocris. Cell culture reagents and other chemicals were obtained from Sigma-Aldrich.

Drosophila Cell Culture and Transfections—The Drosophila Schneider cell line S2 was propagated at 22.5 °C in Falcon flasks into 10 ml of Shields and Sang medium supplemented with 10% heat-inactivated fetal calf serum (Abcys), 100 units/ml penicillin, and 100 µg/ml streptomycin. Most experiments were carried out with a stably dEAAT2-transformed cell line (6) named dEAAT2-S2 that was maintained by hygromycin resistance. Transient co-transfections were performed using the Effectene reagent (Qiagen) in 100-mm plates according to the manufacturer's protocol. These transfections were carried out with the GAL4/UAS-coupled inducible gene expression system as described (26) by co-transfecting a plasmid containing UAS upstream of the cDNA of interest with a plasmid containing the GAL4 sequence under control of the metallothionein promoter (pMT-GAL4). Control cells were transfected with the pMT-GAL4 vector alone. A pUAS-GFP plasmid was co-transfected to monitor transfection efficiencies between the different plates. One day after transfection, CuSO4 was added to each plate at a 500 µM concentration, and the plates were incubated at 25 °C. Two days later, the cells were counted, dispatched at 2 x 106 cell density/ml in 6-well plates, and used for uptake experiments.

DNA Constructions—Construction of the pUAS-dEAAT1 and pUAS-hEAAT2 plasmids were described previously (3). To generate the pUAS-dEAAT2 plasmid, a 2192-bp SspI fragment from the dEAAT2 cDNA was inserted into the dephosphorylated pUAST vector (27) previously digested by EcoRI and blunted. A BamHI-NotI fragment containing the enhanced GFP sequence was excised from the pEGFP-N1 vector (Clontech) and inserted into the BamHI-NotI sites of the pUAST plasmid to make the pUAS-GFP plasmid.

Amino Acid Uptakes—Transport assays were conducted in 6-well plates as described previously (6). The standard incubation medium contained 96 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.3 mM KH2PO4, 1.2 mM MgSO4, 10 mM glucose, 62.5 mM D-mannitol, 10 mM HEPES, pH 7.2, and 1 µCi of D-[2,3-3H]aspartic acid or [1,2-3H]taurine supplemented with the corresponding unlabeled amino acids to 1 µM final concentration. After 2 min at room temperature, the uptake was stopped by washing the cells with 9 ml of total cold Na+-free buffer in which NaCl was replaced by 250 mM D-mannitol. The cells were finally air-dried and lysed with 500 µl of 1 N NaOH. Aliquots were taken from each well for liquid scintillation counting and protein determination by the Lowry method. Experiments were always performed in parallel with native and transfected S2 cells.

The Na+ dependence of taurine transport was determined by substituting osmolar equivalents of D-mannitol for NaCl. For competition experiments, the competitor was applied at the indicated concentrations together with 1 µM tritiated substrate.

For efflux studies, dEAAT2-S2 cells were preloaded for 30 min with 1 µM D-[3H]aspartate or 1 µM [3H]taurine in standard uptake medium and then rapidly washed out with 5 ml of ice-cold Na+-free buffer. Efflux was started by replacing the ice-cold Na+-free buffer with 500 µl of either Na+-free buffer (control) or standard uptake medium at room temperature, both containing 300 µM unlabeled D-aspartate or taurine. After 2 min of efflux, plates were placed on ice, and a sample of 100 µl of the extracellular medium was collected from each well for liquid scintillation counting.

For influx measurements, dEAAT2-S2 cells were preloaded for 30 min with 1 µM unlabeled D-aspartate or taurine in standard uptake medium and subsequently treated as in efflux, except that the external medium (Na+-free buffer for the control or standard buffer for the assay) was supplemented with 300 µM D-aspartate or taurine containing 1 µCi of their respective radiotracer. After 2 min of influx reaction at room temperature, plates were placed on ice. The cells were washed with 9 ml of cold Na+-free buffer, and radioactivity content was determined in the usual manner.

HPLC Measurements of Extracellular Amino Acid—The dEAAT2-S2 cells that were not preloaded were placed in 1 ml of standard uptake medium, and 10 µl of extracellular medium were taken at different time points after the addition of 300 µM taurine or D-aspartate in the medium (t = 0). At the end of the experiment (t = 180 min), all samples were frozen and stored at -80 °C until used for HPLC detection as described in (28).

Data Analysis—All results are expressed as the mean ± S.E. of separate experiments measured in triplicates. Data were analyzed using Microsoft Excel and MacCurveFit (Kevin Raner) software. Statistical analysis was performed using SigmaStat software or StatXAct for non-parametric tests as described in each figure legend.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Taurine Selectively Inhibits dEAAT2-mediated Aspartate Transport—Fig. 1 shows the effects of increasing concentrations of various neuroactive molecules on D-aspartate transport by the stably transfected dEAAT2-S2 cells. We found that 1 µM D-[3H]aspartate uptake was strongly inhibited by taurine in a concentration-dependent manner, i.e. the presence of 10 µM, 100 µM, and 1 mM taurine resulted in decreases of 28, 76, and 98%, respectively. Other compounds including GABA, histamine, and octopamine, whose transporters belong to the same family as the mammalian taurine transporters (29), or glutamine had no inhibitory effect. Only high concentrations of L-cysteine and L-cystine (~100 µM) partly reduced D-[3H]aspartate uptake. These results indicate that taurine has a specific ability to block aspartate transport mediated by dEAAT2.



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  		FIG. 1.
Effects of different neuroactive compounds on D-aspartate transport by dEAAT2. Tritiated D-aspartate (1 µM) uptakes into the dEAAT2-S2 cell line were performed in the presence of increasing concentrations (1, 10, 100, and 1000 µM) of the unlabeled potential competitors taurine, GABA, histamine (HIS), octopamine (OCT), L-glutamine (L-Gln), L-cysteine (L-Cys), and L-cystine. Results are expressed as the percentage of controls performed in the absence of competitor amino acids. The dark shaded bar corresponds to the control (Cont) of the taurine experiments. Background uptake into non-transfected S2 cells (2–11% of the total) was determined in parallel and subtracted from each data point. Results represent the mean ± S.E. of 2–5 independent experiments. Asterisks show significant variations as compared with control (ANOVA followed by Tukey test for multiple comparison procedure). **, p < 0.01; ***, p < 0.001.

 
We then compared the effect of taurine on D-aspartate uptake by dEAAT2 and two others EAATs, namely the Drosophila dEAAT1 and the human hEAAT2 glutamate/aspartate transporters. Transient transfections of these different transporters into S2 cells with the UAS-GAL4 system were performed in parallel as described under "Experimental Procedures." D-[3H]Aspartate uptakes were then assayed in the presence of increasing levels of taurine (Fig. 2). The dEAAT1- and hEAAT2-expressing cells captured D-aspartate with similar efficiency in the absence or presence of taurine at whichever concentration was used. In contrast, 10 µM taurine significantly (p < 0.01) inhibited 1 µM D-[3H]aspartate uptake by dEAAT2. At higher taurine concentrations (100 µM and 1 mM), the D-aspartate transport into dEAAT2-expressing cells became not statistically different from the nonspecific uptake in control cells. Therefore, inhibition of D-aspartate uptake by taurine may be a specific property of the dEAAT2 transporter in the EAAT family.



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  		FIG. 2.
Effect of taurine on D-aspartate transport by different EAATs. Uptakes of 1 µM tritiated D-aspartate into S2 cells was measured in 95 mM Na+ buffer in the presence of increasing concentrations of unlabeled taurine. Cells were transiently transfected as described under "Experimental Procedures" with either the pMT-GAL4 plasmid only (pMT) or pMT-GAL4 with pUAS-dEAAT1, pUAS-dEAAT2, or pUAS-hEAAT2. Results represent the mean ± S.E. of three or four independent transfection experiments and are expressed as percentages of D-aspartate uptake by dEAAT2-expressing cells in the absence of taurine. Results were statistically tested by ANOVA and Fischer multiple comparison test. Significant differences for dEAAT2-versus pMT-expressing cells are indicated. **, p < 0.01; ***, p < 0.001; ns, not significant.

 
dEAAT2 Is a High Affinity Taurine Transporter—We then observed that the dEAAT2-S2 cells were able to take up tritiated taurine from the external medium. The kinetic parameters of taurine transport by dEAAT2 were determined from dose-response experiments. As shown in Fig. 3, taurine uptake into dEAAT2-S2 cells was dose-dependent, reached saturation at high concentrations, and fitted well with the Michaelis-Menten function. Taurine transport was linear within the first 10 min of incubation (data not shown). Determined by Lineweaver-Burk plot data analyses, the mean Km value for taurine uptake was 45.74 ± 5.28 µM (n = 7), indicating a high affinity transport. This Km was somewhat higher but not statistically different from the Km of dEAAT2 for D-aspartate, which is 32.52 ± 5.77 µM (n = 7) (Table II). In contrast, the Vmax of dEAAT2 for taurine transport (293.06 ± 32.76 pmol/min/mg protein) was significantly greater (p < 0.01) than that for D-aspartate transport (151.32 ± 29.81 pmol/min/mg protein). Relative efficacy (Vmax/Km) was comparable for the two substrates. Therefore, dEAAT2 can be described as a high affinity taurine/aspartate transporter in Drosophila.



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  		FIG. 3.
Transport kinetics of taurine transport by dEAAT2. Uptakes carried out by dEAAT2 (closed circles) and into non-transfected S2 cells (open circles) were assessed over a concentration range of 0.82 µM to 1.25 mM unlabeled taurine plus 0.074 µM [3H]taurine. Uptake carried out by dEAAT2 was obtained by subtracting uptake into non-transfected cells from the data obtained with dEAAT2-S2 cells. Curves were obtained by fitting the Michaelis-Menten function to the data. In the inset is the Lineweaver-Burk plot of the same data for dEAAT2 transport. A representative experiment is shown. Results were expressed as the mean ± S.E. of triplicate determinations. Four additional independent experiments yielded similar results.

 


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  		TABLE II
Taurine and D-aspartate are reciprocal competitive inhibitors of dEAAT2 transport

The transport of each labeled amino acid substrate into dEAAT2-S2 cells was determined over a concentration range of 30-500 µM. Results are the mean ± S.E. of data obtained in seven (in the absence of the other substrate) or three (in the presence of 10 µM or 1 mM unlabeled alternative substrate) independent experiments. dEAAT2-specific uptakes were obtained by subtracting uptake into non-transfected cells from the results obtained with dEAAT2-S2 cells. The Km and Vmax values were determined from Lineweaver-Burk plots of the data. The variation index corresponds to the ratio of uptake parameters relative to the control value in the absence of the other substrate. Statistical differences were tested by Kruskal-Wallis tests.

 
Sodium Dependence and Pharmacology of Taurine Transport by dEAAT2—The Na+ dependence of taurine uptake was investigated in both a Na+-free medium and a standard uptake medium. Fig. 4A shows that taurine uptake in dEAAT2-S2 cells was highly dependent on the presence of Na+ ions, whereas the low background level of taurine uptake (10.5%) observed in control cells was not Na+-dependent. Moreover, 300 µM TBOA, a specific, non-transportable inhibitor of all known EAATs (30), abolished the Na+-dependent component of taurine uptake (Fig. 4B), indicating that dEAAT2 triggered the whole Na+-dependent uptake of taurine in these cells. Further pharmacological properties of the dEAAT2-mediated taurine transport were analyzed with antagonists of the mammalian taurine transporters (Table I). In the presence of 1 µM [3H]taurine, significant inhibition of taurine uptake by dEAAT2 was observed only with 1 mM hypotaurine, whereas 10 µM hypotaurine is sufficient to block taurine transport by the mammalian transporters (11, 12). The other substances tested, {beta}-alanine and guanidinoethyl sulfonate, which are known to be slightly less potent than hypotaurine, had no effect in our experiments. Therefore, taurine transport by dEAAT2 is pharmacologically distinct from taurine transport by the structurally unrelated mammalian transporters.



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  		FIG. 4.
Sodium ion dependence of taurine transport by dEAAT2. A, Na+ requirement of taurine uptake by dEAAT2-S2 (gray bars) and non-transfected S2 (white bars) cells determined with 1 µM [3H]taurine in the presence or absence of 96 mM Na+ ions in the external medium. Results are the mean ± S.E. of data obtained in three independent experiments. In the uptake medium without Na+, Na+ ions were replaced by osmolar equivalent of D-mannitol. Statistical differences were tested by ANOVA and the Tukey test for multiple comparison procedure. ***, p < 0.001. B, same experiment performed in the presence of 300 µM TBOA, a non-transportable EAAT inhibitor.

 


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  		TABLE I
Effect of mammalian taurine transport inhibitors on dEAAT2-mediated taurine uptake

The uptake of 1 µM tritiated taurine into the dEAAT2-S2 cell line was tested in the absence (control) and presence of mammalian taurine transport inhibitors at the indicated concentrations. Results are expressed as percentages of the control and the mean ± S.E. of data obtained in two or three independent experiments. Values obtained with non-transfected S2 cells were subtracted from the corresponding dEAAT2-S2 samples.

 
Taurine and Aspartate Are Mutual Competitive Inhibitors of Transport by dEAAT2—To determine the nature of the interactions between the two substrates, we tested the dose effect of taurine on the kinetic properties of D-aspartate uptake and the converse. The parameters of tritiated amino acid transport were determined over a concentration range of 30–500 µM. Results are summarized in Table II. D-aspartate and taurine were found to be competitive inhibitors of each other, as the presence of the other unlabeled substrate led to an increase in Km values that was significant at 1 mM with no statistically significant change in Vmax values.

dEAAT2 Mediates Asymmetric Heteroexchange—Because some transporters can mediate exchanges between extracellular and intracellular pools of amino acids (31, 32), we examined this possibility for dEAAT2. First, we tested the ability of dEAAT2 to mediate efflux of aspartate or taurine by HPLC monitoring in the presence of exogenously supplied taurine or D-aspartate, respectively. As illustrated in Fig. 5, the addition of taurine in the extracellular medium produced a time-dependent efflux of aspartate whereas no release was observed in untreated dEAAT2-S2 cells, indicating that the transporter can mediate heteroexchange. In contrast, the addition of aspartate in the extracellular medium did not elicit taurine efflux (data not shown). However, this result was expected because the dEAAT2-S2 cells do not natively contain detectable levels of taurine. We then performed outward and inward reciprocal measures of radiolabeled D-aspartate and taurine transport after the preloading of each amino acid substrate (Fig. 6). For efflux studies, dEAAT2-S2 cells were preloaded with 1 µM tritiated amino acid and subsequently superfused with the other unlabeled amino acid (300 µM). Influx rates were measured by the incubation of unlabeled amino acid-preloaded cells in the other tritiated amino acid (300 µM). High levels of extracellular taurine triggered outward and inward fluxes of the tritiated amino acids, because D-[3H]aspartate efflux and [3H]taurine influx were observed. The taurine influx rate was found to be very similar to the D-aspartate efflux rate. This suggests that the amount of released D-aspartate was equal to the amount of taurine taken up into cells. In contrast, the presence of D-aspartate on the external side of the cell membrane and taurine inside induced weak fluxes of either radioactive amino acid, suggesting an inhibition of the transporter in these conditions. Therefore, dEAAT2 can mediate heteroexchange of its two substrates, but only when taurine is outside and aspartate inside.



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  		FIG. 5.
dEAAT2 mediates heteroexchange of its amino acid substrates. Extracellular aspartate concentrations were measured by HPLC at different time points (0, 15, 30, 60, 90, and 180 min) after incubating dEAAT2-S2 cells in standard uptake medium (open circles) or in the same medium containing 300 µM taurine (filled circles). Data are mean ± S.E. of values obtained in three independent experiments. Asterisks show significant differences as compared with respective controls (repeated measures ANOVA followed by Tukey test for multiple comparison procedure. ***, p < 0.001.

 



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  		FIG. 6.
Asymmetry of dEAAT2-mediated heteroexchange. dEAAT2-S2 cells were preloaded and assayed for efflux (hatched bars) and influx (gray bars) transports of the indicated amino acid as described under "Experimental Procedures." Efflux data correspond to the Na+-dependent exchange of intracellular tritiated amino acid (1 µM) driven by the presence of the other unlabeled amino acid (300 µM) transport in the external medium. Influx data correspond to the Na+-dependent transport of external tritiated amino acid (300 µM) into cells preloaded with the other unlabeled amino acid substrate (1 µM). Left bars, taurine outside, D-aspartate inside; right bars, D-aspartate outside, taurine inside. Results are mean ± S.E. of determinations obtained in 3–6 independent experiments. Asterisks show significant differences after ANOVA and Tukey test for multiple comparison procedure. **, p < 0.01; ***, p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We reported previously that the Drosophila transporter dEAAT2 is able to take up D-or L-aspartate, but not glutamate, with high affinity (6). Here we report a novel unexpected feature of this protein, i.e. its ability to transport taurine with as high affinity and efficacy as aspartate.

We observed that taurine is a potent and specific competitor of D-aspartate uptake by dEAAT2. This property is not shared by other neuroactive molecules such as GABA, the predominant inhibitory neurotransmitter in mammalian and insect brain, octopamine, a neurotransmitter or neuromodulator in invertebrates (33), and histamine, which is required for photoreceptor synaptic transmission in insects (34). Similarly, glutamine, a putative precursor of neuronal pools of glutamate (35), is not a competitor for dEAAT2-mediated aspartate uptake. Only high levels of L-cysteine and L-cystine partly inhibited D-aspartate transport by dEAAT2. L-Cysteine is known as a substrate of EAAC1, its human homologue hEAAT3 (31, 36), and the neutral ASCTs (37). Studies on mammalian cultured neurons and astrocytes showed that L-cystine uptake occurs by Na+-independent and Na+-dependent processes, including EAATs (38–40). Therefore, these amino acids could act as low affinity substrates of dEAAT2. Alternatively, high concentrations of L-cysteine and L-cystine could trigger taurine synthesis in S2 cells, which would inhibit dEAAT2-mediated aspartate uptake by transinhibition (see below). Indeed, L-cysteine was shown to be a direct metabolic precursor of taurine in astroglial cells (41).

The effect of taurine on D-aspartate uptake appears to be specific to dEAAT2, as it was not observed with two other transporters of the family, namely the Drosophila glutamate transporter dEAAT1 and hEAAT2, the major EAAT protein in the human brain. These three transporters were able to take up D-aspartate with similar efficiency after transient transfection in S2 cells. However, only the D-aspartate transport mediated by dEAAT2 was inhibited by taurine in a dose-dependent manner.

High affinity transport of [3H]taurine by dEAAT2 was demonstrated that has strict dependence on extracellular Na+ ions. Kinetic analysis shows that dEAAT2 discriminates between aspartate and glutamate (6) but not between aspartate and taurine. To our knowledge, this is the first observation that a protein of the EAAT family can accept taurine as substrate. Furthermore, the taurine transport mediated by dEAAT2 is clearly distinct from mammalian taurine transport. First, the two known mammalian taurine transporters are not structurally related to the EAAT proteins. Second, mammalian taurine transport inhibitors are not competitors for taurine uptake by dEAAT2. These findings suggest that the site for taurine binding on the dEAAT2 protein is not accessible to the usual taurine inhibitors but is conformed such that the specific EAAT inhibitor TBOA (30) can efficiently reduce taurine transport. These observations indicate that dEAAT2 possesses comparable pharmacology but quite distinct substrate specificity compared with other EAATs.

dEAAT2 transports D-aspartate and taurine with comparable relative efficacy, and the two substrates are reciprocal competitive inhibitors. Indeed the Km for taurine uptake presents a significant increase when D-aspartate concentration increases and vice versa, whereas their respective Vmax values are not significantly modified. Moreover, there is no apparent difference in transport inhibition potency between D-aspartate and taurine as attested by the similar Km variation indexes. Overall, these data suggest that the sites to which D-aspartate and taurine bind on the transporter have an equivalent accessibility and could be the same.

The vertebrate glutamate/neutral transporters can behave as amino acid exchangers (2, 42). A glutamate-cysteine exchange through EAAC1 (human EAAT3) has been described (31), and cysteine uptake appears to be predominantly mediated by this transporter in neuron cultures (36). The neutral transporters ASCT1 and ASCT2 were reported to function exclusively as amino acid exchangers (43, 44), whereas EAATs mediate both uptake and exchange modes at various levels. Our results indicate that dEAAT2 is able to function in two modes, exchange and uptake. Although further experiments are needed to confirm this possibility, we can postulate that the transport of taurine by dEAAT2 could be mediated either by uptake or by heteroexchange in brain cells containing aspartate. However the transport of aspartate may occur by uptake only, because taurine outward/aspartate inward heteroexchange is not carried out by dEAAT2. Furthermore, aspartate uptake itself is inhibited under these conditions, suggesting a mechanism of transinhibition (45, 46) by intracellular taurine that would efficiently block dEAAT2-mediated transport.

In insects, taurine is a major free amino acid. Its presence has been detected by immunostaining in nerve fibers of the honeybee central and peripheral nervous systems (47, 48). Taurine is generally reported to interact with neurotransmitter systems, and taurine-immunopositive cells have been co-localized in octopaminergic neurons (49), histaminergic photoreceptors (9), and GABAergic neurons (10). These reports suggest that, in insects, taurine could function as a neuromodulator co-released with amines. A taurine immunoreactivity, spatially distinct from that of two other candidate neurotransmitters, aspartate and glutamate, has been recently described in the cockroach and Drosophila mushroom bodies (50, 51). The mushroom bodies are an integrative brain structure involved in learning and memory in insects. These studies show that taurine distribution is comparable with the expression of a neurotransmitter in the insect brain. This is also in agreement with the various roles of taurine suggested by vertebrate studies on developmental plasticity and the long term potentiation of synaptic transmission (16, 17, 25).

From our results, we conclude that dEAAT2 mediates taurine and aspartate transport in the Drosophila nervous system by a common mechanism, these two amino acids being equally favorite substrates of this transporter. Although the precise functions of these two amino acids in the insect nervous system physiology are still unknown, dEAAT2 seems to be a good candidate gene to modulate the function of taurine in the fly brain.


   FOOTNOTES
 
* This work was supported by an Action Concertée Incitative grant from the Ministère de la Recherche and funding from the Association Française contre les Myopathies and Fondation pour la Recherche Médicale (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 33-491-26-96-06; Fax: 33-491-82-06-82; E-mail: birman{at}ibdm.univ-mrs.fr.

1 The abbreviations used are: EAAT, excitatory amino acid transporter; ANOVA, analysis of variance; ASCT, alanine-serine-cysteine transporter; dEAAT, Drosophila EAAT; GABA, {gamma}-aminobutyric acid; GFP, green fluorescent protein; HPLC, high performance liquid chromatography; TBOA, DL-threo-{beta}-benzyloxyaspartate; UAS, upstream activating sequence. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Lydia Kerkerian-Le Goff and Laurence Had-Aïssouni for discussions and comments on the manuscript, Prof. André Nieoullon for use of the radioactivity counter in his laboratory, and Drs. Andrea Brand, Gerald Rubin, and Susan Amara for the pUAST, pMT-GAL4, and pSK-hEAAT2 plasmids, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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