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First published online September 7, 2006
doi: 10.1242/10.1242/jcs.03151
Research Article |
1 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences
2 Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China
* Authors for correspondence (e-mail: baolan{at}sibs.ac.cn; gpei{at}sibs.ac.cn)
Accepted 22 June 2006
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-aminobutyric acid (GABA) and to epileptogenesis. However, the mechanisms that regulate EAAC1 endocytic sorting and function remain largely unknown. Here, we first demonstrate that EAAC1 undergoes internalization through the clathrin-mediated pathway and further show that syntaxin 1A, a key molecule in synaptic exocytosis, potentiates EAAC1 internalization, thus leading to the functional inhibition of EAAC1. In the presence of the transmembrane domain of syntaxin 1A, its H3 coiled-coil domain of syntaxin 1A is necessary and sufficient for the inhibition of EAAC1. Furthermore, specific suppression of endogenous syntaxin 1A significantly blocked EAAC1 endocytic sorting and lysosomal degradation promoted by kainic acid, a drug for kindling the animal model of human temporal lobe epilepsy in rat, indicating a potential role of syntaxin 1A in epileptogenesis. These findings provide new evidence that syntaxin 1A serves as an intrinsic enhancer to EAAC1 endocytic sorting and further suggest that syntaxin 1A is conversant with both `ins' and `outs' of synaptic neurotransmission.
Key words: EAAC1, Syntaxin 1A, Endocytic sorting, Glutamate transport, Kainic acid, Surface expression
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; Maragakis and Rothstein, 2004; Tanaka et al., 1997). The excitatory amino-acid carrier 1 (EAAC1) belongs to the excitatory amino-acid transporter (EAAT) family, which also includes the astroglial glutamate-aspartic-acid transporter (GLAST) and the glutamate transporter-1 (GLT-1), the neuronal EAAT4 and EAAT5 in the mammalian central nervous system (Danbolt, 2001; Sims and Robinson, 1999). EAAC1 is most notably located in the somata, dendrites and axons of many neurons, especially those in the hippocampus and cerebellum (Furuta et al., 1997; He et al., 2000; Rothstein et al., 1994). Strikingly, EAAC1 protein is also highly concentrated in presynaptic GABAergic terminals, where it participates in the neurosynthesis of -aminobutyric acid (GABA) (Conti et al., 1998; He et al., 2000; Mathews and Diamond, 2003; Rothstein et al., 1994). Genetic EAAC1 suppression markedly leads to epileptiform phenotypes, characterized by electrographic seizures, staring-freezing episodes and paresis (Rothstein et al., 1996; Sepkuty et al., 2002), underscoring the important role of this neuronal transporter in neurological disorders. Therefore, EAAC1 activity markedly affects neurotransmitter homeostasis and synaptic transmission.
Considering its central role in shaping glutamatergic and GABAergic signaling, the neuronal glutamate transporter EAAC1 is a likely target for cellular regulation. Indeed, activation of protein kinase C (PKC) or platelet-derived growth factor receptor accelerates the delivery of EAAC1 to the cell surface and, at the same time, activation of PKC also inhibits the internalization of EAAC1 (Fournier et al., 2004). In the kainic acid (KA)-kindled model of epilepsy, the expression of EAAC1 is downregulated in the dentate gyrus, entorhinal cortex layer and hippocampus (Furuta et al., 2003; Ghijsen et al., 1999; Gorter et al., 2002; Simantov et al., 1999), especially with perinuclear deposits of EAAC1 (Furuta et al., 2003). Given the determinant role of trafficking in regulation of neurotransmitter transporters (Deken et al., 2000; Geerlings et al., 2001; Robinson, 2002), a greater understanding of the molecules that regulate EAAC1 trafficking is likely to shed light on this pivotal synaptic modulator.
Syntaxin 1A is a neuronal plasma membrane protein that belongs to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family (Bennett et al., 1993). Syntaxin 1A is involved in vesicle trafficking, docking and/or fusion, and plays a key role in neurotransmitter release (Sudhof, 2000). Syntaxin 1A also directly interacts with and functionally regulates ion channels such as Ca2+ channels (Sheng et al., 1994), cystic fibrosis transmembrane regulator Cl- channels (Naren et al., 1997), K+ channels (Fili et al., 2001), epithelial Na+ channels (Saxena et al., 1999), and Na+/Cl- dependent transporters, such as the GABA (Deken et al., 2000), norepinephrine (Sung et al., 2003), serotonin (Haase et al., 2001) and glycine transporters (Geerlings et al., 2000). A recurring theme in these modulations is that channels and transporters are redistributed to and from the plasma membrane (Deken et al., 2000). However, whether syntaxin 1A modulates trafficking of the transporters for excitatory neurotransmitters remains an open question and the underlying mechanisms have not been elucidated.
Here, we show that syntaxin 1A specifically potentiates the clathrin-mediated and dynamin-dependent EAAC1 internalization, and consequently leads to functional inhibition of glutamate transport. Furthermore, syntaxin 1A functions as an intrinsic enhancer to the EAAC1 endocytic sorting evoked by KA, a drug used broadly in kindling the animal model of human epilepsy. Our findings indicate a new mechanism of syntaxin 1A in regulating the trafficking of membrane proteins and suggest a potential role of syntaxin 1A in epileptogenesis.
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; Geerlings et al., 2001). Here, we assessed in C6 glioma cells the effect of syntaxin 1A on the trafficking of EAAC1, a Na+/K+-dependent neuronal glutamate transporter. It is established that the C6 glioma cell line endogenously expresses syntaxin 1A (supplementary material Fig. 1A) and EAAC1, but not other glutamate transporters (Davis et al., 1998; Dowd et al., 1996; Palos et al., 1996). This cell line has thus been used as a model system to study the regulation of EAAC1 activity and surface expression (Robinson, 2002). Our results showed that the surface expression of endogenous EAAC1 decreased to 58.3±2.1% in syntaxin1A-transfected C6 glioma cells compared with ß-gal-transfected control cells (Fig. 1A). In addition, the change of EAAC1 homomer was identical to that of EAAC1 monomer (Fig. 1A), excluding the possibility that EAAC1 was redistributed between monomer and homomer. Although there was a concurrent overall decrease of EAAC1 protein (Fig. 1A), the unchanged mRNA levels of EAAC1 excluded the possible effects of syntaxin1A on EAAC1 gene expression (Table 1). However, it is difficult to tease out whether the decreased EAAC1 surface expression is due to the altered overall EAAC1 expression or trafficking. To address this issue, we treated the cells with degradation inhibitors 48 hours after transfection. Thereby, we inhibited EAAC1 degradation and detected the changes in the pool of surface and intracellular EAAC1, which were defined as the ratio of surface or intracellular EAAC1 versus total amount of EAAC1 protein expression. Our data showed that in syntaxin-1A-transfected C6 glioma cells the surface EAAC1 pool was decreased, while the intracellular EAAC1 pool was increased (Table 1), suggesting that the decreased EAAC1 surface expression was due to the altered EAAC1 trafficking. These results provide compelling evidence that syntaxin 1A plays a regulatory role in EAAC1 trafficking.
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Considering the effect of syntaxin 1A on the delivery of several transporters and ion channels to the cell surface (Blakely and Sung, 2000; Fili et al., 2001; Geerlings et al., 2001; Saxena et al., 1999; Sung et al., 2003), we addressed this possibility by examining the delivery efficiency of EAAC1. To measure the delivery of EAAC1 to the cell surface, C6 glioma cells were incubated under trafficking-permissive conditions (37°C) in the presence of Sulfo-NHS-biotin for varied time. Under these conditions, the membrane impermeant Sulfo-NHS-biotin should label transporters that cycle through the cell surface (Fournier et al., 2004). In each experiment, the amount of EAAC1 biotinylated under conditions not permissive to trafficking (4°C) was also examined, and the EAAC1 delivery efficiency was expressed as a percentage of transporters delivered to the cell surface versus the intracellular pool of EAAC1. Our results showed that the delivery efficiency of EAAC1 was not altered in syntaxin-1A-transfected C6 glioma cells (Fig. 1B), suggesting that syntaxin 1A regulates the endocytic sorting of EAAC1.
Further studies from the reversible biotinylation confirmed that, compared with cells only expressing endogenous syntaxin 1A, the amount of internalized EAAC1 was more significant in C6 glioma cells transfected with syntaxin 1A (19.7±1.9% versus 36.8±3.0% at 5 minutes, and 26.4±3.3% versus 61.8±3.5%, respectively, at 30 minutes, P<0.01,) (Fig. 1C), despite the reduction in overall EAAC1 available for internalization. In addition, the effect of syntaxin 1A on EAAC1 internalization was unique, because neither the astroglia transporter GLT-1 nor EAAT4 (another neuronal glutamate transporter) showed changes of surface expression by syntaxin 1A in the co-transfected human embryonic kidney 293 (HEK293) cells (supplementary material Fig. 1B,C). Moreover, this unique regulatory role of syntaxin 1A was also supported by the findings that neither syntaxin 4 (another syntaxin isoform localized on the cell surface) nor other neuronal SNAREs, including SNAP-25 and VAMP-2, modulated the surface expression of EAAC1 (supplementary material Fig. 1D). These results suggest that the decreased EAAC1 surface expression is attributable to the promoted internalization of the transporter by syntaxin 1A rather than the altered delivery rate of EAAC1 to the cell surface. Thus, syntaxin 1A serves as an enhancer to EAAC1 endocytic sorting, leading to the reduction of EAAC1 resident on the cell surface.
Syntaxin 1A decreases EAAC1-mediated glutamate transport
Given that the regulated trafficking of neurotransmitter transporters contributes to the tuning of their transport activities (Robinson, 2002), we then tested whether syntaxin 1A modulated the transport activity of EAAC1 by studying the sodium-dependent transport of [3H]glutamate in C6 glioma cells. Glutamate transport was significantly decreased (64.4±9.4%) in cells transfected with syntaxin 1A (Fig. 2A), compared with cells that only expressed endogenous syntaxin 1A. Furthermore, we carried out kinetics analyses to evaluate the biochemical nature of the altered transport activity. The syntaxin-1A-transfected cells showed a decrease in maximal velocity (Vmax=348 pmol/mg protein/minute) without a shift in affinity (Km=30.0 µM), compared with those cells only expressing endogenous syntaxin 1A (Vmax=526 pmol/mg protein/minute, Km=31.0 µM) (Fig. 2B). These data suggest that the decrease in transport activity is attributable to the reduction of the transporters remaining on the surface, which supports the finding that syntaxin 1A downregulates EAAC1 surface expression.
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We then used HEK293 cells to detect the unique regulation of syntaxin 1A on EAAC1. The results also confirmed the progressive decrease in EAAC1 transport activity when expression of syntaxin 1A was increased (Fig. 2E). Moreover, syntaxin 1A did not alter the transport activity of EAAT4 or GLT-1 in the co-transfected HEK293 cells (Fig. 2F). This unique regulatory role of syntaxin 1A was also supported by the findings that the EAAC1 transport activity was not modulated by several other SNARE proteins, including syntaxin 4, SNAP-25 and VAMP-2 (Fig. 2G). Taken together, our studies imply that syntaxin 1A specifically and negatively modulates EAAC1 transport activity by facilitating EAAC1 internalization.
EAAC1 internalization is mediated by clathrin-coated pits in coordination with syntaxin 1A
Until now the endocytic pathway for EAAC1 remained unclear. We therefore inquired whether EAAC1 is internalized via the clathrin-mediated endocytic pathway, a classic internalization pathway for several membrane transporters (Daniels and Amara, 1999; Deken et al., 2003). We used hypertonic media to induce abnormal clathrin polymerization. To control for the effect of sucrose on exocytosis (Li et al., 2001), we first incubated C6 glioma cells for 15 minutes in high-potassium buffer and then for 5 minutes with various concentrations of sucrose (Deken et al., 2003). Surface biotinylation analyses showed that the surface expression of EAAC1 was increased after sucrose treatment, and incubation with 0.45 M sucrose caused an approximately twofold increase in EAAC1 surface expression (Fig. 3A), whereas incubation with 0.15 M and 0.6 M sucrose caused a lesser increase (155.1±11.6%, 119.0±20.3%, respectively). In parallel, the transport activity of EAAC1 was significantly increased after incubation with 0.45 M sucrose (Fig. 3A). Due to the multiple cellular effects of hypertonicity on exocytosis and endocytosis (Heuser and Anderson, 1989), we then examined the effect of K44A dynamin, a dominant-negative form of dynamin that inhibits endocytosis via clathrin-coated pits (Damke et al., 1994), on EAAC1 internalization. We transfected wild-type dynamin and the K44A dynamin into C6 glioma cells to detect the surface expression of EAAC1. Our results showed that cells transfected with wild-type dynamin displayed a significant decrease in the surface expression of EAAC1, whereas transfection with K44A dynamin led to the reinstallation of the EAAC1 surface expression, indicating that EAAC1 internalization is impaired (Fig. 3B, Table 2). Moreover, the functional uptake of glutamate mediated by EAAC1 was decreased by transfection of dynamin and kept unchangeable by transfection of K44A dynamin (Table 2), consistent with the levels in EAAC1 surface expression. Therefore, these data suggest that EAAC1 is internalized in a clathrin-mediated and dynamin-dependent manner.
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To further distinguish the mechanism of the syntaxin-1A-potentiated EAAC1 internalization, we suppressed endogenous syntaxin 1A expression in dynamin-transfected C6 glioma cells. Our data showed that suppression of endogenous syntaxin 1A significantly rescued the dynamin-induced decrease in EAAC1 surface expression (9.8±6.5% decrease compared with 50.4±3.4% decrease in cells transfected with nonspecific siRNA, P<0.01) (Fig. 3C). In addition, syntaxin 1A siRNA had no effect on dynamin expression (data not shown). Thus, these data suggest that syntaxin 1A coordinates with dynamin for EAAC1 internalization.
The H3 coiled-coil and transmembrane domains of syntaxin 1A are necessary and sufficient for interaction with and inhibition of EAAC1
There is no evidence that syntaxin 1A endogenously interacts with EAAC1, especially in mammalian cells. Therefore, we examined the endogenous interaction between syntaxin 1A and EAAC1 to establish a possible molecular mechanism underlying the syntaxin-1A-mediated modulation of EAAC1. Initial experiments were performed in HEK293 cells and, as shown in Fig. 4A, syntaxin 1A was coimmunoprecipitated with EAAC1 in the cell extract prepared from the co-transfected cells and vice versa. To further study the protein interaction in vivo, we tested the association of syntaxin 1A with EAAC1 in the hippocampus extract of the mice and found that syntaxin 1A coimmunoprecipitated with EAAC1 (Fig. 4B). The identical result was obtained from C6 glioma cells, endogenously expressing these two molecules. These data confirm that syntaxin 1A interacts with EAAC1 in mammalian system, especially in vivo. Considering its central role in regulating the endocytic sorting of EAAC1, syntaxin 1A might associate with EAAC1 on the cell surface. Therefore, we used BS3, an impermeable regent that crosslinks the surface proteins to determine the association of proteins on the cell surface. As shown in Fig. 4C, a high-molecular-mass band of 
100 kDa was detected in the BS3-crosslinked samples by EAAC1 and syntaxin 1A antibodies, suggesting that the association of EAAC1 and syntaxin 1A is on the cell surface. Thus, syntaxin 1A interacts with EAAC1 on the cell surface and intrinsically regulates EAAC1 endocytic sorting.
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H3) nor the mutant only containing the H3 domain (amino acids 194-266, Syn1A H3) interacted with EAAC1 (data not shown). In order to dissect the function of those domains, we examined the effect of different syntaxin 1A mutants on EAAC1 transport activity and surface expression. Our data showed that HEK293 cells co-transfected with Syn1A H3-TMD showed an approximately 42% reduction in EAAC1-mediated glutamate transport, comparable to the reduction induced by syntaxin 1A. Neither EAAC1 co-transfected with Syn1A H3 nor co-transfected with Syn1A H3 resulted in the reduction of glutamate transport (Fig. 4F). And the EAAC1 surface expression was also significantly decreased in the cells co-transfected with Syn1A H3-TMD (Fig. 4F). All the mutants had a comparable expression level (data not shown). In addition, the C6 glioma cells transfected with hemagglutinin (HA)-tagged syntaxin 1A displayed a similar inhibition in EAAC1-mediated glutamate uptake (Fig. 4F) to those transfected with syntaxin 1A (Fig. 2A), suggesting that the HA-tag did not interfere with the regulation of syntaxin 1A on EAAC1. These data demonstrate that the H3 and transmembrane domains of syntaxin 1A are necessary and sufficient for its interaction with EAAC1, leading to the downregulation of EAAC1 surface expression and transport activity.
Requirement of syntaxin 1A in KA-promoted internalization of EAAC1
Recent findings suggest that EAAC1 accumulates in the intracellular compartment in KA-kindled epilepsy animal model (Furuta et al., 2003). However, regulation of EAAC1 internalization and the underlying molecular mechanisms are still unknown and, therefore, we investigated the regulation of EAAC1 internalization upon stimulation with KA using cell surface biotinylation and reversible biotinylation assay. We observed that in C6 glioma cells there was an approximately 17% and 48% reduction in EAAC1 surface expression with 10 µM KA stimulation for 5 minutes and 30 minutes, respectively (Fig. 5A,B), concomitant with an approximately 45% inhibition of EAAC1 transport activity for a 30-minute stimulation with 10 µM KA (Fig. 5B). Furthermore, the effect of KA on EAAC1 surface expression was strongly antagonized by K44A dynamin (Fig. 5A), implying that KA promoted the dynamin-dependent internalization of EAAC1. Indeed, compared with untreated control cells, we observed a dramatically promoted internalization of EAAC1 that occurred with KA stimulation at 5 minutes (33.6±2.3% versus 50.8±3.0%, P<0.01) and a more significant response at 30 minutes (51.4±3.8% versus 77.8±3.7%, P<0.01) (Fig. 5C). These results support the notion that KA promotes the clathrin-mediated internalization of EAAC1 leading to functional inhibition of EAAC1.
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Syntaxin 1A enhances EAAC1 sorting via the endosome and/or lysosome pathway upon stimulation with KA
Since EAAC1 was internalized in response to KA stimulation, we examined the sorting pathway of the internalized EAAC1. We found that KA stimulation triggered the internalized EAAC1 to accumulate on the perinuclear vesicular structure and to display extensive colocalization with the early endosome marker EEA1 (Pearson's correlation, Rr=0.5744) and the acidic organelle probe LysoTracker Red DND-99 (Rr=0.6572) (Fig. 6A,B), concomitant with a weaker staining of EAAC1 along the surface at 30 minutes (Fig. 6A,B). By marked contrast, there was far less colocalization of EAAC1 with EEA1 (Rr=0.3473) or with LysoTracker Red DND-99 (Rr=0.2478) without KA stimulation, concurrent with a stronger staining of EAAC1 along the cell surface (Fig. 6A,B). These results further support that KA stimulation promotes the EAAC1 internalization and indicate that the internalized EAAC1 undergoes the endocytic sorting pathway from early endosomes to lysosomes.
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Given that the internalized transporters are driven into the endosomal and/or lysosomal pathway, we tested whether long-term exposure to KA would lead to degradation of EAAC1. The C6 glioma cells were biotinylated on ice and then stimulated for 6 hours with 10 µM KA with or without lysosomal degradation inhibitors at 37°C. Long-term KA stimulation significantly decreased the amount of biotinylated EAAC1 (51.1±3.3% of overall surface EAAC1 at 6 hours), and was totally blocked by the lysosomal protease inhibitor leupeptin (112.5±7.5%) and the lysosomotropic amines chloroquine (104.5±10.3%), and partially by ammonium chloride (NH4Cl) (94.0±13.5%) (Fig. 6C). These results confirm that KA stimulation causes degradation of EAAC1 by trafficking to lysosomes.
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Effect of KA on EAAC1 endocytic sorting in primary neuronal cultures
Our studies strongly suggest that KA stimulates EAAC1 internalization, leading to degradation in lysosomes in the C6 glioma cells. Furthermore, we assessed the effect of KA on endocytic sorting of EAAC1 in primary neuronal cultures. We observed that, compared with untreated control cells, in primary cultured hippocampal neurons the internalization of EAAC1 was dramatically promoted after 5-minute KA stimulation (36.9±2.6% versus 75.3±4.6%, P<0.01) and even more significantly after 30-minute stimulation (56.9±3.7% versus 92.7±8.2%, P<0.05) (Fig. 7A). Accordingly, a 30-minute stimulation with KA induced an approximately 40% reduction in EAAC1 surface expression (Fig. 7B). Therefore, KA has a similar effect on EAAC1 endocytic sorting in a system that has a cellular environment, presumably more like that observed in vivo.
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), but our study demonstrates that syntaxin 1A also functions as an intrinsic enhancer to EAAC1 endocytic sorting. Although the precise mechanism of syntaxin 1A in EAAC1 internalization is yet still unknown, it has been reported that syntaxin 1A is associated with dynamin in adrenal chromaffin cells (Galas et al., 2000) and yeast (Peters et al., 2004), and also with synaptotagmin, which is involved in the assembly of clathrin-coated pits (Chapman et al., 1995; Haucke and De Camilli, 1999; von Poser et al., 2000). Therefore, we can speculate that syntaxin 1A functions as a potential scaffold protein to coordinate the control of exocytosis and endocytosis. In fact, SNARE proteins are known to undergo clathrin-mediated endocytosis after synaptic exocytosis (Heuser, 1989; Royle and Lagnado, 2003), and thus it is possible that EAAC1 is co-internalized with syntaxin 1A through their interaction. Our results are consistent with the notion that syntaxin 1A is conversant with both `ins' and `outs' of chemical signaling in the brain (Blakely and Sung, 2000).
Several early studies suggest that the surface expression of some neurotransmitter transporters can be regulated by a number of signaling molecules under physiological conditions (Robinson, 2002), and some researchers have isolated the transporter-containing synaptic-like vesicles, which are potential mediators of transporter trafficking in axon terminals (Deken et al., 2003; Geerlings et al., 2001). Hence, syntaxin 1A might play a necessary role in the exocytosis of these transporters, which is consistent with its role as a regulator of membrane fusion. Although our current study showed that overexpression of syntaxin 1A had no effect on the constitutive exocytosis of EAAC1, syntaxin 1A could be involved in regulated exocytosis when some signaling pathways are activated. Recent studies show that EAAC1 is redistributed to the cell surface after the activation of signalling cascades, such as the PKC, tyrosine kinase and phosphatidylinositol 3-kinase pathways (Robinson, 2002). Furthermore, the basal and regulated delivery of EAAC1 to the cell surface originates from distinct intracellular pools (Fournier et al., 2004). Thus, whether syntaxin 1A is required for those regulations is worthy of further investigation, and our studies raise interesting questions about the direction of EAAC1 trafficking in different physiological and pathological situations.
Essential role of syntaxin 1A in the regulation of EAAC1-mediated glutamate transport
In a recent study, syntaxin 1A has been shown to reduce the EAAC1-mediated current in Xenopus oocytes (Zhu et al., 2005). Here, we revealed that syntaxin 1A negatively regulates the EAAC1-mediated glutamate transport in the mammalian system, and further demonstrated that the syntaxin-1A-induced decrease in glutamate uptake is associated with the reduced cell surface expression of EAAC1 in virtue of the promoted internalization. Moreover, specific suppression of endogenous syntaxin 1A protein expression improved EAAC1 transport activity, indicating that syntaxin 1A serves as an intrinsic regulator of EAAC1-mediated glutamate transport. Although the degree of the decreased EAAC1 surface expression was too excessive to account for the decrease in glutamate uptake, this discrepancy might be reconciled if the catalytic efficiency of EAAC1 is also regulated, or if some silent transporters are internalized from the cell surface (Somwar et al., 2001). Recent studies suggest that the cell surface expression and the intrinsic activity of some transporters can be independently regulated, suggesting that this dual mode of regulation is a general phenomenon (Gonzalez et al., 2002).
We further demonstrate that the interaction between syntaxin 1A and EAAC1 is pivotal for this inhibition, by finding that the syntaxin 1A H3-domain-deletion mutant cannot properly associate with EAAC1 and failed to downregulate EAAC1 transport activity. However, because of its mis-localization (data not shown), the H3 domain by itself could not induce EAAC1 inhibition without the assistance of the transmembrane domain (Kasai and Akagawa, 2001; Lewis et al., 2001). In fact, our data show that, at least in the presence of the H3 and transmembrane domains, syntaxin 1A associated with EAAC1 on the cell surface, leading to the reduction of its surface expression and the inhibition of glutamate transport. Thus, these observations suggest that syntaxin 1A plays an essential role in modulating glutamate transport by regulating EAAC1 trafficking.
Until now, few molecules have been reported to directly regulate the function of the glutamate transporter. Recently, an EAAC1-associated protein, GTRAP3-18 has been identified to downregulate EAAC1 transport activity (Lin et al., 2001). Since GTRAP3-18 modulates EAAC1 transport activity by lowering substrate affinity without altering EAAC1 trafficking (Lin et al., 2001), our findings suggest a distinct mechanism by which syntaxin 1A, another EAAC1-associated protein, potentiates EAAC1 trafficking and inhibits EAAC1-mediated glutamate transport without altering its substrate affinity.
A possible functional link between syntaxin 1A and epilepsy
It is well known that EAAC1 is localized in inhibitory GABAergic neurons (Conti et al., 1998; He et al., 2000; Rothstein et al., 1994), where glutamate is converted to the important inhibitory neurotransmitter GABA, and thus serves as a major supply for GABA synthesis. In epilepsy, EAAC1 expression levels are known to be altered, which is considered a key factor in epileptogenesis (Ghijsen et al., 1999; Gorter et al., 2002; Simantov et al., 1999). The decreased expression levels of EAAC1 not only lead to the dysfunction in clearing synaptic glutamate but also result in the impairment of GABA synthesis (Maragakis and Rothstein, 2004; Sepkuty et al., 2002), and thus disrupts the balance between glutamate and GABA in the synaptic cleft. Furthermore, previous studies have shown that hippocampal GABA levels are reduced to 50% in EAAC1-knockdown mice (Rothstein et al., 1996; Sepkuty et al., 2002) and the neuropil staining of EAAC1 is decreased in the KA-kindled rat epilepsy model (Furuta et al., 2003). Our current findings illustrate that KA induced a decrease in EAAC1 surface expression and an increase in EAAC1 endocytic sorting, and lysosomal degradation both in C6 glioma cells and in primary cultured neurons, suggesting a possible mechanism in KA-kindled epilepsy. However, the molecular mechanism in KA-promoted endocytic sorting of EAAC1 remains an open question. Our unpublished data show that KA also concurrently induced syntaxin 1A to accumulate along the cell surface, possibly because KA functions as the agonist of the kainite receptor, a subtype of the ionotropic glutamate receptor, to induce Ca2+ influx and evoke exocytosis (Huettner, 2003). It might be that accumulation of syntaxin 1A along the cell surface greatly facilitates its association with EAAC1 and thus promotes the EAAC1 internalization and lysosomal degradation.
Increasing evidence show that the dysfunctions in endocytosis are common themes in many neurological disorders, especially neurodegenerative diseases (Cataldo et al., 2000; Nixon, 2005; Smith et al., 2005; Vanoni et al., 2004), thus interference of abnormal endocytosis may be potential therapeutic strategies in neurological disease. Our study demonstrates that syntaxin 1A is an intrinsic regulator of KA-induced EAAC1 endocytic sorting. It further reveals that suppression of expression of endogenous syntaxin 1A significantly antagonized the KA-promoted EAAC1 internalization and degradation and, hence, largely reinstated EAAC1 expression at the surface, implying a promising approach for the treatment of epilepsy. However, owing to the multiple roles of syntaxin 1A in synaptic activity, the specific disruption of the association between syntaxin 1A and EAAC1 by the competing peptide seems to be an even better solution.
In conclusion, the results presented here demonstrate that syntaxin 1A, the key molecule for exocytosis, is crucially involved in modulating the endocytic sorting of the neuronal glutamate transporter EAAC1. Thus, our study not only delineates a regulatory mechanism for the internalization of a specific cell surface transporter but also sheds light on understanding epileptogenesis.
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). The sequences of the siRNA for rat syntaxin 1A were taken from GenBank (accession number NM_053788, nucleotides 471-492). The sequence of the nonspecific siRNA was 5'-ggccgcaaagaccttgtcctta-3'.
Cell culture and transfection
C6 glioma cells and human embryonic kidney 293 (HEK293) cells were obtained from American Type Culture Collection (Manassas, VA). The C6 glioma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (both Invitrogen, Carlsbad, CA). The constructed cDNA and the siRNA constructs were transfected into C6 glioma cells with Lipofectamine or lipofectamine 2000 (Invitrogen) as described previously (Xu et al., 2004). HEK293 cells were maintained in minimal essential medium (MEM) (Invitrogen) supplemented with 10% fetal bovine serum. Transient transfection of HEK293 cells was done using the calcium phosphate method. Forty-eight hours after transfection, cells were collected for different assays.
Primary neuron-enriched cultures were derived from postnatal, day 0 to 1, Sprague-Dawley rat hippocampi. Hippocampi were isolated, trypsinized for 20 minutes at 37°C, triturated in a Pasteur pipette, and then plated on poly-L-lysine-coated dishes. Neuronal cultures were maintained in Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with 2% B27 (Invitrogen). For all experiments, neurons were used at 14 days in vitro.
Drug treatment
C6 glioma cells or primary cultured neurons were rinsed twice with PBS, and 10 µM KA (Sigma, St Louis, MO) was applied for different durations, then [3H]glutamate uptake was assayed and cell-surface biotin labeling was performed as described below.
Measurement of Na+-dependent transport activity
The transport assays were performed in C6 glioma cells as described (Davis et al., 1998). The cells were grown as a monolayer in 24-well plates and uptake of [3H]glutamate was measured while cells were in a 37°C water bath. For that, cells were incubated in choline buffer for 10 minutes followed by incubation with 10 nM [3H]glutamate (Amersham Biosciences, UK) and 10 µM unlabeled glutamate in sodium buffer or choline buffer for 10 minutes. Radioactive uptake was stopped by adding ice-cold choline buffer. Cells were then solubilized in 200 µl of 100 mM sodium hydroxide, and 100 µl of lysate was analyzed for radioactivity in a scintillation counter. The Na+-dependent uptake was defined as the difference in radioactivity accumulated in sodium buffer and in choline buffer. Under these conditions the uptake is linear with time. Protein content was measured using the BCA kit (Pierce, Rockford, IL). Data were processed and analyzed with unpaired t-test and are given as the mean ± s.e.m.
Cell surface biotinylation and western blotting
The amount of EAAC1 on the surface was determined as previously described (Fournier et al., 2004) with some modifications. Briefly, C6 glioma cells or primary cultured neurons were incubated with sulfo-NHS-biotin (Pierce) for 30 minutes at 4°C, then lysed for 1 hour in immunoprecipitation buffer (50 mM Tris, 5 mM EDTA, 5 mM EGTA pH 7.5) (Lin et al., 2001) containing complete protease inhibitor cocktail (Roche, Indianapolis, IN), 0.1% SDS and 1% Triton X-100. Protein concentrations were determined using the BCA kit, and equivalent amounts of protein were precipitated overnight with immunopure immobilized streptavidin (Pierce). After efficient washing (5 times) with washing buffer (50 mM Tris, 5 mM EDTA, 5 mM EGTA, 150 mM NaCl, 1% Triton X-100, pH 7.5), beads were incubated in SDS-PAGE loading buffer for 30 minutes at 50°C.
Samples were analyzed by on SDS-PAGE, proteins were transferred, probed with antibodies and visualized by enhanced chemiluminescence (Amersham Biosciences,). Antibodies against EAAC1 (1:1000, ADI, San Antonio, TX), syntaxin 1A (1:10,000; Synaptic Systems, Germany; 1:1000; Sigma) and actin (1:1000; Sigma) were used. Immunoreactive bands were quantified with ScionImage software (Scion, Frederick, MD) and quantification was based on at least three independent experiments. The data were processed and analyzed with unpaired t-test and are given as the mean ± s.e.m.
For the detection of surface EAAC1 and intracellular EAAC1, the C6 glioma cells were, 48 hours after transfection, incubated with the lysosomal and proteasomal degradation inhibitors, including leupeptin and MG132, for 6 hours followed by surface biotinylation. Pools of surface and intracellular EAAC1 were obtained by the following formula: [surface EAAC1] / [total EAAC1] or [intracellular EAAC1] / [total EAAC1], for which surface, intracellular and total EAAC1 concentrations were directly measured.
Quantitative real time reverse transcriptase PCR
Transfected C6 glioma cells were lysed in TRIzol reagent (Invitrogen). Total RNA was isolated and 4 µg were used to generate cDNA using random primers and Superscript III (Invitrogen). Real-time RT-PCR was performed on Mx3000P (Stratagene, La Jolla, CA). The following forward and reverse primer pairs were used for specific amplification: rat EAAC1, 5'-aacccttccagttacattcc-3' and 5'-aaacgcatcacccagaac-3'; rat TATA-box-binding protein, 5'-tgcacaggagccaagagtgaa-3' and 5'-cacatcacagctccccacca-3'. Expression values were normalized against those from control TATA-box-binding protein, and data were processed and analyzed with unpaired t-test and are given as the mean ± s.e.m.
Internalization of EAAC1
Reversible biotinylation was performed as previously described (Fournier et al., 2004). C6 glioma cells and primary cultured neurons were labeled with NHS-SS-biotin (Pierce) for 30 minutes at 4°C, and the biotinylation was stopped in PBS containing 100 mM glycine. At time zero (t=0), the cells left for the total pool of surface EAAC1 control and the strip control were kept at 4°C. Meanwhile, the cells for internalization analysis were incubated with pre-warmed (37°C) DMEM with or without 10 µM KA for different durations. To halt internalization, the cells were immerged in ice-cold sodium-Tris buffer (150 mM NaCl, 1 mM EDTA, 0.2% BSA, 20 mM Tris pH 8.6) for 10 minutes. The cell-surface-bound NHS-SS-biotin was then stripped by incubating cells in sodium-Tris buffer containing freshly dissolved 50 mM GSH for 40 minutes. Then the cells were lysed and the biotinylated proteins were isolated and analyzed as described above. Strip efficiencies were typically 90% of total labeled protein at t=0.
Delivery of EAAC1 to the cell surface
The amount of EAAC1 delivered to the cell surface was measured as previously described (Fournier et al., 2004) with some modifications. In brief, C6 glioma cells were incubated with sulfo-NHS-biotin for 10 minutes at 4°C. At t=0, the cells left for the measurement of the total pool of surface EAAC1 in the steady-state (not permissive to trafficking) were kept at 4°C. Meanwhile, cells for delivery analysis were quickly rinsed with pre-warmed DMEM, and then incubated under conditions permissive for trafficking (37°C) in the presence of sulfo-NHS-biotin and degradation inhibitors for different durations. Five percent of the intracellular lysate and 25% of the surface fraction were analyzed by western blotting as described above. The delivery efficiency (%) was obtained by the following formula: 100 x [Exo(t)/25%] / [Intra/5%]. The Exo(t) was calculated as (Tt-T0), of which T0 was the biotinylated transporter level directly measured in the steady-state, Tt was that measured at 5 or 30 minutes under trafficking permissive condition.
Immunoprecipitation
The cells were lysed in immunoprecipitate buffer, and the supernatants were incubated with mouse anti-EAAC1 (Chemicon, Temecula, CA) or mouse anti-syntaxin 1A antibodies overnight at 4°C, followed by incubation with protein A-sepharose beads (Amersham Biosciences). For the mouse brain, immunoprecipitation was performed as described previously (Lin et al., 2001). In summary, the hippocampus region was excised from the brain and washed in cold buffer A [50 mM Tris pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5 mM dithiothreitol (DTT)]. The tissue was weighed and then homogenized in ice-cold buffer B (50 mM Tris pH 7.5, 10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA, 0.5 mM DTT) containing protease inhibitors in a glass-Teflon homogenizer. The supernatant fraction was incubated with EAAC1 antibody or the preimmune serum. The immunoprecipitated proteins and 5% of total lysate were analyzed on the same gel by western blotting as described above.
Surface protein cross-linking with BS3
C6 glioma cells were collected and incubated with 2 mM BS3 (Pierce) to crosslink the surface proteins. Incubation proceeded for 1 hour at 4°C with gentle agitation. After rinsing with ice-cold PBS containing glycine to stop the crosslinking, the cells were lysed and immunoprecipitated with anti-EAAC1 antibody overnight at 4°C, followed by incubation with protein-A-sepharose beads. The surface EAAC1-syntaxin-1A complex was detected by EAAC1 and syntaxin 1A antibodies.
Immunocytochemistry
C6 glioma cells were grown to confluence treated with 10 µM KA for 30 minutes following pretreatment with 500 nM PMA (Davis et al., 1998; Furuta et al., 2003) and processed for immunofluorescence. Briefly, the cells were fixed in freshly prepared 4% paraformaldehyde in PBS for 10 minutes at 4°C, then permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature. Cells were incubated with antibodies against EAAC1 (1:100), and EEA1 (1:100, BD Biosciences, San Jose, CA) overnight, and followed by corresponding secondary antibodies conjugated to Fluorescein or Rhodamine (1:100; Jackson ImmunoResearch, West Grove, PA). LysoTracker Red DND99 dye (Invitrogen) was applied to the medium before cells were fixed. The images were acquired using a Leica SP2 confocal microscopy (Leica, Germany). The image analysis and algorithm generation were performed using the Image-Pro Plus 5.1 software (Media Cybernetics, Silver Spring, MD). Pearson's correlation is calculated according to the following formula:
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