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3776 Research Article Introduction Glutamate transporters mediate high-affinity excitatory neurotransmitter reuptake, the fundamental mechanism maintaining extracellular glutamate levels, preventing excitotoxicity and averting neural damage associated with epilepsy (Danbolt, 2001; 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 Ca 2+ 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 The neuronal glutamate transporter, excitatory amino- acid carrier 1 (EAAC1), plays an important role in the modulation of neurotransmission and contributes to synthesis of the inhibitory neurotransmitter - 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. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/18/3776/DC1 Key words: EAAC1, Syntaxin 1A, Endocytic sorting, Glutamate transport, Kainic acid, Surface expression Summary Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport Yong-Xin Yu 1,2 , Li Shen 1 , Peng Xia 1 , Ya-Wei Tang 1 , Lan Bao 1, * and Gang Pei 1, * 1 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences and 2 Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China *Authors for correspondence (e-mail: [email protected]; [email protected]) Accepted 22 June 2006 Journal of Cell Science 119, 3776-3787 Published by The Company of Biologists 2006 doi:10.1242/jcs.03151 Journal of Cell Science

Syntaxin 1A promotes the endocytic sorting of EAAC1 ... · The neuronal glutamate transporter, excitatory amino-acid carrier 1 (EAAC1), plays an important role in the modulation of

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Page 1: Syntaxin 1A promotes the endocytic sorting of EAAC1 ... · The neuronal glutamate transporter, excitatory amino-acid carrier 1 (EAAC1), plays an important role in the modulation of

3776 Research Article

IntroductionGlutamate transporters mediate high-affinity excitatoryneurotransmitter reuptake, the fundamental mechanismmaintaining extracellular glutamate levels, preventingexcitotoxicity and averting neural damage associated withepilepsy (Danbolt, 2001; 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 astroglialglutamate–aspartic-acid transporter (GLAST) and theglutamate transporter-1 (GLT-1), the neuronal EAAT4 andEAAT5 in the mammalian central nervous system (Danbolt,2001; Sims and Robinson, 1999). EAAC1 is most notablylocated in the somata, dendrites and axons of many neurons,especially those in the hippocampus and cerebellum (Furuta etal., 1997; He et al., 2000; Rothstein et al., 1994). Strikingly,EAAC1 protein is also highly concentrated in presynapticGABAergic terminals, where it participates in theneurosynthesis of �-aminobutyric acid (GABA) (Conti et al.,1998; He et al., 2000; Mathews and Diamond, 2003; Rothsteinet al., 1994). Genetic EAAC1 suppression markedly leads toepileptiform phenotypes, characterized by electrographicseizures, staring-freezing episodes and paresis (Rothstein et al.,1996; Sepkuty et al., 2002), underscoring the important role ofthis neuronal transporter in neurological disorders. Therefore,EAAC1 activity markedly affects neurotransmitterhomeostasis and synaptic transmission.

Considering its central role in shaping glutamatergic and

GABAergic signaling, the neuronal glutamate transporterEAAC1 is a likely target for cellular regulation. Indeed,activation of protein kinase C (PKC) or platelet-derived growthfactor receptor accelerates the delivery of EAAC1 to the cellsurface and, at the same time, activation of PKC also inhibitsthe internalization of EAAC1 (Fournier et al., 2004). In thekainic acid (KA)-kindled model of epilepsy, the expression ofEAAC1 is downregulated in the dentate gyrus, entorhinalcortex layer and hippocampus (Furuta et al., 2003; Ghijsen etal., 1999; Gorter et al., 2002; Simantov et al., 1999), especiallywith perinuclear deposits of EAAC1 (Furuta et al., 2003).Given the determinant role of trafficking in regulation ofneurotransmitter transporters (Deken et al., 2000; Geerlings etal., 2001; Robinson, 2002), a greater understanding of themolecules that regulate EAAC1 trafficking is likely to shedlight on this pivotal synaptic modulator.

Syntaxin 1A is a neuronal plasma membrane protein thatbelongs to the soluble N-ethylmaleimide-sensitive factorattachment protein receptor (SNARE) family (Bennett et al.,1993). Syntaxin 1A is involved in vesicle trafficking, dockingand/or fusion, and plays a key role in neurotransmitter release(Sudhof, 2000). Syntaxin 1A also directly interacts with andfunctionally regulates ion channels such as Ca2+ channels(Sheng et al., 1994), cystic fibrosis transmembrane regulatorCl– channels (Naren et al., 1997), K+ channels (Fili et al.,2001), epithelial Na+ channels (Saxena et al., 1999), andNa+/Cl– dependent transporters, such as the GABA (Deken etal., 2000), norepinephrine (Sung et al., 2003), serotonin (Haase

The neuronal glutamate transporter, excitatory amino-acid carrier 1 (EAAC1), plays an important role inthe modulation of neurotransmission and contributesto synthesis of the inhibitory neurotransmitter ��-aminobutyric acid (GABA) and to epileptogenesis.However, the mechanisms that regulate EAAC1 endocyticsorting and function remain largely unknown. Here, wefirst demonstrate that EAAC1 undergoes internalizationthrough the clathrin-mediated pathway and further showthat syntaxin 1A, a key molecule in synaptic exocytosis,potentiates EAAC1 internalization, thus leading to thefunctional inhibition of EAAC1. In the presence of thetransmembrane domain of syntaxin 1A, its H3 coiled-coildomain of syntaxin 1A is necessary and sufficient for theinhibition of EAAC1. Furthermore, specific suppression of

endogenous syntaxin 1A significantly blocked EAAC1endocytic sorting and lysosomal degradation promoted bykainic acid, a drug for kindling the animal model of humantemporal lobe epilepsy in rat, indicating a potential role ofsyntaxin 1A in epileptogenesis. These findings provide newevidence that syntaxin 1A serves as an intrinsic enhancerto EAAC1 endocytic sorting and further suggest thatsyntaxin 1A is conversant with both ‘ins’ and ‘outs’ ofsynaptic neurotransmission.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/18/3776/DC1

Key words: EAAC1, Syntaxin 1A, Endocytic sorting, Glutamatetransport, Kainic acid, Surface expression

Summary

Syntaxin 1A promotes the endocytic sorting of EAAC1leading to inhibition of glutamate transportYong-Xin Yu1,2, Li Shen1, Peng Xia1, Ya-Wei Tang1, Lan Bao1,* and Gang Pei1,*1Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy ofSciences and 2Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People’s Republic of China*Authors for correspondence (e-mail: [email protected]; [email protected])

Accepted 22 June 2006Journal of Cell Science 119, 3776-3787 Published by The Company of Biologists 2006doi:10.1242/jcs.03151

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et al., 2001) and glycine transporters (Geerlings et al., 2000).A recurring theme in these modulations is that channels andtransporters are redistributed to and from the plasma membrane(Deken et al., 2000). However, whether syntaxin 1A modulatestrafficking of the transporters for excitatory neurotransmittersremains an open question and the underlying mechanisms havenot been elucidated.

Here, we show that syntaxin 1A specifically potentiatesthe clathrin-mediated and dynamin-dependent EAAC1internalization, and consequently leads to functional inhibitionof glutamate transport. Furthermore, syntaxin 1A functions asan intrinsic enhancer to the EAAC1 endocytic sorting evokedby KA, a drug used broadly in kindling the animal model ofhuman epilepsy. Our findings indicate a new mechanism ofsyntaxin 1A in regulating the trafficking of membrane proteinsand suggest a potential role of syntaxin 1A in epileptogenesis.

ResultsSyntaxin 1A specifically potentiates the EAAC1endocytic sortingIt is well known that syntaxin 1A regulates the trafficking ofseveral Na+/Cl–-dependent transporters, such as GABA orglycine transporters (Deken et al., 2000; Geerlings et al., 2001).Here, we assessed in C6 glioma cells the effect of syntaxin 1Aon the trafficking of EAAC1, a Na+/K+-dependent neuronalglutamate transporter. It is established that the C6 glioma cellline endogenously expresses syntaxin 1A (supplementarymaterial Fig. 1A) and EAAC1, but not other glutamatetransporters (Davis et al., 1998; Dowd et al., 1996; Paloset al., 1996). This cell line has thus been used as a modelsystem to study the regulation of EAAC1 activity andsurface expression (Robinson, 2002). Our results showedthat the surface expression of endogenous EAAC1decreased to 58.3±2.1% in syntaxin1A-transfected C6glioma cells compared with �-gal-transfected controlcells (Fig. 1A). In addition, the change of EAAC1homomer was identical to that of EAAC1 monomer (Fig.1A), excluding the possibility that EAAC1 was

redistributed between monomer and homomer. Although therewas a concurrent overall decrease of EAAC1 protein (Fig. 1A),the unchanged mRNA levels of EAAC1 excluded the possibleeffects of syntaxin1A on EAAC1 gene expression (Table 1).However, it is difficult to tease out whether the decreasedEAAC1 surface expression is due to the altered overall EAAC1expression or trafficking. To address this issue, we treated thecells with degradation inhibitors 48 hours after transfection.Thereby, we inhibited EAAC1 degradation and detected thechanges in the pool of surface and intracellular EAAC1, whichwere defined as the ratio of surface or intracellular EAAC1versus total amount of EAAC1 protein expression. Our datashowed that in syntaxin-1A-transfected C6 glioma cells thesurface EAAC1 pool was decreased, while the intracellularEAAC1 pool was increased (Table 1), suggesting that thedecreased EAAC1 surface expression was due to the alteredEAAC1 trafficking. These results provide compelling evidencethat syntaxin 1A plays a regulatory role in EAAC1 trafficking.

Considering the effect of syntaxin 1A on the delivery ofseveral 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 thispossibility by examining the delivery efficiency of EAAC1. Tomeasure the delivery of EAAC1 to the cell surface, C6 gliomacells 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

Fig. 1. Syntaxin 1A facilitates EAAC1 endocytic sorting.(A) Representative immunoblot and quantitative analysisshowing that in C6 glioma cells transfected with syntaxin 1A(Syn1A), the expression of endogenous EAAC1 is decreasedin both total lysate and cell surface fractions. Actin served asan internal control for protein loading. Data are plotted as apercentage of EAAC1 in the cells transfected with �-galwithin each fraction. **P<0.01 compared with cellstransfected with �-gal (n=3). (B) Representative immunoblotand quantitative analysis show that the delivery efficiency ofEAAC1 is not altered in Syn1A-transfected C6 glioma cells.The data are plotted as a percentage of the increased cellsurface fraction versus the intracellular EAAC1 pool availablefor surface delivery (n=3). (C) Representative immunoblot andquantitative analysis of internalized EAAC1 show that theinternalization of EAAC1 is potentiated in Syn1A-transfectedC6 glioma cells (upper panel), despite syntaxin 1A decreasingthe overall EAAC1 surface pool available for internalization(lower panel). The band intensities for the internalizedEAAC1 are plotted as a percentage of the total surfaceEAAC1 available for internalization within each transfectiongroup. **P<0.01 compared with the cells transfected with �-gal at the same time point (n=3).

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surface (Fournier et al., 2004). In each experiment, the amountof EAAC1 biotinylated under conditions not permissive totrafficking (4°C) was also examined, and the EAAC1 deliveryefficiency was expressed as a percentage of transportersdelivered to the cell surface versus the intracellular pool ofEAAC1. Our results showed that the delivery efficiency ofEAAC1 was not altered in syntaxin-1A-transfected C6 gliomacells (Fig. 1B), suggesting that syntaxin 1A regulates theendocytic sorting of EAAC1.

Further studies from the reversible biotinylation confirmedthat, compared with cells only expressing endogenous syntaxin1A, the amount of internalized EAAC1 was more significantin C6 glioma cells transfected with syntaxin 1A (19.7±1.9%versus 36.8±3.0% at 5 minutes, and 26.4±3.3% versus61.8±3.5%, respectively, at 30 minutes, P<0.01,) (Fig. 1C),despite the reduction in overall EAAC1 available forinternalization. In addition, the effect of syntaxin 1A onEAAC1 internalization was unique, because neither theastroglia transporter GLT-1 nor EAAT4 (another neuronalglutamate transporter) showed changes of surface expressionby syntaxin 1A in the co-transfected human embryonic kidney293 (HEK293) cells (supplementary material Fig. 1B,C).Moreover, this unique regulatory role of syntaxin 1A was alsosupported by the findings that neither syntaxin 4 (anothersyntaxin isoform localized on the cell surface) nor otherneuronal SNAREs, including SNAP-25 and VAMP-2,modulated the surface expression of EAAC1 (supplementarymaterial Fig. 1D). These results suggest that the decreasedEAAC1 surface expression is attributable to the promotedinternalization of the transporter by syntaxin 1A rather than thealtered delivery rate of EAAC1 to the cell surface. Thus,syntaxin 1A serves as an enhancer to EAAC1 endocyticsorting, leading to the reduction of EAAC1 resident on the cellsurface.

Syntaxin 1A decreases EAAC1-mediated glutamatetransportGiven that the regulated trafficking of neurotransmittertransporters contributes to the tuning of their transportactivities (Robinson, 2002), we then tested whether syntaxin1A modulated the transport activity of EAAC1 by studying thesodium-dependent transport of [3H]glutamate in C6 gliomacells. Glutamate transport was significantly decreased(64.4±9.4%) in cells transfected with syntaxin 1A (Fig. 2A),compared with cells that only expressed endogenous syntaxin1A. Furthermore, we carried out kinetics analyses to evaluatethe biochemical nature of the altered transport activity. Thesyntaxin-1A-transfected cells showed a decrease in maximalvelocity (Vmax=348 pmol/mg protein/minute) without a shift inaffinity (Km=30.0 �M), compared with those cells onlyexpressing endogenous syntaxin 1A (Vmax=526 pmol/mgprotein/minute, Km=31.0 �M) (Fig. 2B). These data suggestthat the decrease in transport activity is attributable to the

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reduction of the transporters remaining on the surface, whichsupports the finding that syntaxin 1A downregulates EAAC1surface expression.

On the basis of these results, we thought that syntaxin 1Amight tonically modulate EAAC1 activity. To test this, we usedsyntaxin-1A-specific small interference RNA (siRNA) tosuppress its endogenous expression in C6 glioma cells.Notably, the endogenous syntaxin 1A expression wassuppressed specifically by its siRNA in C6 glioma cells(supplementary material Fig. 1A). Our data showed that theglutamate transport activity was significantly elevated(117.9±1.6%) (Fig. 2C), correlating with the reduction inendogenous syntaxin 1A protein levels. Kinetics analyses ofEAAC1 transport activity showed an increase in maximalvelocity (Vmax=521 pmol/mg protein/minute) without a shift inaffinity (Km=27.5 �M), compared with the cells transfectedwith nonspecific siRNA (Vmax=414 pmol/mg protein/minute,Km=26.6 �M), which served as a control for the homogeneityof transfection (Fig. 2D). Coherently, we found that aftersuppression of endogenous syntaxin 1A EAAC1 surfaceexpression was significantly increased (supplementary materialFig. 2A), without altering the EAAC1 gene expression (Table1). Furthermore, the surface EAAC1 pool was increased (Table1), consistent with the observation that in cells whoseendogenous syntaxin 1A expression was suppressedinternalization of EAAC1 was strongly impaired comparedwith cells transfected with the nonspecific siRNA (at 5minutes: 8.2±1.4% compared with 14.0±2.2%, respectively,P<0.05; at 30 minutes: 11.7±2.1% compared with 29.9±4.2%,respectively, P<0.01) (supplementary material Fig. 2B). Thesedata indicate that syntaxin 1A functions as an intrinsicregulator for EAAC1 internalization leading to thedownregulation of glutamate transport.

We then used HEK293 cells to detect the unique regulationof syntaxin 1A on EAAC1. The results also confirmed theprogressive decrease in EAAC1 transport activity whenexpression of syntaxin 1A was increased (Fig. 2E). Moreover,syntaxin 1A did not alter the transport activity of EAAT4 orGLT-1 in the co-transfected HEK293 cells (Fig. 2F). Thisunique regulatory role of syntaxin 1A was also supported bythe findings that the EAAC1 transport activity was notmodulated by several other SNARE proteins, includingsyntaxin 4, SNAP-25 and VAMP-2 (Fig. 2G). Taken together,our studies imply that syntaxin 1A specifically and negativelymodulates EAAC1 transport activity by facilitating EAAC1internalization.

EAAC1 internalization is mediated by clathrin-coatedpits in coordination with syntaxin 1AUntil now the endocytic pathway for EAAC1 remainedunclear. We therefore inquired whether EAAC1 isinternalized via the clathrin-mediated endocytic pathway, aclassic internalization pathway for several membrane

Table 1. Effects of syntaxin 1A on EAAC1 trafficking in transfected C6 glioma cellsEAAC1 mRNA Surface pool Intracellular pool

Transfection (% of control) (% of control) (% of control)

Syntaxin 1A 105.8±9.7 (4) 43.0±3.4** (4) 133.5±7.4** (4)Syntaxin 1A siRNA 110.9±5.5 (5) 164.0±13.7* (3) 74.6±6.9* (3)

*P<0.05, **P< 0.01 compared with mock-transfected control cells. Numbers in parentheses give numbers of independent observations.

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transporters (Daniels and Amara, 1999; Deken et al., 2003).We used hypertonic media to induce abnormal clathrinpolymerization. To control for the effect of sucrose onexocytosis (Li et al., 2001), we first incubated C6 glioma cellsfor 15 minutes in high-potassium buffer and then for 5minutes with various concentrations of sucrose (Deken et al.,2003). Surface biotinylation analyses showed that the surfaceexpression of EAAC1 was increased after sucrose treatment,and incubation with 0.45 M sucrose caused an approximatelytwofold increase in EAAC1 surface expression (Fig. 3A),whereas incubation with 0.15 M and 0.6 M sucrose caused alesser increase (155.1±11.6%, 119.0±20.3%, respectively). Inparallel, the transport activity of EAAC1 was significantlyincreased after incubation with 0.45 M sucrose (Fig. 3A). Due

to the multiple cellular effects of hypertonicity on exocytosisand endocytosis (Heuser and Anderson, 1989), we thenexamined the effect of K44A dynamin, a dominant-negativeform of dynamin that inhibits endocytosis via clathrin-coatedpits (Damke et al., 1994), on EAAC1 internalization. Wetransfected wild-type dynamin and the K44A dynamin intoC6 glioma cells to detect the surface expression of EAAC1.Our results showed that cells transfected with wild-typedynamin displayed a significant decrease in the surfaceexpression of EAAC1, whereas transfection with K44Adynamin led to the reinstallation of the EAAC1 surfaceexpression, indicating that EAAC1 internalization is impaired(Fig. 3B, Table 2). Moreover, the functional uptake ofglutamate mediated by EAAC1 was decreased by transfection

Fig. 2. Syntaxin 1A specifically inhibits EAAC1-mediated glutamate transport. In C6 glioma cells, the transfection of Syn1A and specificSyn1A siRNA (Syn1A siRNA) are indicated (A-D). In HEK293 cells, Syn1A are co-transfected pairwise with EAAC1, EAAT4 and GLT1;EAAC1 is co-transfected pairwise with syntaxin 4, SNAP-25 and VAMP-2 as indicated (E-G). (A) Syntaxin 1A significantly inhibits EAAC1transport activity in C6 glioma cells. **P<0.01 compared with the cells transfected with �-gal (n=4). (B) Kinetics analysis of glutamatetransport in C6 glioma cells transfected with Syn1A. (Left panel) Saturation analysis of glutamate transport shows that Syn1A decreasestransport maximal velocity compared with the cells transfected with �-gal, whereas Km is nearly identical. (Right panel) Eadie-Hofsteetransformations of these data (n=4). (C) Specific siRNA of Syn1A increases EAAC1 transport activity in C6 glioma cells. **P<0.01 comparedwith the cells transfected with non-specific siRNA (NS siRNA) (n=5). (D) Kinetics analysis of glutamate transport in C6 glioma cellstransfected with Syn1A siRNA. (Left panel) Saturation analysis shows that Syn1A siRNA increases EAAC1 transport maximal velocitycompared with the cells transfected with NS siRNA, whereas Km is nearly identical. (Right panel) Eadie-Hofstee transformations of these data(n=5). (E) EAAC1 transport activity is decreased progressively with increasing amounts of Syn1A plasmid. The HEK293 cells are triple-transfected with EAAC1, Syn1A, and �-gal expression plasmids as indicated. **P<0.01 compared with control (n=3). (F) Syn1A has no effecton the transport activity of EAAT4 or GLT1 (n=3). (G) The transport activity of EAAC1 is not regulated by syntaxin 4, SNAP-25 or VAMP-2(n=3).

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of dynamin and kept unchangeable by transfection of K44Adynamin (Table 2), consistent with the levels in EAAC1surface expression. Therefore, these data suggest that EAAC1is internalized in a clathrin-mediated and dynamin-dependentmanner.

To further distinguish the mechanism of the syntaxin-1A-potentiated EAAC1 internalization, we suppressed endogenoussyntaxin 1A expression in dynamin-transfected C6 gliomacells. Our data showed that suppression of endogenoussyntaxin 1A significantly rescued the dynamin-induceddecrease in EAAC1 surface expression (9.8±6.5% decreasecompared with 50.4±3.4% decrease in cells transfected withnonspecific siRNA, P<0.01) (Fig. 3C). In addition, syntaxin1A siRNA had no effect on dynamin expression (data notshown). Thus, these data suggest that syntaxin 1A coordinateswith dynamin for EAAC1 internalization.

The H3 coiled-coil and transmembrane domains ofsyntaxin 1A are necessary and sufficient for interactionwith and inhibition of EAAC1There is no evidence that syntaxin 1A endogenously interactswith EAAC1, especially in mammalian cells. Therefore, weexamined the endogenous interaction between syntaxin 1A andEAAC1 to establish a possible molecular mechanismunderlying the syntaxin-1A-mediated modulation of EAAC1.Initial experiments were performed in HEK293 cells and, asshown in Fig. 4A, syntaxin 1A was coimmunoprecipitated withEAAC1 in the cell extract prepared from the co-transfectedcells and vice versa. To further study the protein interaction invivo, we tested the association of syntaxin 1A with EAAC1 inthe hippocampus extract of the mice and found that syntaxin1A coimmunoprecipitated with EAAC1 (Fig. 4B). Theidentical result was obtained from C6 glioma cells,

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endogenously expressing these two molecules. These dataconfirm that syntaxin 1A interacts with EAAC1 in mammaliansystem, especially in vivo. Considering its central role inregulating the endocytic sorting of EAAC1, syntaxin 1A mightassociate with EAAC1 on the cell surface. Therefore, we usedBS3, an impermeable regent that crosslinks the surface proteinsto determine the association of proteins on the cell surface. Asshown in Fig. 4C, a high-molecular-mass band of ~100 kDawas detected in the BS3-crosslinked samples by EAAC1 andsyntaxin 1A antibodies, suggesting that the association ofEAAC1 and syntaxin 1A is on the cell surface. Thus, syntaxin1A interacts with EAAC1 on the cell surface and intrinsicallyregulates EAAC1 endocytic sorting.

We next constructed a series of syntaxin 1A mutants withdomain truncations to map the region where syntaxin 1A bindswith EAAC1 (Fig. 4D). Our data revealed that the syntaxin 1Amutant containing the coiled-coil H3 domain and thetransmembrane domain (amino acids 194-288; Syn1A H3-TMD) interacted with EAAC1 (Fig. 4E), whereas neither themutant without the H3 domain (Syn1A �H3) nor the mutantonly containing the H3 domain (amino acids 194–266, Syn1AH3) interacted with EAAC1 (data not shown). In order todissect the function of those domains, we examined the effectof different syntaxin 1A mutants on EAAC1 transport activityand surface expression. Our data showed that HEK293 cellsco-transfected with Syn1A H3-TMD showed an approximately42% reduction in EAAC1-mediated glutamate transport,comparable to the reduction induced by syntaxin 1A. NeitherEAAC1 co-transfected with Syn1A �H3 nor co-transfectedwith Syn1A H3 resulted in the reduction of glutamate transport(Fig. 4F). And the EAAC1 surface expression was alsosignificantly decreased in the cells co-transfected with Syn1AH3-TMD (Fig. 4F). All the mutants had a comparable

Fig. 3. Regulation of the clathrin-mediated EAAC1internalization by syntaxin 1A. (A) Representativeimmunoblot and quantitative analysis showing thathypertonic medium containing 0.45 M sucroseincreases EAAC1 surface expression and glutamatetransport activity. **P<0.01 compared with thecontrol (n=3). (B) The C6 glioma cells are transfectedwith �-gal, wild-type dynamin (Dyn), or K44Adynamin (K44A) constructs. Representativeimmunoblot showing that the EAAC1 surfaceexpression is decreased by transfection of dynaminbut not by K44A dynamin. (C) Representativeimmunoblot of surface biotinylated-EAAC1 showsthat Syn1A siRNA significantly rescues the dynamin-induced decrease in EAAC1 surface expression.

Table 2. Effects of dynamin on EAAC1 surface expression and transport activity in transfected C6 glioma cellsTotal EAAC1 Surface EAAC1 Glutamate uptake

Transfection (% of control) (% of control) (% of control)

Dynamin 104.9±5.0 47.7±4.1** 56.7±8.3**K44A dynamin 92.5±8.2 101.7±4.7 103.5±4.6

**P<0.01 compared with mock-transfected control cells. Surface biotinylation, n=4; glutamate uptake, n=3.

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expression level (data not shown). In addition, the C6 gliomacells transfected with hemagglutinin (HA)-tagged syntaxin 1Adisplayed a similar inhibition in EAAC1-mediated glutamateuptake (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 demonstratethat the H3 and transmembrane domains of syntaxin 1A arenecessary and sufficient for its interaction with EAAC1,leading to the downregulation of EAAC1 surface expressionand transport activity.

Fig. 4. Syntaxin 1A H3 and transmembrane domains mediate the association with EAAC1. (A) Coimmunoprecipitation (IP) andimmunoblotting (IB) show that in HEK293 cells co-transfected with Syn1A and EAAC1, Syn1A is found in EAAC1-antibody-precipitatedproteins and vice versa. The lysate and the immunoprecipitated proteins are analyzed on the same gel. Data represent three independentexperiments. (B) Coimmunoprecipitation and immunoblotting show that in the mouse hippocampal extracts and C6 glioma cells, Syn1A isfound in the EAAC1 antibody-precipitated proteins and indicate that Syn1A interacts with EAAC1 in vivo. The lysate and theimmunoprecipitated proteins are analyzed on the same gel. The data represent three independent experiments. (C) Representative immunoblotshowing that a high-molecular-mass band at ~100 kDa is detected by both EAAC1 (left panel) and Syn1A (right panel) antibodies in EAAC1antibody-precipitated proteins from the C6 glioma cells cross-linked with BS3. Data represent three independent experiments. (D) Diagramillustrates wild-type and mutants of Syn1A including H3 and transmembrane domains (Syn1A H3-TMD), H3 domain (Syn1A H3), and H3domain deletion (Syn1A �H3), and summarizes the results of the interaction and regulation of EAAC1 by these molecules tagged with HA inHEK293 cells. (E) In HEK293 cells co-transfected with EAAC1 and HA-Syn1A H3-TMD, HA-immunoreactive band is found at the positionof Syn1A H3-TMD in EAAC1-antibody-precipitated protein. The lysate and the immunoprecipitated proteins are analyzed on the same gel.The data represent three independent experiments. (F) [3H]glutamate uptake shows that Syn1A H3-TMD has a similar inhibition on EAAC1transport activity as Syn1A, whereas Syn1A �H3 and Syn1A H3 have no effect on EAAC1 transport activity. **P<0.01 compared with thecells co-transfected with �-gal (n=3). Consistently, representative immunoblot shows that the cell surface expression of EAAC1 is decreased byco-transfection of Syn1A H3-TMD, whereas Syn1A �H3 and Syn1A H3 have no effect on EAAC1 surface expression.

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Requirement of syntaxin 1A in KA-promotedinternalization of EAAC1Recent findings suggest that EAAC1 accumulates in theintracellular compartment in KA-kindled epilepsy animalmodel (Furuta et al., 2003). However, regulation of EAAC1internalization and the underlying molecular mechanisms arestill unknown and, therefore, we investigated the regulation ofEAAC1 internalization upon stimulation with KA using cellsurface biotinylation and reversible biotinylation assay. Weobserved that in C6 glioma cells there was an approximately17% and 48% reduction in EAAC1 surface expression with 10�M 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-minutestimulation with 10 �M KA (Fig. 5B). Furthermore, the effectof KA on EAAC1 surface expression was strongly antagonizedby K44A dynamin (Fig. 5A), implying that KA promoted thedynamin-dependent internalization of EAAC1. Indeed,compared with untreated control cells, we observed adramatically promoted internalization of EAAC1 that occurredwith KA stimulation at 5 minutes (33.6±2.3% versus50.8±3.0%, P<0.01) and a more significant response at 30minutes (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 functionalinhibition of EAAC1.

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To examine whether syntaxin 1A participates in the KA-promoted internalization of EAAC1, we suppressed theendogenous syntaxin 1A expression and found that the KA-promoted EAAC1 internalization was significantly abolished,despite the increasing pool of EAAC1 available forinternalization (Fig. 5E, Table 3). Consequently, there wasmore EAAC1 remaining on the cell surface upon KAstimulation after the suppression of endogenous syntaxin 1A(Fig. 5D, Table 3). These results indicate that syntaxin 1A isintrinsically involved in KA-promoted EAAC1 internalization.

Syntaxin 1A enhances EAAC1 sorting via the endosomeand/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 internalizedEAAC1 to accumulate on the perinuclear vesicular structureand to display extensive colocalization with the earlyendosome 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 stainingof EAAC1 along the surface at 30 minutes (Fig. 6A,B). Bymarked contrast, there was far less colocalization of EAAC1with EEA1 (Rr=0.3473) or with LysoTracker Red DND-99(Rr=0.2478) without KA stimulation, concurrent with astronger staining of EAAC1 along the cell surface (Fig. 6A,B).

Fig. 5. Essential role of syntaxin 1A in KA-promoted EAAC1 internalization. (A)Representative immunoblot and quantitativeanalysis showing that stimulation with 10 �MKA causes a decrease in EAAC1 surfaceexpression in C6 glioma cells, which isrescued by transfection of K44A dynamin.*P<0.05, **P<0.01 compared with the cellswithout KA stimulation (n=3). (B) Stimulationwith KA for 30 minutes inhibits the EAAC1-mediated glutamate transport in C6 gliomacells (upper panel), which is correlated with adecrease in the surface expression of EAAC1(lower panel). **P<0.01 versus the cellswithout KA stimulation (n=3). (C)Representative immunoblot and quantitativeanalysis of internalized EAAC1 upon KAstimulation for various time show constitutiveinternalization of EAAC1, and that KAstimulation significantly promotes EAAC1internalization in C6 glioma cells. **P<0.01compared with cells without KA stimulation atidentical time points (n=4). (D) Representativeimmunoblot of surface EAAC1 expressionupon KA stimulation for various time showingthat Syn1A siRNA rescues KA-inducedreduction in EAAC1 surface expression in C6glioma cells. (E) Representative immunoblotof internalized EAAC1 upon KA stimulationfor various time showing that Syn1A siRNAabolishes the KA-promoted EAAC1internalization in C6 glioma cells.

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These results further support that KA stimulation promotes theEAAC1 internalization and indicate that the internalizedEAAC1 undergoes the endocytic sorting pathway from earlyendosomes to lysosomes.

Given that the internalized transporters are driven into theendosomal 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 thenstimulated for 6 hours with 10 �M KA with or withoutlysosomal degradation inhibitors at 37°C. Long-term KAstimulation significantly decreased the amount of biotinylatedEAAC1 (51.1±3.3% of overall surface EAAC1 at 6 hours), and

Table 3. Syntaxin 1A is involved in the KA-induced internalization of EAAC1Surface EAAC1 (% of control) Internalized EAAC1†

Transfection KA 5 minutes KA 30 minutes KA 5 minutes KA 30 minutes

NS siRNA 71.4±1.9* 55.3±2.3** 35.5±8.0 58.6±8.7Syntaxin 1A siRNA 97.3±4.7 91.5±5.2 12.4±4.2*** 18.8±5.4****

*P<0.05, **P<0.01 compared with cells without KA stimulation (n=3). ***P<0.05, ****P<0.01 compared with cells transfected with NS siRNA after 5-minute and 30-minute stimulation with KA, respectively (n=3).†Levels of internalized EAAC1 are plotted as percentage of total surface EAAC1 available for internalization within each transfection group.

Fig. 6. KA stimulation promotes thedegradation of internalized EAAC1 inthe lysosome. (A,B) Double-immunofluorescence labeling showingthat internalized EAAC1 is colocalizedwith the early endosomal marker EEA1(A) and the lysosomal probeLysoTracker Red DND-99 (B) in C6glioma cells upon a 30-minutestimulation with 10 �M KA. Bars, 10�m. Scatterplot graphs of the individualpixels from the paired images and thePearson’s correlations calculated byImage-Pro-Plus software, suggestingthat the colocalization of EAAC1 withEEA1 or Lysotracker Red DND-99 issignificantly enhanced after KAstimulation. (C) Representativeimmunoblot and quantitative analysisshow that incubation with 10 �M KAfor 6 hours leads to the degradation ofthe internalized EAAC1, which isblocked by the lysosomal degradationinhibitor leupeptin (100 �M) or thecompartment acidification blockersNH4Cl (50 mM) and chloroquine (200�M). **P<0.01 compared with thecells without KA stimulation (n=4). (D)Representative immunoblot andquantitative analysis showing that upon10 �M KA stimulation the degradationof the internalized EAAC1 increases ina time-dependent manner. Additionally,Syn1A siRNA blocks the KA-promotedEAAC1 degradation. *P<0.05,**P<0.01 compared with the cellstransfected with NS siRNA (n=4).

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was totally blocked by the lysosomal protease inhibitorleupeptin (112.5±7.5%) and the lysosomotropic amineschloroquine (104.5±10.3%), and partially by ammoniumchloride (NH4Cl) (94.0±13.5%) (Fig. 6C). These resultsconfirm that KA stimulation causes degradation of EAAC1 bytrafficking to lysosomes.

We then explored the role of syntaxin 1A in the lysosomaldegradation of EAAC1. In C6 glioma cells treated with KA,the biotinylated EAAC1 at the surface was degraded in a time-dependent manner, whereas suppression of endogenoussyntaxin 1A expression markedly blocked the KA-inducedEAAC1 degradation (Fig. 6D). Thus, the rescue of EAAC1from degradation might be attributable to the impaired EAAC1internalization. Our studies provide the first identification ofthe endosome and/or lysosome pathway for EAAC1 traffickingand suggests that syntaxin 1A is pivotal for endocytic sortingof EAAC1.

Effect of KA on EAAC1 endocytic sorting in primaryneuronal culturesOur studies strongly suggest that KA stimulates EAAC1internalization, leading to degradation in lysosomes in the C6glioma cells. Furthermore, we assessed the effect of KA onendocytic sorting of EAAC1 in primary neuronal cultures. Weobserved that, compared with untreated control cells, inprimary cultured hippocampal neurons the internalization

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of EAAC1 was dramatically promoted after 5-minute KAstimulation (36.9±2.6% versus 75.3±4.6%, P<0.01) and evenmore 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 asystem that has a cellular environment, presumably more likethat observed in vivo.

DiscussionSyntaxin 1A potentiates the clathrin-mediatedinternalization of EAAC1Our study provides the first direct evidence that EAAC1, aneuronal glutamate transporter, undergoes clathrin-mediatedand dynamin-dependent internalization, and then reveals anunanticipated function for syntaxin 1A as a crucial regulatorfor EAAC1 internalization. It is well established that syntaxin1A is a key player in the synaptic exocytosis (Sudhof, 2000),but our study demonstrates that syntaxin 1A also functions asan intrinsic enhancer to EAAC1 endocytic sorting. Althoughthe precise mechanism of syntaxin 1A in EAAC1internalization is yet still unknown, it has been reported thatsyntaxin 1A is associated with dynamin in adrenal chromaffincells (Galas et al., 2000) and yeast (Peters et al., 2004), andalso with synaptotagmin, which is involved in the assembly ofclathrin-coated pits (Chapman et al., 1995; Haucke and DeCamilli, 1999; von Poser et al., 2000). Therefore, we canspeculate that syntaxin 1A functions as a potential scaffoldprotein 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 EAAC1is co-internalized with syntaxin 1A through their interaction.Our results are consistent with the notion that syntaxin 1A isconversant with both ‘ins’ and ‘outs’ of chemical signaling inthe brain (Blakely and Sung, 2000).

Several early studies suggest that the surface expression ofsome neurotransmitter transporters can be regulated by anumber of signaling molecules under physiological conditions(Robinson, 2002), and some researchers have isolated thetransporter-containing synaptic-like vesicles, which arepotential mediators of transporter trafficking in axon terminals(Deken et al., 2003; Geerlings et al., 2001). Hence, syntaxin1A might play a necessary role in the exocytosis of thesetransporters, which is consistent with its role as a regulator ofmembrane fusion. Although our current study showed thatoverexpression of syntaxin 1A had no effect on theconstitutive exocytosis of EAAC1, syntaxin 1A could beinvolved in regulated exocytosis when some signalingpathways are activated. Recent studies show that EAAC1 isredistributed to the cell surface after the activation ofsignalling cascades, such as the PKC, tyrosine kinase andphosphatidylinositol 3-kinase pathways (Robinson, 2002).Furthermore, the basal and regulated delivery of EAAC1 tothe cell surface originates from distinct intracellular pools(Fournier et al., 2004). Thus, whether syntaxin 1A is requiredfor those regulations is worthy of further investigation, and ourstudies raise interesting questions about the direction ofEAAC1 trafficking in different physiological and pathologicalsituations.

Fig. 7. KA stimulates EAAC1 internalization in primary culturedneurons. (A) Representative immunoblot and quantitative analysis ofinternalized EAAC1 upon KA stimulation in the primary culturedhippocampal neurons for various time showing that there is aconstitutive internalization of EAAC1 that is significantly promotedby KA stimulation. *P<0.05, **P<0.01 compared with the cellswithout KA stimulation at identical time points (n=3). (B)Representative immunoblot and quantitative analysis show thatstimulation with 10 �M KA decreases EAAC1 surface expression inthe primary cultured hippocampal neurons. *P<0.05, **P<0.01compared with the cells without KA stimulation (n=3).

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Essential role of syntaxin 1A in the regulation of EAAC1-mediated glutamate transportIn a recent study, syntaxin 1A has been shown to reduce theEAAC1-mediated current in Xenopus oocytes (Zhu et al.,2005). Here, we revealed that syntaxin 1A negatively regulatesthe EAAC1-mediated glutamate transport in the mammaliansystem, and further demonstrated that the syntaxin-1A-induceddecrease in glutamate uptake is associated with the reduced cellsurface expression of EAAC1 in virtue of the promotedinternalization. Moreover, specific suppression of endogenoussyntaxin 1A protein expression improved EAAC1 transportactivity, indicating that syntaxin 1A serves as an intrinsicregulator of EAAC1-mediated glutamate transport. Althoughthe degree of the decreased EAAC1 surface expression was tooexcessive to account for the decrease in glutamate uptake, thisdiscrepancy might be reconciled if the catalytic efficiency ofEAAC1 is also regulated, or if some silent transporters areinternalized from the cell surface (Somwar et al., 2001). Recentstudies suggest that the cell surface expression and the intrinsicactivity of some transporters can be independently regulated,suggesting that this dual mode of regulation is a generalphenomenon (Gonzalez et al., 2002).

We further demonstrate that the interaction between syntaxin1A and EAAC1 is pivotal for this inhibition, by finding thatthe syntaxin 1A H3-domain-deletion mutant cannot properlyassociate with EAAC1 and failed to downregulate EAAC1transport activity. However, because of its mis-localization(data not shown), the H3 domain by itself could not induceEAAC1 inhibition without the assistance of the transmembranedomain (Kasai and Akagawa, 2001; Lewis et al., 2001). In fact,our data show that, at least in the presence of the H3 andtransmembrane domains, syntaxin 1A associated with EAAC1on the cell surface, leading to the reduction of its surfaceexpression and the inhibition of glutamate transport. Thus,these observations suggest that syntaxin 1A plays an essentialrole in modulating glutamate transport by regulating EAAC1trafficking.

Until now, few molecules have been reported to directlyregulate the function of the glutamate transporter. Recently, anEAAC1-associated protein, GTRAP3-18 has been identified todownregulate EAAC1 transport activity (Lin et al., 2001).Since GTRAP3-18 modulates EAAC1 transport activity bylowering substrate affinity without altering EAAC1 trafficking(Lin et al., 2001), our findings suggest a distinct mechanismby which syntaxin 1A, another EAAC1-associated protein,potentiates EAAC1 trafficking and inhibits EAAC1-mediatedglutamate transport without altering its substrate affinity.

A possible functional link between syntaxin 1A andepilepsyIt is well known that EAAC1 is localized in inhibitoryGABAergic neurons (Conti et al., 1998; He et al., 2000;Rothstein et al., 1994), where glutamate is converted to theimportant inhibitory neurotransmitter GABA, and thus servesas a major supply for GABA synthesis. In epilepsy, EAAC1expression levels are known to be altered, which is considereda key factor in epileptogenesis (Ghijsen et al., 1999; Gorter etal., 2002; Simantov et al., 1999). The decreased expressionlevels of EAAC1 not only lead to the dysfunction in clearingsynaptic glutamate but also result in the impairment of GABAsynthesis (Maragakis and Rothstein, 2004; Sepkuty et al.,

2002), and thus disrupts the balance between glutamate andGABA in the synaptic cleft. Furthermore, previous studieshave shown that hippocampal GABA levels are reduced to 50%in EAAC1-knockdown mice (Rothstein et al., 1996; Sepkutyet al., 2002) and the neuropil staining of EAAC1 is decreasedin the KA-kindled rat epilepsy model (Furuta et al., 2003). Ourcurrent findings illustrate that KA induced a decrease inEAAC1 surface expression and an increase in EAAC1endocytic sorting, and lysosomal degradation both in C6glioma cells and in primary cultured neurons, suggesting apossible mechanism in KA-kindled epilepsy. However, themolecular mechanism in KA-promoted endocytic sorting ofEAAC1 remains an open question. Our unpublished data showthat KA also concurrently induced syntaxin 1A to accumulatealong the cell surface, possibly because KA functions as theagonist of the kainite receptor, a subtype of the ionotropicglutamate receptor, to induce Ca2+ influx and evoke exocytosis(Huettner, 2003). It might be that accumulation of syntaxin 1Aalong the cell surface greatly facilitates its association withEAAC1 and thus promotes the EAAC1 internalization andlysosomal degradation.

Increasing evidence show that the dysfunctions inendocytosis are common themes in many neurologicaldisorders, especially neurodegenerative diseases (Cataldo etal., 2000; Nixon, 2005; Smith et al., 2005; Vanoni et al., 2004),thus interference of abnormal endocytosis may be potentialtherapeutic strategies in neurological disease. Our studydemonstrates that syntaxin 1A is an intrinsic regulator of KA-induced EAAC1 endocytic sorting. It further reveals thatsuppression of expression of endogenous syntaxin 1Asignificantly antagonized the KA-promoted EAAC1internalization and degradation and, hence, largely reinstatedEAAC1 expression at the surface, implying a promisingapproach for the treatment of epilepsy. However, owing to themultiple roles of syntaxin 1A in synaptic activity, the specificdisruption of the association between syntaxin 1A and EAAC1by the competing peptide seems to be an even better solution.

In conclusion, the results presented here demonstrate thatsyntaxin 1A, the key molecule for exocytosis, is cruciallyinvolved in modulating the endocytic sorting of the neuronalglutamate transporter EAAC1. Thus, our study not onlydelineates a regulatory mechanism for the internalization of aspecific cell surface transporter but also sheds light onunderstanding epileptogenesis.

Materials and MethodsPlasmidsPlasmids cDNA encoding syntaxin 1A and its different truncation mutants weregifts from Kevin L. Kirk (University of Alabama at Birmingham, AL) andAnjaparavanda P. Naren (University of Tennessee, Memphis, TN), and weresubcloned into modified pcDNA3 vector in-frame with HA at the N-terminus. ThesiRNA plasmid for syntaxin 1A was constructed as previously described (Sun et al.,2002). The sequences of the siRNA for rat syntaxin 1A were taken from GenBank(accession number NM_053788, nucleotides 471-492). The sequence of thenonspecific siRNA was 5�-ggccgcaaagaccttgtcctta-3�.

Cell culture and transfectionC6 glioma cells and human embryonic kidney 293 (HEK293) cells were obtainedfrom American Type Culture Collection (Manassas, VA). The C6 glioma cells weremaintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with5% fetal bovine serum (both Invitrogen, Carlsbad, CA). The constructed cDNA andthe siRNA constructs were transfected into C6 glioma cells with Lipofectamine orlipofectamine 2000 (Invitrogen) as described previously (Xu et al., 2004). HEK293cells were maintained in minimal essential medium (MEM) (Invitrogen)supplemented with 10% fetal bovine serum. Transient transfection of HEK293 cells

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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 20minutes 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 allexperiments, neurons were used at 14 days in vitro.

Drug treatmentC6 glioma cells or primary cultured neurons were rinsed twice with PBS, and 10�M KA (Sigma, St Louis, MO) was applied for different durations, then[3H]glutamate uptake was assayed and cell-surface biotin labeling was performedas described below.

Measurement of Na+-dependent transport activityThe 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, cellswere incubated in choline buffer for 10 minutes followed by incubation with 10 nM[3H]glutamate (Amersham Biosciences, UK) and 10 �M unlabeled glutamate insodium buffer or choline buffer for 10 minutes. Radioactive uptake was stopped byadding ice-cold choline buffer. Cells were then solubilized in 200 �l of 100 mMsodium hydroxide, and 100 �l of lysate was analyzed for radioactivity in ascintillation counter. The Na+-dependent uptake was defined as the difference inradioactivity accumulated in sodium buffer and in choline buffer. Under theseconditions the uptake is linear with time. Protein content was measured using theBCA kit (Pierce, Rockford, IL). Data were processed and analyzed with unpairedt-test and are given as the mean ± s.e.m.

Cell surface biotinylation and western blottingThe amount of EAAC1 on the surface was determined as previously described(Fournier et al., 2004) with some modifications. Briefly, C6 glioma cells or primarycultured neurons were incubated with sulfo-NHS-biotin (Pierce) for 30 minutes at4°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 inhibitorcocktail (Roche, Indianapolis, IN), 0.1% SDS and 1% Triton X-100. Proteinconcentrations were determined using the BCA kit, and equivalent amounts ofprotein were precipitated overnight with immunopure immobilized streptavidin(Pierce). After efficient washing (5 times) with washing buffer (50 mM Tris, 5 mMEDTA, 5 mM EGTA, 150 mM NaCl, 1% Triton X-100, pH 7.5), beads wereincubated in SDS-PAGE loading buffer for 30 minutes at 50°C.

Samples were analyzed by on SDS-PAGE, proteins were transferred, probedwith antibodies and visualized by enhanced chemiluminescence (AmershamBiosciences,). 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 withScionImage software (Scion, Frederick, MD) and quantification was based on atleast three independent experiments. The data were processed and analyzed withunpaired t-test and are given as the mean ± s.e.m.

For the detection of surface EAAC1 and intracellular EAAC1, the C6 glioma cellswere, 48 hours after transfection, incubated with the lysosomal and proteasomaldegradation inhibitors, including leupeptin and MG132, for 6 hours followed bysurface biotinylation. Pools of surface and intracellular EAAC1 were obtained bythe following formula: [surface EAAC1] / [total EAAC1] or [intracellular EAAC1]/ [total EAAC1], for which surface, intracellular and total EAAC1 concentrationswere directly measured.

Quantitative real time reverse transcriptase PCRTransfected C6 glioma cells were lysed in TRIzol reagent (Invitrogen). Total RNAwas isolated and 4 �g were used to generate cDNA using random primers andSuperscript III (Invitrogen). Real-time RT-PCR was performed on Mx3000P(Stratagene, La Jolla, CA). The following forward and reverse primer pairs wereused 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 againstthose from control TATA-box-binding protein, and data were processed andanalyzed with unpaired t-test and are given as the mean ± s.e.m.

Internalization of EAAC1Reversible 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 PBScontaining 100 mM glycine. At time zero (t=0), the cells left for the total pool ofsurface EAAC1 control and the strip control were kept at 4°C. Meanwhile, the cellsfor internalization analysis were incubated with pre-warmed (37°C) DMEM withor without 10 �M KA for different durations. To halt internalization, the cells wereimmerged 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 wasthen stripped by incubating cells in sodium-Tris buffer containing freshly dissolved50 mM GSH for 40 minutes. Then the cells were lysed and the biotinylated proteinswere isolated and analyzed as described above. Strip efficiencies were typically 90%of total labeled protein at t=0.

Delivery of EAAC1 to the cell surfaceThe amount of EAAC1 delivered to the cell surface was measured as previouslydescribed (Fournier et al., 2004) with some modifications. In brief, C6 glioma cellswere incubated with sulfo-NHS-biotin for 10 minutes at 4°C. At t=0, the cells leftfor the measurement of the total pool of surface EAAC1 in the steady-state (notpermissive to trafficking) were kept at 4°C. Meanwhile, cells for delivery analysiswere quickly rinsed with pre-warmed DMEM, and then incubated under conditionspermissive for trafficking (37°C) in the presence of sulfo-NHS-biotin anddegradation inhibitors for different durations. Five percent of the intracellular lysateand 25% of the surface fraction were analyzed by western blotting as describedabove. The delivery efficiency (%) was obtained by the following formula: 100 �[Exo(t)/25%] / [Intra/5%]. The Exo(t) was calculated as (Tt–T0), of which T0 wasthe biotinylated transporter level directly measured in the steady-state, Tt was thatmeasured at 5 or 30 minutes under trafficking permissive condition.

ImmunoprecipitationThe cells were lysed in immunoprecipitate buffer, and the supernatants wereincubated with mouse anti-EAAC1 (Chemicon, Temecula, CA) or mouse anti-syntaxin 1A antibodies overnight at 4°C, followed by incubation with proteinA-sepharose beads (Amersham Biosciences). For the mouse brain,immunoprecipitation was performed as described previously (Lin et al., 2001). Insummary, the hippocampus region was excised from the brain and washed in coldbuffer 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 (50mM Tris pH 7.5, 10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA, 0.5 mMDTT) containing protease inhibitors in a glass-Teflon homogenizer. The supernatantfraction was incubated with EAAC1 antibody or the preimmune serum. Theimmunoprecipitated proteins and 5% of total lysate were analyzed on the same gelby western blotting as described above.

Surface protein cross-linking with BS3

C6 glioma cells were collected and incubated with 2 mM BS3 (Pierce) to crosslinkthe 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 cellswere lysed and immunoprecipitated with anti-EAAC1 antibody overnight at4°C, followed by incubation with protein-A–sepharose beads. The surfaceEAAC1–syntaxin-1A complex was detected by EAAC1 and syntaxin 1A antibodies.

ImmunocytochemistryC6 glioma cells were grown to confluence treated with 10 �M KA for 30 minutesfollowing pretreatment with 500 nM PMA (Davis et al., 1998; Furuta et al., 2003)and processed for immunofluorescence. Briefly, the cells were fixed in freshlyprepared 4% paraformaldehyde in PBS for 10 minutes at 4°C, then permeabilizedwith 0.2% Triton X-100 for 10 minutes at room temperature. Cells were incubatedwith antibodies against EAAC1 (1:100), and EEA1 (1:100, BD Biosciences, SanJose, CA) overnight, and followed by corresponding secondary antibodiesconjugated to Fluorescein or Rhodamine (1:100; Jackson ImmunoResearch, WestGrove, PA). LysoTracker Red DND99 dye (Invitrogen) was applied to the mediumbefore cells were fixed. The images were acquired using a Leica SP2 confocalmicroscopy (Leica, Germany). The image analysis and algorithm generation wereperformed using the Image-Pro Plus 5.1 software (Media Cybernetics, Silver Spring,MD). Pearson’s correlation is calculated according to the following formula:

where S1 is the signal intensity of pixels in the first channel and S2 the signalintensity of pixels in the second channel, S1av and S2av are average intensities offirst and second channels, respectively.

We thank Kevin L. Kirk for providing syntaxin 1A mutant cDNAs,Anjaparavanda P. Naren for providing syntaxin 1A cDNA, and JianFei for providing EAAC1 and GLT-1 cDNAs. We also appreciate thehelp of Nan-Jie Xu for critical comments on the manuscript, and Ya-Lan Wu, Shun-Mei Xin, Xiao-Hui Zhao and Yu-Ting Li for theirtechnical assistance. This work was supported by grants from Ministryof Science and Technology (2003CB515405 and 2005CB522406), theNational Natural Science Foundation of China (30021003,30325024), Chinese Academy of Sciences (KSCX1-SW), ShanghaiScience and Technology Committee (03DZ19213).

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Page 12: Syntaxin 1A promotes the endocytic sorting of EAAC1 ... · The neuronal glutamate transporter, excitatory amino-acid carrier 1 (EAAC1), plays an important role in the modulation of

3787Syntaxin 1A promotes EAAC1 endocytic sorting

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