Rapid Trafficking of the Neuronal Glutamate Transporter, EAAC1
2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês
10.1074/jbc.m404032200
ISSN1083-351X
AutoresKeith M. Fournier, Marco I. González, Michael B. Robinson,
Tópico(s)Ion channel regulation and function
ResumoThe neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, γ-aminobutyric acid. There is evidence for a large intracellular pool of EAAC1 from which transporter is redistributed to the cell surface following activation of protein kinase C (PKC) or platelet-derived growth factor (PDGF) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAAC1 to and from the plasma membrane and to examine the effects of phorbol ester and PDGF on these events. Biotinylation of cell surface protein under trafficking-permissive conditions (37 °C) resulted in a 2-fold increase in the amount of biotinylated EAAC1 within 15 min in C6 glioma and in primary neuronal cultures, suggesting that EAAC1 has a half-life of ∼5–7 min for residence at the plasma membrane. Both phorbol ester and PDGF increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAAC1 was observed in C6 glioma. Phorbol ester, but not PDGF, blocked this measure of internalization. Incubation at 18 °C, which blocks some forms of intracellular membrane trafficking, inhibited PKC- and PDGF-dependent redistribution of EAAC1 but had no effect on basal trafficking of EAAC1. These studies suggest that both PKC and PDGF accelerate delivery of EAAC1 to the cell surface and that PKC has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAAC1 exist in distinct compartments. The neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, γ-aminobutyric acid. There is evidence for a large intracellular pool of EAAC1 from which transporter is redistributed to the cell surface following activation of protein kinase C (PKC) or platelet-derived growth factor (PDGF) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAAC1 to and from the plasma membrane and to examine the effects of phorbol ester and PDGF on these events. Biotinylation of cell surface protein under trafficking-permissive conditions (37 °C) resulted in a 2-fold increase in the amount of biotinylated EAAC1 within 15 min in C6 glioma and in primary neuronal cultures, suggesting that EAAC1 has a half-life of ∼5–7 min for residence at the plasma membrane. Both phorbol ester and PDGF increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAAC1 was observed in C6 glioma. Phorbol ester, but not PDGF, blocked this measure of internalization. Incubation at 18 °C, which blocks some forms of intracellular membrane trafficking, inhibited PKC- and PDGF-dependent redistribution of EAAC1 but had no effect on basal trafficking of EAAC1. These studies suggest that both PKC and PDGF accelerate delivery of EAAC1 to the cell surface and that PKC has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAAC1 exist in distinct compartments. A family of Na+-dependent transporters both ensures appropriate excitatory signaling and limits the excitotoxic potential of glutamate in the mammalian central nervous system. This family includes members that are generally expressed in astrocytes (GLT-1 and GLAST) or neurons (EAAC1 and EAAT4) (for reviews, see Refs. 1Amara S.G. Sonders M.S. Zahniser N.R. Povlock S.L. Daniels G.M. Adv. Pharmacol. 1998; 42: 164-168Crossref PubMed Scopus (39) Google Scholar, 2Sims K.D. Robinson M.B. Crit. Rev. Neurobiol. 1999; 13: 169-197Crossref PubMed Scopus (146) Google Scholar, 3Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3668) Google Scholar). EAAC1 is enriched on the post-synaptic processes of pyramidal cells in cortex and hippocampus, two pathways that display remarkable synaptic plasticity and exquisite sensitivity to excitotoxic insults (4Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1443) Google Scholar, 5Maragakis N.J. Rothstein J.D. Arch. Neurol. 2001; 58: 365-370Crossref PubMed Scopus (256) Google Scholar). There is evidence that EAAC1 limits spillover between excitatory synapses in hippocampus (6Diamond J.S. J. Neurosci. 2001; 21: 8328-8338Crossref PubMed Google Scholar, 7Diamond J.S. Nat. Neurosci. 2002; 5: 291-292Crossref PubMed Scopus (46) Google Scholar). EAAC1 is also found on inhibitory interneurons (8Conti F. DiBiasi S. Minelli A. Rothstein J.D. Melone M. Cerebral Cortex. 1998; 8: 108-116Crossref PubMed Scopus (185) Google Scholar) where it appears to provide precursor for the synthesis of the inhibitory neurotransmitter, γ-aminobutyric acid (9Mathews G.C. Diamond J.S. J. Neurosci. 2003; 23: 2040-2048Crossref PubMed Google Scholar). Antisense oligonucleotide knockdown of EAAC1 disrupts γ-aminobutyric acid synthesis and causes an epilepsy-like phenotype (10Sepkuty J.P. Cohen A.S. Eccles C. Rafiq A. Behar K. Ganel R. Coulter D.A. Rothstein J.D. J. Neurosci. 2002; 22: 6372-6379Crossref PubMed Google Scholar). In addition to these physiologic roles, there is evidence that EAAC1 expression is altered under various pathologic conditions (11Crino P.B. Jin H. Shumate M.D. Robinson M.B. Coulter D.A. Brooks-Kayal A.R. Epilepsia. 2002; 43: 211-218Crossref PubMed Scopus (114) Google Scholar, 12Miller H.P. Levey A.I. Rothstein J.D. Tzingounis A.V. Conn P.J. J. Neurochem. 1997; 68: 1564-1570Crossref PubMed Scopus (124) Google Scholar, 13Ueda Y. Doi T. Tokumaru J. Yokoyama H. Nakajima A. Mitsuyama Y. Ohya-Nishiguchi H. Kamada H. Willmore L.J. J. Neurochem. 2001; 76: 892-900Crossref PubMed Scopus (98) Google Scholar, 14Rossi D.J. Oshima T. Attwell D. Nature. 2000; 403: 316-321Crossref PubMed Scopus (1198) Google Scholar). The activities of the GLUT4 subtype of glucose transporter and of several neurotransmitter transporters can be rapidly altered by changing the number of transporters present at the plasma membrane (for reviews, see Refs. 15Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (906) Google Scholar, 16Richerson G.B. Wu Y. J. Neurophysiol. 2003; 90: 1363-1374Crossref PubMed Scopus (249) Google Scholar, 17Beckman M.L. Quick M.W. J. Membr. Biol. 1998; 164: 1-10Crossref PubMed Scopus (77) Google Scholar, 18Robinson M.B. J. Neurochem. 2002; 80: 1-11Crossref PubMed Scopus (174) Google Scholar). In both C6 glioma and primary neuronal cultures, less than 30% of total EAAC1 is found on the cell surface, with the remainder on intracellular vesicles of unknown identity (19Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (115) Google Scholar, 20González M.I. Kazanietz M.G. Robinson M.B. Mol. Pharmacol. 2002; 62: 901-910Crossref PubMed Scopus (90) Google Scholar). Similarly, there is evidence for an intracellular pool of EAAC1 in vivo (8Conti F. DiBiasi S. Minelli A. Rothstein J.D. Melone M. Cerebral Cortex. 1998; 8: 108-116Crossref PubMed Scopus (185) Google Scholar). In C6 glioma, activation of PKC 1The abbreviations used are: PKC, protein kinase C; GLUT4, glucose transporter 4; PMA, phorbol 12-myristate 13-acetate; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; MesNa, 2-mercaptoethanesulfonic acid; BSA, bovine serum albumin; TfR, transferrin receptor; PBS, phosphate-buffered saline; ANOVA, analysis of variance; DAT, dopamine transporter; GAT-1, γ-aminobutyric acid transporter subtype 1; NET, norepinephrine transporter; NHS-biotin, N-hydroxysulfosuccinimidobiotin; SERT, serotonin transporter; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. 1The abbreviations used are: PKC, protein kinase C; GLUT4, glucose transporter 4; PMA, phorbol 12-myristate 13-acetate; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; DMEM, Dulbecco's modified Eagle's medium; MesNa, 2-mercaptoethanesulfonic acid; BSA, bovine serum albumin; TfR, transferrin receptor; PBS, phosphate-buffered saline; ANOVA, analysis of variance; DAT, dopamine transporter; GAT-1, γ-aminobutyric acid transporter subtype 1; NET, norepinephrine transporter; NHS-biotin, N-hydroxysulfosuccinimidobiotin; SERT, serotonin transporter; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. with PMA causes a rapid (within minutes) increase in EAAC1-mediated activity (21Dowd L.A. Robinson M.B. J. Neurochem. 1996; 67: 508-516Crossref PubMed Scopus (126) Google Scholar). This effect is associated with a ∼2-fold increase in cell surface EAAC1 that is independent of synthesis of new transporters but is instead related to a redistribution of EAAC1 from a subcellular compartment to the cell surface (22Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar). PDGF also causes an increase in both EAAC1-mediated activity and cell surface expression in C6 glioma that is blocked with inhibitors of PI3K. These effects of PDGF are not blocked by PKC antagonists, suggesting that two independent signaling pathways regulate EAAC1 trafficking (19Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (115) Google Scholar). Transfection of C6 glioma with the neurotensin receptor subtype 1 and activation of this receptor also cause a redistribution of EAAC1 that is not blocked by inhibitors of either PKC or PI3K (23Najimi M. Maloteaux J.M. Hermans E. FEB Lett. 2002; 523: 224-228Crossref PubMed Scopus (31) Google Scholar). There is evidence for an association between regulated trafficking of EAAC1 and models of learning and memory. Levenson and coworkers (24Levenson J. Weeber E. Selcher J.C. Kategaya L.S. Sweatt J.D. Eskin A. Nat. Neurosci. 2001; 5: 155-161Crossref Scopus (124) Google Scholar) have shown a redistribution of EAAC1 from an intracellular pool to the cell surface in both long term potentiation and contextual fear conditioning. However, the mechanisms by which these events regulate EAAC1 trafficking have not been identified. To begin to define the mechanisms by which PMA and PDGF might regulate EAAC1 cell surface expression, we examined the kinetics of EAAC1 trafficking to or from the cell surface and the effects of phorbol ester or PDGF on these events. Here we report that there is robust basal trafficking of the transporter and that both PMA and PDGF increased the delivery of EAAC1 to the membrane. PMA, but not PDGF, also inhibited net internalization of EAAC1. At a lower temperature (18 °C), basal delivery of EAAC1 to the plasma membrane was unaffected, but the cell surface increases induced by either PMA or PDGF were blocked. Basal delivery of EAAC1 to the plasma membrane was comparably rapid in primary neuronal cultures, where the cellular milieu is more likely to mimic that observed in vivo. Based on these findings, we conclude that: (i) EAAC1 trafficking to and from the cell surface in both C6 and neurons is rapid compared with many other membrane proteins; (ii) although both PKC and PDGF increase cell surface expression, they appear to have differential effects on delivery and internalization of the transporter; and (iii) basal and regulated delivery of EAAC1 to the plasma membrane may originate from distinct intracellular pools. Materials—C6 glioma were obtained from the American Type Culture Collection (Rockville, MD). DMEM, trypsin-EDTA, Neurobasal medium, B27 supplement, l-glutamine, and penicillin-streptomycin were purchased from Invitrogen. Fetal bovine serum was purchased from HyClone (Logan, UT). Culture plates were purchased from Corning (Cambridge, MA). GenePorter transfection reagent was purchased from Gene Therapy Systems (San Diego, CA). BSA, MesNa, PMA, and polyclonal anti-actin antibody were purchased from Sigma-Aldrich. PDGF was purchased from Calbiochem. Radioisotopes were from PerkinElmer Life Sciences. N-Hydroxysulfosuccinimidobiotin (NHS-biotin), N-hydroxysulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (NHS-SS-biotin), UltraLink immobilized monomeric avidin, and the bicinchoninic acid (BCA) protein assay kit were purchased from Pierce. Anti-rabbit and anti-mouse horseradish peroxidase IgG, rainbow molecular weight marker, and enhanced chemiluminescence kits were purchased from Amersham Biosciences. Monoclonal anti-transferrin receptor antibody was purchased from Zymed Laboratories Inc. laboratories (South San Francisco, CA). Polyclonal anti-EAAC1 and GLT1 antibodies were the kind gifts of Dr. Jeffrey D. Rothstein (Johns Hopkins University). Cell Culture—C6 glioma were grown in DMEM supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 at 37 °C. Cells were passaged less than 30 times and used between 75 and 85% confluence. There was no evidence for passage-dependent changes in morphology or effects measured in the present study. Multiple growth factors are present in fetal bovine serum, and, thus, to examine the effects of a single growth factor it was necessary to remove serum with preincubation for 2 h in DMEM containing (0.5%) BSA (19Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (115) Google Scholar). The cells were then rinsed twice with and treated in plain DMEM. Primary neuron-enriched cultures were derived from embryonic (days 18–19) Sprague-Dawley rat cortex. After removal of the meninges, cortex was isolated, trypsinized for 20 min at 37 °C, and triturated in a Pasteur pipette. The cells were then plated in 10-cm dishes at a density of 4 × 106 cells/dish. Neuronal cultures were maintained in Neurobasal medium supplemented with 2% B27 in an incubator with 5% CO2 at 37 °C. For all experiments, neurons were used at 14 days in vitro and treated in conditioned media. Transfection of C6 Glioma Cells—C6 glioma were grown to 40–60% confluence and transfected ∼24 h before experimental use. Cells were cotransfected with 2 μg of green fluorescent protein plasmid DNA and 8 μg of GLT-1 plasmid DNA using 50 μl of GenePorter cationic lipid reagent per the manufacturer's instructions. After a 3- to 5-h incubation with the transfection mixture in 5 ml of DMEM, 5 ml of modified medium (DMEM containing 20% fetal bovine serum, 4 mm glutamine, 200 units/ml penicillin, and 200 μg/ml streptomycin) was added to the mixture for an additional 15 h. The media was changed to C6 media prior to serum starvation and experimental use. Green fluorescent protein was included as a control for the homogeneity of transfection across multiple plates. Transfection efficiency was consistently between 10 and 15% of cells. Measurement of Na+-dependent Transport Activity—Na+-dependent l-Glu transport activity was measured as previously described (25Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar). Briefly, C6 glioma were grown in 12-well plates to 70–90% confluence, and cells were preincubated for 2 h in DMEM containing (0.5%) BSA prior to treatment. Cells were first rinsed twice (1 ml per well) with plain DMEM either pre-warmed to 37 °C or pre-cooled to 18 °C and treated with PDGF (10 μg/ml) or vehicle (4 mm HCl with 0.01% BSA) at these temperatures for 15 min. Both sets of cells were then rinsed twice with either sodium- or choline-containing buffer at 18 °C. Uptake was initiated by rinsing into the same buffers containing l-[3H]glutamate (0.5 μm) and terminated after 5 min by rinsing into ice-cold choline-containing buffer (3 washes), after which the cells were solubilized with 0.1 n NaOH. Radioactivity was determined using a Beckman scintillation counter. Na+-dependent transport activity was normalized to the amount of protein in each well. Biotinylation of Cell Surface Proteins—Both C6 glioma and neuronal cell monolayers were first rinsed twice with ice-cold PBS (pH 7.35) containing 0.1 mm CaCl2 and 0.1 mm MgCl2. The cells were then incubated in 2 ml of biotinylation solution (1 mg/ml NHS-biotin in PBS/Ca2+/Mg2+) for 30 min at 4 °C with gentle shaking. The biotinylation solution was removed, and excess biotinylating reagent was quenched by rinsing the cells twice with PBS/Ca2+/Mg2+ containing 100 mm glycine, and incubation in PBS/Ca2+/Mg2+/glycine for 30 min at 4 °C with gentle shaking. Cells were then rinsed twice with PBS/Ca2+/Mg2+ before being lysed in 1 ml of radioimmune precipitation assay buffer containing protease inhibitors for 30 min. The cells were scraped from the plates, and the lysates were centrifuged for 15 min at 12,500 rpm. After removal of the cellular debris, an aliquot of lysate was frozen for protein analysis. In addition, an aliquot was mixed with an equal volume of SDS-PAGE (4×) loading buffer and labeled as the “lysate” fraction. Another aliquot (300 μl) was incubated overnight with 250 μl of UltraLink monomeric avidin-coated Sepharose beads solution. After centrifugation for 15 min, an aliquot of the resulting supernatant was diluted into an equal volume of (4×) loading buffer and labeled as the “non-biotinylated” fraction. The beads were washed twice with radioimmune precipitation assay buffer, and then sequentially with a “high-salt” (50 mm Tris, 5 mm EDTA, 500 mm NaCl, 0.1% Triton X-100, pH 7.5) solution, and a “low-salt” solution (50 mm Tris, pH 7.5), with centrifugation between each wash as per the manufacturer's instructions. The beads were then incubated for 10 min at room temperature in (2×) SDS-PAGE loading buffer (600 μl) followed by an additional incubation of 30 min at 37 °C. After centrifugation, the resulting supernatant was collected as the “biotinylated” fraction. Delivery of EAAC1 to the Plasma Membrane—Biotinylation under trafficking-permissive conditions was carried out as follows. C6 glioma or primary neurons were rinsed twice with PBS/Ca2+/Mg2+ solution at 37 °Cor18 °C, and then incubated with 4 ml of biotinylation solution at the same temperature for different periods of time. In some experiments, cells were treated with either PMA or PDGF during biotinylation. After rinsing cells into ice-cold PBS/Ca2+/Mg2+ solution containing glycine to stop trafficking, biotinylated proteins were extracted as described above. Internalization of EAAC1—Reversible biotinylation was performed as previously described (26Loder M.K. Melikian H.E. J. Biol. Chem. 2003; 278: 22168-22174Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). After the preincubation with DMEM/BSA solution, cells were rinsed twice with PBS/Ca2+/Mg2+ solution at 4 °C and then cell surface proteins were labeled with the reversible biotinylating reagent NHS-SS-biotin (2 ml of 1 mg/ml). After 30 min, excess biotinylating reagent was quenched with PBS/Ca2+/Mg2+/glycine as described above. Cells were rapidly rinsed twice with pre-warmed (37 °C) plain DMEM and incubated for varying periods of time. To halt internalization, cells were rinsed twice with ice-cold sodium-Tris (NT) buffer (150 mm NaCl, 1 mm EDTA, 0.2% BSA, 20 mm Tris, pH 8.6) followed by an incubation for an additional 10 min. Cell surface-bound biotinylating reagent was stripped by incubating the cells twice for 25 min in NT buffer containing freshly dissolved 50 mm MesNa. MesNa was only used for 6 months after date of purchase. In each experiment, one plate labeled “No MesNa” did not undergo re-warming or subsequent stripping of biotinylating reagent to provide a measure of the total pool of cell surface transporter available for internalization. A second plate, labeled “t = 0,” did not undergo re-warming before having cell surface-bound biotinylating reagent stripped, to control for the efficiency of the MesNa reagent. Both of these controls were rinsed into NT buffer, and all stripping occurred at the same time. After removal of cell surface-bound biotinylating reagent, cells were rinsed twice with PBS/Ca2+/Mg2+ buffer, and biotinylated proteins were extracted as described above. Western Blot Analysis—Aliquots of lysate from individual samples containing equal amounts of protein (10–20 μg) were loaded in an 8% SDS-polyacrylamide gel. Equal volumes of all three fractions were analyzed, such that if the yield from the extraction of biotinylated proteins was 100%, the sum of the amount of immunoreactivity in the non-biotinylated and biotinylated fractions should equal the amount of immunoreactivity in the lysate. When the number of samples exceeded the number of lanes available in a single mini-gel, then two gels were prepared with aliquots of lysate from each sample present in both gels. Proteins were transferred to polyvinylidene difluoride membranes. These membranes were blocked with Tris buffer containing (0.1%) Tween 20 and 1% nonfat milk and probed with specific antibodies to EAAC1 (1:75), GLT-1 (1:10,000), or transferrin receptor (TfR: 1 μg/ml). All blots were also probed with an anti-actin antibody (1:5,000) to control for possible cell lysis prior to biotinylation. Proteins were visualized using ECL as per the manufacturer's instructions. Immunoreactivity was quantified using National Institutes of Health Image software (available at rsb.info.nih.gov/nih-image/). In some cases, different exposures of the film were used to quantify different immunoreactive bands to ensure that the signal was within the linear range. Immunoreactivity of each sample was normalized to the amount of actin in the lysate fraction of that sample from the same gel and expressed as a percentage of the amount of immunoreactivity observed in the control sample within each fraction. Data are presented as the (means ± S.E. of the mean) and compared using ANOVA. Time Course for PMA- and PDGF-induced Increases in Cell Surface EAAC1—Before determining if PMA or PDGF affect the rates of delivery or removal of EAAC1 from cell surface, it was necessary to examine the time course for the effects of these agents on cell surface expression. C6 glioma, a brain-derived cell line that endogenously expresses EAAC1 and none of the other Na+-dependent glutamate transporters (22Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar), were treated with the phorbol ester, PMA, for different periods of time. Cell surface expression of EAAC1 was examined using a membrane-impermeant biotinylating reagent followed by batch extraction of the biotinylated proteins with avidin-coated beads. Under control conditions, ∼20% of EAAC1 was found in the biotinylated fraction; this is consistent with the large pool of intracellular immunoreactivity observed with confocal microscopy (20González M.I. Kazanietz M.G. Robinson M.B. Mol. Pharmacol. 2002; 62: 901-910Crossref PubMed Scopus (90) Google Scholar, 22Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar). Under identical conditions, the same biotinylating reagent captures greater than 80% of the homologous transporter, GLT-1, providing evidence that the reaction conditions are appropriate for efficient labeling of cell surface proteins (27Kalandadze A. Wu Y. Robinson M.B. J. Biol. Chem. 2002; 277: 45741-45750Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). PMA caused a significant increase in EAAC1 cell surface expression within 2 min and a nearly 2-fold increase in cell surface expression within 15 min (Fig. 1, A and B). The change in surface expression was maximal at 15 min and was not significantly different from control with 60 min of treatment. PDGF increased cell surface expression of EAAC1 with a similar time-course having a maximal effect at 15 min. 2A. L. Sheldon and M. B. Robinson, unpublished observations. Notably, neither PMA nor PDGF caused a complete redistribution of EAAC1 from the intracellular pools to the cell surface. It is not known if this observation is related to “desensitization/down-regulation” of the signaling pathway or depletion of a specific “regulated pool” of EAAC1 that can be redistributed to the cell surface. These studies identify 15 min as a critical time for determining if PMA or PDGF affect delivery or removal of EAAC1 from the cell surface. Rate of Delivery of EAAC1 to the Cell Surface under Basal Conditions—Other membrane proteins, including the TfR, DAT, GAT-1, the choline transporter, and GLUT4, have been reported to recycle rapidly to and from the cell surface (for reviews, see Refs. 15Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (906) Google Scholar, 18Robinson M.B. J. Neurochem. 2002; 80: 1-11Crossref PubMed Scopus (174) Google Scholar, 28Buckley K.M. Melikian H.E. Provoda C.J. Waring M.T. J. Physiol. 2000; 525: 11-19Crossref PubMed Scopus (50) Google Scholar). To measure the rate of delivery of EAAC1 to the cell surface, C6 glioma were incubated under trafficking permissive conditions (37 °C) in the presence of the biotinylating reagent. Under these conditions, the membrane-impermeant biotinylating reagent should label transporters that cycle through the plasma membrane. In each experiment, the amount of EAAC1 that was biotinylated under conditions not permissive to trafficking (4 °C) was also examined, and the amount of transporter delivered to the cell surface was expressed as a percentage of this steady-state measure of transporter surface expression. At 37 °C, there was a time-dependent increase in the amount of biotinylated protein (Fig. 2, A and B). There was no evidence for changes in the amount of biotinylated actin, providing evidence that this increase in biotinylated EAAC1 cannot be attributed to labeling of intracellular pools of EAAC1. The increase in biotinylated EAAC1 was accompanied by a decrease in the amount of transporter in the non-biotinylated fraction and no change in the total amount of EAAC1 immunoreactivity in the cell lysates (Fig. 2, A and B). Within 15 min, there was nearly a 2-fold increase in the amount of biotinylated EAAC1, implying that the entire population of cell surface transporter was replaced over this time period to maintain the steady-state level of cell surface EAAC1. There was a continued increase in the amount of biotinylated transporter at 30 and 60 min consistent with this rapid rate of turnover of cell surface EAAC1. To test the validity of this approach, the rate of delivery of TfR was examined. Consistent with previous studies in other cellular systems, the amount of biotinylated TfR increased in a time-dependent manner with nearly the entire population being labeled within 30 min (Fig. 2C). Although the acylation reaction between the activated carboxylic acid (NHS-ester) of the biotinylating reagent and the primary amine (typically lysine) from a cell surface protein is thought to be quite fast (29Morpurgo M. Bayer E.A. Wilchek M. J. Biochem. Biophys. Methods. 1999; 38: 17-28Crossref PubMed Scopus (57) Google Scholar, 30Miller B.T. Collins T.J. Rogers M.E. Kurosky A. Peptides. 1997; 18: 1585-1595Crossref PubMed Scopus (38) Google Scholar), labeling of transporter that cycles through the plasma membrane may not be 100% efficient. Therefore, these values may represent an underestimate of the rate of delivery/removal of transporter to and from the cell surface under basal conditions but are consistent with a half-life for turnover of no longer than ∼5–7 min. Rate of Internalization of EAAC1—As a complementary approach, EAAC1 internalization was examined using a reversible biotinylation strategy. C6 glioma cells were incubated with a disulfide bond-containing biotinylating reagent at 4 °C to label cell surface proteins. After re-warming to trafficking-permissive conditions for various periods of time, cells were cooled and cell surface-bound biotinylating reagent was removed by treatment with MesNa, a membrane-impermeant reducing agent. Using this approach, 21.5 ± 6.9% of EAAC1 was biotinylated at steady state, consistent with our other studies; this sets the upper limit for the amount of transporter that could become inaccessible to MesNa (internalized). With no re-warming, slightly more than 80% of the biotinylating reagent can be stripped (Fig. 3, A and B; 0 min at 37 °C before MesNa). The 20% of biotinylating reagent that cannot be removed with MesNa is presumably related to incomplete stripping of cell surface biotinylating reagent by MesNa, but may also be related to inaccessibility of this pool to MesNa (31Schmidt A. Hannah M.J. Huttner W.B. J. Cell Biol. 1997; 137: 445-458Crossref PubMed Scopus (103) Google Scholar). With this approach, we observed a time-dependent increase in the amount of biotinylated transporter that became inaccessible to MesNa (Fig. 3, A and B). In contrast to the half-life estimated from measurement of delivery of transporter to the cell surface, only slightly greater than 50% of the biotinylated EAAC1 appeared to be internalized within 15 min. It is possible that internalized transporter rapidly recycles, that C6 glioma contain a reducing environment that cleaves the disulfide bond in the spacer arm of the biotinylating reagent, or that having only 20% of the transporter on the cell surface under steady state limits detection of internalized transporter at early time points. To address these possibilities, C6 glioma cells were treated with PMA to increase the pool of EAAC1 available for internalization from the cell surface. After incubation with PMA for 15 min, cells underwen
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