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• W2005242158 endingPage "17024" @default.
• W2005242158 startingPage "17017" @default.
• W2005242158 abstract "Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na++ K+)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning α-helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the non-transportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step. Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na++ K+)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning α-helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the non-transportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step. Uptake of glutamate into nerve terminals and glial cells serves to keep its extracellular concentration below neurotoxic levels and helps in conjunction with diffusion to terminate its action in synaptic transmission (cf. 20Kanner B.I. Schuldiner S. CRC Crit. Rev. Biochem. 1987; 22: 1-38Crossref PubMed Scopus (404) Google Scholar; 25Nicholls D.G. Attwell D. Trends Pharmacol. Sci. 1990; 11: 462-468Abstract Full Text PDF PubMed Scopus (974) Google Scholar; Mennerick and Zorumski, 1994; Tong and Jahr, 1994). The process is catalyzed by electrogenic sodium- and potassium-coupled L-glutamate transporters. Although not established definitely, the stoichiometry is likely to be two sodium ions accompanying each glutamate anion, while one potassium and one hydroxyl ion are transported out (Kanner and Sharon, 1978; 35Stallcup W.B. Bulloch K. Baetge E.E. J. Neurochem. 1979; 32: 57-65Crossref PubMed Scopus (77) Google Scholar; 6Bouvier M. Szatkowski M. Amato A. Attwell D. Nature. 1992; 335: 433-435Google Scholar). Three related glutamate transporters (∼55% homology) have been cloned from rat brain, GLAST (36Storck T. Schulte S. Hofmann K. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10955-10959Crossref PubMed Scopus (1098) Google Scholar), EAAC-1 (17Kanai Y. Hediger M.A. Nature. 1992; 360: 467-471Crossref PubMed Scopus (1198) Google Scholar), and GLT-1 (28Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1136) Google Scholar). These three glutamate transporters form a small family which also includes a sodium-coupled small neutral amino acid transporter (1Arriza J.L. Kavanaugh M.P. Fairman W.A. Wu Y.-N. Murdoch G.H. North R.A. Amara S.G. J. Biol. Chem. 1993; 268: 15329-15332Abstract Full Text PDF PubMed Google Scholar; 34Shafqat S. Tamarappoo B.K. Kilberg M.S. Puranam R.S. McNamara J.D. Guadano-Ferraz A. Fremeau Jr., R.T. J. Biol. Chem. 1993; 268: 15351-15355Abstract Full Text PDF PubMed Google Scholar) as well as bacterial dicarboxylic acid and proton-coupled glutamate transporters (15Jiang J. Gu B. Albright L.M. Nixon B.T. J. Bacteriol. 1989; 171: 5244-5253Crossref PubMed Google Scholar; 38Tolner B. Poolman B. Wallace B. Konings W. J. Bacteriol. 1992; 174: 2391-2393Crossref PubMed Google Scholar). The GLT-1 protein has been purified to near homogeneity and reconstituted (9Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar, 10Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (370) Google Scholar). It represents around 0.6% of the protein of crude synaptosomal fractions (9Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar) and is exclusively located in fine astroglial process of rat brain (10Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (370) Google Scholar). The cloning and characterization of the human homologues of these proteins has recently been described (2Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar). The relative role of diffusion and uptake of L-glutamate in the determination of its time course in the synaptic cleft is a subject of intensive investigation (14Isaacson J.S. Nicoll R.A. J. Neurophys. 1993; 70: 2187-2191Crossref PubMed Scopus (131) Google Scholar; 33Sarantis M. Ballerini L. Miller B. Silver R.A. Edwards M. Attwell D. Neuron. 1993; 11: 541-549Abstract Full Text PDF PubMed Scopus (149) Google Scholar; 24Mennerick S. Zorumski C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (290) Google Scholar; 5Barbour B. Keller B.U. Llano I. Marty A. Neuron. 1994; 12: 1331-1343Abstract Full Text PDF PubMed Scopus (316) Google Scholar; 39Tong G. Jahr C.E. Neuron. 1994; 13: 1195-1203Abstract Full Text PDF PubMed Scopus (306) Google Scholar). The turnover number of the transporter has been estimated to be only a few per second (21Kanner B.I. Sharon I. Biochemistry. 1978; 17: 3949-3953Crossref PubMed Scopus (261) Google Scholar). This is much too slow to account for the decay of L-glutamate in hippocampal synapses (8Clements J.D. Lester R.A.J. Tong G. Jahr C.E. Westbrook G.L. Science. 1992; 258: 1498-1501Crossref PubMed Scopus (805) Google Scholar). However, recent evidence indicates a possible role of glutamate reuptake in this process, and it was pointed out that the transporters could do so by binding the transmitter rapidly (39Tong G. Jahr C.E. Neuron. 1994; 13: 1195-1203Abstract Full Text PDF PubMed Scopus (306) Google Scholar). Binding of glutamate, a partial reaction of transport, could be much faster than the overall transport cycle. In order to probe the feasibility of this concept, we have investigated the conformational changes of the (Na++ K+)-coupled glutamate transporter GLT-1, expected to occur during its translocation cycle. Its susceptibility to trypsin, in the presence and absence of its substrates, was monitored using sequence-directed antibodies. Our data indicate that substrate binding causes changes in trypsin-sensitive sites throughout the protein and also provide the first experimental data on its topology. We provide evidence that there are at least two distinct transporter-glutamate complexes. In the first the position of glutamate can be occupied by the non-transportable analogue dihydrokainate. The transporter-glutamate complex, but not the transporter-dihydrokainate complex, can undergo a sodium-dependent conformational change which represents or is highly associated with the transport step. Standard proteins for Tricine1 1The abbreviations used are: TricineN-tris(hydroxymethyl)methylglycinePAGEpolyacrylamide gel electrophoresisGABAγ-aminobutyric acid. -SDS-PAGE and Sephadex G-50 were obtained from Pharmacia LKB Biotechnol. Inc. Trypsin (diphenylcarbamyl chloride treated, from bovine pancreas, type XI), trypsin inhibitor (type I-S, from soybean), albumin bovine (fraction V), valinomycin, asolectin (soybean phospholipids, catalogue no. P.5038), cholic acid, and Tricine were purchased from Sigma. Nigericin was purchased from Calbiochem. Affi-Gel 15 and 10 were obtained from Bio-Rad. Nitrocellulose membranes (0.2 μm) were obtained from Hoefer Scientific Instruments. Tween 20 was purchased form J. T. Baker Inc. L-[3H]Glutamate (60 Ci/mmol) was obtained from American Radiochemical Corporation and 125I-Protein A (130 μCi/ml) was obtained from DuPont NEN. All other reagents were analytical grade. N-tris(hydroxymethyl)methylglycine polyacrylamide gel electrophoresis γ-aminobutyric acid. Asolectin was purified by acetone extraction (Kagawa and Racker, 1971), crude bovine brain lipids were extracted as described (11Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), and cholic acid was recrystallized from 70% ethanol (16Kagawa Y. Racker E. J. Biol. Chem. 1971; 246: 5477-5487Abstract Full Text PDF Google Scholar) and neutralized with NaOH to pH 7.4. X-ray films were from AGFA. Membrane vesicles from rat brain were prepared as described (9Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar), except that the resuspension buffer contained 100 mM LiPi, pH 7.4, 5 mM Tris-SO4, 1 mM MgSO4, 0.5 mM Na-EDTA, and 1% glycerol. Crude membrane vesicles (10-20 mg/ml) were rapidly thawed at 37°C in a water bath, and after addition of 10 ml of either of the following loading buffer the incubation was continued for 10 more min. The composition of these buffers was 135 mM NaCl and 10 mM NaPi, pH 7.4; 135 mM KCl and 10 mM KPi, pH 7.4; 135 mM LiCl and 10 mM LiPi pH 7.4; or 135 mM Tris-HCl and 10 mM Tris-Pi, pH 7.4. Then the vesicles were spun down by centrifugation at 4°C for 15 min at 37,000 × g, and the pellets were resuspended in the same solution at a protein concentration of 10 to 20 mg/ml. Subsequently, 30 μl of this suspension was diluted with 500 μl of the same solution with or without additions as detailed in the legends to the figures and incubated at 37°C for 10 min. Unless indicated otherwise in the figure legends, L-glutamate was used at 1 mM. Then, 40 μl of a freshly prepared trypsin solution (dissolved in water) was added such that the protein ratio trypsin/membrane vesicles was 1:5. After the indicated times of incubation, the reaction was terminated by adding 600 μl of a solution of trypsin inhibitor (1 mg/ml). Immediately thereafter the mixture was diluted with 10 ml of ice-cold loading buffer containing 3% bovine serum albumin. Zero time points were performed by adding the trypsin inhibitor prior to trypsin, followed by immediate dilution in the ice-cold stop solution. After centrifugation at 15,000 revolutions/min for 15 min, the pellets were washed with 10 ml of ice-cold sodium containing loading buffer and resuspended in the same buffer at a protein concentration of 3 mg/ml. The membrane vesicles were then processed for immunoblotting or transport as described below. This was performed as described (31Reimerdes E.H. Klostermeyer H. Methods Enzymol. 1976; 45B: 26-28Crossref Scopus (91) Google Scholar). Antibodies were raised in rabbits against peptides corresponding to residues 17-32 (P-692, part of the amino terminus, RMHDSHLSSEEPKHRN), residues 151-167 (P-693, part of the putative external loop between transmembrane domains 3 and 4, KQLGPGKKNDEVSSLDA), to residues 496-512 (P-694, part of the carboxyl terminus, SKSELDTISDQHRMHED). The protocol described (Mabjeesh and Kanner, 1992) was followed. The antisera were characterized against the HTP peak, a purified preparation of the L-glutamate transporter (9Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar). They were affinity purified on Affi-Gel 10 coupled to P-692 or to Affi-Gel 15 coupled to either P-693 or P-694. The antibodies were tested against membrane vesicles from rat brain, using a 1:300 dilution. The 73 kDa was the only band detected by the anti-P-692 and the anti P-693 antibodies. In the case of the anti-P-694 antibodies, a minor 35 kDa band was also revealed in some preparations, probably representing a proteolytic fragment of the transporter. The immunoreactivity of all the bands observed with the affinity purified antibodies was abolished by 50 nmol of the peptides used to raise them. To membrane vesicles (120 μl, 3 mg/ml) were added (in this order) 15 μl of saturated ammonium sulfate; 168 μl of a 1:1 mixture of asolectin and brain lipids, 27 μmol total, suspended in 100 mM KPi, pH 7.4, 1% glycerol, 10 mM Tris SO4 pH 7.4, 0.5 mM NaEDTA, pH 7.4, 1 mM MgSO4; 42 μl of NaCl 3 M and 22.5 μl of 20% cholic acid (neutralized with NaOH to pH 7.4. After incubation for 10 min on ice, the mixture was applied to three dried Sephadex minicolumns equilibrated with the above potassium-containing medium as described (30Radian R. Kanner B.I. J. Biol. Chem. 1985; 260: 11859-11865Abstract Full Text PDF PubMed Google Scholar). After they were collected by centrifugation, the proteoliposomes (20 μl) were assayed for (Na++ K+)-coupled L-[3H]glutamate transport as described (Gordon and Kanner, 1988), except that millipore filters with 0.45-μm pore size were used. After the assay, the proteins were quantified according to 26Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7134) Google Scholar. Glutamate transport in membrane vesicles (without reconstitution) was done as described (Kanner and Sharon, 1978). Similar amounts of membrane suspensions (determined by the method of 7Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216334) Google Scholar) were subjected to Tricine-SDS-PAGE and immunoblotted using the affinity purified antibodies, used at a 1:300 dilution, as described (Mabjeesh and Kanner, 1993). The standard proteins indicated in the figures are in kilodaltons. Antibodies were raised against peptides corresponding to regions of the GLT-1 transporter predicted to be extramembranous (28Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1136) Google Scholar) as illustrated in Fig. 1A. The peptides comprised residues 17-32 (part of the amino terminus, P-692), residues 151-167 (part of loop 3-4, the loop connecting putative transmembrane helices III and IV which contains the predicted N-glycosylation sites, P-693), and residues 496-512 (part of the carboxyl-terminal, P-694). In order to increase the probability that the antibodies raised against the individual peptides would also recognize the intact protein, the hydrophilicity and antigenicity profile of these peptides was determined according to the methods described (12Hopp T.P. Woods K.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3824-3828Crossref PubMed Scopus (2912) Google Scholar, 13Hopp T.P. Woods K.R. Mol. Immunol. 1983; 20: 483-489Crossref PubMed Scopus (252) Google Scholar). Indeed, with affinity purified antibodies raised against all three peptides, an intense band of around 73 kDa can be observed in the immunoblot of rat brain membranes (Fig. 1B, lanes 2, 5, and 8) which has a similar mobility as that observed with an antibody raised against the highly purified glutamate transporter (Fig. 1B, lane 1, 10Danbolt N.C. Storm-Mathisen J. Kanner B.I. Neuroscience. 1992; 51: 295-310Crossref PubMed Scopus (370) Google Scholar). The specificity of each of these antipeptide antibodies is illustrated by the ability of 50 nmol of the corresponding free peptide (homologous peptide) to inhibit the immunoreactivity (Fig. 1B, lanes 3, 6, and 9). Heterologous peptides do not inhibit (Fig. 1B, lanes 4, 7, and 10). Similar results are obtained using the HTP-peak fractions (9Danbolt N.C. Pines G. Kanner B.I. Biochemistry. 1990; 29: 6734-6740Crossref PubMed Scopus (185) Google Scholar), a highly purified glutamate transporter preparation from rat brain (data not shown). The antibody raised against peptide P-694 was recently also shown to specifically react with the cloned and expressed glutamate transporter GLT-1 (40Zhang Y. Pines G. Kanner B.I. J. Biol. Chem. 1994; 269: 19573-19577Abstract Full Text PDF PubMed Google Scholar). Membrane vesicles from rat brain have been incubated with trypsin for various times. After stopping the reaction with trypsin inhibitor and washing, the membrane proteins are separated on Tricine-SDS-PAGE and transferred to nitrocellulose. The fragments from the glutamate transporter which contain the amino terminus have been visualized with the anti-P-692 antibody (Fig. 2A). All bands visualized with this antibody, as well as with those described below, are specific. They were competed for by homologous but not by heterologous peptides (data not shown). In the presence of sodium ions, the band of 73 kDa corresponding to the intact transporter almost completely disappears, and bands of 30 and 16 kDa are generated. The kinetics suggest that the 30 kDa band emerges first, and subsequently the 16 kDa band is derived from it (Fig. 2A). The rate of conversion of the 30 to 16 kDa band is increased if in addition to sodium, glutamate is present as well (Fig. 2A). A pattern similar to that observed in the presence of sodium is also seen in the presence of potassium, except that now the effect of glutamate is smaller (Fig. 2A). The 30 kDa band is also generated in a lithium-containing medium, but its rate of conversion to the 16 kDa band is greatly slowed down whether glutamate is present or absent (Fig. 2A). Under these conditions the generation of the 30 kDa band from the full-length transporter is also retarded (Fig. 2A). In a control experiment we established that trypsin activity is not affected by sodium, potassium, and lithium ions and also not by glutamate (data not shown). The small effect of glutamate in potassium- and lithium-containing media is due to the fact that glutamate was added as its sodium salt, as it is not observed when the Tris salt of glutamate is used (data not shown). We have also examined the functional consequences of the trypsin treatment on the transporter. We have used reconstitution to optimally express the transporters in the membrane vesicle preparation which were still active after the proteolysis. After stopping the reaction and extensive washing, the vesicles were solubilized with cholate and immediately reconstituted with a mixture of asolectin and brain lipids (Radian and Kanner, 1985) so that the internal medium contained potassium phosphate. The reconstituted proteoliposomes were diluted into the transport medium containing sodium chloride, L-[3H]glutamate and the potassium-specific ionophore, valinomycin. These conditions (inwardly directed sodium and outwardly directed potassium gradients and an interior negative membrane potential) generate the maximal driving force for L-glutamate accumulation. It can be observed that the kinetics of inactivation of transport (Fig. 2B, a typical experiment, which was run in parallel to the one depicted in Fig. 2A) is only partly correlated to the conversion of the 30 to 16 kDa bands observed in Fig. 2A. Thus, the rate of inactivation when proteolysis is carried out in the presence of sodium is only slightly faster than when it is done in the presence of lithium (Fig. 2B). Interestingly, the rate of inactivation is increased in the presence of glutamate, but only when sodium is present (Fig. 2B). It is obvious that the correlation between the disappearance of the intact transporter (Fig. 2A) and the inactivation of transport activity (Fig. 2B) is partial. This discrepancy will be addressed under “Discussion.” The specificity of the effect of glutamate on the proteolysis of the 30 kDa amino-terminal fragment is shown in Fig. 3A. The conditions were chosen such that with sodium alone, both the 30 kDa as well as the 16 kDa bands are observed. Similar to the data shown in Fig. 2A, L-glutamate accelerates the proteolytic cleavage of the 30 kDa band (Fig. 3A, lane 3). The same effect is observed with L-aspartate (lane 7), D-aspartate (lane 8). All of these are substrates which are translocated by the glutamate transporter. On the other hand, the neurotransmitters GABA (lane 5), dopamine (lane 6), and serotonin (lane 9) did not exhibit the effect (Fig. 3A). The same result was obtained with D-glutamate (lane 4). This is in agreement with the well-known stereospecificity of the glutamate transporter (3Balcar V.J. Johnston G.A. J. Neurobiol. 1972; 3: 295-301Crossref PubMed Scopus (125) Google Scholar). The effect of glutamate is dependent on its concentration. The half-maximal effect was observed at around 10 μM (Fig. 3B) which is somewhat higher than the observed Km for transport (around 2 μM; Kanner and Sharon, 1978). However, under transport conditions, sodium outside, potassium inside (rather than sodium on both sides), the half-maximal effect of glutamate was shifted to lower concentrations, around 5 μM. The data presented thus far can be explained by a sodium- and glutamate-dependent conformational change of the transporter, which causes two trypsin-sensitive sites, located in the amino-terminal half of the transporter, to become more exposed. The data presented in Fig. 4, in which an antibody against the carboxyl terminus was used to detect other fragments, indicate that this is also the case for at least one more site, located in the carboxyl-terminal part. In the presence of sodium alone trypsin treatment of the transporter results in the generation of a major 43 kDa band, as well as a minor band of 10 kDa. At longer times the minor 10 kDa band disappears, and part of the 43 kDa band seems to be converted into one of 35 kDa. In the presence of glutamate, the intensity of the 43 kDa band is reduced while the intensity of the 35 kDa band is increased. This phenomenon is a function of its concentration, just like that observed with the anti-P-692 antibody (data not shown). Also this effect appears to be a kinetic one as the phenomenon also occurs in the absence of glutamate but at longer times (data not shown). Again, the stimulation of the conversion of the 43 to 35 kDa band is observed with amino acid substrates of the transporter and not with those solutes which are not (data not shown). It should be pointed out that, unlike fragments containing the amino-terminal epitope P-692 (Fig. 2A and 3) or epitope P-693 (Fig. 8), those containing the carboxyl-terminal epitope P-694 give rise to a distinct pattern at the upper part of the gel (Fig. 4). This part contains a smeared background, probably reflecting fragments in different states of aggregation (Fig. 4). Although it is impossible to convey this feature, seen on the autoradiograph, to the photograph paper, it should be emphasized that superimposed on the background, the residual 73 kDa band corresponding to the intact transporter is present at strongly reduced levels.Figure 8:Immunoreactivity of the tryptic amino-terminal fragments with an antibody raised against the external N-glycosylated domain. The blot shown inFig. 2A was stripped with 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-mercaptoethanol for 45 min at 50°C. After washing in phosphate-buffered saline containing 0.2% Tween-20, the blot was reprobed with the anti-P693 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As with the amino-terminal fragments, the ionic composition of the medium influences the trypsinization pattern, but in a slightly different way (Fig. 4). The conversion rate is increased by glutamate provided sodium is present. In the presence of lithium, a similar pattern is observed to the one with sodium, except for the lack of stimulation by L-glutamate (Fig. 4). On the other hand, in the presence of potassium, regardless of the presence of glutamate, the rate of conversion is similar to that observed in the presence of sodium and L-glutamate (Fig. 4). Dihydrokainic acid is a competitive inhibitor of the GLT-1 transporter (28Pines G. Danbolt N.C. Bjoras M. Zhang Y. Bendahan A. Eide L. Koepsell H. Storm-Mathisen J. Seeberg E. Kanner B. Nature. 1992; 360: 464-467Crossref PubMed Scopus (1136) Google Scholar; 2Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar) but appears not to be transported by it (29Pocock J.M. Murphie H.M. Nicholls D.G. J. Neurochem. 1988; 50: 745-751Crossref PubMed Scopus (54) Google Scholar; 4Barbour B. Brew H. Attwell D. J. Physiol. 1991; 436: 169-193Crossref PubMed Scopus (163) Google Scholar; 2Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar). Thus, this compound may allow us to distinguish between the possibility that the effects of L-glutamate on the trypsinization pattern of the GLT-1 transporter are due to the mere binding of the substrate or one invoking a conformational change of the transporter, subsequent to the binding step. The experiment depicted in Fig. 5A, in which the anti-P-694 antibody is used, indicates that dihydroxykainate does not induce the sodium-dependent increase of conversion of the 43- to 35-kDa fragment. In this regard, it behaves like GABA, which is not a substrate, rather than like L-glutamate (Fig. 5A). However, while an excess of GABA does not affect the ability of L-glutamate to speed up the conversion of the 43 to the 35 kDa band, excess dihydroxykainate inhibits this action of L-glutamate (Fig. 5A). Similar results are obtained with the anti-P-692 antibody (Fig. 5B). The conversion of the 30 kDa band to one of 16 kDa containing the P-692 epitope observed in the presence of sodium and potassium was greatly retarded when lithium containing media were used (Fig. 2A). Sodium and potassium are both substrates of the transporter. The retardation observed in the presence of lithium could be due to the fact that this ion is not translocated by the transporter. An alternative possibility is that lithium is not inert but is somehow interacting with the transporter and that this interaction is of a different nature than that with transportable ions. This was tested using substitution by Tris. It can be seen that with Tris present, the conversion of the 30 to the 16 kDa band is much more similar to that observed in a sodium or potassium medium than to that in lithium medium (Fig. 2A and 6). The conversion to the 16 kDa band was hardly influenced by L-glutamate, in contrast to the situation in sodium media. These observations suggest that the effects of lithium are due to a specific interaction of this cation with the transporter. Evidence for this at the functional level is seen in the experiment depicted in Fig. 7. The initial rate of glutamate transport depends on the sodium concentration in a sigmoid fashion when the ionic strength is maintained by Tris (Fig. 7). However, when lithium is used, the sodium dependence assumes the form of a rectangular hyperbola (Fig. 7). The small amount of uptake observed at zero-sodium may be due to the fact that glutamate (present at 10 μM) was added as its sodium salt. With choline substitution the sigmoid concentration dependence is observed as well (data not shown). T" @default.
• W2005242158 created "2016-06-24" @default.
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• W2005242158 date "1995-07-01" @default.
• W2005242158 modified "2023-10-18" @default.
• W2005242158 title "Conformational Changes Monitored on the Glutamate Transporter GLT-1 Indicate the Existence of Two Neurotransmitter-bound States" @default.
• W2005242158 cites W1486976896 @default.
• W2005242158 cites W1512720139 @default.
• W2005242158 cites W1525819460 @default.
• W2005242158 cites W1544803716 @default.
• W2005242158 cites W1582586031 @default.
• W2005242158 cites W1594934722 @default.
• W2005242158 cites W1604834760 @default.
• W2005242158 cites W1877229434 @default.
• W2005242158 cites W1902585184 @default.
• W2005242158 cites W1966548233 @default.
• W2005242158 cites W1969400861 @default.
• W2005242158 cites W1976754780 @default.
• W2005242158 cites W1977634657 @default.
• W2005242158 cites W1981837661 @default.
• W2005242158 cites W1983197885 @default.
• W2005242158 cites W1991701465 @default.
• W2005242158 cites W1993391624 @default.
• W2005242158 cites W1998478437 @default.
• W2005242158 cites W1998527909 @default.
• W2005242158 cites W1999613366 @default.
• W2005242158 cites W2002905697 @default.
• W2005242158 cites W2003059369 @default.
• W2005242158 cites W2003410399 @default.
• W2005242158 cites W2018606012 @default.
• W2005242158 cites W2019709628 @default.
• W2005242158 cites W2027905503 @default.
• W2005242158 cites W2045037622 @default.
• W2005242158 cites W2053418558 @default.
• W2005242158 cites W2066419715 @default.
• W2005242158 cites W2079812049 @default.
• W2005242158 cites W2094089753 @default.
• W2005242158 cites W2094843626 @default.
• W2005242158 cites W2102131116 @default.
• W2005242158 cites W2157853053 @default.
• W2005242158 cites W2168526937 @default.
• W2005242158 cites W21862234 @default.
• W2005242158 cites W4293247451 @default.
• W2005242158 cites W989281374 @default.
• W2005242158 doi "https://doi.org/10.1074/jbc.270.28.17017" @default.
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