Vesicular Glutamate Transporter Contains Two Independent Transport Machineries

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial FoF1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (ΔΨ)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for ∼4 mm Cl-, was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in l-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ΔΨ-dependent l-glutamate uptake and a Na+-dependent Pi uptake. Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial FoF1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (ΔΨ)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for ∼4 mm Cl-, was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg184, His128, and Glu191 showed a dramatic loss in l-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a ΔΨ-dependent l-glutamate uptake and a Na+-dependent Pi uptake. Vesicular storage and subsequent exocytosis of l-glutamate is the major pathway for excitatory signal transmission in the central nervous system (1Maycox P.R. Hell J.W. Jahn R. Trends Neurosci. 1990; 13: 83-87Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 2Reimer R.J. Edwards R.H. Pflugers. Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar, 3Fremeau R.T. Voglmaier S. Seal R.P. Edwards R.H. Trends Neurosci. 2004; 27: 98-103Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Vesicular glutamate transporters (VGLUTs) 2The abbreviations used are: VGLUT, vesicular glutamate transporter; MFS, major facilitator superfamilies; MOPS, 4-morpholinopropanesulfonic acid; PDB, Protein Data Bank; TM, transmembrane; WT, wild type. 2The abbreviations used are: VGLUT, vesicular glutamate transporter; MFS, major facilitator superfamilies; MOPS, 4-morpholinopropanesulfonic acid; PDB, Protein Data Bank; TM, transmembrane; WT, wild type. are essential for the vesicular storage of l-glutamate through active transport of l-glutamate into synaptic vesicles at the expense of ΔμH+ established by vacuolar H+-ATPase (V-ATPase) (1Maycox P.R. Hell J.W. Jahn R. Trends Neurosci. 1990; 13: 83-87Abstract Full Text PDF PubMed Scopus (198) Google Scholar). There are three isoforms of VGLUT, denoted VGLUT1, VGLUT2, and VGLUT3 on the basis of the order of their discovery (2Reimer R.J. Edwards R.H. Pflugers. Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar, 4Fremeau R.T. Kam K. Qureshi T. Johnson J. Copenhagen D.R. Storm-Mathisen J. Chaudhry F.A. Nicoll R.A. Edwards R.H. Science. 2004; 304: 815-1819Crossref Scopus (382) Google Scholar, 5Moriyama Y. Yamamoto A. J. Biochem. 2004; 135: 155-163Crossref PubMed Scopus (67) Google Scholar, 6Takamori S. Neurosci. Res. 2006; 55: 343-351Crossref PubMed Scopus (203) Google Scholar). VGLUT1 and VGLUT2 show a complementary expression pattern in essentially all known glutamatergic neurons, suggesting that the two VGLUTs are involved in glutamatergic neurotransmission (7Fremeau R.T. Troyer M.D. Pahner I. Nygaard G.O. Tran C.H. Reimer R.J. Bellocchio E.E. Fortin D. Storm-Mathisen J. Edwards R.H. Neuron. 2001; 31: 247-260Abstract Full Text Full Text PDF PubMed Scopus (995) Google Scholar, 8Herzog E. Bellenchi G.C. Gras C. Bernard V. Ravassard P. Bedet C. Gasnier B. Giros B. El Mestikawy S. J. Neurosci. 2001; 21: RC181Crossref PubMed Google Scholar, 9Takamori S. Rhee J.S. Rosenmund C. Jahn R. J. Neurosci. 2001; 21: RC182 1-6Crossref Google Scholar). In fact, VGLUT1 knock-out mice exhibit a loss of secretion of l-glutamate and glutamatergic neurotransmission in neurons that normally express VGLUT1 (4Fremeau R.T. Kam K. Qureshi T. Johnson J. Copenhagen D.R. Storm-Mathisen J. Chaudhry F.A. Nicoll R.A. Edwards R.H. Science. 2004; 304: 815-1819Crossref Scopus (382) Google Scholar, 10Wojcik S.M. Rhee J.S. Herzog E. Sigler A. Jahn R. Takamori S. Brose N. Rosenmund C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7158-7163Crossref PubMed Scopus (385) Google Scholar). In contrast, VGLUT3 is expressed in neurons that are usually classified as non-glutamatergic neurons and astrocytes suggesting the involvement of VGLUT3 in a novel mode of l-glutamate signaling (11Fremeau R.T. Burman J. Qureshi T. Tran C.H. Proctor J. Johnson J. Zhang H. Sulzer D. Copenhagen D.R. Storm-Mathisen J. Reimer R.J. Chaudhry F.A. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14488-14493Crossref PubMed Scopus (480) Google Scholar, 12Gras C. Herzog E. Bellenchi G.C. Bernard V. Ravassard P. Pohl M. Gasnier B. Giros B. El Mestikawy S. J. Neurosci. 2002; 22: 5442-5451Crossref PubMed Google Scholar, 13Takamori S. Malherbe P. Broger C. Jahn R. EMBO Rep. 2002; 3: 798-803Crossref PubMed Scopus (184) Google Scholar). VGLUTs are also expressed in peripheral non-neuronal cells, associated with a wide variety of secretory vesicles and are responsible for glutamate-mediated regulation in various cellular processes (5Moriyama Y. Yamamoto A. J. Biochem. 2004; 135: 155-163Crossref PubMed Scopus (67) Google Scholar). VGLUTs belong to the SLC17/type I anion transport family, one of the major facilitator superfamilies (MFS), and are not related to other neurotransmitter transporters such as vesicular acetylcholine transporter and vesicular monoamine transporter (2Reimer R.J. Edwards R.H. Pflugers. Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar, 14Schuldiner S. Shirvan A. Linial M. Physiol. Rev. 1995; 75: 369-392Crossref PubMed Scopus (266) Google Scholar). VGLUT exhibits unique transport properties when compared with other vesicular neurotransmitter transporters. For one, VGLUT is activated by low concentrations of Cl- (∼4 mm) through a putative Cl- binding site (15Naito S. Ueda T. J. Neurochem. 1985; 44: 99-109Crossref PubMed Scopus (376) Google Scholar, 16Maycox P.R. Deckwerth T. Hell J.W. Jahn R. J. Biol. Chem. 1988; 263: 15423-15428Abstract Full Text PDF PubMed Google Scholar, 17Hartinger J. Jahn R. J. Biol. Chem. 1993; 268: 23122-23127Abstract Full Text PDF PubMed Google Scholar, 18Moriyama Y. Yamamoto A. J. Biol. Chem. 1995; 270: 22314-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Furthermore, VGLUT requires membrane potential (positive inside) as a driving force, transports l-glutamate electrophoretically, shows strict substrate recognition and does not recognize d,l-aspartate as a substrate (2Reimer R.J. Edwards R.H. Pflugers. Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar, 5Moriyama Y. Yamamoto A. J. Biochem. 2004; 135: 155-163Crossref PubMed Scopus (67) Google Scholar, 15Naito S. Ueda T. J. Neurochem. 1985; 44: 99-109Crossref PubMed Scopus (376) Google Scholar, 16Maycox P.R. Deckwerth T. Hell J.W. Jahn R. J. Biol. Chem. 1988; 263: 15423-15428Abstract Full Text PDF PubMed Google Scholar, 17Hartinger J. Jahn R. J. Biol. Chem. 1993; 268: 23122-23127Abstract Full Text PDF PubMed Google Scholar, 18Moriyama Y. Yamamoto A. J. Biol. Chem. 1995; 270: 22314-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). VGLUT also exhibits Na+-driven Pi transport when expressed in oocytes, like Na-Pi co-transporter 1 (NPT1), another SLC17/type 1 family (19Ni B. Rosteck P.R. Nadi N.S. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5607-5611Crossref PubMed Scopus (276) Google Scholar, 20Aihara Y. Mashima H. Onda H. Hisano S. Kasuya H. Hori T. Yamada S. Tomura H. Yamada Y. Inoue I. Kojima I. Takeda J. J. Neurochem. 2000; 74: 2622-2625Crossref PubMed Scopus (239) Google Scholar). However, no information on the transport mechanism and mechanistic relationship between the transport of l-glutamate and Pi is currently available. The amino acid residues involved in substrate recognition, transport, and regulation also have not been identified. In general, site-directed mutagenesis is a powerful tool for analyzing the structure and function relationship of transporter proteins. Recombinant VGLUT activity can be assessed as ATP-dependent l-glutamate uptake in membrane vesicle fractions and/or in detergent-permeabilized cells (2Reimer R.J. Edwards R.H. Pflugers. Arch. 2004; 447: 629-635Crossref PubMed Scopus (149) Google Scholar, 3Fremeau R.T. Voglmaier S. Seal R.P. Edwards R.H. Trends Neurosci. 2004; 27: 98-103Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 5Moriyama Y. Yamamoto A. J. Biochem. 2004; 135: 155-163Crossref PubMed Scopus (67) Google Scholar). However, VGLUT activity measured by this procedure is too low to allow quantitative evaluation of the effect of mutations. Moreover, it is frequently observed that mutations disturb targeting of the protein to the correct cellular organelle. For instance, sialin, another member of the SLC17 family, is a lysosomal sialic acid/H+ co-transporter and targeted to the plasma membrane when mutations are introduced into its dileucine motif (21Morin P. Sagne C. Gasnier B. EMBO J. 2004; 23: 4560-4570Crossref PubMed Scopus (82) Google Scholar, 22Wreden C.C. Wlizla M. Reimer R.J. J. Biol. Chem. 2005; 280: 1408-1416Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Because the VGLUT assay procedure is based on the assumption that the transporter is targeted to acidic organelles and that endogenous ATP fully energizes V-ATPase to drive the uptake, it is not certain whether this assay is valid for mutant VGLUT. Consequently, information on mutant VGLUTs has yet to be published. In the present work, we developed a procedure to assess the activity of wild and mutant VGLUT with purified and thus, well-defined components. This procedure includes mutagenesis, expression of wild and mutant VGLUT2 in insect cells, purification, and co-reconstitution with bacterial F-ATPase. F-ATPase is a well-defined electrogenic proton pump with a pharmacological profile distinct from that of V-ATPase N. Physiol. Rev. PubMed Scopus Google Scholar). The proteoliposomes purified VGLUT2 and F-ATPase exhibit the ATP-dependent l-glutamate uptake to that of synaptic VGLUT2 has an absolute requirement for Cl- for the l-glutamate transport, three amino acid residues in the are responsible for the l-glutamate transport activity, and the transport for l-glutamate and Pi are independent of each wild and mutant VGLUT2 M. M. R. S. S. Takeda J. Yamamoto A. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google the and expression to the For the VGLUT2 was by with the and and into the and of and used to cells to For the VGLUT2 was by the and and into a VGLUT2 was from the to a and The VGLUT2 an cells with Recombinant was from cells and used for of cells to cells used for expression of VGLUT2 cells in with and at cells in with mm at by at a of of and a for and for the cells for membrane introduced to by The was by The and cells in a mm mm 1 and 1 and by with a at for to and the was at for 1 The was in mm 1 and 1 The membrane was by addition of to at for the was to 1 of and for at with The was into a and with mm mm and VGLUT2 was from the with of the mm The purified VGLUT2 was at F-ATPase was expressed in cells and purified by as Y. A. H. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of purified VGLUT2 and bacterial F-ATPase into was by the in Y. A. H. M. M. J. Biol. Chem. Full Text PDF PubMed Google In of VGLUT2 was with of F-ATPase and at and at this for at The was by the in the and with reconstitution mm mm and mm proteoliposomes by at for 1 at and in of mm mm and mm for phosphate transport assay F-ATPase. as was in mm mm The was in a into and at proteoliposomes for 1 at in a mm mm mm and was by the of to a of 1 The was on a with for the of VGLUT2 R. M. H. Biol. 2006; PubMed Scopus Google Scholar). proteoliposomes of in mm mm mm and and for at ATP was to a of mm and the for a The assay was by addition of and at the and through a at for 1 Y. M. Arch. Biochem. 1993; PubMed Scopus Google Scholar). of the was For Na+-driven Pi transport, was by the addition of proteoliposomes to the mm mm and the the on transport are expressed as of VGLUT2 was by A. J. Biol. 1993; PubMed Scopus Google based on the structure of transporter Y. J. M. Science. PubMed Scopus Google Res. 1994; 22: PubMed Scopus Google Scholar). in the of and as Y. A. H. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of inside) and (positive inside) measured by with and a as Y. Yamamoto A. J. Biol. Chem. 1995; 270: 22314-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Y. A. H. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Protein was as a C. Biochem. PubMed Scopus Google Scholar). of proteoliposomes by of with as a and of a system for the expression and purification of wild and mutant Upon the cells expressed VGLUT2 as by expression was at an of 1 The membrane vesicles from insect cells did not show ATP-dependent l-glutamate uptake the assay of the membrane with VGLUT2 was purified to by The of which was by was for cells and for cells with of and we used cells for purification of VGLUT2 was into with purified bacterial F-ATPase the The proteoliposomes VGLUT2 as as F-ATPase of the of VGLUT2 in proteoliposomes was to with that of the VGLUT2 was with the to the of the VGLUT as the driving of l-glutamate uptake, of a is for the in vitro transport used F-ATPase as a of Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a of VGLUT the uptake to a low that VGLUT2 was an for the l-glutamate uptake of F-ATPase l-glutamate uptake to the as ATP, suggesting that l-glutamate uptake was the of active and transport through VGLUT Because the of the proteoliposomes to of the of l-glutamate membrane was of the l-glutamate was also by the addition of 1 this proton of l-glutamate in proteoliposomes and the of various on l-glutamate uptake uptake was measured in the of various as vesicles with for addition of uptake at was in the of uptake of the was For F-ATPase VGLUT2, the of as the was 1 mm 1 1 Evans mm 1 mm 1 mm in a The l-glutamate uptake was inhibited by a F-ATPase but not by a V-ATPase a from the of V-ATPase to F-ATPase ATP-dependent and independent l-glutamate uptake exhibited The and of ATP-dependent l-glutamate uptake mm and for The and of the purified system and l-glutamate uptake in synaptic H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The for the is The purified VGLUT2 can be at for at a loss of the transport of properties of VGLUT have been the such as the requirement for as the driving force, to to Evans blue, and the on Cl- (1Maycox P.R. Hell J.W. Jahn R. Trends Neurosci. 1990; 13: 83-87Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 15Naito S. Ueda T. J. Neurochem. 1985; 44: 99-109Crossref PubMed Scopus (376) Google Scholar, 16Maycox P.R. Deckwerth T. Hell J.W. Jahn R. J. Biol. Chem. 1988; 263: 15423-15428Abstract Full Text PDF PubMed Google Scholar, 17Hartinger J. Jahn R. J. Biol. Chem. 1993; 268: 23122-23127Abstract Full Text PDF PubMed Google Scholar, 18Moriyama Y. Yamamoto A. J. Biol. Chem. 1995; 270: 22314-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, properties with synaptic vesicle fractions that other that with of the the properties of VGLUT2 The ATP-dependent l-glutamate uptake was sensitive to that ΔμH+ the uptake. the l-glutamate uptake to of the in the of showed of and the Furthermore, a of but not of did not the l-glutamate uptake. that was a driving for uptake was concentrations of d,l-aspartate did not l-glutamate uptake. Evans blue, a known of VGLUT S. J. Neurochem. 1995; PubMed Scopus Google Scholar, E. S. R.J. Chem. 2005; PubMed Scopus Google l-glutamate uptake. Furthermore, of VGLUT2 with 1 mm to l-glutamate uptake, suggesting that VGLUT2 does not have residues that are for the transport for of VGLUT is its on Cl- for l-glutamate uptake in synaptic vesicles (15Naito S. Ueda T. J. Neurochem. 1985; 44: 99-109Crossref PubMed Scopus (376) Google Scholar, 16Maycox P.R. Deckwerth T. Hell J.W. Jahn R. J. Biol. Chem. 1988; 263: 15423-15428Abstract Full Text PDF PubMed Google Scholar, 17Hartinger J. Jahn R. J. Biol. Chem. 1993; 268: 23122-23127Abstract Full Text PDF PubMed Google Scholar, 18Moriyama Y. Yamamoto A. J. Biol. Chem. 1995; 270: 22314-22320Abstract Full Text Full Text PDF PubMed Scopus (85) Google ATP-dependent l-glutamate uptake is by low concentrations of Cl- (∼4 mm) and inhibited at concentrations of In purified an absolute requirement of Cl- was essentially no ATP-dependent l-glutamate uptake is observed in the of the ATP-dependent uptake activity the addition of Cl- and was at ∼4 with concentrations of Cl- the ATP-dependent of as by was in the of Cl- and with Cl- as by is in the of Cl- and with Cl- Thus, VGLUT2 requires Cl- for the l-glutamate uptake, and the Cl- effect is of a of of VGLUT2 by Cl- and driving by Cl- of relationship of membrane transporters have the of the amino acid residues in The of VGLUT2 R. M. H. Biol. 2006; PubMed Scopus Google amino acid residues have been that are in putative of the SLC17 VGLUT (19Ni B. Rosteck P.R. Nadi N.S. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5607-5611Crossref PubMed Scopus (276) Google Scholar, 20Aihara Y. Mashima H. Onda H. Hisano S. Kasuya H. Hori T. Yamada S. Tomura H. Yamada Y. Inoue I. Kojima I. Takeda J. J. Neurochem. 2000; 74: 2622-2625Crossref PubMed Scopus (239) Google Scholar, A. N. J. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. N. M. P. H. 23: PubMed Scopus Google Scholar). on amino acid and Glu191 in in in and in and introduced mutations in amino acid residues VGLUTs purified to and into by the used for the wild protein uptake activity was into of mutations on l-glutamate uptake. of mutations on uptake at in vesicles was measured in l-glutamate in the of ATP and of ATP purification of mutant proteins. mutant protein was by of the is in the all of the SLC17 family of this with in an loss of ATP-dependent l-glutamate transport activity at l-glutamate concentrations to ATP-dependent transport activity of the mutant was not to the mutant VGLUTs and also in their activity mutant showed activity other Because the activity to the transport of l-glutamate through VGLUT2, this the active and transport of wild and mutant VGLUT2s and mutant VGLUT2 purified and with F-ATPase as in the The ATP-dependent l-glutamate uptake was measured at various l-glutamate concentrations as in and the in a of in also in loss of In to Arg184, this is in of the VGLUT family, suggesting a role for l-glutamate of in which is in and (19Ni B. Rosteck P.R. Nadi N.S. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5607-5611Crossref PubMed Scopus (276) Google Scholar, 20Aihara Y. Mashima H. Onda H. Hisano S. Kasuya H. Hori T. Yamada S. Tomura H. Yamada Y. Inoue I. Kojima I. Takeda J. J. Neurochem. 2000; 74: 2622-2625Crossref PubMed Scopus (239) Google Scholar, A. N. J. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. N. M. P. H. 23: PubMed Scopus Google with the ATP-dependent uptake activity to of the wild type. of Glu191 in which is in VGLUTs and sialin, with and the ATP-dependent activity to and of the wild uptake in mutant was also at l-glutamate concentrations to Because the l-glutamate uptake activity of the and to that of the wild residues not for transport activity of and suggesting their role in the for the requirement for Cl-, the and did not show l-glutamate uptake at Cl- to that loss of l-glutamate transport activity in mutations was not to Cl- and exhibited the Cl- for l-glutamate uptake as wild VGLUT2 not suggesting that residues are not involved in Cl- Pi Pi transport activity be another of this activity expression of VGLUT in (19Ni B. Rosteck P.R. Nadi N.S. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5607-5611Crossref PubMed Scopus (276) Google Scholar, 20Aihara Y. Mashima H. Onda H. Hisano S. Kasuya H. Hori T. Yamada S. Tomura H. Yamada Y. Inoue I. Kojima I. Takeda J. J. Neurochem. 2000; 74: 2622-2625Crossref PubMed Scopus (239) Google Scholar). whether proteoliposomes wild VGLUT2 exhibit Na+-dependent Pi transport a was in proteoliposomes VGLUT2, Pi uptake was observed with and of mm and and that Na+-driven Pi transport activity is intrinsic of the properties of the Na+-dependent Pi uptake. The activity was not inhibited by of by Evans Cl- at to mm did not Na+-dependent Pi uptake activity of the mutations a effect on Na+-dependent Pi uptake and the of various on Na+-dependent Pi uptake Pi uptake was measured in the of various as For uptake was measured in mm The activity of the was For the VGLUT2, the of was to the mm mm 1 Evans in a The of an in vitro assay system the of the molecular mechanism of vesicular l-glutamate In the present we established an in vitro assay system with the purified protein that to the relationship of VGLUT. observed l-glutamate uptake in the of ATP in assay system. Because the of l-glutamate transport was when VGLUT2 was from this to the of l-glutamate through The activity of mutant VGLUT2s also this on this the of and active transports to the l-glutamate the membrane This was and is in with the l-glutamate synaptic vesicle E. P.R. M. P. Jahn R. Neuron. 3: Full Text PDF PubMed Scopus Google Scholar). that of VGLUT observed in synaptic vesicles are intrinsic to VGLUT. to Evans and to d,l-aspartate. the other two in the the for l-glutamate and the requirement for for l-glutamate transport in system in with that in synaptic major of is the of l-glutamate binding to VGLUT. Because l-glutamate is an anion at the of acidic in the and the of have the and of l-glutamate the vesicle Thus, the in the proteoliposomes be by a in the l-glutamate binding of The vesicle and between and have also the through potential of the for the requirement for Cl-, VGLUT2 exhibited essentially no ATP-dependent transport in the of Cl- but a at mm in the Because was not at this the that Cl- with VGLUT2 and l-glutamate transport Thus, VGLUT2 is a Because Cl- activated active transport the of l-glutamate through VGLUT2, Cl- be involved in the active transport of amino acid residues is to the molecular mechanism of the transporters. showed that was the in The loss of l-glutamate transport activity by the of with that the of a is for l-glutamate Because is in all of SLC17 anion this amino acid is a for the recognition of the of of with in a loss in This is in of the VGLUT and thus, play a role in the transport of The of activity by the of Glu191 with also the of a at this of l-glutamate transport by with that of of this is for Because l-glutamate transport was sensitive to with to residues and the major role of three residues on l-glutamate transport to be to active in the of the bacterial transporter and the structure of transporters Y. J. M. Science. PubMed Scopus Google Scholar, J. I. V. S. PubMed Scopus Google Scholar, J. I. V. S. Science. PubMed Scopus Google Scholar). Because SLC17 to the the structure of VGLUT2 is to be to that of this transporter Thus, based on the structure of we a of in Arg184, His128, and which are for l-glutamate transport as are all the of central Because amino acid residues are present in the as the substrate binding site of transporter Y. J. M. Science. PubMed Scopus Google Scholar, J. I. V. S. PubMed Scopus Google Scholar, J. I. V. S. Science. PubMed Scopus Google residues in l-glutamate binding through the of an which was as the transport mechanism of bacterial transporters J. I. V. S. PubMed Scopus Google Scholar, J. I. V. S. Science. PubMed Scopus Google a the substrate binding site to one the other of is that VGLUTs have a mechanism for Because of the residues in the putative substrate binding site of VGLUT2 are in binding of the l-glutamate anion and/or Cl- the substrate binding and of the substrate binding site of VGLUT2, to the of Na+-dependent Pi transport is another intrinsic of Because is essential for l-glutamate transport, we that this amino acid is also for Pi mutations in the amino acid did not Pi transport at all and Glu191 also not for Pi transport that amino acid residues responsible for the transport of l-glutamate are at in from for Pi In addition to the l-glutamate and Pi transports in their as to requirement for Cl- and to and Evans blue. Thus, Na+-driven Pi transport from l-glutamate transport in of driving force, Cl- to and amino acid on we that VGLUT2 two independent transport machineries a we currently have no information on amino acid residues essential to Pi transport, it is that residues in the in this as for l-glutamate a is in in In we have that VGLUT2 two independent machineries for the uptake of l-glutamate and the Na+-dependent uptake of with in system for the and on VGLUT. M. and M. for with of mutant with