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Molecular and Functional Analysis of a Novel Neuronal Vesicular Glutamate Transporter*

  • Liqun Bai
    Footnotes
    Affiliations
    Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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  • Hua Xu
    Affiliations
    Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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  • James F. Collins
    Affiliations
    Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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  • Fayez K. Ghishan
    Correspondence
    To whom correspondence should be addressed: Dept. of Pediatrics, Director, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-5170; Fax: 520-626-4141; E-mail: [email protected]
    Affiliations
    Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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  • Author Footnotes
    * This work was supported by NIDDK, National Institutes of Health Grant 2R01-R37DK-33209 and the W. M. Keck Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF 324864.
    ‡ Present address: Tucson Hospital Medical Education Program, 5301 E. Grant Rd., Tucson, AZ 85733. E-mail: [email protected]
Open AccessPublished:September 28, 2001DOI:https://doi.org/10.1074/jbc.M104578200
      Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Packaging and storage of glutamate into glutamatergic neuronal vesicles requires ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. VGLUT1, the first identified vesicular glutamate transporter, is only expressed in a subset of glutamatergic neurons. We report here the molecular cloning and functional characterization of a novel glutamate transporter, VGLUT2, from mouse brain. VGLUT2 has all major functional characteristics of a synaptic vesicle glutamate transporter, including ATP dependence, chloride stimulation, substrate specificity, and substrate affinity. It has 75 and 79% amino acid identity with human and rat VGLUT1, respectively. However, expression patterns of VGLUT2 in brain are different from that of VGLUT1. In addition, VGLUT2 activity is dependent on both membrane potential and pH gradient of the electrochemical proton gradient, whereas VGLUT1 is primarily dependent on only membrane potential. The presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together play an important role in vesicular glutamate transport in glutamatergic neurons.
      ΔμH+
      proton electrochemical gradient
      V-ATPase
      vacuolar H+-ATPase
      VGLUT1
      vesicular glutamate transporter isoform 1
      VGLUT2
      vesicular glutamate transporter isoform 2
      Δψ
      membrane potential
      ΔpH
      proton gradient
      Km
      apparent affinity constant
      Vmax
      maximal velocity
      EST
      expressed sequence tag
      bp
      base pair(s)
      DCCD
      N,N′-dicyclohexylcarbodiimide
      DIDS
      4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
      Neurotransmission depends on the regulated exocytotic release of vesicular transmitter molecules to the synaptic cleft, where they interact with postsynaptic receptors that subsequently transduce the information. Two types of neurotransmitter transporters have been identified based on membrane localization on plasma membrane or vesicular membrane. Removal of the transmitter from the synaptic cleft results in termination of the signal, and this requires destruction of transmitter or reuptake of transmitter back to the presynaptic terminal or glial cells via a sodium-dependent uptake system on the plasma membrane (
      • Masson J.
      • Sagne C.
      • Hamon M.
      • Mestikaway S.E.
      ). Packaging and storage of neurotransmitters into specialized secretory vesicles in neurons ensures their regulated release. This storage is also crucial for protecting the neurotransmmitter molecules from leakage or intraneuronal metabolism and for protecting the neuron from possible toxic effects. This process is mediated by specific transporters on the vesicular membranes. At least four different types of vesicular transporters have been functionally identified that are specific for transport of classic neurotransmitters: monoamines, acetylcholine, γ-aminobutyric acid (GABA), and glutamate (
      • Fykse E.M.
      • Fonnum F.
      ,
      • Reimer R.J.
      • Fon E.A.
      • Edwards R.H.
      ). Unlike the plasma membrane transporters, which rely on a sodium gradient across the plasma membrane, all of these vesicular transport processes depend on the proton electrochemical gradient (ΔμH+)1generated by a Mg2+-activated vacuolar H+-ATPase (V-ATPase) on the vesicular membrane (
      • Forgac M.
      ). When protons are pumped into the vesicular lumen, a proton gradient (ΔpH) and a membrane potential (Δψ) occur across the membrane to form ΔμH+, which favors the exchange of luminal protons for cytoplasmic transmitter. The transport of monoamines and acetylcholine rely predominantly on ΔpH (
      • Knoth J.
      • Zallakian M.
      • Njus D.
      ,
      • Anderson D.C.
      • King S.C.
      • Parsons S.M.
      ), whereas the accumulation of GABA depends on both Δψ and ΔpH (
      • Kish P.E.
      • Fischer-Bovenkerk C.
      • Ueda T.
      ,
      • Hell J.W.
      • Maycox P.R.
      • Jahn R.
      ). In the case of glutamate, there is disagreement over whether the transport of glutamate is driven by Δψ only (
      • Maycox P.R.
      • Deckwerth T.
      • Hell J.W.
      • Jahn R.
      ,
      • Cidon S.
      • Sihra T.
      ,
      • Moriyama Y.
      • Maeda M.
      • Futai M.
      ) or by both Δψ and ΔpH components of ΔμH+ (
      • Naito S.
      • Ueda T.
      ,
      • Tabb J.S.
      • Kish P.E.
      • Van Dyke R.
      • Ueda T.
      ).
      In addition to the differences in driving force, kinetic properties, substrate specificity, and ion dependence also clearly distinguish vesicular glutamate transport from high affinity plasma membrane glutamate transport. The vesicular ATP-dependent glutamate transporter is specific for glutamate, is stimulated by millimolar concentrations of chloride, and has a low affinity for uptake (Km about 1–2 mm) (
      • Naito S.
      • Ueda T.
      ,
      • Kish P.E.
      • Ueda T.
      ). These functional characteristics are in contrast to those of the plasma membrane glutamate transporter, which is sodium dependent, accepts aspartate as well as glutamate, and has a high affinity for glutamate with Km in the 3–20 μm range (
      • Kanner B.I.
      • Sharon I.
      ,
      • Logan W.J.
      • Synder S.H.
      ).
      Although vesicular transporters for other neurotransmitters have been intensively studied at mechanistic, biochemical, and molecular levels, molecular cloning of vesicular glutamate transporters has only occurred recently. BNPI, a brain-specific sodium-dependent phosphate cotransporter originally characterized as a plasma membrane transporter, was recently localized on vesicular membranes of small synaptic vesicles in neurons (
      • Bellocchio E.E.
      • Hu H.
      • Pohorille A.
      • Chan J.
      • Pickel V.M.
      • Edwards R.H.
      ,
      • Takmori S.
      • Rhee J.S.
      • Rosenmund C.
      • Jahn R.
      ) and further functionally characterized as a vesicular glutamate transporter called VGLUT1 (
      • Takmori S.
      • Rhee J.S.
      • Rosenmund C.
      • Jahn R.
      ,
      • Bellocchio E.E.
      • Reimer R.J.
      • Fremeau Jr., R.T.
      • Edwards R.H.
      ). Uptake of glutamate by VGLUT1 has all of the functional characteristics previously reported for vesicular glutamate transporter with Δψ as the predominant driving force. However, only a subset of glutamatergic neurons expresses VGLUT1 (
      • Bellocchio E.E.
      • Hu H.
      • Pohorille A.
      • Chan J.
      • Pickel V.M.
      • Edwards R.H.
      ,
      • Ni B.
      • Wu X.
      • Yan G.M.
      • Wang J.
      • Paul S.M.
      ). Moreover, inCaenorhabditis elegans, loss-of-function mutations in the VGLUT1 orthologue eat-4, lead to impairment but not to a complete loss of glutamate-mediated neurotransmission (
      • Raizen D.M.
      • Avery L.
      ,
      • Lee R.Y.
      • Sawin E.R.
      • Chalfie M.
      • Horvitz H.R.
      • Avery L.
      ). These findings imply that an additional vesicular glutamate transporter(s) may exist. To identify new vesicular glutamate transporter isoforms, we searched the GenbankTM Expressed Sequence Tags (EST) data base using human and rat VGLUT1 sequences (
      • Ni B.
      • Du Y.
      • Wu X.
      • DeHoff B.S.
      • Rosteck Jr., P.R.
      • Paul S.M.
      ,
      • Ni B.
      • Roseteck P.R.
      • Nadi N.S.
      • Paul S.M.
      ), and we found a mouse EST clone, which has ∼49% homology with human and rat VGLUT1. We report here the molecular cloning and functional characterization of VGLUT2, the second identified vesicular glutamate transporter with different transport characteristics and cellular localization as compared with VGLUT1.

      DISCUSSION

      Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It has been shown that glutamate can be accumulated into synaptic vesicles to concentrations of at least 60 mm through an ATP-dependent mechanism (
      • Burger P.M.
      • Mehl E.
      • Cameron P.L.
      • Maycox P.R.
      • Baumer M.
      • Lottspeich F.
      • De Camilli P.
      • Jahn R.
      ,
      • Naito S.
      • Ueda T.
      ). The vesicular transport system, in contrast to the sodium-dependent plasma membrane transport system, is specific for glutamate, has a low affinity for glutamate, and is stimulated by physiologically relevant concentrations of chloride (
      • Naito S.
      • Ueda T.
      ,
      • Tabb J.S.
      • Kish P.E.
      • Van Dyke R.
      • Ueda T.
      ,
      • Carlson M.D.
      • Kish P.E.
      • Ueda T.
      ,
      • Fisher-Bovenkerk C.
      • Kish P.E.
      • Ueda T.
      ). These unique properties of the vesicular glutamate transporter are conserved in such diverse vertebrates as fish, reptiles, amphibians, and mammals (
      • Tabb J.S.
      • Ueda T.
      ). Consistent with these observations, glutamate was transported by VGLUT2 in the absence of sodium. Furthermore, the transport required ATP and was inhibited by specific inhibitors of the V-ATPase. Glutamate transported by VGLUT2 is saturable with a Km of 1.1 mm, which is consistent with characteristics of the vesicular glutamate transporter. In addition, VGLUT2 is specific for glutamate and does not transport aspartate or other amino acids.
      The requirement of low Cl concentrations is a significant property of vesicular glutamate transport. The presence of chloride at low concentrations (1–5 mm) is essential for the uptake of glutamate, with substantially lower transport activity observed at higher and lower levels (
      • Maycox P.R.
      • Deckwerth T.
      • Hell J.W.
      • Jahn R.
      ,
      • Tabb J.S.
      • Kish P.E.
      • Van Dyke R.
      • Ueda T.
      ,
      • Carlson M.D.
      • Kish P.E.
      • Ueda T.
      ). This biphasic effect of chloride and the DIDS-sensitive chloride dependence of glutamate uptake, have suggested that the vesicular glutamate transporter possesses an anion binding site, distinct from the substrate binding site, which regulates transporter activity (
      • Hartinger J.
      • Jahn R.
      ). Chloride showed a similar effect on glutamate uptake by VGLUT2. We also observed a 70% inhibition in VGLUT2-mediated glutamate uptake by 1 μm DIDS, which is consistent with previous findings. Taken together, these functional characteristics suggest that VGLUT2 functions as a vesicular glutamate transporter.
      Based on the high homology (97% at the amino acid level) between mouse VGLUT2 and human DNPI, we surmised that DNPI is the human homologue of mouse VGLUT2. By using Xenopus oocytes expressing hDNPI, Aihara et al. (
      • Aihara Y.
      • Mashima H.
      • Onda H.
      • Hisano S.
      • Kasuya H.
      • Hori T.
      • Yamada S.
      • Tomura H.
      • Yamada Y.
      • Inoue I.
      • Kojima I.
      • Takeda J.
      ) observed an ∼75% increase in sodium-dependent phosphate uptake. However, in our substrate specificity experiments, high concentrations of phosphate did not inhibit glutamate uptake by VGLUT2, suggesting that phosphate was not transported by the same mechanism as glutamate. This is a similar situation to VGLUT1, where weak sodium-dependent phosphate uptake was observed in oocyte plasma membranes expressing VGLUT1 (
      • Ni B.
      • Du Y.
      • Wu X.
      • DeHoff B.S.
      • Rosteck Jr., P.R.
      • Paul S.M.
      ,
      • Ni B.
      • Roseteck P.R.
      • Nadi N.S.
      • Paul S.M.
      ), and relatively strong ATP-dependent glutamate uptake was observed in vesicular membranes expressing VGLUT1 (
      • Takmori S.
      • Rhee J.S.
      • Rosenmund C.
      • Jahn R.
      ,
      • Bellocchio E.E.
      • Reimer R.J.
      • Fremeau Jr., R.T.
      • Edwards R.H.
      ). The relationship between glutamate and phosphate transport by VGLUT1 and VGLUT2 remains unclear. As has been proposed for VGLUT1 (
      • Bellocchio E.E.
      • Reimer R.J.
      • Fremeau Jr., R.T.
      • Edwards R.H.
      ), VGLUT2 may be a bifunctional transporter, which functions as a phosphate transporter at the plasma membrane and a glutamate transporter in synaptic vesicles.
      Whereas it is known that vesicular glutamate transport is driven by an electrochemical proton gradient generated by V-ATPase, the precise manner in which the glutamate transporter and V-ATPase operate is currently under debate. To assess the driving forces of glutamate transport mediated by VGLUT2, we tested the effect of different ionophores on VGLUT2-mediated glutamate uptake. In the presence of 4 mm KCl, the K+ ionophore valinomycin, which reduces only Δψ without changing ΔpH, deceased glutamate uptake by VGLUT2 by 50%. Nigericin, an electroneutral ionophore that exchanges K+ for H+, eliminates ΔpH whereas allowing Δψ to increase in the presence of 4 mm KCl and subsaturating concentrations of glutamate, inhibited glutamate uptake by VGLUT2 by 40%. This result is consistent with previous findings that both the Δψ and ΔpH components are driving forces for the transport of glutamate into synaptic vesicles (
      • Tabb J.S.
      • Kish P.E.
      • Van Dyke R.
      • Ueda T.
      ). However, this functional characteristic is distinct from VGLUT1, because Δψ was suggested as the primary driving force for VGLUT1 (
      • Takmori S.
      • Rhee J.S.
      • Rosenmund C.
      • Jahn R.
      ,
      • Bellocchio E.E.
      • Reimer R.J.
      • Fremeau Jr., R.T.
      • Edwards R.H.
      ). Thus, one possible explanation of the previous controversial results observed in synaptic vesicles could be the differing functions of two closely related isoforms. Further elucidation of molecular differences between these two isoforms may answer this question.
      Another interesting finding is that the two vesicular glutamate transporters have different expression patterns in brain. VGLUT1 is only expressed in a subset of glutamatergic neurons in the cerebral cortex, hippocampus, and cerebellum (
      • Ni B.
      • Wu X.
      • Yan G.M.
      • Wang J.
      • Paul S.M.
      ), whereas VGLUT2 is expressed in cerebral cortex, hippocampus, and the thalamus (Fig. 5). Thalamic nuclei use glutamate as their neurotransmitter but they lack VGLUT1 mRNA and protein (
      • Bellocchio E.E.
      • Hu H.
      • Pohorille A.
      • Chan J.
      • Pickel V.M.
      • Edwards R.H.
      ,
      • Ni B.
      • Wu X.
      • Yan G.M.
      • Wang J.
      • Paul S.M.
      ). Thus, VGLUT2 appears to be an important glutamate transporter in thalamic neuronal vesicles. In addition, DNPI, the human homologue of VGLUT2, was highly expressed in the fetal brain when VGLUT1 was not expressed (
      • Aihara Y.
      • Mashima H.
      • Onda H.
      • Hisano S.
      • Kasuya H.
      • Hori T.
      • Yamada S.
      • Tomura H.
      • Yamada Y.
      • Inoue I.
      • Kojima I.
      • Takeda J.
      ), suggesting that VGLUT2 rather than VGLUT1 might be important in the initial steps of neuronal differentiation.
      In summary, we have cloned and functionally characterized a novel vesicular glutamate transporter expressed in the mouse brain. The VGLUT2 cDNA encodes a protein of 582 amino acids with 75% identity with human VGLUT1, the first identified vesicular glutamate transporter. All of the major functional characteristics of VGLUT2, such as ATP dependence, chloride stimulation, substrate specificity, substrate affinity, and mode of energization, are consistent with that of a glutamate transporter previously characterized from synaptic, vesicular membranes. Thus, VGLUT2 functions as a vesicular glutamate transporter. Identification of two isoforms with different functional characteristics provides molecular information for structure-function studies of vesicular glutamate transporters. In addition, the presence of VGLUT2 in brain regions lacking VGLUT1 suggests that the two isoforms together might account for glutamate transport into synaptic vesicles in glutamatergic neurons.

      Acknowledgments

      We thank Dr. Naomi E. Rance (Department of Pathology, University of Arizona) and Dr. Bruce M. Coull (Department of Neurology, University of Arizona) for assistance with interpreting thein situ hybridization results.

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