Neurochemical Research

, Volume 34, Issue 5, pp 807–818 | Cite as

Synaptic Vesicle-bound Pyruvate Kinase can Support Vesicular Glutamate Uptake

Original Paper


Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [3H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. “ATP-trapping” experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.


Glycolysis Energy metabolism Neurotransmitter Refilling VGLUT Nerve terminal 



1-Aminocyclopentane-1,3-dicarboxylic acid






Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone


Glyceraldehyde-3-phosphate dehydrogenase




3-Phosphoglycerate kinase


Sodium dodecyl sulfate


V-type proton-pump ATPase


Vesicular glutamate transporter



This work was supported by National Institutes of Health grants RO1 NS 42200 (TU) and RO1 MH 071384 (TU), and a grant from Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan) (TU). We thank Dr. Bernhard Erni and Dr. Luis Fernando Garcia-Alles (University of Bern, Switzerland) for kindly providing (Z)-Cl-PEP and related compounds, and Dr. Kathleen Buckley (Harvard University) for kindly providing a hybridoma clone for production of an anti-SV2 monoclonal antibody. We are also grateful to Dr. Minor J. Coon (University of Michigan) for kind permission to use the Cary 3E spectrophotometer, to Dr. David G. Bole for assistance in initial glutamate uptake assays and critical reading of the manuscript, and to Ms. Mary Roth for excellent assistance in preparation of the manuscript.


  1. 1.
    Sokoloff L (1977) Relation between physiological function and energy metabolism in the central nervous system. J Neurochem 29:13–26. doi: 10.1111/j.1471-4159.1977.tb03919.x CrossRefPubMedGoogle Scholar
  2. 2.
    Fox PT, Raichle ME, Mintun MA et al (1988) Nonoxidative glucose consumption during focal physiologic neural activity. Science 241:462–464. doi: 10.1126/science.3260686 CrossRefPubMedGoogle Scholar
  3. 3.
    McNay EC, Fries TM, Gold PE (2000) Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task. Proc Natl Acad Sci USA 97:2881–2885. doi: 10.1073/pnas.050583697 CrossRefPubMedGoogle Scholar
  4. 4.
    Sommerfield AJ, Deary IJ, McAulay V et al (2003) Moderate hypoglycemia impairs multiple memory functions in healthy adults. Neuropsychol 17:125–132. doi: 10.1037/0894-4105.17.1.125 CrossRefGoogle Scholar
  5. 5.
    Siesjo BK (1978) Brain energy metabolism. John Wiley & Sons, Inc., New YorkGoogle Scholar
  6. 6.
    Lewis LD, Ljunggren B, Ratcheson RA et al (1974) Cerebral energy state in insulin-induced hypoglycemia, related to blood glucose and to EEG. J Neurochem 23:673–679. doi: 10.1111/j.1471-4159.1974.tb04390.x CrossRefPubMedGoogle Scholar
  7. 7.
    Dirks B, Hanke H, Krieglstein J et al (1980) Studies on the linkage of energy metabolism and activity in the isolated perfused rat brain. J Neurochem 35:311–317. doi: 10.1111/j.1471-4159.1980.tb06266.x CrossRefPubMedGoogle Scholar
  8. 8.
    Ghajar JBG, Plum F, Duffy TE (1982) Cerebral oxidative metabolism and blood flow during acute hypoglycemia and recovery in unanesthetized rats. J Neurochem 38:397–409. doi: 10.1111/j.1471-4159.1982.tb08643.x CrossRefPubMedGoogle Scholar
  9. 9.
    Bachelard HS, Cox DWG, Drower J (1984) Sensitivity of guinea-pig hippocampal granule cell field potentials to hexoses in vitro: an effect on cell excitability? J Physiol 352:91–102PubMedGoogle Scholar
  10. 10.
    Fleck MW, Henze DA, Barrionuevo G et al (1993) Aspartate and glutamate mediate excitatory synaptic transmission in area CA1 of the hippocampus. J Neurosci 13:3944–3955PubMedGoogle Scholar
  11. 11.
    Yamane K, Yokono K, Okada Y (2000) Anaerobic glycolysis is crucial for the maintenance of neural activity in guinea pig hippocampal slices. J Neurosci Methods 103:163–171. doi: 10.1016/S0165-0270(00)00312-5 CrossRefPubMedGoogle Scholar
  12. 12.
    Okada Y, Lipton P (2007) Glucose, oxidative energy metabolism, and neural function in brian slices-glycolysis plays a key role in neural activity. In: Laitha A, Gibson G, Dienel GA (eds) Handbook of neurochemistry and molecular neurobiology.Brain energetics. Integration of molecular and cellular processes, 3rd edn. Springer-Verlag, Heidelberg, pp 17–40Google Scholar
  13. 13.
    Cox DWG, Bachelard HS (1982) Attenuation of evoked field potentials from dentate granule cells by low glucose, pyruvate, malate, and sodium fluoride. Brain Res 239:527–534. doi: 10.1016/0006-8993(82)90527-3 CrossRefPubMedGoogle Scholar
  14. 14.
    Cox DWG, Morris PG, Feeney J et al (1983) 31P-n.m.r. studies on cerebral energy metabolism under conditions of hypoglycaemia and hypoxia in vitro. Biochem J 212:365–370PubMedGoogle Scholar
  15. 15.
    Kanatani T, Mizuno K, Okada Y (1995) Effects of glycolytic metabolites on preservation of high energy phosphate level and synaptic transmission in the granule cells of guinea pig hippocampal slices. Experientia 51:213–216. doi: 10.1007/BF01931098 CrossRefPubMedGoogle Scholar
  16. 16.
    Ikemoto A, Bole DG, Ueda T (2003) Glycolysis and glutamate accumulation into synaptic vesicles: role of glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase. J Biol Chem 278:5929–5930. doi: 10.1074/jbc.M211617200 CrossRefPubMedGoogle Scholar
  17. 17.
    Collingridge GL, Bliss TVP (1987) NMDA receptors––their role in long-term potentiation. Trends Neurosci 10:288–293. doi: 10.1016/0166-2236(87)90175-5 CrossRefGoogle Scholar
  18. 18.
    Cotman CW, Monaghan DT, Ganong AH (1988) Excitatory amino acid neurotransmission: NMDA receptors and Hebb-type synaptic plasticity. Annu Rev Neurosci 11:61–80. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  19. 19.
    Watkins JC, Evans RH (1981) Excitatory amino acid transmitters. Annu Rev Pharmacol Toxicol 21:165–204. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  20. 20.
    Cotman CW, Foster A, Lanthorn T (1981) An overview of glutamate as a neurotransmitter. In: DiChiara G, Gessa GL (eds) Glutamate as a neurotransmitter. Raven Press, New York, pp 1–27Google Scholar
  21. 21.
    Fonnum F (1984) Glutamate: a neurotransmitter in mammalian brain. J Neurochem 42:1–11. doi: 10.1111/j.1471-4159.1984.tb09689.x CrossRefPubMedGoogle Scholar
  22. 22.
    Ueda T (1986) Glutamate transport in the synaptic vesicle. In: Roberts PJ, Storm-Mathisen J, Bradford HF (eds) Excitatory amino acids. Macmillan, London, pp 173–195Google Scholar
  23. 23.
    Nicholls DG (1989) Release of glutamate, aspartate, and γ-aminobutyric acid from isolated nerve terminals. J Neurochem 52:331–341. doi: 10.1111/j.1471-4159.1989.tb09126.x CrossRefPubMedGoogle Scholar
  24. 24.
    Maycox PR, Hell JW, Jahn R (1990) Amino acid neurotransmission: spotlight on synaptic vesicles. Trends Neurosci 13:83–87. doi: 10.1016/0166-2236(90)90178-D CrossRefPubMedGoogle Scholar
  25. 25.
    Özkan ED, Ueda T (1998) Glutamate transport and storage in synaptic vesicles. Jpn J Pharmacol 77:1–10. doi: 10.1254/jjp.77.1 CrossRefPubMedGoogle Scholar
  26. 26.
    Reimer RJ, Fremeau RT, Bellocchio EE et al (2001) The essence of excitation. Curr Opin Cell Biol 13:417–421. doi: 10.1016/S0955-0674(00)00230-1 CrossRefPubMedGoogle Scholar
  27. 27.
    Takamori S, Rhee JS, Rosenmund C et al (2000) Identification of a vesicular glutamate transporter that defines a glutamatargic phenotype in neurons. Nature 407:189–194. doi: 10.1038/35025070 CrossRefPubMedGoogle Scholar
  28. 28.
    Otis TS (2001) Vesicular glutamate transporters incognito. Neuron 29:11–14. doi: 10.1016/S0896-6273(01)00176-3 CrossRefPubMedGoogle Scholar
  29. 29.
    Naito S, Ueda T (1983) Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles. J Biol Chem 258:696–699PubMedGoogle Scholar
  30. 30.
    Naito S, Ueda T (1985) Characterization of glutamate uptake into synaptic vesicles. J Neurochem 44:99–109. doi: 10.1111/j.1471-4159.1985.tb07118.x CrossRefPubMedGoogle Scholar
  31. 31.
    Maycox PR, Deckwerth T, Hell JW et al (1988) Glutamate uptake by brain synaptic vesicles. J Biol Chem 263:15423–15428PubMedGoogle Scholar
  32. 32.
    Hell JW, Maycox PR, Jahn R (1990) Energy dependence and functional reconstitution of the γ-aminobutyric acid carrier from synaptic vesicles. J Biol Chem 265:2111–2117PubMedGoogle Scholar
  33. 33.
    Tabb JS, Ueda T (1991) Phylogenetic studies on the synaptic vesicle glutamate transport system. J Neurosci 11:1822–1828PubMedGoogle Scholar
  34. 34.
    Tabb JS, Kish PE, Van Dyke R et al (1992) Glutamate transport into synaptic vesicles: roles of membrane potential, pH gradient, and intravesicular pH. J Biol Chem 267:15412–15418PubMedGoogle Scholar
  35. 35.
    Wolosker H, de Souza DO, de Meis L (1996) Regulation of glutamate transport into synaptic vesicles by chloride and proton gradient. J Biol Chem 271:11726–11731. doi: 10.1074/jbc.271.20.11726 CrossRefPubMedGoogle Scholar
  36. 36.
    Bellocchio EE, Reimer RJ, Fremeau RT et al (2000) Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter. Science 289:957–960. doi: 10.1126/science.289.5481.957 CrossRefPubMedGoogle Scholar
  37. 37.
    Shepherd GM, Harris KM (1998) Three-dimensional structure and composition of CA3 → CA1 axons in rat hippocampal slices: implications for presynaptic connectivity and compartmentalization. J Neurosci 18:8300–8310PubMedGoogle Scholar
  38. 38.
    Buckley K, Kelly RB (1985) Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol 100:1284–1294. doi: 10.1083/jcb.100.4.1284 CrossRefPubMedGoogle Scholar
  39. 39.
    Garcia-Alles LF, Erni B (2002) Synthesis of phosphoenol pyruvate (PEP) analogues and evaluation as inhibitors of PEP-utilizing enzymes. Eur J Biochem 269:3226–3236. doi: 10.1046/j.1432-1033.2002.02995.x CrossRefPubMedGoogle Scholar
  40. 40.
    Kish PE, Ueda T (1989) Glutamate accumulation into synaptic vesicles. Methods Enzymol 174:9–25. doi: 10.1016/0076-6879(89)74005-2 CrossRefPubMedGoogle Scholar
  41. 41.
    Ueda T, Greengard P, Berzins K et al (1979) Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase. J Cell Biol 83:308–319. doi: 10.1083/jcb.83.2.308 CrossRefPubMedGoogle Scholar
  42. 42.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  43. 43.
    Kochhar S, Mehta PK, Christen P (1989) Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography. Anal Biochem 179:182–185. doi: 10.1016/0003-2697(89)90221-2 CrossRefPubMedGoogle Scholar
  44. 44.
    Bücher T, Pfleiderer G (1955) Pyruvate kinase from muscle. Methods Enzymol 1:435–440. doi: 10.1016/0076-6879(55)01071-9 CrossRefGoogle Scholar
  45. 45.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi: 10.1038/227680a0 CrossRefPubMedGoogle Scholar
  46. 46.
    Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  47. 47.
    Ogita K, Hirata K, Bole DG et al (2001) Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal. J Neurochem 77:34–42PubMedCrossRefGoogle Scholar
  48. 48.
    Bellocchio EE, Hu H, Pohorille A et al (1998) The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission. J Neurosci 18:8648–8659PubMedGoogle Scholar
  49. 49.
    Fremeau RT Jr, Troyer MD, Pahner I et al (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31:247–260. doi: 10.1016/S0896-6273(01)00344-0 CrossRefPubMedGoogle Scholar
  50. 50.
    Herzog E, Bellenchi GC, Gras C et al (2001) The existence of a second vesicular glutamate transporter specifies subpopulations of glutamatergic neurons. J Neurosci 21:181Google Scholar
  51. 51.
    Navone F, Jahn R, Di Gioia G et al (1986) Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J Cell Biol 103:2511–2527. doi: 10.1083/jcb.103.6.2511 CrossRefPubMedGoogle Scholar
  52. 52.
    Kayne F (1973) Pyruvate kinase. In: Boyer P (ed) The enzymes, vol 8A. Academic Press, New York, pp 353–382Google Scholar
  53. 53.
    Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci USA 85:7972–7976. doi: 10.1073/pnas.85.21.7972 CrossRefPubMedGoogle Scholar
  54. 54.
    Fykse EM, Christensen H, Fonnum F (1989) Comparison of the properties of γ-aminobutyric acid and L-glutamate uptake into synaptic vesicles isolated from rat brain. J Neurochem 52:946–951. doi: 10.1111/j.1471-4159.1989.tb02546.x CrossRefPubMedGoogle Scholar
  55. 55.
    Winter HC, Ueda T (1993) Glutamate uptake system in the presynaptic vesicle: glutamic acid analogs as inhibitors and alternate substrates. Neurochem Res 18:79–85. doi: 10.1007/BF00966925 CrossRefPubMedGoogle Scholar
  56. 56.
    Winter HC, Ueda T (2008) The glutamate uptake system in synaptic vesicles: further characterization of structural requirements for inhibitors and substrates. Neurochem Res 33:223–231. doi: 10.1007/s11064-007-9493-8 CrossRefPubMedGoogle Scholar
  57. 57.
    Xu KY, Zweier JL, Becker LC (1995) Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport. Circ Res 77:88–97PubMedGoogle Scholar
  58. 58.
    Morciano M, Burre J, Corvey C et al (2005) Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis. J Neurochem 95:1732–1745. doi: 10.1111/j.1471-4159.2005.03506.x CrossRefPubMedGoogle Scholar
  59. 59.
    Takamori S, Holt M, Stenius K et al (2006) Molecular anatomy of a trafficking organelle. Cell 127:831–846. doi: 10.1016/j.cell.2006.10.030 CrossRefPubMedGoogle Scholar
  60. 60.
    Blondeau F, Ritter B, Allaire PD et al (2004) Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc Natl Acad Sci USA 101:3833–3838. doi: 10.1073/pnas.0308186101 CrossRefPubMedGoogle Scholar
  61. 61.
    Pollack GH (2001) Cells, gels, and the engine of life. Ebner & Sons, SeattleGoogle Scholar
  62. 62.
    Coughenour HD, Spaulding RS, Thompson CM (2004) The synaptic vesicle proteome: a comparative study in membrane protein identification. Proteomics 4:3141–3155. doi: 10.1002/pmic.200300817 CrossRefPubMedGoogle Scholar
  63. 63.
    Pappas GD, Waxman SG (1972) Synaptic fine structure-morphological correlates of chemical and electrotonic transmission. In: Pappas GD, Purpura DP (eds) Structure and function of synapses. Raven Press, New York, pp 1–43Google Scholar
  64. 64.
    Pyle JL, Kavalali ET, Piedras-Renteria ES et al (2000) Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron 28:221–231. doi: 10.1016/S0896-6273(00)00098-2 CrossRefPubMedGoogle Scholar
  65. 65.
    Pellerin L, Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci USA 91:10625–10629. doi: 10.1073/pnas.91.22.10625 CrossRefPubMedGoogle Scholar
  66. 66.
    Rikhy R, Ramaswami M, Kirshnan KS (2003) A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation. Genetics 165:1243–1253PubMedGoogle Scholar
  67. 67.
    Hagopian K, Ramsey JJ, Weindruch R (2003) Influence of age and caloric restriction on liver glycolytic enzyme activities and metabolite concentrations in mice. Exp Gerontol 38:253–266. doi: 10.1016/S0531-5565(02)00203-6 CrossRefPubMedGoogle Scholar
  68. 68.
    Poon HF, Shepherd HM, Reed TT et al (2006) Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis. Neurobiol Aging 27:1020–1034. doi: 10.1016/j.neurobiolaging.2005.05.014 CrossRefPubMedGoogle Scholar
  69. 69.
    Ueda T, Ikemoto A (2007) Synaptic vesicle-associated glycolytic ATP-generating enzymes: coupling to neurotransmitter accumulation. In: Laitha A, Gibson GE, Dienel GA (eds) Handbook of neurochemistry and molecular neurobiology. Brain energetics. Integration of cellular and molecular processes, 3rd edn. Springer-Verlag, Heidelberg, pp 241–259Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Atsuhiko Ishida
    • 1
    • 2
    • 3
  • Yasuko Noda
    • 1
    • 4
  • Tetsufumi Ueda
    • 1
    • 5
    • 6
  1. 1.Molecular and Behavioral Neuroscience InstituteUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Department of BiochemistryAsahikawa Medical CollegeAsahikawaJapan
  3. 3.Graduate School of Integrated Arts and SciencesHiroshima UniversityHigashi-HiroshimaJapan
  4. 4.Department of Anti-Aging Food SciencesOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical SciencesOkayamaJapan
  5. 5.Department of PharmacologyUniversity of Michigan Medical SchoolAnn ArborUSA
  6. 6.Department of PsychiatryUniversity of Michigan Medical SchoolAnn ArborUSA

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