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Synaptic Vesicle-bound Pyruvate Kinase can Support Vesicular Glutamate Uptake

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Abstract

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-trap**” 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.

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Abbreviations

ACPD:

1-Aminocyclopentane-1,3-dicarboxylic acid

Cl-PEP:

3-Chlorophosphoenolpyruvate

DTT:

Dithiothreitol

FCCP:

Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

PEP:

Phosphoenolpyruvate

3-PGK:

3-Phosphoglycerate kinase

SDS:

Sodium dodecyl sulfate

V-ATPase:

V-type proton-pump ATPase

VGLUT:

Vesicular glutamate transporter

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Acknowledgments

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.

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  1. Atsuhiko Ishida

    Present address: Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521, Japan

  2. Yasuko Noda

    Present address: Department of Anti-Aging Food Sciences, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, 700-8558, Japan

Authors and Affiliations

  1. Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School, BSRB, 109 Zina Pitcher Place, Ann Arbor, MI, 48109-2200, USA

    Atsuhiko Ishida, Yasuko Noda & Tetsufumi Ueda

  2. Department of Biochemistry, Asahikawa Medical College, Asahikawa, 078-8510, Japan

    Atsuhiko Ishida

  3. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, 48109-2200, USA

    Tetsufumi Ueda

  4. Department of Psychiatry, University of Michigan Medical School, Ann Arbor, MI, 48109-2200, USA

    Tetsufumi Ueda

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  1. Atsuhiko Ishida
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Correspondence to Tetsufumi Ueda.

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Atsuhiko Ishida—On leave from the Department of Biochemistry, Asahikawa Medical College, Asahikawa, Japan.

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Ishida, A., Noda, Y. & Ueda, T. Synaptic Vesicle-bound Pyruvate Kinase can Support Vesicular Glutamate Uptake. Neurochem Res 34, 807–818 (2009). https://doi.org/10.1007/s11064-008-9833-3

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