The objective of this research is to broaden understanding of the ATP-dependent glutamate uptake system in the synaptic vesicle, particularly to investigate its regulation. There is compelling evidence that glutamate serves as a major excitatory neurotransmitter in the central nervous system. The ATP-dependent vesicular uptake is highly specific for glutamate and considered to play an important role in determining the neurotransmitter role of glutamate. Recently, we have obtained evidence that vesicular glutamate uptake is inhibited by a proteinaceous substance. I propose to (a) purify the inhibitory substance by classical and high pressure (HPLC) liquid chromatography; (b) determine physicochemical properties by SDS-polyacrylamide gel electrophoresis, gel filtration, sucrose density gradient centrifugation and amino acid analysis; (c) determine the mechanism of inhibition by examining its effects on a proton-pump ATPase, membrane potential, chloride influx, and glutamate efflux; (d) examine the role of protein phosphorylation in the inhibition by SDS-gel electrophoresis and autoradiography; (e) identify a peptide fragment which retains the inhibitory activity by HPLC; and (f) identify the protein(s) with which the inhibitor interacts, by inhibitor-conjugated affinity chromatography. This research will not only enhance understanding of the vesicular glutamate uptake system, but also may ultimately provide new insights into the investigation of those types of neuropathophysiology where abnormal glutamate transmission is implicated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS026884-02
Application #
3412991
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1988-12-01
Project End
1995-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Tamura, Yutaka; Ogita, Kiyokazu; Ueda, Tetsufumi (2014) A new VGLUT-specific potent inhibitor: pharmacophore of Brilliant Yellow. Neurochem Res 39:117-28
Winter, Harry C; Ueda, Tetsufumi (2008) The glutamate uptake system in presynaptic vesicles: further characterization of structural requirements for inhibitors and substrates. Neurochem Res 33:223-31
Amano, Taku; Matsubayashi, Hiroaki; Ozkan, Eric D et al. (2002) Aberrant reduction of an inhibitory protein factor in a rat epileptic model. Epilepsy Res 51:81-91
Tamura, Y; Ozkan, E D; Bole, D G et al. (2001) IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca(2+)-dependent, evoked release of glutamate, GABA and serotonin. J Neurochem 76:1153-64
Ozkan, E D; Ueda, T (1998) Glutamate transport and storage in synaptic vesicles. Jpn J Pharmacol 77:1-10
Lewis, S M; Ueda, T (1998) Solubilization and reconstitution of synaptic vesicle glutamate transport system. Methods Enzymol 296:125-44
Lewis, S M; Lee, F S; Todorova, M et al. (1997) Synaptic vesicle glutamate uptake in epileptic (EL) mice. Neurochem Int 31:581-5
Ozkan, E D; Lee, F S; Ueda, T (1997) A protein factor that inhibits ATP-dependent glutamate and gamma-aminobutyric acid accumulation into synaptic vesicles: purification and initial characterization. Proc Natl Acad Sci U S A 94:4137-42
Tabb, J S; Kish, P E; Van Dyke, R et al. (1992) Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH. J Biol Chem 267:15412-8
Tabb, J S; Ueda, T (1991) Phylogenetic studies on the synaptic vesicle glutamate transport system. J Neurosci 11:1822-8

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