Within the central nervous system extracellular and synaptic concentrations of most classical neurotransmitters are tightly regulated by specific, high affinity transporters that mediate the rapid reuptake into the presynaptic terminal and surrounding glial cells. Transporters for the excitatory amino acid neurotransmitters are positioned to have a major influence on both synaptic signalling and on the neurotoxic actions mediated by glutamate and aspartate, yet they are often ignored in many studies examining the role of these neurotransmitters in the CNS. The purpose of this proposal is to better define the physiological functions of excitatory amino acid transmitter transporters by 1) establishing the kinetic properties of cloned transporters that determine their capacity to take up and release glutamate, 2) identifying the structural and topological features required for transport, 3) determining their regional and cellular localization in human brain and 4) examining how signal transduction mechanisms act to modulate the reuptake process. Initial experiments will be directed at a detailed characterization of the kinetics, ion dependence, electrogenicity of three cloned excitatory amino acid transporters that have been expressed in oocytes and transfected cells. Alterations in transmembrane ion gradients, the driving forces for reuptake can have dramatic effects on the direction of transport, and thus, under a variety of conditions such as those which occur during ischemia, these changes can lead to net glutamate release through transporter reversal. The development of an electrophysiological assay for examining glutamate efflux and the ionic requirements of transport, as well as mammalian cell expression system to study the flux of radiolabeled substrates should provide insight into how the carriers function normally and how they may contribute to mechanisms of neuronal excitotoxicity. Additional goals of these studies will be to address the regional and cellular localization of carriers in the human CNS to further evaluate their potential contribution to neurodegenerative disease. Although the project is focused initially on three human carriers that have been cloned in the applicant's laboratory, it will be expanded to address the role of additional carrier subtypes and cDNAs encoding different glutamate transporters as they are identified. The importance of understanding the function, localization and regulation of different amino acid transporter subtypes is underscored by the many clinical and experimental studies which have implicated abnormal or inadequate transmitter reaccumulation in degenerative disorders such as ALS, Huntington's disease, ischemia-induced neurotoxicity, and Alzheimer's dementia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS033273-01A1
Application #
2271972
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1995-06-01
Project End
2000-05-31
Budget Start
1995-06-01
Budget End
1996-05-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Organized Research Units
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Leighton, Barbara H; Seal, Rebecca P; Watts, Spencer D et al. (2006) Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1. J Biol Chem 281:29788-96
Shimamoto, Keiko; Sakai, Ryuichi; Takaoka, Kiyo et al. (2004) Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Mol Pharmacol 65:1008-15
Cheng, Chialin; Glover, Greta; Banker, Gary et al. (2002) A novel sorting motif in the glutamate transporter excitatory amino acid transporter 3 directs its targeting in Madin-Darby canine kidney cells and hippocampal neurons. J Neurosci 22:10643-52
Leighton, Barbara H; Seal, Rebecca P; Shimamoto, Keiko et al. (2002) A hydrophobic domain in glutamate transporters forms an extracellular helix associated with the permeation pathway for substrates. J Biol Chem 277:29847-55
Shigeri, Y; Shimamoto, K; Yasuda-Kamatani, Y et al. (2001) Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5). J Neurochem 79:297-302
Seal, R P; Leighton, B H; Amara, S G (2000) A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines. Neuron 25:695-706
Fairman, W A; Sonders, M S; Murdoch, G H et al. (1998) Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4. Nat Neurosci 1:105-13
Eliasof, S; Arriza, J L; Leighton, B H et al. (1998) Excitatory amino acid transporters of the salamander retina: identification, localization, and function. J Neurosci 18:698-712
Seal, R P; Amara, S G (1998) A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation. Neuron 21:1487-98
Eliasof, S; Arriza, J L; Leighton, B H et al. (1998) Localization and function of five glutamate transporters cloned from the salamander retina. Vision Res 38:1443-54

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