Excitatory amino acid transporters (EAATs) in the CNS maintain extracellular glutamate concentrations below excitotoxic levels and contribute to the clearance of glutamate released during neurotransmission. Over the previous funding period our laboratory took advantage of a highly functional cysteineless version of EAAT1, to identify the structural features required for substrate transport and ion permeation using cysteine substitutions together with sulfhydryl modifying reagents. In this competing renewal application we plan to assess proximity of different residues during the transport cycle using introduced cysteine pairs and crosslinking reagents. Studies will continue to emphasize kinetic, biochemical, pharmacological and electrophysiological analyses of EAAT function. In a second aim these approaches will be combined with experiments using computational methods to model the conformational dynamics of glutamate transporters. Gaussian network modeling (GNM) and molecular dynamic (MD) simulations are techniques ideally suited for the study of large, multifunctional structures such as ion channels and neurotransmitter transporters. To date, methods that treat such multimeric proteins have been restricted to atomic interactions or limited, sub- nanosecond time ranges, which are too localized or fast compared to the phenomena that are observable in our experiments. The use of these two complementary methods provide a robust way of identifying critical interactions, which then can be tested by structure-function experiments designed to alter the structure and mobility of the domain of interest.
A third aim will explore the mechanism of action of a neuroprotective compound purified from a spider venom, which appears to enhance transport activity by altering a less studied transition step in the transport cycle, the reorientation of the unoccupied carrier to the outside. This compound, which acts selectively on the major glial carrier EAAT2, increases glutamate influx but not efflux, and provides proof of principle for the development of allosteric activators of EAATs with therapeutic potential. The importance of understanding the structure, function, and dynamics of excitatory amino acid transporters is underscored by clinical and experimental studies, which have implicated increases in extracellular glutamate concentration 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 Mental Health (NIMH)
Type
Research Project (R01)
Project #
3R01MH080726-14S1
Application #
8597082
Study Section
Biophysics of Neural Systems Study Section (BPNS)
Program Officer
Nadler, Laurie S
Project Start
1995-06-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
14
Fiscal Year
2013
Total Cost
$537,485
Indirect Cost
$180,514
Name
University of Pittsburgh
Department
Biology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Fontana, Andreia Cristina Karklin; de Oliveira Beleboni, Rene; Wojewodzic, Marcin Wlodzimierz et al. (2007) Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom. Mol Pharmacol 72:1228-37