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This article is part of the supplement: 18th Scientific Symposium of the Austrian Pharmacological Society (APHAR)

Open Access Meeting abstract

LRET-based distance measurements in the mammalian glutamate transporter EAAT3

Kusumika Saha1, SanthoshKannan Venkatesan1, Azmat Sohail1, Thomas Stockner1, Simon Bulling1, Gerhard F Ecker2, Harald H Sitte1* and Walter Sandtner1

Author Affiliations

1 Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University Vienna, 1090 Vienna, Austria

2 Pharmacoinformatics Research Group, Department of Medicinal Chemistry, University of Vienna, 1090 Vienna, Austria

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BMC Pharmacology and Toxicology 2012, 13(Suppl 1):A52  doi:10.1186/2050-6511-13-S1-A52

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/2050-6511/13/S1/A52


Published:17 September 2012

© 2012 Saha et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

EAAT3 (excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. It is distributed in neuronal membranes and is selectively enriched in the neurons of the hippocampus, cerebellum and the basal ganglia. It belongs to the family of soluble carrier family 1 member 1 (SLC1A1) and is expressed in kidney, a wide variety of epithelial tissues, brain and eyes.

Methods

The project utilizes the high-resolution crystal structure of GltPh, the bacterial orthologue to mammalian glutamate transporters. GltPh provides a structural framework for the determination of the helical movement in EAAT3. The structural rearrangement of the protein is caused by the helical movements which will be assessed by distance measurements using the technique of lanthanide resonance energy transfer (LRET). The protein will be expressed in Xenopus laevis oocytes. Lanthanide binding tags (LBT) will be inserted into the protein to chelate the lanthanide terbium which serves as the donor element. Cysteines will be reacted to an acceptor dye (bodipy FL).

Expected results

The measured distances will allow us to obtain new insights into the structure-function relationship of the glutamate transporters which can be further investigated using different substrates and inhibitors.

Discussion

The results obtained in this project will allow us to better understand pathophysiological conditions associated with mutations in EAAT3, for instance mutations causing human dicarboxylic aminoaciduria.

Acknowledgements

The study is supported by grants F3506 and W1232 of the PhD program MolTag (Molecular Drug Targets) of the University of Vienna, the Medical University of Vienna and the Vienna University of Technology.