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

Open Access Meeting abstract

Deciphering structural rearrangements during transport process in the bacterial transporter GltPh, homolog to mammalian glutamate transporter

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

Author Affiliations

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

2 Department of Medicinal Chemistry, University of Vienna, 1090 Vienna, Austria

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BMC Pharmacology and Toxicology 2012, 13(Suppl 1):A57  doi:10.1186/2050-6511-13-S1-A57


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/2050-6511/13/S1/A57


Published:17 September 2012

© 2012 Venkatesan 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

Glutamate transporters are integral membrane proteins that catalyze the concentrative uptake of glutamate from the synapse by harnessing pre-existing ion gradients. In the central nervous system glutamate transporters are essential for normal development and function; they also are implicated in stroke, epilepsy and neurodegenerative diseases. The crystal structure of a eukaryotic glutamate transporter homologue from Pyrococcus horikoshii, is available at various conformations providing a structural framework for the determination of substrate and inhibitor binding to the transporter. In this study we aim to measure structural changes upon transport using lanthanide resonance energy transfer (LRET).

Methods

Site-directed mutagenesis was employed to insert genetically encoded lanthanide binding tags (LBT) into the the protein to perform LRET measurements. Thus generated LBT mutants were expressed and purified, and the functionality of the mutants was assessed by radioligand binding assay.

Results

Models for insertion of LBT were derived from the available crystal structures of the transporter. The wild-type and mutant proteins were expressed and purified using affinity column chromatography. Donor decay signals were recorded for LBT insertion mutants to confirm the insertion of tags. Furthermore, radioligand binding assays were performed with the mutants and they were found to be functional.

Conclusions

Taken together these mutants serve as the starting point to probe the conformational changes that were observed in previously solved crystal structures in reconstituted proteoliposomes. This could help us to integrate the structure-function relationship in the mammalian counterparts.

Acknowledgements

The study was 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.