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

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Decrypting structural and functional changes in LeuTAa at atomic level employing LRET

Azmat Sohail1, Simon Bulling1, Peggy Stolt-Bergner2, Oliver Kudlacek1, Gerhard F Ecker3, Michael Freissmuth1, Thomas Stockner1, Harald H Sitte1* and Walter Sandtner1

Author affiliations

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

2 Research Institute of Molecular Pathology, Campus Vienna Biocenter, 1030 Vienna, Austria

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

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Citation and License

BMC Pharmacology and Toxicology 2012, 13(Suppl 1):A53  doi:10.1186/2050-6511-13-S1-A53

The electronic version of this article is the complete one and can be found online at:

Published:17 September 2012

© 2012 Sohail et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Neurotransmitter:sodium symporters (NSS) are integral membrane proteins that mediate the reuptake of monoamine neurotransmitters previously released into the synaptic cleft. They are of pharmacological significance because they are the target of many clinically important drugs. LeuTAa, a leucine/alanine transporter is a bacterial homolog to NSS. Crystal structures of LeuTAa with open to outward, occluded and inward-facing states have already been resolved at high resolution. Hence, LeuTAa serves as a good paradigm for exploring the structure-function relationship of NSS proteins.

Methods and results

To investigate the structure-function relation in LeuT at atomic level we employ lanthanide-based resonance energy transfer (LRET). LRET-based measurements require the introduction of an LBT (lanthanide binding tag) to accommodate terbium as the donor element and fluorophores chemically linked to a cysteine residue as the acceptor element. LBT tags and cysteine are introduced at selected positions in LeuTAa. Introduction of an LBT tag may lead to a functionally disturbed host protein. So to screen functional LBT-LeuT mutants we established the scintillation proximity assay in the lab. To date, after screening functional LBT mutants of LeuTAa, we have measured the intramolecular distances at atomic level of LeuT in micelles. In order to validate these distances we want to see the distance changes after reconstitution into liposomes, a more native environment that allows to establish a sodium gradient; this is important since the NSS operates along a chemical gradient.


Our LRET measurements are expected to help us validate or propose models of substrate transport. Our future plan focusses on the reconstitution of LeuTAa in liposomes to allow for distance measurements in a more native environment.


Supported by the Austrian Science Fund FWF (grant SFB3506 to H.H.S.) and partially funded by HEC (A.S.).