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Lanthanide resonance energy transfer‐based distance measurements in the mammalian glutamate transporter excitatory amino acid transporter 3 (1064.12)
Author(s) -
Saha Kusumika,
Venkatesan Santhosh,
Sohail Azmat,
Stockner Thomas,
Sandtner Walter,
Ecker Gerhard,
Sitte Harald
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1064.12
Subject(s) - transporter , chemistry , glutamate receptor , symporter , glutamate aspartate transporter , förster resonance energy transfer , pyrococcus horikoshii , acceptor , biophysics , amino acid , excitatory postsynaptic potential , excitatory amino acid transporter , glutamic acid , neurotransmission , biochemistry , biology , fluorescence , physics , receptor , enzyme , quantum mechanics , gene , condensed matter physics
EAAT3 (Excitatory amino acid transporter 3) mediates the regulation of synaptic transmission by reuptake of glutamate in the synaptic cleft. The motivation of the study is to gain insight into the structure function relation of EAAT3. The project utilizes the high resolution crystal structure of GltPh, the bacterial orthologue of Pyrococcus horykoshi to mammalian glutamate transporters. 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). LRET relies on Förster's theory of resonance energy transfer where there is distance dependent transfer of energy between the donor fluorochrome to an acceptor fluorochrome. The protein is expressed in Xenopus laevis oocytes. Lanthanide binding tags (LBT) has been inserted into the protein to chelate the lanthanide terbium which serves as the donor element and aminoacid residues in the protein are mutated to cysteine which then reacts to an acceptor dye serving as acceptor. The measured distances allow us to understand the structure function relationship of the glutamate transporters and can be further investigated using different substrates and inhibitors. The results obtained in this project will allow us to better understand pathophysiological conditions associated with loss of function in EAAT3, for instance in ischemia. Grant Funding Source : F3506 and W1232 MolTag