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Elucidating Conformational Changes in the γ‐Aminobutyric Acid (GABA)Transporter
Author(s) -
Meinild AnneKristine,
Loo Donald,
MacAulay Nanna
Publication year - 2008
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.22.1_supplement.936.11
Subject(s) - gaba transporter , chemistry , fluorescence , transporter , cysteine , xenopus , biophysics , homology modeling , voltage clamp , amino acid , neurotransmitter transporter , rhodamine , membrane potential , biochemistry , biology , physics , quantum mechanics , gene , enzyme
In addition to the Na + ‐coupled GABA transport, the Na + /Cl − dependent GABA transporter, GAT‐1, also has a Li + ‐induced leak. Recently, the amino acids directly involved in the interaction with the Na + and Li + ions have been identified (Zhou et al, 2006, JBC Vol. 281). In the present study, we obtain insight into the structural dynamics of the two transport modes of GAT‐1 by applying the voltage clamp fluorometry method (VCF) to wild type GAT‐1 expressed in Xenopus oocytes. We have taken advantage of the only accessible cysteine in wild type rat GAT‐1, C74, and labeled this residue with a cysteine‐reactive fluorophor, tetramethyl rhodamine‐6‐maleinamide. Our fluorescent read‐outs show that the transporter undergoes different voltage dependent conformational changes in the presence of the different ions in the external solution: in Na + buffer the V ½ for fluorescence is −2 ± 9 mV whereas in Li + buffer it is −71 ± 12 mV. The time courses of these fluorescent changes are also highly dependent on the cation: in Na + the τ max is 120 ms and in Li + it is 40 ms. The fluorescence data for Li + correlates with the Li + induced currents: these are highly voltage dependent and only present at hyperpolarized potentials (<−50 mV). The study demonstrates the advantage of applying the VCF method in combination with homology modeling of the neurotransmitter transporters to the newly crystallized bacterial homolog, LeuT.