Abstract
Glutamate transporters catalyze concentrative uptake of the neurotransmitter into glial cells and neurons. Their transport cycle involves binding and release of the substrate on the extra- and intracellular sides of the plasma membranes and translocation of the substrate-binding site across the lipid bilayers. The energy of the ionic gradients, mainly sodium, fuels the cycle. Here, we used a cross-linking approach to trap a glutamate transporter homolog from Pyrococcus horikoshii in key conformational states with the substrate-binding site facing either the extracellular or the intracellular side of the membrane to study binding thermodynamics. We show that the chemical potential of sodium ions in solution is exclusively coupled to substrate binding and release, not to substrate translocation. Despite the transporter's structural symmetry, the binding mechanisms are distinct on the opposite sides of the membrane and more complex than the current models suggest.
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Acknowledgements
The Rockefeller University high-throughput screening facility is acknowledged for the use of their ITC instrument, contributing to the ITC studies. US National Institutes of Health grants R01 NS064357 (O.B.) and S10 RR026970 (O.B.) are acknowledged for supporting the research and providing funding for ITC instrumentation, respectively. The US National Synchrotron Light Source X29 beamline was used for data collection, and we thank the staff for help and continuous support. We thank H.J. Appel for his advice on the use RH421 dye. We also thank A. Accardi for critical reading of the manuscript.
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N.R. and O.B. designed the experiments. N.R. and S.O. conducted the experiments, and N.R. and O.B. analyzed the data and wrote the manuscript.
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Reyes, N., Oh, S. & Boudker, O. Binding thermodynamics of a glutamate transporter homolog. Nat Struct Mol Biol 20, 634–640 (2013). https://doi.org/10.1038/nsmb.2548
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DOI: https://doi.org/10.1038/nsmb.2548
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