Distinct roles of the Na+ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, GltPh

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of gluta...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 119; no. 19; p. 1
Main Authors Riederer, Erika A, Moënne-Loccoz, Pierre, Valiyaveetil, Francis I
Format Journal Article
LanguageEnglish
Published Washington National Academy of Sciences 10.05.2022
Subjects
Allosteric properties
Assaying
Binding sites
Biological Sciences
Cell membranes
Fluorescence
Glutamic acid transporter
Homology
Sodium
Spectroscopy
Substrates
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Summary:Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh, but the specific roles of the individual Na+ sites in the binding process have not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies, we also propose an alternate pathway for the coupled binding of Na+ and Asp.
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Edited by Ernest Wright, David Geffen School of Medicine at UCLA, Los Angeles, CA; received November 29, 2021; accepted March 18, 2022
Author contributions: E.A.R. and F.I.V. designed research; E.A.R. performed research; E.A.R., P.M.-L., and F.I.V. analyzed data and wrote the paper.
ISSN:0027-8424
1091-6490
1091-6490
DOI:10.1073/pnas.2121653119