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Transport dynamics in a glutamate transporter homologue

Nature volume 502, pages 114–118 (2013)Cite this article

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Abstract

Glutamate transporters are integral membrane proteins that catalyse neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons1. Their mechanism of action involves transitions between extracellular (outward)-facing and intracellular (inward)-facing conformations, whereby substrate binding sites become accessible to either side of the membrane2. This process has been proposed to entail transmembrane movements of three discrete transport domains within a trimeric scaffold3. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging4, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods5,6. We propose that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent transmembrane movements and may be rate determining in the transport cycle.

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Figure 1: FRET efficiency changes reflect relative orientations of the transport domains.
Figure 2: Dynamics in the apo and substrate bound transporter.
Figure 3: Na+ ions and Asp favour the outward facing state.
Figure 4: Modulation of dynamics by substrate and inhibitor binding.

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Acknowledgements

We would like to thank D. S. Terry for his help with the design of smFRET experiments and discussions; Z. Zhou for the synthesis of cyanine fluorophores; E. Georgieva for initial biochemical characterization of the single-cysteine mutants; G. Verdon, A. Accardi and N. Reyes for helpful discussions and comments on the manuscript. The work was supported in part by the National Institute of Health grants 5U54GM087519 and R01NS064357.

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  1. Department of Physiology and Biophysics, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10064, USA,

    Nurunisa Akyuz, Roger B. Altman, Scott C. Blanchard & Olga Boudker

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  1. Nurunisa Akyuz
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  2. Roger B. Altman
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  3. Scott C. Blanchard
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Contributions

N.A. purified GltPh mutants, carried out the experiments and analysed the data. R.B.A. prepared reagents for smFRET experiments. N.A., O.B. and S.C.B. together designed, analysed and interpreted the experiments and wrote the manuscript.

Corresponding authors

Correspondence to Scott C. Blanchard or Olga Boudker.

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The authors declare no competing financial interests.

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Supplementary Information file contains Supplementary Figures 1-10, Supplementary Methods, Supplementary Tables 1-2, Supplementary Discussion and Supplementary References. (PDF 3709 kb)

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Akyuz, N., Altman, R., Blanchard, S. et al. Transport dynamics in a glutamate transporter homologue. Nature 502, 114–118 (2013). https://doi.org/10.1038/nature12265

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