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Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters


Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids1. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh2,3,4,5,6. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na+ ions2 as well as an uncoupled flux of Cl ions7. Recent crystal structures of GltPh3,5,6,8 revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.

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Figure 1: Experimental setup.
Figure 2: Single-molecule FRET dynamics.
Figure 3: Independent subunit dynamics within GltPh trimmers.
Figure 4: Branching ratios and dwell times for various GltPh conformations.


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The authors would like to thank M. Punter for developing software and algorithms for data analysis, V. Krasnikov for his help with the design, construction and maintenance of the single-molecule fluorescence microscopes and I. Küsters for sharing experience on liposome tethering and single-molecule imaging of membrane proteins. A.M.v.O. acknowledges funding from the Netherlands Organization for Scientific Research (NWO; Vici 680-47-607) and the European Research Council (ERC Starting 281098). D.J.S. acknowledges funding from the Netherlands Organization for Scientific Research (NWO; Vidi 700.54.423, Vici 865.11.001) and the European Research Council (ERC Starting 282083). I.H. acknowledges the Deutsche Forschungsgemeinschaft (HA 6322/1-1) for providing funding.

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Authors and Affiliations



G.B.E., I.H., J.M.H.G., D.J.S. and A.M.v.O. designed experiments, G.B.E. and I.H. performed experiments, J.M.H.G. performed the FRET level transition calculations, G.B.E., D.J.S. and A.M.v.O. wrote the manuscript, all authors contributed to the interpretation of the data.

Corresponding authors

Correspondence to Dirk Jan Slotboom or Antoine M. van Oijen.

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The authors declare no competing financial interests.

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Erkens, G., Hänelt, I., Goudsmits, J. et al. Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters. Nature 502, 119–123 (2013).

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