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. 2018 Oct 18;7:e40110. doi: 10.7554/eLife.40110

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© 2018, Cirri et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 1. — (a-b) Residues exchanged for consensus amino acids in EAAT1_CO (a) and EAAT1_COCO (b) are mapped into the structure of the EAAT1_CRYST (PDB 5LLM) trimer viewed from the extracellular medium (left panel), as well as the scaffold (cyan) and the transport (orange) domains viewed from the membrane. These domains are depicted separately for clarity of display, including two views of the transport domain separated ~ 180° from each other, showing its interface with the scaffold domain (left) and the membrane (right), respectively. Spheres correspond to the alpha carbon atoms of residues that were exchanged by conservative (grey) and non-conservative (black) consensus mutations. (c–d), Radioactive L-glutamate uptake in HEK293 cells expressing the transporters (c), including control cells transfected with a vector lacking EAAT1 genes, and in liposomes with purified reconstituted transporters (d). EAAT1_WT data in (d) was originally published in ref. (29). Yellow circles depict the liposomal bilayer separating sodium- (Na⁺), potassium- (K⁺), and choline-based (Ch⁺) solutions. (e), Rate of L-glutamate uptake by purified EAAT1_CO (blue) and EAAT1_COCO (red) reconstituted in liposomes, as a function of L-glutamate concentration. Solid lines indicate Michaelis-Menten fits to the data with K_m values 30.7 ± 25.6 and 18.8 ± 8.3 μM, and V_max values 18.3 ± 4.3 and 12.8 ± 1.4 pmol μg⁻¹ min⁻¹ for EAAT1_CO (blue) and EAAT1_COCO (red), respectively. Plots in c–e) depict an average of at least three independent experiments performed with duplicate measurements, and error bars represent s.e.m.

Figure 1—figure supplement 1. — (a-b) Residues exchanged for consensus amino acids in EAAT1_CO (a) and EAAT1_COCO (b) are mapped into the structure of the EAAT1_CRYST (PDB 5LLM) trimer viewed from the extracellular medium (left panel), as well as the scaffold (cyan) and the transport (orange) domains viewed from the membrane. These domains are depicted separately for clarity of display, including two views of the transport domain separated ~ 180° from each other, showing its interface with the scaffold domain (left) and the membrane (right), respectively. Spheres correspond to the alpha carbon atoms of residues that were exchanged by conservative (grey) and non-conservative (black) consensus mutations. (c–d), Radioactive L-glutamate uptake in HEK293 cells expressing the transporters (c), including control cells transfected with a vector lacking EAAT1 genes, and in liposomes with purified reconstituted transporters (d). EAAT1_WT data in (d) was originally published in ref. (29). Yellow circles depict the liposomal bilayer separating sodium- (Na⁺), potassium- (K⁺), and choline-based (Ch⁺) solutions. (e), Rate of L-glutamate uptake by purified EAAT1_CO (blue) and EAAT1_COCO (red) reconstituted in liposomes, as a function of L-glutamate concentration. Solid lines indicate Michaelis-Menten fits to the data with K_m values 30.7 ± 25.6 and 18.8 ± 8.3 μM, and V_max values 18.3 ± 4.3 and 12.8 ± 1.4 pmol μg⁻¹ min⁻¹ for EAAT1_CO (blue) and EAAT1_COCO (red), respectively. Plots in c–e) depict an average of at least three independent experiments performed with duplicate measurements, and error bars represent s.e.m.