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Fig. 1. The structure of a GltPh protomer, and sequence alignments of ASCTs and EAATs and GltPh
a GltPh protomer (PDB: 2NWX) shown in the plane of the membrane, with the trimerization domain (TM1, 2, 4 and 5) in grey and the transport domain: TM3 (blue), TM6 (green), TM7 (orange), TM8 (purple), HP1 (yellow) and HP2 (red). L-Aspartate and two bound Na+ ions (dark grey spheres) are shown. Close-up view of the substrate-binding site is shown with b R397 and c Y236, L239 and G396 shown in stick representation. In c, the protein has been rotated for better visualisation of the TM3 and TM6 interface. Images were made using Pymol64. d Sequence alignment of part of TM8 in EAAT1-3, ASCT1-2 and GltPh, where conserved residues are highlighted in black, and mutated residues are highlighted by blue boxes
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Fig. 2. Mutations in ASCT1 and ASCT2 affect substrate and inhibitor selectivity a Current–voltage relationships elicited by 300 µM L-serine in Cl−-containing buffer (open squares) and NO3
−-containing buffer (closed squares), at pH 7.5 in wild-type ASCT1. Concentration–response curves are shown for L-serine b and L-glutamine c in ASCT1 (red, closed squares), ASCT2 (blue, closed triangles), A1-T458S (red diamonds), A1-T459C (red, closed circles), A1-T458S/T459C (red, open squares) and A2-S481T/C482T (blue, open circles) at+60 mV. As L-glutamine concentration–response curves for ASCT1, A1-T458S and A2-S481T/C482T was not determined, data were normalised to the predicted maximal current for L-serine for each transporter. d Concentration–response curves are shown for GPNA in ASCT1 (red, closed squares), ASCT2 (blue, closed triangles), A1-T459C (red, closed circles), A1-T458S (red diamonds), A1-T458S/T459C (red, open squares) and A2-S481T/C482T (blue, open circles). GPNA was co-applied with an EC50 value of L-serine for the respective transporters and normalised to the current generated by the application of L-serine alone. L-[3H]serine e and L-[3H]glutamine f uptake into oocytes expressing wild-type and mutant ASCT1 (red), ASCT2 (blue) transporters and uninjected control oocytes (black). Oocytes were incubated in Cl−-containing buffer with 10 µM L-[3H]substrate at room temperature, pH 7.5 for 10 min. Values presented are mean ± S.E.M, (n ≥ 3)
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Fig. 3. Mutations in GltPh alter the substrate selectivity to prefer neutral amino acids L-[3H]aspartate (100 nM; green triangles), L-[3H]serine (1 µM; red circles), L-[3H]alanine (1 µM; blue squares) and L-[3H]glutamine (1 µM; purple diamonds) transport by wild-type GltPh
a, GltPh-R397C c and GltPh-G396S/R397C e in the presence of an inwardly directed Na+ gradient at pH 7.5. Uptake of each substrate in the absence of a Na+ gradient is shown in open, black symbols, of which multiple are overlayed. b L-[3H]aspartate concentration–response for wild-type GltPh. L-[3H]serine (red circles) and L-[3H]alanine (blue squares) concentration responses are shown for GltPh-R397C d and GltPh-G396S/R397C f. Values presented are mean ± S.E.M, (n ≥ 3)
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Fig. 4. Neutral amino acid and inhibitor selectivity in wild-type and mutant GltPh Initial rates of uptake in the presence of 100 nM L-[3H]aspartate (black bar) for wild type a or 10 µM L-[3H]serine (black bars) for GltPh-R397C b and GltPh-G396S/R397C c and 100-fold unlabelled competitor as indicated on the x-axis (white bars). Values are normalised to initial rates of L-[3H]substrate uptake alone, n = 3 ± SEM. Concentration–response curves are shown for TBOA (closed circles), benzylserine (closed triangles), benzylcysteine (open triangles) and GPNA (open squares) in wild-type GltPh
d, GltPh-R397C e and GltPh-G396S/R397C f. Values shown represent mean ± SEM, n ≥ 3
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Fig. 5. Substrate-binding site in wild-type and mutant GltPh View of a single protomer of the wild-type GltPh bound to L-Aspartate a and GltPh-R397C bound to L-Cysteine c. The protomer is divided into the ‘scaffold domain’ (TM1, TM2, TM4 and TM5, coloured in grey) and the ‘transport domain’ (TM3 in blue, TM6 in green, TM7 in orange, TM8 in magenta, HP1 in yellow and HP2 in red). Bound substrate is shown in stick representation and Na1 and Na2 are shown as dark grey spheres. Close-up view of the L-Aspartate b and the L-Cysteine d binding sites, respectively. Interactions within 3.5 Å between bound substrate and residues in the GltPh-binding site are shown as dashed lines. Averaged 2fo–fc electron density maps at 1σ are shown in dark grey mesh and averaged fo–fc electron density maps at 3σ sigma are shown in green
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Fig. 6. Inhibitor-binding site in wild-type and mutant GltPh. View of a single protomer of the wild-type GltPh bound to TBOA (PDB:2NWW, a and GltPh-R397C bound to benzylcysteine c. The protomer is divided into the ‘scaffold domain’ (TM1, TM2, TM4 and TM5, coloured in grey) and the ‘transport domain’ (TM3 in blue, TM6 in green, TM7 in orange, TM8 in magenta, HP1 in yellow and HP2 in red). Bound inhibitor is shown in stick representation and Na1 and Na2 are shown as dark grey spheres. Close-up view of the TBOA b and benzylcysteine d binding sites. Interactions within 3.5 Å between bound inhibitor and residues in the GltPh binding site are shown as dashed lines. Averaged 2fo–fc electron density maps at 1σ in dark grey mesh are shown for benzylcysteine and C397
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Fig. 7. Binding pocket of GltPh-R397C Surface representation of the binding pocket of GltPh-R397C bound to benzylcysteine (green sticks). TBOA bound to GltPh (PDB:2NWW; cyan sticks) is superimposed in the binding site. ‘Pocket A’ and ‘Pocket B’ indicated by orange circles
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