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Figure 1. Amino acid sequence alignment of representative members of the glutamate transporter family and substrate transport by replacement mutants at these positions. (A) Amino acid sequence alignment of the representative members of the glutamate transporter family. EAAC, excitatory amino acid carrier; GLT, glutamate transporter; ASCT, alanine serine cysteine transporter; DctA Ec, dicarboxylate transporter Escherichia coli; DctA Rle, dicarboxylate transporter Rhizobium leguminosarum. (B) Uptake of d-[3H]-aspartate in Xenopus laevis oocytes expressing mutants in the stretch 444DRFRTVV450 (EAAC1 numbering) was done as described under Materials and Methods. Except for arginine-447, each residue of this stretch in EAAC1 was replaced by the equivalent residue of DctA Ec. Aspartate-444 of EAAC1 was changed not only to serine, but also to the other residues indicated. The values are corrected for those obtained with uninjected oocytes and are given as percent of WT uptake. Data shown are mean ± SEM, n = 3. (C) Oocytes expressing WT or the mutants D444S, D444C, and D444E were voltage clamped and gravity perfused with ND96 recording solution (see Materials and Methods) with and without 2 mM L-aspartate. The voltage was stepped from â25 mV to voltages between â100 and +40 mV, in increments of 10 mV. Each potential was held clamped for 250 ms, and the steady-state current from 210 to 240 ms at each potential was averaged. The current in the absence of L-aspartate was subtracted from that in its presence (I) and plotted against the holding potential (Vhold). The values shown are mean ± SEM from three oocytes from different batches that had similar expression levels.
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Figure 2. Currents in oocytes expressing D444S-EAAC1 transporters. Currents recorded in the absence of substrate during 250-ms voltage pulses from â25 mV to voltages between â100 and +40 mV were subtracted from currents in the same medium containing 2 mM succinate (A and B). In C and D, currents in the presence and absence of 10 mM succinate (C) or 160 μM d,l-TBOA (D) were recorded. The current traces in C and D are from the same cell. For these two panels, the opposite subtraction procedure of that used in A and B was followed. Currents from representative oocytes (n = 3) are shown. The zero current level is indicated by the stippled line.
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Figure 3. Specificity and sodium dependence of the inhibition of transient currents by succinate in D444S-EAAC1. Oocytes expressing D444S-EAAC1 were perfused in the presence and absence of succinate 5 mM (A, C, and D) or GABA 5 mM (B) in media containing either sodium 96 mM (A and B) lithium 96 mM (C), or choline 96 mM (D), and net isolated transient currents were visualized by subtracting the currents in the presence of succinate or GABA from those in their absence. A representative oocyte is shown (n = 3).
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Figure 4. Blockade of charge movements in D444S-EAAC1 by succinate and acidic amino acids. The charge moved was calculated from the isolated transient currentâtime integrals as described under Materials and Methods. The fraction of transient currents blocked at each concentration of the indicated compound was normalized to the transient currents isolated by saturating (10 mM) concentrations of L-glutamate (circles), L-aspartate (triangles), D-aspartate (diamonds), and succinate (squares) and is shown in A. The currentâtime integrals of the transients ranged from 1.0 to 2.8 nC. Data shown are mean ± SEM of four to six oocytes. The currents blocked by 10 mM D- and L-aspartate in the same oocyte are shown in B and C, respectively.
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Figure 5. Blockade of charge movements in D444C-EAAC1 by succinate and acidic amino acids. The charge movements were analyzed as described in the legend to Fig. 4. The isolated transient currents were normalized to the transient currents isolated by saturating concentrations (10 mM) of the same compounds (same symbols as in Fig. 4 A) and are shown in A. The currentâtime integrals of the transients ranged from 1.5 to 7.0 nC. Data shown are mean ± SEM of three to eight oocytes. The currents blocked by 10 mM L-aspartate and 10 μM d,l-TBOA in the same oocyte, typical of three, are shown in B and C, respectively.
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Figure 6. The effects of MTSES and DTT on the transient currents by D444C. Currents recorded in the presence of 2 mM L-aspartate were subtracted from currents recorded in its absence in oocytes expressing either D444C-EAAC1 (AâC) or D444S-EAAC1 (DâF). The transient currents were measured before (A and D) or after the application of 2 mM MTSES (B and E) and after MTSES and 50 mM DTT (C and F). Typical oocytes are shown (n = 3).
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Figure 7. Transient currents by D444E-EAAC1 transporters. The concentration dependence of the suppression of the charge movements by L-aspartate (A) or succinate (B) was determined as described under Materials and Methods. Charge moved was normalized to that observed at 1 mM of L-aspartate and 15 mM succinate, respectively. The currentâtime integrals of the transients ranged from 2.1 to 4.6 nC. Data shown are mean ± SEM of three oocytes. Currents recorded in the presence of L-aspartate at 2 mM (C) or 50 μM (E), of d,l-TBOA at 10 μM (D), or of succinate at 2 mM (F) were subtracted from currents recorded in their absence in oocytes expressing D444E-EAAC1. Representative oocytes are shown (n = 3). The same cell was used in C and D and the same is true for E and F.
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Figure 8. Substrate-induced steady-state currents by WT and D444E transporters in the presence of thiocyanate. Oocytes expressing either WT-EAAC1 (A) or D444E-EAAC1 (B) were voltage clamped and gravity perfused with NaSCN-based buffer as described under Materials and Methods in the absence or presence of 2 mM L-aspartate (triangles), succinate (circles), and GABA (squares) or 10 (A) or 30 μM (B) d,l-TBOA (inverted triangles). The voltage was stepped from â25 mV to voltages between â100 and +40 mV. The currents in the absence of substrate/blocker were subtracted from those in its presence and normalized to the L-aspartateâelicited current at +40 mV (I), and plotted against the holding potential (Vhold). In the three oocytes where currents by L-aspartate and d,l-TBOA were compared directly, the L-aspartateâinduced currents ranged from +625 to +837 nA at +40 mV in oocytes expressing WT-EAAC1, whereas in oocytes expressing D444E-EAAC1, the currents suppressed by L-aspartate ranged from +460 to +700 nA. The corresponding values for the currents blocked by d,l-TBOA were +270 to +366 nA for WT-EAAC1 and +600 to +820 nA for D444E-EAAC1. Data shown are mean ± SEM of three oocytes, except for L-aspartate (n = 6).
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Figure 9. Anion selectivity and sodium dependence of D444E transporters. Steady-state currents in oocytes expressing D444E in the presence of 2 mM L-aspartate minus those in its absence were monitored in a chloride-based ND96 external medium (squares) or in media where 9.6 mM NaCl was replaced by an equimolar concentration of the sodium salts of iodide (circles), nitrate (triangles), perchlorate (inverted triangles), or thiocyanate (diamonds). The currents shown are normalized to the value in the thiocyanate medium at +40 mV. The absolute values of the currents in 9.6 mM sodium thiocyanate plus 86.4 mM NaCl ranged from 220 to 320 nA (A). The sodium dependence of the currents suppressed by 2 mM L-aspartate was determined using media containing NaCl at concentrations ranging from 0 to 86 mM (iso-osmotic compensation by choline chloride), 10 mM KSCN, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM Tris-HEPES, pH 7.5. The magnitude of these currents suppressed by L-aspartate did not change when its concentration was increased to 4 mM, indicating that the concentration of the acidic amino acid was saturating, even at low sodium concentrations. The absolute values of the currents suppressed by L-aspartate in the presence of 86 mM NaCl at +40 mV ranged from 125 to 254 nA (B). Data shown in each of the panels are mean ± SEM of three oocytes.
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Figure 10. Uptake of d-[3H]-aspartate in HeLa cells expressing mutants D444C and D444E. Uptake of d-[3H]-aspartate in HeLa cells transfected with WT-EAAC1, D444C, D444E, and the empty vector were performed in NaCl-containing medium in the absence (open bars) or presence (hatched bars) of 5 mM succinate as described under Materials and Methods. Values were corrected for uptake in cells transfected with the empty vector and given as percent of uptake relative to WT-EAAC1. Values represent the mean ± SEM of three separate experiments, each done in triplicate. The absolute counts of D444C were â¼2.5-fold of those by cells expressing the empty vector, and in the case of D444E, the corresponding value was â¼1.4-fold.
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