Article Figures & Data
Figures
- Figure 1.
Activation of EAAT4 anion channels by transporter substrates. A, C, Pulse protocols and representative whole-cell current traces recorded from tsA201 cells expressing EAAT4 transporters. Cells were dialyzed with NaCl- (A) or NaNO3- (C) based internal solutions and perfused with the indicated solutions. Dashed lines represent 0 current amplitude. B, D, Mean current–voltage relationships in the absence of Na+ with K+ as the main extracellular cation (filled circles; n = 5), in the presence of 140 mm Na+ (open circles; n = 13 in B and n = 12 in D), and in the presence of 140 mm Na+ and 0.5 mm glutamate (Glut; inverted filled triangles; n = 25 in B and n = 13 in D). Open triangles give mean current amplitudes from cells perfused with 0.2 mm l-TBOA (n = 5), and the solid line represent current amplitudes from untransfected tsA201 cells measured in 140 mm Na+-containing external solutions in the presence of 0.5 mm glutamate (n = 5).
- Figure 2.
Sodium dependence of EAAT4 anion currents. A, C, Pulse protocols and representative whole-cell current traces recorded from tsA201 cells expressing EAAT4 transporters at different external sodium concentrations. Cells were dialyzed with either an NaCl-(A) or NaNO3-(C) based internal solution, respectively. B, D, Sodium dependences of isochronal anion current amplitudes measured at +165 and −175 mV, respectively. Means ± SEM; n = 6 (B) and n = 5 (D). The solid lines represent fits with the Hill equation with a coefficient of 3. Insets, Plot of apparent dissociation constants (KD values) for various voltages versus the test potentials. In all experiments, 0.5 mm glutamate (Glut) is present, and the extracellular [Na+] was modified by equimolar substitution of Na+ by K+.
- Figure 3.
Glutamate dependence of EAAT4 anion currents and glutamate uptake. A, C, Pulse protocols and representative whole-cell current traces recorded from tsA201 cells expressing EAAT4 transporters at different glutamate (Glut) concentrations. Cells were dialyzed with either 115 mm NaCl- (A) or NaNO3-(C) based internal solutions and externally perfused with 140 NaNO3- and NaCl-based solutions, respectively. B, D, Glutamate dependence of anion currents for NaCl- (B) and NaNO3-based (D) internal solutions. Means ± SEM; n = 5 (B) and n = 8 (D), respectively. Lines represent fits with the Hill equation with different fixed Hill coefficients (n = 1, dashed line; n = 2, dotted line; and n = 3, solid line). Insets give the voltage dependences of the apparent dissociation constants for glutamate (KD values). E, Glutamate dependence of the normalized radioactive glutamate uptake into oocytes expressing EAAT4 glutamate transporters in 96 mm NaSCN external-based solution. Means ± SEM from eight oocytes. Fits with the Hill equation with different Hill coefficients are shown as dashed (n = 1), dotted (n = 2), and solid (n = 3) lines. In the experiments shown in A–D, the extracellular [Na+] was 140 mm and in E was 96 mm.
- Figure 4.
Sodium and glutamate interact in activating EAAT4 anion channels. A, Sodium dependence of EAAT4 anion currents determined for two external glutamate concentrations (5 cells each). B, Glutamate dependence of EAAT4 anion currents for two external sodium concentrations: 40 mm Na+, filled circles (n = 6) and 140 mm Na+, open circles (n = 8). Solid lines represent fits with Hill equations. Insets give the voltage dependence of apparent dissociation constants. Cells were internally dialyzed with NaNO3-based solution and externally perfused with NaCl-based solution. The external [Na+] was changed by equimolar substitution of NaCl by KCl.
- Figure 5.
Effects of two point mutations close to the putative glutamate binding site on coupled and uncoupled transport. A, Alignment of the region containing G464 and Q467 in various human and rat EAAT isoforms. B, Localization of the residues corresponding to G464 (black) and Q467 (gray) of EAAT4 in the ribbon presentation of the three-dimensional structure of the P. horikoshii glutamate transporter (Yernool et al., 2004), viewed from the external membrane site. C, Glutamate dependence of radioactive glutamate uptake by WT and mutant EAAT4. Means ± SEM from eight cells for each glutamate concentration. D, Ratio of uptake current to anion current amplitudes from at least five cells for WT and mutant EAAT4 transporters at 0.5 mm glutamate. E, Anion permeability ratios for WT and mutant EAAT4 in the presence of 0.5 mm glutamate (Glut) for at least four cells.
- Figure 6.
G464S and Q467S modify the intersubunit cooperativity in the EAAT4 anion channel activation. Plots of the normalized anion currents at −155 mV versus the glutamate (Glut) (A) and the Na+ (B) concentration for WT, G464S, and Q467S EAAT4. Means ± SEM from at least three (A) or four (B) cells, respectively. The cells were dialyzed with an NaNO3-based standard internal and perfused with an NaCl-based standard external solution.
- Figure 7.
G464S and Q467S modify gating of EAAT4 anion channels. Representative current recordings for WT and mutant transporters for external (A) or internal (C) NO3−. Recordings in the left column were done in the absence of glutamate and those in the right column after applying 0.5 mm l-glutamate (Glut). B, D, Voltage dependence of the relative open probability for WT, G464S, and Q467S EAAT4 at the same anion conditions as in the corresponding left panel. Means ± SEM from at least six cells (B, D).
- Figure 8.
Mixed heterotrimers consisting of WT and R501C EAAT4 exhibit functional properties that are intermediate to WT and R501C homotrimers. A, Representative current responses to voltage steps to −95 and +125 mV, respectively, from a cell cotransfected with WT and R501C EAAT4 in the presence of 1 mm serine (dotted line), 1 mm glutamate (dashed line), and both together (solid line). The bold solid line gives the sum of the current amplitudes observed after application of individual substrates. B, Voltage dependence of current amplitudes from the experiment shown in A. C, Voltage dependence of normalized mean current amplitudes during the application of both substrates (filled triangles) compared with the predicted value assuming independence of individual subunits forming an anion conduction pathway (open inverted triangles). Means ± SEM from 10 cells. *p < 0.05 and **p < 0.01 indicate a significant difference between measured and predicted values.
Tables
- Table 1.
Gating properties for EAAT4 wild-type and mutants in the absence and presence of glutamate for two anion conditions
V50 (mV) Po min Time constant (ms) n Fast Slow int NaCl−/ext NaNO3− 140 mm Na+ WT EAAT4 42.0 ± 0.5 0.2 ± 0.003 3.8 ± 0.20 41.3 ± 1.75 19 G464S EAAT4 −107.9 ± 15.8** 0.13 ± 0.005 3.5 ± 0.1 36.9 ± 1.74 3 Q467S EAAT4 −97.9 ± 3.5** 0.12 ± 0.012 4.4 ± 0.03 40.1 ± 0.47 3 140 mm Na++ 0.5 mm glutamate WT EAAT4 108.6 ± 3.6 0.47 ± 0.018 2.6 ± 0.14 10.1 ± 0.59 12 G464S EAAT4 66.2 ± 2.1** 0.33 ± 0.014 3.4 ± 0.05** 33.1 ± 0.92** 9 Q467S EAAT4 78.1 ± 1.0** 0.38 ± 0.006 5.9 ± 0.21** 31.0 ± 0.26** 3 int NaNO3− /ext NaCl− 140 mm Na+ WT EAAT4 −4.6 ± 1.17 0.37 ± 0.008 181.5 ± 3.82 11 G464S EAAT4 −125.1 ± 22.7** 0.23 ± 0.013 114.2 ± 10.08** 6 Q467S EAAT4 51.9 ± 12.9** 0.68 ± 0.053 5 140 mm Na++ 0.5 mm glutamate WT EAAT4 −69.9 ± 2.8 0.50 ± 0.015 24.5 ± 0.29 342.2 ± 6.05 14 G464S EAAT4 −58.2 ± 6.2 0.53 ± 0.031 30.6 ± 0.43** 1690 ± 140.2** 7 Q467S EAAT4 −101.2 ± 8.7** 0.70 ± 0.037 2032 ± 62.0** 9 -
↵**p < 0.01 indicate a significant difference from WT. int, Internal; ext, external.
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Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental Figure 1. R501C changes the substrate sensitivity of EAAT4. A,B, Representative current responses to a voltage steps to +125 mV from cells transfected with WT (A) and R501C (B) EAAT4 without application of substrate (dotted line), in the presence of 1 mM serine (dashed line) and 1 mM glutamate (solid line), respectively. C, D, Voltage dependence of mean current amplitudes � SEM (n = 5;C and 6;D) upon the application of serine (?), glutamate (?) and without any of these two substrates (?). WT current amplitudes at 1 mM serine and R501C current amplitudes at 1 mM glutamate were not different from control values without substrate (p>0.05 for WT and p>0.1 for R501C EAAT4, respectively).
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