R477H presents strong pH dependence for substrate transport. Uptake of l-[³H]glutamate into oocytes expressing WT EAAT1 (A) and R477H (B) was measured at pH 5.5 (black), 6.5 (orange), 7.5 (blue), and 8.5 (red) over a course of 10 min. Oocytes expressing WT EAAT1 were incubated in 10 µM l-[³H]glutamate, and equivalent concentrations of l-[³H]glutamate were used for oocytes expressing R477H to account for the shifts in K_0.5 for glutamate observed in this mutant transporter. See Materials and methods for further details. (C and D) Uptake of l-[³H]glutamate, into oocytes expressing WT EAAT1 (squares) and R477H (circles), was measured over a 120-min time period at pH 8.5 (red) and pH 5.5 (black). 10 µM l-[³H]glutamate was used for WT EAAT1 at both pH 5.5 and 8.5 and R477H at pH 8.5. 0.2 µM l-[³H]glutamate was used for R477H at pH 5.5 to account for differences in substrate affinity. (D) Uptake for l-[³H]glutamate into oocytes expressing R477H at pH 5.5 was lower than at pH 8.5 but is significantly above that of uninjected oocytes. A and B represent l-[³H]glutamate of WT EAAT1– and R477H-expressing oocytes; background uptake is presented in C and D. Background refers to l-[³H]glutamate uptake into uninjected oocytes for each condition. Data shown represent the mean ± SEM (n ≥ 7). Where error bars cannot be seen, they lie within the symbol. Note the scale difference in the y axes.