FIGURE 9.

Na⁺-dependent [³H]DA uptake by DAT-WT and the DAT-R60A, DAT-Y335A, and DAT-D436A mutants. [³H]DA uptake by DAT-WT (■) is increased with increasing [Na⁺] and reaches saturation with an EC₅₀ = 27 ± 2 mm Na⁺ and a Hill slope of 2.0 ± 0.23 (n = 6). [³H]DA uptake by DAT-R60A (▵) and DAT-D436A (▿) exhibits Na⁺ dependence indistinguishable from WT traces (n = 7 and 3, respectively). The uptake by DAT-Y335A (○) also increases with increasing [Na⁺] but does not saturate within the range of tested Na⁺ concentrations (n = 4). To obtain a constant ionic strength in the uptake buffer, the NaCl is titrated against choline chloride. Data are means ± S.E. (error bars) of experiments performed in triplicate on COS7 cells transiently expressing DAT-WT or the indicated mutations herein and normalized to their respective uptake in 200 mm Na⁺. Kinetic constants were determined using a single-site fitting model in GraphPad Prism version 5.0.