Fig. 2.

Functional DA efflux properties of hNET mutants. A, cells were loaded with 5 μM [³H]DA for 20 min at room temperature and washed with KRH. Basal efflux was measured in hNET (■), T58A-hNET (▴), and T58D-hNET (♢) as described under Materials and Methods. Efflux is expressed as the percentage of total [³H]DA present in the cell at the start of the experiment (n = 3). In a two-way ANOVA considering time and mutants, p < 0.001 for time, p < 0.01 for mutants, and p < 0.001 for interaction between time and mutants. In Bonferroni post-test, ***, p < 0.001 for hNET or T58A-hNET compared with T58D-hNET. B, AMPH-stimulated DA efflux was assessed by superfusion of the cells as described under Materials and Methods. Cells were loaded with DA, washed with KRH for 30 min, and then challenged with AMPH for 2 min. Fractions (800 μl) were collected, and DA content was analyzed by high-performance liquid chromatography and electrochemical detection. Data are expressed as DA concentration in each fraction as a percentage of total amount of DA taken up in the cells (n = 4). In a two-way ANOVA comparing time with mutants, p < 0.0001 for time, p < 0.01 for mutants, and p < 0.001 for interaction between time and mutants. In Bonferroni post-test, ***, p < 0.001; **, p < 0.01.