Figure 5.

Mutation of Tyr470 alters transporter conformational transitions. Tyr470 mutation of DAT affects zinc regulation of DA uptake (A) and [³H]WIN 35,428 binding (B). CHO cells transfected with WT or Y470H-hDAT were incubated with KRH buffer alone (control) or with ZnCI₂ (10 μM, final concentration) followed by [³H]DA uptake or [³H]WIN 35,428 binding (n = 4). The histogram shows [³H]DA uptake and [³H]WIN 35,428 binding expressed as mean ± S.E.M. of the respective controls set to 100% for the mutant. *p < 0.05 compared to control. ^#p < 0.05 compared to WT hDAT with ZnCI₂. (C) Functional DA efflux properties of WT hDAT and mutant. CHO cells transfected WT or Y470H-hDAT were preincubated with [³H]DA (0.05 μM, final concentration) at room temperature for 20 min. After incubation, cells were washed and incubated with fresh buffer at indicated time points. Subsequently, the buffer was separated from cells, and radioactivity in the buffer and remaining in the cells was counted. Each fractional [³H]DA efflux in WT hDAT and Y470H-hDAT was expressed as percentage of total [³H] in the cells at the start of the experiment. Fractional [³H]DA efflux at 1, 10, 20, 30 and 40 min are expressed as the percentage of total [³H]DA with preloading with 0.05 μM (WT hDAT: 15379 ± 1800 dpm and Y470H-hDAT: 2488 ± 150 dpm) present in the cells at the start of the experiment (n = 4). ^×p < 0.05 and ^××p < 0.01, compared to WT hDAT (Bonferroni t-test). (D) Functional DA efflux properties of WT hDAT in the presence or absence of Tat_1-72. CHO cells transfected with WT hDAT were preincubated with released Tat_1-72 (1 ng/mg) followed by DA efflux assay. Fractional [³H]DA efflux at 1, 10, 20, 30, 40 and 50 min are expressed as the percentage of total [³H]DA with preloading with 0.05 μM (control: 14200 ± 1448 dpm and released Tat: 10102 ± 1505 dpm) present in the cells at the start of the experiment (n = 6). ^p < 0.05 and ^^p < 0.01, compared to WT hDAT in the absence of Tat (Bonferroni t-test).