Figure 6.

TM4-9 mutant displays increased cocaine binding and altered kinetics of the substrate transport. A, PAE/YFP-HA-DAT (wtDAT) and PAE/TM4-9 cells were incubated with 100 nM JHC 1-64 in F12 supplemented with 10 mM Hepes (pH7.4) for 15 min at room temperature. 3-D images were acquired from fixed cells through 488 nm (YFP) and 561 nm (JHC 1–64) channels. Maximum intensity projections of representative 3D images are shown. Scale bar, 10 μm. B, the ratio of JHC 1-64 and YFP fluorescence intensities was calculated from experiments shown in (A) as described in “Experimental procedures”. Mean values (±SD; n = 10) are presented. The p values were calculated for TM4-9 versus wtDAT using two-tail, unpaired t test. C, PAE/YFP-HA-DAT (wtDAT) and PAE/TM4-9 cells were incubated with 100 nM [3-H]β-CFT at 20 °C for 15 min. Mean values (±SD; n = 3) are presented. The p values were calculated for TM4-9 versus wtDAT using two-tail, paired t test. D, PAE/YFP-DAT (wtDAT) and PAE/TM4-9 cells were incubated with 50 nM [3-H]DA and 0.2 to 20 μM cold DA at 20 °C for 10 min. An example of the DA uptake concentration dependence is shown. The amount of bound [3-H]DA was normalized by the amount of YFP-HA-DAT (in arbitrary units) determined by Western blotting with the GFP antibody. Each data point is a mean of triplicate wells. Statistical deviations are not shown if they are smaller than the size of the symbol. Inset shows a detailed view of the part of the graph depicting differences in the DA uptake between WT and mutant DAT at low DA concentrations. Mean values of Km and Vmax were determined from 5 independent experiments. Vmax values are expressed as percent of the Vmax value calculated for WT YFP-HA-DAT in each experiment. The p values for TM4-9 versus wtDAT were calculated using two-tail, paired t test. AL, AIM-100–like; DAT, dopamine transporter; PAE, porcine aortic endothelial; TM, tansmembrane; YFP-HA-DAT, YFP- and HA-epitope tagged DAT.