Figure 4.

Mutations in TM4 and TM9 inhibit the effects of AIM-100 and ALs on DAT.A, alignment of the TM4 and TM9 sequences of several SLC6 transporters and the TM4-9 mutant of human DAT. B, PAE/YFP-HA-DAT and PAE/TM4-9 cells were incubated with vehicle or AIM-100, AL3, AL4, or AL8 (all 20 μM) for 2 h at 37 °C, and lysates were analyzed by SDS-PAGE and immunoblotting with the GFP antibody. Representative experiment is shown. Blots of WT YFP-HA-DAT and TM4-9 are from the same gel. C, quantification of the fraction of trimeric species (T) of the total YFP-HA-DAT or TM4-9 DAT immunoreactivities. The mean values of the T fraction of total (±SD) were measured in four independent experiments. The p values were calculated for TM4-9 versus wtDAT using two-tail, paired t test. D, PAE/YFP-HA-DAT and PAE/TM4-9 cells were incubated with HA11 for 60 min at 37 °C and then incubated with vehicle (DMSO), 20 μM AIM-100 for 2 h or 1 μM PMA for 30 min, all at 37 °C. After fixation, cultures were stained with secondary anti-mouse antibodies conjugated with Cy3 (surface HA-DAT), permeabilized with Triton X-100, and stained with secondary anti-mouse conjugated with Cy5 (internalized HA-DAT). 3D images were acquired through 488 (YFP, green), 561 (Cy3, cyan), and 640 nm (Cy5, red) channels. Maximum intensity projections of representative 3D images are shown. Scale bars, 10 μm. E, Cy5/Cy3 ratios were calculated in images exemplified in (E). Results are presented as mean values of the ratio (±S.D.; n = 4). The p values were calculated for TM4-9 versus wtDAT using two-tail, paired t test. n.s., difference is not significant (p > 0.05). AL, AIM-100–like; DAT, dopamine transporter; DMSO, dimethyl sulfoxide; im-M, immature monomers; im-T, immature trimers; M, monomers; PAE, porcine aortic endothelial; PMA, phorbol 12-myristate 13-acetate; T, trimers; TM, transmembrane; YFP-HA-DAT, YFP- and HA-epitope tagged DAT.