Figure 5. Cocaine inhibits AIM-100-induced endocytosis, oligomerization and clustering of DAT.
(A) PAE/YFP-HA-DAT cells were incubated without or with 4 µM cocaine for 30 min, and further incubated with vehicle (DMSO) or 20 µM AIM-100 for 1.5 hr at 37°C in the same media. 3D images were acquired from living cells through the 515 channel. Individual confocal sections through the middle of the cell are shown. (B) PAE/YFP-HA-DAT cells were incubated with HA11 for 30 min at 37°C, and then incubated with vehicle (PBS) or 10 µM cocaine (coc) for 10 min, and further incubated with vehicle (DMSO) or 20 µM AIM-100 in the presence of vehicle (PBS) or 1 µM cocaine for 2 hr at 37°C. After fixation, cultures were stained with secondary anti-mouse antibodies conjugated with Cy5 (surface HA-DAT), permeabilized with Triton X-100 and stained with secondary anti-mouse conjugated with Cy3 (internalized HA-DAT). 3D images were acquired through 488 (YFP, not shown), 561 (Cy3, red) and 640 nm (Cy5, cyan) channels. Individual confocal sections are presented. (C) Cy3/Cy5 ratios were calculated in experiments exemplified in (B). Results are presented as mean values of the ratio (±S.D.; n = 3-5). (D) PAE/YFP-DAT cells were incubated with vehicle or 10 µM cocaine for 10 min at 37°C, and further incubated with vehicle or 20 µM AIM-100 for 2 hr at 37°C, and lysates were analyzed by immunoblotting with the GFP antibody. Representative experiment is shown. M, monomers; O, oligomers. The mean values of the O/M ratios (±S.D.) (below the blot) were measured in three independent experiments. (E) Time-lapse TIR-FM imaging of PAE/YFP-HA-DAT cells that were pre-incubated or not with 10 µM cocaine for 5 min at 37°C, and then incubated with AIM-100 (20 µM) for 1 hr at 37°C. Individual time frames before (0’) and 30 min after addition of AIM-100 are shown. Scale bars are 10 µm. (F) PAE/YFP-DAT and PAE/W63A-YFP-HA-DAT cells were incubated with DMSO (veh) or AIM-100 as in (D), and lysates were analyzed by western blotting with the GFP antibody. Representative experiment of three independent experiments is shown. M, monomers; O, oligomers. (G) PAE/W63A-YFP-HA-DAT cells were incubated with HA11, cocaine and AIM-100 as in (B). After fixation, cultures were stained with secondary antibodies as in (B). 3D images were acquired through 488 (YFP, not shown), 561 (Cy3, red) and 640 nm (Cy5, cyan) channels. Maximum intensity projections of z-stack of confocal images are presented. Scale bars, 10 µm. (H) Cy3/Cy5 ratios were calculated in experiments exemplified in (E). Results are presented as mean values of the ratio (±SD, n = 4-5). n.s., p=0.146.
Figure 5—figure supplement 1. AIM-100 non-competitively inhibits CFT binding.
