Figure 4.

Modulation of DAT conformation by σ₁R agonists in the presence of cocaine. A, schematic of the Drosophila DAT structure with cocaine (yellow) bound (PDB 4XP4) (7). Highlighted positions corresponding to Cys-306 (red), Thr-316 (green), and TM6a (blue) in human DAT. Part of TM11 was omitted for clarity. B and C, potentiation of cocaine-induced accessibility changes of Cys-306 in DAT by σ₁R agonists. Transfected HEK293 cells expressing DAT and epitope-tagged σ₁R were preincubated with PRE-084 or (+)-pentazocine for 1 h at 37 °C, washed with PBSCM, and labeled with maleimide-PEG2-biotin at 4 °C for 45 min in the presence or absence of cocaine. Biotinylated proteins were enriched from cell lysates with NeutrAvidin beads, subjected to SDS-PAGE, and detected on immunoblot using DAT-specific antibodies (see details under “Experimental procedures”). D, σ₁R agonists did not change cell expression levels of DAT. Transfected cells were incubated with σ₁R drugs for 1 h at 37 °C, washed with PBSCM, and labeled with sulfo-NHS-SS-biotin at 4 °C for 45 min. E, σ₁R antagonist CM304 blocked the effects of PRE-084 on cocaine-induced accessibility changes of Cys-306 in DAT. Cells were incubated with CM304 for 15 min before PRE-084 treatment for 1 h at 37 °C with CM304 also present. F, potentiation of cocaine-induced accessibility changes of T316C/C306A DAT by σ₁R agonists. All bar graphs (B–F) show summarized results (mean ± S.E.) from multiple experiments (n) with representative immunoblots of labeled DAT and lysate DAT signals. *, p < 0.05; ***, p < 0.001, one-way ANOVA with post-hoc Bonferroni's multiple comparison test. NS, not significant. (+)pent, (+)-pentazocine.