| In the present study, the DUIPs of cocaine and other DAT ligands at wildtype DAT CHO cells fluctuated as a function of cell age, while their binding affinities remained static. Curiously, neither the DUIP nor the binding affinity of amphetamine fluctuated with cell age. The DUIP fluctuations of cocaine also extended to the N2A neuronal cells, demonstrating its physiological relevance. The loss of correlation between DUIP and binding affinity at DAT might be a unique property of DAT blockers that is not exhibited by DAT substrates. A plausible explanation for the DUIP fluctuations with cell age is the existence of two different DAT populations on the cell surface, the relative distribution of which changes with cell age under the influence of intracellular events such as DAT-protein interactions or alterations in the phosphorylation state. Several proteins interact with the N- and C-terminal regions of the DAT. Studies employing GFP fusion polypeptides of the DAT Nor C-terminal fragments, which would act as decoys by intercepting DAT modulators, showed that the DAT N- or C-terminal interactions with intracellular proteins do not appear to be responsible for the DUIP shift. The role of phosphorylation state changes of DAT in DUIP fluctuations was also investigated. Studies employing a mutant DAT lacking the first 20 N-terminal amino acids demonstrated that N-terminal phosphorylation of DAT, specifically involving the first five serines is not associated with the DUIP shift. |
