The dopamine transporter (DAT) has been identified as the cocaine receptor most linked with psychostimulant reward and reinforcement in the brain. Cloning DAT CDNAS from rat and man by Addiction Research Center molecular neurobiologists in previous Fys has allowed testing of the roles of discrete residues in the recognition of dopamine, cocaine and their analogs using site-directed mutagenesis. These approaches have allowed elucidation of residues likely to be involved in post- translational modifications of the transporter, and in the functions mediated by posttranslational alterations. These approaches were especially explored in order to seek transporter regions and functions selectively involved in cocaine binding and not in dopamine transport, a potential aid in development of anti-cocaine therapeutics. Cysteine and asparagine residues that may be required for post- translational maturation of DAT structure and polar or highly conserved residues within transmembrane regions have been identified during this FY. Mutant analyses revealed that each of the four sites for N-linked glycosylation appears to be utilized, and that significant amounts of glycosylation are essential for full transporter function. Cysteine residues at positions 180 and 189 were each found to each be necessary for DAT function, indicating the possible presence of an intramolecular disulfide bond that could provide secondary and tertiary structural constraints to this portion of the transporter. Studies with phorbol esters and forskolin indicated that phosphorylation by protein kinase C, but not protein kinase A, reduced transporter function in both cocaine analog recognition and dopamine uptake. Mechanisms were different, however: velocity of transport, and affinity of binding were altered.
