The role of conserved tryptophan and acidic residues in the human dopaminetransporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans an d acidic residues that are completely or highly conserved among Na+/Cl--dep endent transporters. We have explored the roles of these residues using non conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184 L) lacked plasma membrane immunostaining and were not functional. Both DA u ptake and cocaine analog (i.e. 2 beta -carbomethoxy-3 beta-(4-fluorophenyl) tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted th e relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold . In particular, mutation of D345 in the third intracellular loop still all owed considerable [H-3]DA uptake, but caused undetectable [H-3]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine a nalog [I-125]-3 beta-(p-chlorophenyl)tropane-2 beta -carboxylic acid ([I-12 5]RTI-82). Unexpectedly. in this mutant, cocaine-like drugs remained potent inhibitors of [H-3]DA uptake. CFT solely raised the K-m of [H-3]DA uptake in wild-type hDAT, but increased K-m and decreased V-max in D345N, suggesti ng different mechanisms of inhibition. The data taken together indicate tha t mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding o f cocaine to the transporter may not be the only way by which cocaine analo gs inhibit DA uptake.