Abstract

Dopamine transporter (DAT) is a major target of cocaine, one of the most abused drugs. Major efforts have been focused on defining residues in DAT involved in cocaine binding. We have isolated theDrosophila melanogaster DAT (dDAT) cDNA, which is 10-fold less sensitive to cocaine than the mammalian DATs. Replacing transmembrane domain 2 (TM2) of mouse DAT (mDAT) with dDAT sequence reduced cocaine sensitivity. The reciprocal construct exhibited increased cocaine sensitivity. Switching residue 105 in TM2, a phenylalanine conserved in all mammalian DATs, to methionine, the corresponding residue in dDAT, resulted in a functional transporter with cocaine sensitivity 4-fold lower. Replacing F105 with alanine, leucine, isoleucine, serine, threonine, asparagine, or glutamine resulted in transporters with low transport activity. In contrast, changing F105 to the other aromatic residues tyrosine or tryptophan retained more than 75% transport activity and high cocaine sensitivity. Most significantly, the reciprocal construct, switching the methionine in dDAT at the corresponding residue to phenylalanine, increased cocaine sensitivity 3-fold. Finally, the mDAT mutant with a cysteine at this position had normal transport activity but exhibited cocaine sensitivity that was 15-fold lower. These results suggest that F105 in mDAT contributes to high-affinity cocaine binding. The functional cocaine-insensitive mutants provide tools for the study of the mechanism of cocaine addiction.