Dopamine transporter: Transmembrane phenylalanine mutations can selectively influence dopamine uptake and cocaine analog recognition

Cocaine blocks the normal role of the dopamine transporter (DAT) in termina ting dopamine signaling through molecular interactions that are only partia lly understood. Cocaine analog structure-activity studies have suggested ro les for both cationic and aromatic interactions among DAT, dopamine, and co caine. We hypothesized that phenylalanine residues lying in putative DAT tr ansmembrane (TM) domains were good candidates to contribute to aromatic and /or cationic interactions among DAT, dopamine, and cocaine. To test this id ea, we characterized the influences of alanine substitution for each of 29 phenylalanine residues lying in or near a putative DAT TM domain. Cells exp ress 22 mutants at near wild-type levels, manifest by DAT immunohistochemis try and binding of the radiolabeled cocaine analog [H-3](-)-2-beta-carbomet hoxy-3-beta-(4-fluorophenyl)tropane (CFT). Seven mutants fail to express at normal levels. Four mutations selectively reduce cocaine analog affinities . Alanine substitutions at Phe(76), Phe(98), Phe(390), and Phe(361) located in TM domains 1 and 2, the fourth extracellular loop near TM 4 and in TM 7 , displayed normal affinities for dopamine but 3- to 8-fold reductions in a ffinities for CFT. One TM 3 mutation, F(155)A, selectively decreased dopami ne affinity to less than 3% of wild-type levels while reducing CFT affinity less than 3-fold. In a current DAT structural model, each of the residues at which alanine substitution selectively reduces cocaine analog or dopamin e affinities faces a central transporter cavity, whereas mutations that inf luence expression levels are more likely to lie at potential helix/helix in terfaces. Specific, overlapping sets of phenylalanine residues contribute s electively to DAT recognition of dopamine and cocaine.