TYROSINE-533 OF RAT DOPAMINE TRANSPORTER - INVOLVEMENT IN INTERACTIONS WITH 1-METHYL-4-PHENYLPYRIDINIUM AND COCAINE

To improve our understanding of structure-function relationships for n eurotransmitter transporters, we performed site-directed mutagenesis o f the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino a cid of human DAT is phenylalanine. Alanine substitution of tyrosine-53 3 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridin ium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increas ed the velocity of MPP+ uptake but decreased DAT's affinity for MPP+. Cocaine's potency in inhibiting dopamine uptake was unchanged with Y53 3A, but increased with Y533F. Differences in the uptake kinetics and i nhibitory potency of cocaine between rat and human DATs were similar t o the differences observed between the wild-type and Y533F mutants DAT s. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivi ties to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in huma ns and rats. (C) 1998 Elsevier Science B.V.