THE DOPAMINE TRANSPORTER CARBOXYL-TERMINAL TAIL - TRUNCATIONS SUBSTITUTION MUTANTS SELECTIVELY CONFER HIGH-AFFINITY DOPAMINE UPTAKE WHILE ATTENUATING RECOGNITION OF THE LIGAND-BINDING DOMAIN/

In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [H-3]dopamine uptake kinetics and [H-3]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH termi nus was truncated or substituted, Complete truncation of the carboxyl tail from Ser(582) allowed for the expression of biphasic [H-3]dopamin e uptake kinetics displaying both a low capacity (V-max similar to-0.4 pmol/10(5) cells/min) high affinity (K-m similar to 300 nM) component and one exhibiting low affinity (K-m similar to 15 mu M) and high cap acity (V-max similar to 5 pmol/10(5) cells/min) with a concomitant 40% decrease in overall apparent V-max relative to wild type (WT) DAT. Tr uncation of the last 22 amino acids or substitution of the DAT-COOH ta il with sequences encoding the intracellular carboxyl-terminal of eith er dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DA T-specific sequence motifs distal to Pro(597). The attenuation of WT t ransport activity, either by lowering levels of DAT expression or by p retreatment of cells with phorbol 12-myristate 13- acetate (1 mu M), d id not affect the kinetics of [H-3]dopamine transport. The estimated a ffinity of dopamine (K-i similar to 180 nM) for all truncated/substitu ted DAT mutants was 10-fold lower than that of WT DAT (similar to 2000 nM) and appears selective for the endogenous substrate, since the est imated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed signif icantly reduced high affinity [H-3]CFT binding interactions with estim ated K-i values for dopamine and numerous other substrates and inhibit ors tested from 10-100-fold lower than that observed for WT DAT, Moreo ver, co-expression of truncated and/or substituted DATs with WT transp orter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of un iphasic low affinity [H-3]dopamine uptake kinetics (K-m similar to 200 0 nM) and high affinity substrate and inhibitor [H-3]CFT binding inter actions attributable to WT DATs were evident. These data clearly sugge st the functional independence and differential regulation of the dopa mine translocation process from the characteristics exhibited by its l igand binding domain. The lack of functional phenotypic ex pression of mutant DAT activities in cells co-expressing WT transporter is consis tent with the contention that native DATs may exist as multisubunit co mplexes, the formation and maintenance of which is dependent upon sequ ences encoded within the carboxyl tail.