Cloning of dopamine, norepinephrine and serotonin transporters from monkeybrain: relevance to cocaine sensitivity

We used RT-PCR to clone monoamine transporters from Macaca mulatta, Macaca fasicularis, is and Saimiri sciureus (dopamine transporter; DAT) and Macaca mulatta (norepinephrine transporter; NET and serotonin transporter; SERT). Monkey DAT, NET and SERT proteins were >98% homologous to human and, when expressed in HEK-293 cells, displayed drug affinities and uptake kinetics t hat were highly correlated with monkey brain or human monoamine transporter s. In contrast to reports of other species, we discovered double (leucine f or phenylalanine 143 and arginine for glutamine 509; Variant I) and single (proline for leucine 355; Variant LI) amino acid variants of DAT. Variant I displayed dopamine transport kinetics and binding affinities for various D AT blockers (including cocaine) versus [H-3] CFT (WIN 35, 428) that were id entical to wild-type DAT (n = 7 drugs; r(2)=0.991). However, we detected a six-fold difference in the affinity of cocaine versus [H-3] cocaine between Variant I (IC50: 488 +/- 102 nM, SEM, n = 3) and wild-type DAT (IC50: 79 /- 8.2 nM, n = 3, P < 0.05). Variant II was localized intracellularly in HE K-293 cells, as detected by confocal microscopy, and had very low levels of binding and dopamine transport. Also discovered was a novel exon 5 splice variant of NET that displayed very low levels of transport and did not bind cocaine. With NetPhos analysis, we detected a number of highly conserved p utative phosphorylation sites on extracellular as well as intracellular loo ps of the DAT, NET, and SERT, which may be functional for internalized tran sporters. The homology and functional similarity of human and monkey monoam ine transporters further support the value of primates in investigating the role of monoamine transporters in substance abuse mechanisms, neuropsychia tric disorders and development of diagnostic and therapeutic agents. (C) 20 01 Elsevier Science B.V. All rights reserved.