Successful cloning efforts have established a family of Na +- and Cl −-dependent transporters for neurotransmitters, amino acids, and other substrates ( Guastella et al., 1990 Pacholczyk et al., 1991 Amara and Kuhar, 1993 Rudnick and Clark, 1993 Nelson and Lill, 1994). The transporters for dopamine, norepinephrine, and serotonin are high-affinity targets for drugs of abuse such as cocaine and amphetamines and for therapeutic drugs used to treat depression, obsessive-compulsive disorder, and other mental diseases ( Ritz et al., 1987 Koe, 1990 Barr et al., 1992 Boyer and Feighner, 1992 Giros and Caron, 1993 Gu et al., 1994 Seeman and Madras, 1998 Smith et al., 1998). Transporters localized near sites of neurotransmitter release terminate the action of these transmitters by reuptake into neurons and glia ( Uhl, 1992 Borowsky and Hoffman, 1995 Rudnick, 1997). However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH 2-terminal region. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH 2-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH 2-terminal regions. To identify possible localization signals responsible for this difference, DAT–NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity.
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