Abstract

The human and Drosophila serotonin transporters (hSERT and dSERT, respectively) were used to explore differences in substrate properties. hSERT and dSERT showed similar K_m values for 5-hydroxytryptamine (5-HT; serotonin) transport (1.2 and 0.9 μM, respectively), suggesting similar recognition of 5-HT by the two species variants. Although dSERT cell surface expression was approximately 8-fold lower than that of hSERT, dSERT does appear to have a 2-fold faster turnover number for inward transport of 5-HT. Interestingly, another substrate, N-methyl-4-phenylpyridinium (MPP⁺), was transported only by hSERT. However, MPP⁺ inhibited 5-HT uptake in both species variants with similar potencies. Two cross-species chimeras, H^1–118D^119–627 and H^1–281D^282–476H^477–638, were also unable to transport MPP⁺, implicating the role of transmembrane domains V to IX in the substrate permeation pathway. Based on exchange experiments, certain substituted-amphetamines also appear to be poor substrates at dSERT. Two-electrode voltage-clamp studies in oocytes confirmed that the amphetamines do not possess substrate-like properties for dSERT. Our data suggest distinct molecular recognition among SERT substrate classes that influence translocation mechanisms.