CLONING, EXPRESSION, AND LOCALIZATION OF A CHLORIDE-FACILITATED, COCAINE-SENSITIVE SEROTONIN TRANSPORTER FROM DROSOPHILA-MELANOGASTER

We report here on the isolation and characterization of a serotonin (5 HT) transporter from Drosophila melanogaster. A 3.1-kb complementary D NA clone (dSERT) was found to encode a protein of 622 amino acid resid ues with a predicted molecular mass of approximate to 69 kDa and a put ative transmembrane topology characteristic of cloned members of the m ammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT dis plays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and s hares with all transporters 104 absolutely conserved amino acid residu es. Upon transient expression in HeLa cells, dSERT exhibited saturable , high-affinity, and sodium-dependent [H-3]5HT uptake with estimated K -m and V-max values of approximate to 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSE RT), 5HT-mediated transport by dSERT was not absolutely dependent on e xtracellular Cl-, while the sodium-dependent uptake of 5HT was facilit ated by increased extracellular Cl- concentrations. dSERT displays a p harmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affini ties of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximate t o 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximat e to 500 nM, while high concentrations (> 10 mu M) of dopamine, norepi nephrine, octopamine, tyramine, and histamine failed to inhibit transp ort. In situ hybridization reveals the selective expression of dSERT m RNA to specific cell bodies in the ventral ganglion of the embryonic a nd larval Drosophila nervous system with a distribution pattern virtua lly identical to that of 5HT-containing neurons. The dSERT gene was ma pped to position 60C on chromosome 2. The availability of the gene enc oding the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and s tructural moths that confer the pharmacologic specificity and genetic regulation of the 5HT transport process.