Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster.

Abstract

We report here on the isolation and characterization of a serotonin (SHT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of w69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl-neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues.Upon tnsient expression in HeLa cells, dSERT exhibited saturabl, high-affinity, and sodium-dependent [3H5HT uptake with esimated K and V. values of w500 nM and 5.2 x 1o-i8 mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), SHT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of MHT was facilitatd by Increased extracellular Cl-concentrations. dSERT disys a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the afnities of various compounds for the inhibiion 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 w30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estmated inhibition constants of 500 nM, while high concentrations (>10 FM) of dopmin, norepinephrine, octopamine, tramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral gagulon of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene e ing the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the phannacologrc specificity and genetic regulation of the 5HT transport process. Serotonin (5HT) plays a vital role in modulation ofa variety of biochemical and physiological functions in the central nervous system, including sleep, appetite, and pain perception, and has been shown to regulate complex behavioral phenomena, such as learning and memory in invertebrates (1, 2). Numerous studies have provided evidence for production of SHT in Drosophila as well as its cell-specific localization in neurons within the central nervous system (3). Released from neuronal terminals into the synaptic cleft, 5HT is believed to mediate its effects in Drosophila via an interaction with specific multiple cell surface 5HT receptors (4, 5), which in turn are coupled to subtype-specific guanine nucleotide-binding proteins (see ref.6 and references therein) activating various membran...

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DrosophilaSerotonin Transporters Have Voltage-Dependent Uptake Coupled to a Serotonin-Gated Ion Channel

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“…In this system, it was also shown that 5 to 12 Na ϩ ions could "escape" into the cell during each transport cycle, thus generating an ionic current in excess of the predicted electroneutrality of coupled transport in mammalian SERTs. Corey et al (1994) and Demchyshyn et al (1994) cloned the Drosophila serotonin transporter (dSERT) and studied its ionic dependence and pharmacological characteristics. In Xenopus oocytes expressing dSERT, Corey et al report that 5HT uptake depends on Na and Cl.…”