Distinct Recognition of Substrates by the Human andDrosophilaSerotonin Transporters

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 M, 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 ϩ ), was transported only by hSERT. However, MPP ϩ inhibited 5-HT uptake in both species variants with similar potencies. Two cross-species chimeras, H 1-118 D 119 -627 and H 1-281 D 282-476 H 477-638 , were also unable to transport MPP ϩ , 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.

Comparative Molecular Field Analysis Using Selectivity Fields Reveals Residues in the Third Transmembrane Helix of the Serotonin Transporter Associated with Substrate and Antagonist Recognition

“…4B). A lack of substrateinduced efflux is characteristic of channels and may indicate that the A169D mutant and dSERT are operating in a channel-like mode (Rodríguez et al, 2003). This finding may suggest that the aspartate in position 169 is involved with gating SERT channel-like properties.…”

“…Previous work by our group has demonstrated that amphetamines were poorly transported by and unable to induce substrate efflux at the dSERT (Rodríguez et al, 2003). Of the five hSERT mutants constructed with a dSERT residue, only the A169D mutant gained a net negative charge.…”

Functional characterization of Rat Plasma Membrane Monoamine Transporter in the Blood–Brain and Blood–Cerebrospinal Fluid Barriers

“…Partial inhibitory effects by tetraethylammonium, guanidine, l-carnitine, and thiamine were observed in TR-BBB13 and TR-CSFB3 cells, but not in PMATexpressing CHO cells, suggesting that endogenous transporters beside PMAT in these brain barrier cells were related to these inhibitory effects. Indeed, MPP + has been reported to be a substrate for not only PMAT but also OCT1-3, 36-38 DAT, 39 SERT, 40 and OCTN2. 41 Of these transporters, rOCTN2 mRNA was expressed at a relatively high level in both TR-BBB13 and TR-CSFB3 cells.…”

Distinct Molecular Recognition of Psychostimulants by Human andDrosophilaSerotonin Transporters

“…When 3-phenyltropane analogs were assayed, the cells were preincubated with drug at 37°C for 10 min. This preincubation was not included when the substituted amphetamines were assayed, because these compounds have been shown to be substrates for SERT, and the intracellular accumulation of drug could affect 5-HT transport (Wall et al, 1995;Rodriguez et al, 2003). [ 3 H]5-HT (ϳ110 Ci/mmol) was diluted to yield a final assay concentration of 20 nM in KRH buffer supplemented with 100 M Lascorbic acid and 100 M pargyline.…”

“…The H 1-281 D 282-476 H 477-638 chimera was generated as described previously (Rodriguez et al, 2003). Briefly, it is a full-length 638 amino acid transporter, constructed from amino acids 1-281 and 477-638 with identity from hSERT and residues 282-476 with identity from dSERT.…”