Molecular cloning and functional characterization of neurotransmitter transporters from the central nervous system of Manduca sexta

Two full length clones encoding for serotonin (MasSERT) and proline (MasPROT) transporters were isolated from a cDNA library from the CNS of Manduca sexta. MasSERT exhibited a Na+ and Cl- dependent high affinity uptake of 5-hydroxy tryptamine (5HT) with Km of 436 nM. Despite of having 53% and 74% amino acid identity to the reported human (hSERT) and fruit fly (dSERT) serotonin transporters, respectively, MasSERT possesses distinct pharmacology compared to its vertebrate and invertebrate homologues. The property of MasSERT being poorly inhibited by fluoxetine (IC 50 1.23 muM) and cocaine (IC50 12.89 muM) directed us to analyze elements that dictate specificity of antagonist binding by carrying out site directed mutagenesis and chimera construction. Analysis of a hSERT mutant, 188A&barbelow;/189LA that carried insertions of unique amino acids found in extracellular loop 2 (EL2) of MasSERT, at the corresponding positions led to the identification of a domain that causes a shift in cocaine sensitivity (IC50 1542 nM) from hSERT (IC 50 0.431 muM) towards MasSERT. Analysis of two chimeras between MasSERT and hSERT emphasized the involvement of transmembrane domain (TMD) 1 and 2 in forming a part of cocaine binding pocket. The chimera hSERT1--146 /MasSERT106--587, which consisted of MasSERT carrying TMD 1 and 2 of hSERT, was strikingly insensitive to cocaine (IC50 180 muM). The chimera MasSERT1--67/hSERT109--630 , which consisted of hSERT carrying TMD 1 of MasSERT, became even more sensitive to cocaine (IC50 0.225 muM) than hSERT. The addition of cocaine insensitive transporters (MasSERT and chimera hSERT1--146 /MasSERT106--587) into the pool of cocaine sensitive monoamine transporters widens the spectrum, which could serve as a basis to delineate SERT antagonist binding sites especially for cocaine. MasPROT, which is expressed in the CNS, flight muscles, midgut, hindgut and Malphigian tubules, exclusively transported L. proline in Xenopus oocytes suggesting that it is an insect homologue of mammalian proline transporters. Manduca sexta GABA transporter MasGAT, which is expressed in the CNS and leg muscles, is pharmacologically distinct from its mammalian homologues and thus makes a promising target for new insecticide development. In order to develop a sensitive and convenient transport assay for rapid screening of natural and synthetic chemicals, MasGAT was expressed in CV-1 cells and the assay conditions were established. Transiently expressed MasGAT in CV-1 cells displayed a high affinity GABA transport with Km of 1.87 muM and similar pharmacological properties as determined previously by expressing it in Xenopus oocytes.