ABSTRACT
A cDNA was cloned from the cabbage looper Trichoplusia ni based on similarity to other cloned dopamine transporters (DATs). The total nucleotide sequence is 3.8 kb in length and contains an open reading frame for a protein of 612 amino acids. The predicted moth DAT protein (TrnDAT) has greatest amino acid sequence identity with Drosophila melanogasterDAT (73%) and Caenorhabditis elegansDAT (51%). TrnDAT shares only 45% amino acid sequence identity with an octopamine transporter (TrnOAT) cloned recently from this moth. The functional properties of TrnDAT and TrnOAT were compared through transient heterologous expression in Sf9 cells. Both transporters have similar transport affinities for DA (Km 2.43 and 2.16 micro m, respectively). However, the competitive substrates octopamine and tyramine are more potent blockers of [3H]dopamine (DA) uptake by TrnOAT than by TrnDAT. D-Amphetamine is a strong inhibitor and l-norepinephrine a weak inhibitor of both transporters. TrnDAT-mediated DA uptake is approximately 100-fold more sensitive to selective blockers of vertebrate transporters of dopamine and norepinephrine, such as nisoxetine, nomifensine and dibenzazepine antidepressants, than TrnOAT-mediated DA uptake. TrnOAT is 10-fold less sensitive to cocaine than TrnDAT. None of the 15 monoamine uptake blockers tested was TrnOAT-selective. In situ hybridization shows that TrnDAT and TrnOAT transcripts are expressed by different sets of neurons in caterpillar brain and ventral nerve cord. These results show that the caterpillar CNS contains both a phenolamine transporter and a catecholamine transporter whereas in the three invertebrates whose genomes have been completely sequenced only a dopamine-selective transporter is found.
Subject(s)
Insect Proteins/metabolism , Membrane Glycoproteins , Membrane Transport Proteins/physiology , Moths/physiology , Nerve Tissue Proteins , Nervous System/metabolism , Octopamine/physiology , Amino Acid Sequence , Animals , Binding, Competitive , Biological Transport , Blotting, Northern , Caenorhabditis elegans/chemistry , Cloning, Molecular , Cocaine/pharmacology , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins , Drosophila melanogaster/chemistry , In Situ Hybridization , Insect Proteins/chemistry , Membrane Transport Proteins/chemistry , Molecular Sequence Data , Octopamine/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A cDNA encoding a high-affinity Na(+)/anion(-)-dependent octopamine transporter (OAT) was isolated via an RT-PCR-based approach from caterpillars of the cabbage looper, Trichoplusia ni. The deduced amino acid sequence of the OAT cDNA predicts a 670 amino acid protein bearing strong homology to previously cloned monoamine transporters. The expression pattern of OAT mRNA in the central nervous system revealed by in situ hybridization closely resembles that of OA-ergic neurons identified by the presence of mRNA for tyramine beta-hydroxylase, a marker enzyme for OA-ergic neurons in invertebrates. In vitro, insect cells infected with OAT-expressing baculovirus accumulated both (3)H-OA and (3)H-dopamine with saturation kinetics typical of carrier-mediated processes. (3)H-dopamine uptake by OAT was most inhibited by tyramine, OA, dopamine and the tricyclic antidepressants desipramine and imipramine. Substitution studies for Na(+) and Cl(-) indicate that OAT has a strong requirement for Na(+) and a less stringent requirement for Cl(-). The pharmacological profile of OAT is distinct from those of other cloned monoamine transporters and makes OAT a potential target for neuro-active pest control agents.
