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  • 【期刊】 Functional comparison of full-length and N-terminal-truncated octopamine transporters from Lepidoptera

    摘要:We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (Km and Ki range, 0.4 μM–2.7 μM) and capacity (3H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity (3H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.
  • 【期刊】 Functional comparison of full-length and N-terminal-truncated octopamine transporters from Lepidoptera

    摘要:We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (Km and Ki range, 0.4 μM–2.7 μM) and capacity (3H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity (3H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.
  • 【期刊】 Molecular Cloning and Functional Expression of an Insect High-Affinity Na+-Dependent Glutamate Transporter

    刊名:European journal of biochemistry / FEBS 作者:B. Cameron Donly ; Alex Richman ; Erika Hawkins ; Heather Mclean ; Stan Caveney 年份:1997
    摘要:Excitatory amino acid transporters in the central and peripheral nervous systems of insects are thought to assist in maintaining glutamate concentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Trichoplusia ni which encodes a high-affinity Na+-dependent glutamate transporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+- and K+-dependent amino acid transporter family. Expression of the insect transporter mRNA was predominantly localized in the caterpillar brain. The function of the TrnEAAT1 protein was analyzed in cultured insect cells using a baculovirus expression system. Cells infected with the recombinant virus were found to exhibit a 50-fold increase in ability to accumulate labeled L-glutamate compared to mock-infected cultures, and this activity was shown to be Na+-dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate analogues accumulated by infected cells. Various glutamate uptake inhibitors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Despite the significant differences between insect and vertebrate physiology, the characteristics of the respective transporters were found to be remarkably similar.
  • 【期刊】 A high-affinity octopamine transporter cloned from the central nervous system of cabbage looper Trichoplusia ni

    刊名:Insect Biochemistry and Molecular Biology 作者:Tabita Malutan ; Heather McLean ; Stan Caveney ; Cam Donly 年份:2002
    摘要: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 3H-OA and 3H-dopamine with saturation kinetics typical of carrier-mediated processes. 3H-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.