Molecular cloning and functional expression of an insect high-affinity Na+-dependent glutamate transporter.
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.
Donly
B C
BC
Pest Management Research Centre, Agriculture and Agri-Food Canada, London, Ontario. donlyc@em.agr.ca
Richman
A
A
Hawkins
E
E
McLean
H
H
Caveney
S
S
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003544:Cysteic Acid / Q000378:metabolism / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism*; D007915:Lepidoptera; D008322:Mammals; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D014018:Tissue Distribution
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D019476:Insect Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012964:Sodium; D003544:Cysteic Acid