MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF AN INSECT HIGH-AFFINITY NA-DEPENDENT GLUTAMATE TRANSPORTER()

Excitatory amino acid transporters in the central and peripheral nervo us systems of insects are thought to assist in maintaining glutamate c oncentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Tri choplusia iii which encodes a high-affinity Na+-dependent glutamate tr ansporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+- and K+-depen dent amino acid transporter family. Expression of the insect transport er mRNA was predominantly localized in the caterpillar brain. The func tion of the TrnEAAT1 protein was analyzed in cultured insect cells usi ng a baculovirus expression system, Cells infected with the recombinan t virus were found to exhibit a 50-fold increase in ability to accumul ate labeled L-glutamate compared to mock-infected cultures, and this a ctivity was shown to be Na+-dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate an alogues accumulated by infected cells, Various glutamate uptake inhibi tors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Desp ite the significant differences between insect and vertebrate physiolo gy, the characteristics of the respective transporters were found to b e remarkably similar.