Identification and characterization of a cDNA encoding a neuronal glutamate transporter from Drosophila melanogaster.
Sodium-dependent glutamate transporters influence neurotransmission in the central nervous system by removing synaptically released glutamate from the extracellular space and by maintaining extracellular glutamate concentrations below neurotoxic levels. In insects, glutamate also serves as the neurotransmitter at the neuromuscular junction, but the mechanism for neurotransmitter clearance at this synapse has not well-established. Here we report that cloning and characterization of a sodium-dependent glutamate transporter, dEAAT, from Drosophila melanogaster. The 479 amino acid dEAAT gene product is 40-50% homologous to mammalian members of this carrier family. A 3.3 kilobase (kb) transcript for dEAAT was detected in adult fly heads and to a lesser extent in bodies by Northern-blot analysis and was also localized to neurons in the central nervous system by in situ hybridization. The transport activity observed following express of dEAAT in Xenopus oocytes or COS-7 cells shows a high affinity for L-glutamate, L-aspartate and D-aspartate, an absolute dependence on external sodium ions, and considerable stereoselectivity for the transport of L-glutamate over D-glutamate. As has been observed for the human carriers, EAAT 4 and EAAT 5, a significant component of the current activated by L-glutamate application to dEAAT-expressing oocytes appears to arise from the activation of a chloride channel associated with the carrier.
Seal
R P
RP
Program in Neuroscience, Oregon Health Sciences University 97201, USA.
Daniels
G M
GM
Wolfgang
W J
WJ
Forte
M A
MA
Amara
S G
SG
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D018118:Chloride Channels / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D017344:Genes, Insect; D006801:Humans; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D018118:Chloride Channels; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger