Substrate-stereoselectivity of a high-affinity glutamate transporter cloned from the CNS of the cockroach Diploptera punctata.
A cDNA encoding a Na(+)-dependent glutamate transporter has been cloned from the brain of the cockroach Diploptera punctata. The cDNA encodes a transporter protein of 481 amino acids, designated DipEAAT1, which when expressed in baculovirus infected insect cells, resulted in a 40-50 fold increase in [(3)H]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the caterpillar Trichoplusia ni. The affinity of these transporters for L-glutamate and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affinities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [(3)H]L-glutamate into cells was inhibited strongly by D- and L- isomers of aspartate and its analogues. DipEAAT1, by contrast, was inhibited weakly by all D- isomers tested. The affinity of DipEAAT1 for [(3)H]D-aspartate was found to be an order of magnitude lower than that of TrnEAAT1, revealing an unusual stereoselectivity for aspartate substrates by the cockroach transporter. The activity of DipEAAT1 was also unaffected by the presence of Zn(++) in the bathing solution, despite the presence of a putative Zn(++)-binding motif conferring Zn(++)-sensitivity on some mammalian glutamate transporters.
Donly
C
C
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford St., London, Ontario, Canada. donlyc@em.agr.ca
Jevnikar
J
J
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; D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D003058:Cockroaches / Q000235:genetics*; D003545:Cysteine / Q000378:metabolism; D015723:Gene Library; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013237:Stereoisomerism; D015032:Zinc / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D005971:Glutamates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D015032:Zinc; D003545:Cysteine