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 transpor ter protein of 481 amino acids, designated DipEAAT1, which when expressed i n baculovirus infected insect cells, resulted in a 40-50 fold increase in [ H-3]L-glutamate uptake. DipEAAT1 mRNA is expressed in the brain, as is the RNA encoding TrnEAAT1, a related transporter recently isolated from the cat erpillar Trichoplusia ni. The affinity of these transporters for L-glutamat e and several structural analogues was compared. Both have a high affinity for L-glutamate, their presumed primary substrate, but quite different affi nities for D-aspartate. TrnEAAT1 was found to be similar to other glutamate transporters in that its ability to transport [H-3]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 [H-3]D-aspartate was found to be an order of magni tude lower than that of TrnEAAT1, revealing an unusual stereoselectivity fo r aspartate substrates by the cockroach transporter. The activity of DipEAA T1 was also unaffected by the presence of Zn++ in the bathing solution, des pite the presence of a putative Zn++-binding motif conferring Zn++-sensitiv ity on some mammalian glutamate transporters. (C) 2000 Elsevier Science Ltd . All rights reserved.