Steroid hormone-inducible expression of the GLT-1 subtype of high-affinity l-glutamate transporter in human embryonic kidney cells.
The cDNA encoding the predominant rat brain high-affinity l-glutamate transporter GLT-1 was isolated and subcloned into the pIND expression vector for the establishment of steroid hormone inducible expression in vitro using the ecdysone-inducible mammalian expression system. Steroid hormone-inducible expression was demonstrable in a stable cell line designated HEK/GLT-1. Treatment of HEK/GLT-1 cells with 10 microM ponasterone A for 24 hincreased the maximum velocity (V(max)) of Na(+)-dependent l-glutamate uptake by greater than 10-fold, as compared with the uninduced cells. Equivalent levels of l-glutamate transport capacity were observed in the uninduced GLT-1 cell line and the host cell line indicating that the expression of GLT-1 was tightly regulated. To confirm that the increased l-glutamate uptake observed in HEK/GLT-1 cells following induction was attributable to the expression of GLT-1, rather than the up-regulation of the endogenously expressed EAAT3 subtype present in the host cells, we evaluated the effects of the selective GLT-1 inhibitors dihydrokainate (DHK) and kainate. Both DHK and kainate produced concentration-dependent inhibition of the l-glutamate uptake into HEK/GLT-1 cells, and the estimated IC(50) values were consistent with those described for the cloned GLT-1. These results demonstrate that the expression of GLT-1 can be tightly regulated in vitro using the ecdysone system.
Dunlop
J
J
Neuroscience Drug Discovery, Wyeth-Ayerst Research, CN-8000, Princeton, New Jersey, 08543-8000, USA.
Lou
Z
Z
McIlvain
H B
HB
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D011994:Recombinant Proteins; D018698:Glutamic Acid; D004441:Ecdysterone; C008999:ponasterone A; D012964:Sodium; D007608:Kainic Acid