Steroid Hormone-Inducible Expression of the GLT-1 Subtype of High-Affinity -Glutamate Transporter in Human Embryonic Kidney Cells

The cDNA encoding the predominant rat brain high-affinity -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 mu M ponasterone A for 24 hincreased the maximum velocity (V sub(max)) of Na super(+)-dependent -glutamate uptake by greater than 10-fold, as compared with the uninduced cells. Equivalent levels of -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 -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 -glutamate uptake into HEK/GLT-1 cells, and the estimated IC sub(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.