Steroid hormone-inducible expression of the GLT-1 subtype of high-affinityL-glutamate transporter in human embryonic kidney cells

The cDNA encoding the predominant rat brain high-affinity L-glutamate trans porter GLT-1 was isolated and subcloned into the pIND expression vector for the establishment of steroid hormone inducible expression in vitro using t he ecdysone-inducible mammalian expression system. Steroid hormone-inducibl e expression was demonstrable in a stable cell line designated HEK/GLT-1. T reatment of HEk/GLT-1 cells with 10 mu M ponasterone A for 24 hincreased th e maximum velocity (V-max) of Na+-dependent L-glutamate uptake by greater t han 10-fold, as compared with the uninduced cells. Equivalent levels of L-g lutamate 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 subtyp e present in the host cells, we evaluated the effects of the selective GLT- 1 inhibitors dihydrokainate (DHK) and kainate. Both DHK, and kainate produc ed concentration-dependent inhibition of the L-glutamate uptake into HEK/GL T-1 cells, and the estimated IC50 values were consistent with those describ ed for the cloned GLT-1. These results demonstrate that the expression of G LT-1 can be tightly regulated in vitro using the ecdysone system. (C) 1999 Academic Press.