J. Dunlop et al., Steroid hormone-inducible expression of the GLT-1 subtype of high-affinityL-glutamate transporter in human embryonic kidney cells, BIOC BIOP R, 265(1), 1999, pp. 101-105
Citations number
18
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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.