1 In this study we have examined the use of the ecdysone-inducible mammalia
n expression system (Invitrogen) for the regulation of expression of the pr
edominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter)
in HEK 293 cells.
2 HEK 293 cells which were stably transformed with the regulatory vector pV
gRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA usi
ng the inducible vector pIND and a clone designated HEK/EAAT2 was selected
for further characterization.
3 Na+-dependent L-glutamate uptake activity (3.2 pmol min(-1) mg(-1)) was o
bserved in EcR 293 cells and this was increased approximately 2 fold in the
uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity
in the absence of exogenous inducing agent. Exposure of HEK/ EAAT2 cells to
the ecdysone analogue Ponasterone A (10 mu M for 24 h) resulted in a great
er than or equal to 10 fold increase in the Na+-dependent activity.
4 L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Mich
aelis-Menten kinetics and Eadie-Hofstee transformation of the saturable upt
ake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5
mu M, V-max 3.8+/-0.9 nmol min(-1) mg(-1) protein.
5 The pharmacological profile of the EAAT2 subtype was characterized using
a series of L-glutamate transport inhibitors and the rank order of inhibito
ry potency was similar to that described previously for the rat homologue G
LT-1 and in synaptosomal preparations from rat cortex.
6 Addition of the EAAT2 modulator arachidonic acid resulted in an enhanceme
nt (155+/-5% control in the presence of 30 mu M) of the L-glutamate transpo
rt capacity in the induced HEK/EAAT2 cells.
7 This study demonstrates that the expression of EAAT2 can be regulated in
a mammalian cell line using the ecdysone-inducible mammalian expression sys