Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter.
1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using 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 observed 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 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 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 inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport 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 system.
Dunlop
J
J
Wyeth Neuroscience, 865 Ridge Road, Princeton, New Jersey, NJ 08543, USA.
Lou
Z
Z
Zhang
Y
Y
McIlvain
H B
HB
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D027322:Amino Acid Transport System X-AG; D016718:Arachidonic Acid / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004440:Ecdysone / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D017981:Receptors, Neurotransmitter / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D018076:DNA, Complementary; D018690:Excitatory Amino Acid Agonists; D027342:Excitatory Amino Acid Transporter 2; D017981:Receptors, Neurotransmitter; D016718:Arachidonic Acid; D004440:Ecdysone; D018698:Glutamic Acid; C020889:dihydrokainic acid; D007608:Kainic Acid