Inducible expression and pharmacology of recombinant NMDA receptors, composed of rat NR1a/NR2B subunits.
An ecdysone-inducible mammalian expression system was used to study expression of recombinant N-methyl-D-aspartate (NMDA) receptors. Human embryonic kidney (HEK) 293 cells expressing the regulatory vector pVgRXR (EcR 293 cells) were transfected with rat NR1a and NR2B cDNAs using the inducible vector pIND (Invitrogen). Inducible expression of the NR2B subunit in cell clone designated EcR/rNR1a2B was investigated using quantitative RT-PCR and flow cytometry based immunocytochemical methods. The mRNA level of the NR2B subunits in EcR/rNRa2B cells was dependent on the concentration of the ecdysone analogue inducing agent, muristerone A (MuA). Similarly, NR2B subunit protein expression was higher in cells pre-treated with the inducing agent. Functionally active NMDA receptors were also detected in EcR/rNR1a2B cells after MuA induction. In presence of the inducing factor, NMDA-evoked ion currents as well as increase in cytoplasmic calcium-concentrations were measured using whole-cell patch clamp and fluorometric calcium measuring techniques. The pharmacological profile of the expressed NMDA receptors was characterised by comparing the inhibitory activity of several NR2B subunit selective NMDA antagonists in EcR/rNR1a2B cells with that observed in primary cultures of rat cortical neurones. Whereas the efficacies of the NR2B subunit selective NMDA antagonists were similar in EcR/rNR1a2B cells and in neurones, their maximal inhibitory effects were significantly higher in cells expressing NR1a/NR2B recombinant receptors. This study demonstrates that recombinant NMDA receptors can be expressed in an inducible way in non-neuronal cell lines using the ecdysone-inducible mammalian expression system. Such cell lines can be suitable tools in high throughput functional screening for potential subtype selective modulators of the NMDA receptor.
Nagy
József
J
Pharmacological and Drug Safety Research, Gedeon Richter Ltd., 10. POB 27, H-1475, Budapest, Hungary. iozsef.nagy@richter.hu
Boros
András
A
Dezso
Péter
P
Kolok
Sándor
S
Fodor
László
L
D000818:Animals; D002460:Cell Line; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004440:Ecdysone / Q000494:pharmacology; D004594:Electrophysiology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects; D019579:Neocortex / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018691:Excitatory Amino Acid Antagonists; C473649:NR1A NMDA receptor, rat; C121001:NR2B NMDA receptor; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D004440:Ecdysone