Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors.
In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.
Kurkó
Dalma
D
Pharmacological and Drug Safety Research, Gedeon Richter Ltd., Budapest, Hungary. d.kurko@richter.hu
Dezso
Péter
P
Boros
András
A
Kolok
Sándor
S
Fodor
László
L
Nagy
József
J
Szombathelyi
Zsolt
Z
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004441:Ecdysterone / Q000031:analogs & derivatives* / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016202:N-Methylaspartate / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D018691:Excitatory Amino Acid Antagonists; D007473:Ion Channels; C473649:NR1A NMDA receptor, rat; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; C055348:muristerone A; D004441:Ecdysterone; D016202:N-Methylaspartate; D002118:Calcium