Development of an inducible NMDA receptor stable cell line with an intracellular Ca2+ reporter

Cytotoxicity associated with NMDA receptor activation has impeded the estab lishment of cell lines expressing recombinant subtypes of this ligand-gated ion channel class. To circumvent this toxicity, we describe in this report the use of a potent inducible promoter in the construction of a cell line stably expressing the NR1a/NR2A subtype of the NMDA receptor. Western blot analysis using subunit selective antibodies revealed that NR2A subunits wer e constitutively expressed in this cell line, whereas expression of NR1a su bunits was tightly regulated by tetracycline. Upon tetracycline removal, el ectrophysiological recordings using the patch clamp technique indicated the expression of functional receptors with biophysical and pharmacological pr operties corresponding to those expected of the NR1a/NR2A subtype. In addit ion, we utilized this cell line with the recombinant membrane targeted Ca2 reporter, aequorin, in a functional assay of NMDA receptor activation. An evaluation of the coupling efficiency of NMDA receptor activation and aequo rin response, as well as the pharmacological profile of this assay, illustr ates the suitability of this cell line and the Ca2+ reporter assay to funct ionally identify novel NMDA receptor antagonists. (C) 1999 Elsevier Science B.V. All rights reserved.