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

Cytotoxicity associated with NMDA receptor activation has impeded the establishment 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 were constitutively expressed in this cell line, whereas expression of NR1a subunits was tightly regulated by tetracycline. Upon tetracycline removal, electrophysiological recordings using the patch clamp technique indicated the expression of functional receptors with biophysical and pharmacological properties corresponding to those expected of the NR1a/NR2A subtype. In addition, 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 aequorin response, as well as the pharmacological profile of this assay, illustrates the suitability of this cell line and the Ca2+ reporter assay to functionally identify novel NMDA receptor antagonists.