Figure 1

Functional expression of NMDAR in HEK293 cells using baculovirus. (A) NR1 and NR2A protein levels in HEK293 cells transduced with baculovirus. HEK293 cells were transduced in the presence of 1 mM Ketamine with different amounts of baculovirus encoding human NR1 and NR2A. Cells were harvested 16 h after transduction, and NR1 and NR2A protein levels were analyzed by immunoblotting. Representative images are shown. Molecular weight standards are indicated. NR2A shows an unspecific band at ~150 kDa. (B) Cell viability of HEK293 cells 16 h after transduction with different amounts of baculovirus encoding NR1 or NR2A. Cell viability was assessed using CellTiterGlo reagent. Data was normalized to non-transduced cells and represents the mean ± standard error of the mean (SEM) of three experiments. (C,D) Cell viability of HEK293 cells 16 h after transduction with baculovirus encoding NR1 or NR2A with concurrent treatment with MK801 (C) or Ketamine (D). Data was normalized to non-transduced cells in the presence of DMSO and represents the mean ± SEM of three experiments. (E) Model for lack of NMDAR activity in functional assay after protection with MK801 or Ketamine. NMDAR-ligands contained or secreted by the cells into the media activate NMDAR and lead to excitotoxicity and desensitization of the channel. Protection with MK801 or Ketamine prevent excitotoxicity, however, both compounds are difficult to wash out and do not prevent occupation of the ligand binding sites with endogenous ligand and prevent NMDAR activity in functional assays.