An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor

The effect of N-glycosylation on the assembly of N-methyl-D-aspartate (NMDA) heteromeric cloned receptors was studied. Thus human embryonic kidney (HEK) 293 cells were cotransfected with N-methyl-D-aspartate R1 (NR1) and N-methyl-D-aspartate R2A (NR2A) clones and the cells grown post-transfection in the presence of tunicamycin (TM). TM treatment resulted in a decrease of the NR1 subunit with M(r) 117 000 with a concomitant increase in a M(r) 97 000 immunoreactive species previously identified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [H-3]MK801 binding to the expressed receptor which was a result of an approximate four-fold reduction in the Dissociation constant (K-D) but with no change in the number of binding sites (B-MAX). NMDA receptor cell surface expression was unchanged following TM treatment but it did result in a decrease in the percentage cell death post-transfection compared to control samples. The removal of TM from the cell culture media resulted in a return to the control K,value for [3H]MK801 binding and partial reglycosylation of newly synthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A receptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A receptor.