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 re sulted in a decrease of the NR1 subunit with M(r) 117 000 with a conco mitant increase in a M(r) 97 000 immunoreactive species previously ide ntified as the non-N-glycosylated NR1 subunit. In parallel, TM caused a dose-dependent inhibition of [H-3]MK801 binding to the expressed rec eptor which was a result of an approximate four-fold reduction in the Dissociation constant (K-D) but with no change in the number of bindin g sites (B-MAX). NMDA receptor cell surface expression was unchanged f ollowing TM treatment but it did result in a decrease in the percentag e cell death post-transfection compared to control samples. The remova l of TM from the cell culture media resulted in a return to the contro l K,value for [3H]MK801 binding and partial reglycosylation of newly s ynthesized NR1 subunit. These results demonstrate that N-glycosylation is requisite for the efficient expression of functional NR1/NR2A rece ptors. Furthermore, they suggest that N-glycosylation may be important for the correct formation of the channel domain of the NR1/NR2A recep tor.