HIGH-LEVEL EXPRESSION OF THE NMDAR1 GLUTAMATE-RECEPTOR SUBUNIT IN ELECTROPORATED COS CELLS

The rat N-methyl-D-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electrop oration, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7- [H-3]dichlorokynurenic acid labeled a single high-affinity site (K-D = 29.6 +/- 6 nM; B-max = 19.4 +/- 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to prev ious reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist ,11-dihydro-5H-dibenzo [a,d]-cyclohepten-5,10-imine ([H-3]MK-801) was not detected in NR1-1a- transfected COS cells. Although immunofluorescent labeling of electrop orated COS cells demonstrated that the NR1-1a protein appears to be as sociated with the cell membrane, neither NMDA nor glutamate effected a n increase in intracellular calcium concentration in fura-2-loaded cel ls, suggesting that homomeric NR1-1a receptors do not act as functiona l ligand-gated ion channels. Therefore, COS cells appear to differ fro m Xenopus oocytes with respect to the transient expression of function al homomeric NR1 receptors. Although expression of NR1-1a is sufficien t to reconstitute a glycine binding site with wild-type affinity for a ntagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when exp ressed in this mammalian cell line.