High Level Expression of the NMDAR1 Glutamate Receptor Subunit in Electroporated COS Cells

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