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 electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 +/- 6 nM; Bmax = 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-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]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 Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.
Ishmael
J E
JE
College of Pharmacy, Oregon State University, Corvallis 97331, USA.
Franklin
P H
PH
Murray
T F
TF
Leid
M
M
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D018274:Electroporation; D018691:Excitatory Amino Acid Antagonists / Q000378:metabolism; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D007700:Kinetics; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D046911:Macromolecular Substances; D011869:Radioligand Assay; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012694:Serine / Q000494:pharmacology; D013237:Stereoisomerism; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D002118:Calcium; D018691:Excitatory Amino Acid Antagonists; D005998:Glycine; D007736:Kynurenic Acid; D046911:Macromolecular Substances; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D012694:Serine; D001616:beta-Galactosidase; C066192:5,7-dichlorokynurenic acid