High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells

Jane E. Ishmael, Paul H. Franklin, Thomas F. Murray, Mark Leid

Research output: Contribution to journalArticle

7 Citations (Scopus)

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- [3H]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 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 NR1-1 a 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.

Original languageEnglish
Pages (from-to)1500-1510
Number of pages11
JournalJournal of Neurochemistry
Volume67
Issue number4
StatePublished - Oct 1996
Externally publishedYes

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Dizocilpine Maleate
COS Cells
Glutamate Receptors
N-Methylaspartate
Glycine
Ligand-Gated Ion Channels
Imines
Fura-2
Cell membranes
N-Methyl-D-Aspartate Receptors
Labeling
Rats
Glutamic Acid
Proteins
Binding Sites
Cells
Calcium
Membranes
Acids
Electroporation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells. / Ishmael, Jane E.; Franklin, Paul H.; Murray, Thomas F.; Leid, Mark.

In: Journal of Neurochemistry, Vol. 67, No. 4, 10.1996, p. 1500-1510.

Research output: Contribution to journalArticle

Ishmael, Jane E. ; Franklin, Paul H. ; Murray, Thomas F. ; Leid, Mark. / High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells. In: Journal of Neurochemistry. 1996 ; Vol. 67, No. 4. pp. 1500-1510.
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