Abstract
The NMDA subtype of excitatory glutamate receptor is a multisubunit, fast-acting, ligand-gated cation channel with a high permeability for Ca2+. NMDA receptors have been shown to be of importance in physiological processes, such as long-term potentiation, and also in pathophysiological conditions, including chronic neuronal degeneration and acute excitotoxity (1). Molecular cloning has identified five genes encoding two types of NMDA receptor subunits, the NR1 and NR2A-D subunits. Additionally, the NR1 subunit gene undergoes alternative splicing to yield eight variant forms, thus providing further heterogeneity. The quaternary structures of NMDA receptors are not known, but most are believed to be heteromeric complexes comprising multiple copies of both NR1 and NR2 subunits in as-yet unknown stoichiometries (summarized in 1). Since the absolute polypeptide compositions of native NMDA receptors are unknown, a convenient model system in which to study the properties of defined cloned NMDA receptor subtypes and thus to compare them with their in vivo counterparts is their expression in mammalian cells. This permits the pharmacological, functional and biochemical characterization of either a single type of NMDA receptor subunit or coexpression of combinations of NR1 and NR2 subunit genes (e.g., 2–4).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sucher, N., Awobuluyi, M., Choi, Y-B., and Lipton, S. A. (1996) NMDA receptors: from genes to channels. TIPS 17, 348–355.
Buller, A. L., Larson, H. H., Schneider, B. E., Beaton, J. A., Morrisett, R. A., and Monaghan, D. T. (1994) The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition. J. Neurosci. 14, 5471–5484.
Laurie, D. J. and Seeburg, P. H. (1994) Ligand binding affinities at recombinant N-methyl-d-aspartate receptors depend on subunit composition. Eur. J. Pharmacol. 268, 335–345.
Chazot, P. L., Coleman, S. K., Cik, M., and Stephenson, F. A. (1994) Molecular characterisation of N-methyl-d-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. J. Biol. Chem. 269, 24,403–24,409.
Behe, P., Stern, P., Wyllie, D. J., Nassar, M., Schoepfer, R., and Colquhoun, D. (1995) Determination of NMDA R1 subunit copy number in recombinant NMDA receptors. Proc. R. Soc. Lond. B. 262, 205–213.
Ehlers, M. D., Tingley, W. G., and Huganir, R. L. (1995) Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. Science 269, 1734–1737.
Cik, M., Chazot, P. L., and Stephenson, F. A. (1994) Expression of NMDAR1-1A (N598Q)/NMDAR2A receptors results in decreased cell mortality. Eur. J. Pharmacol. 266, R1–R3.
Laube, B., Hirai, H., Sturgess, M., Betz, H., and Kuhse, J. (1997) Molecular determinants of agonist discrimination by NMDA receptor subunits. Neuron 18, 493–503.
Kuryatov, A., Laube, B., Betz, H., and Kuhse, J. (1994) Mutational analysis of the glycine-binding site of the NMDA receptor. Neuron 12, 1291–1300.
Mori, H. H., Masaki, T., Yamakura, T., and Mishina, M. (1992) Identification by mutagenesis of a Mg2+-block site of the NMDA receptor channel. Nature 358, 673–675.
Grimwood, S., LeBourdelles, B., and Whiting, P. J. (1995) Recombinant human NMDA homomeric NR1 receptors expressed in mammalian cells form a high-affinity glycine antagonist binding site. J. Neurochem. 64, 525–530.
Lynch, D. R., Anegawa, N. J., Verdoorn, T., and Pritchett, D. B. (1994). N-Methyl-d-aspartate receptors: Different subunit requirements for binding of glutamate antagonists, glycine antagonists, and channel-blocking agents. Mol. Pharmacol. 45, 540–545.
Chazot, P. L., Reiss, C., and Stephenson, F. A. (1997) [3H] MDL 105,519, a novel antagonist for the glycine site of the NMDA receptor Br. J. Pharmacol. (Suppl.) 119, C39.
Brimecombe, J. C., Boeckman, F. A., and Aizenman, E. (1997) Functional consequences of NR2 subunit composition in single recombinant N-methyl-d-aspartate receptors. Proc. Natl. Acad. Sci. USA 94, 11,019–11,024.
Varney, M. A., Jahec, C., Deal, C., Hess, S. D., Daggett, R., Skvoretz, R., et al. (1996) Stable expression and characterisation of recombinant human heteromeric N-methyl-d-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. J. Pharm. Exp. Ther. 279, 367–378.
Wood, M. W., VanDongen, H. M. A., and Vandongen, A. M. J. (1996) The 5′-untranslated region of the N-methyl-d-aspartate receptor NR2A subunit controls efficiency of translation. J. Biol. Chem. 271, 8115–8120.
Cik, M., Chazot, P. L., and Stephenson, F. A. (1993) Optimal expression of cloned NMDAR1/NMDAR2A heteromeric glutamate receptors: a biochemical characterisation. Biochem. J. 296, 877–883.
Stern, P., Cik, M., Colquhoun, D., and Stephenson, F. A. (1994) Single-channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes. J. Physiol. 476, 391–397.
Chazot, P. L., Cik, M., and Stephenson, F. A. (1992) Immunological detection of the NMDAR1 glutamate receptor expressed in human embryonic kidney 293 cells and in rat brain. J. Neurochem. 59, 1176–1178.
Chazot, P. L. and Stephenson, F. A. (1997) Biochemical evidence for the existence of a pool of unassembled C2 exon-containing NR1 subunits of the mammalian forebrain NMDA receptor. J. Neurochem. 68, 507–516.
Chazot, P. L., Cik, M., and Stephenson, F. A. (1995) An investigation into the role of N-glycosylation in the functional expression of a recombinant heteromeric NMDA receptor. Mol. Membr. Biol. 12, 331–337.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Chazot, P.L., Cik, M., Stephenson, F.A. (1999). Transient Expression of Functional NMDA Receptors in Mammalian Cells. In: Li, M. (eds) NMDA Receptor Protocols. Methods in Molecular Biology™, vol 128. Humana Press. https://doi.org/10.1385/1-59259-683-5:33
Download citation
DOI: https://doi.org/10.1385/1-59259-683-5:33
Publisher Name: Humana Press
Print ISBN: 978-0-89603-713-7
Online ISBN: 978-1-59259-683-6
eBook Packages: Springer Protocols