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Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells.
J Pharmacol Exp Ther. 1996 Oct; 279(1):367-78.JP

Abstract

The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/2B are described. In whole-cell electrophysiological recordings, application of NMDA/glycine elicited inward currents at negative holding potentials in human NMDAR1A/2A (hNMDAR1A/2A)- and hNMDAR1A/2B-expressing cells. The current-voltage relationships determined in both cell lines in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heteromeric complexes are formed in these stable cell lines. Stimulation of NMDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressing cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/2A. NMDA/glycine-evoked increases in Ca++ levels were inhibited by CGS 19755, (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These data suggest that cells stably expressing recombinant heteromeric hNMDAR1A/2A and hNMDAR1A/2B represent pharmacologically valid experimental systems to study human NMDA receptors.

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Authors+Show Affiliations

Varney MA
SIBIA Neurosciences, Inc., La Jolla, California, USA.
Jachec C
No affiliation info available
Deal C
No affiliation info available
Hess SD
No affiliation info available
Daggett LP
No affiliation info available
Skvoretz R
No affiliation info available
Urcan M
No affiliation info available
Morrison JH
No affiliation info available
Moran T
No affiliation info available
Johnson EC
No affiliation info available
Veliçelebi G
No affiliation info available

MeSH

AnimalsAntibodies, MonoclonalCalciumCell LineHumansRatsReceptors, N-Methyl-D-AspartateRecombinant ProteinsTransfection

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8859015

Citation

Varney, M A., et al. "Stable Expression and Characterization of Recombinant Human Heteromeric N-methyl-D-aspartate Receptor Subtypes NMDAR1A/2A and NMDAR1A/2B in Mammalian Cells." The Journal of Pharmacology and Experimental Therapeutics, vol. 279, no. 1, 1996, pp. 367-78.
Varney MA, Jachec C, Deal C, et al. Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. J Pharmacol Exp Ther. 1996;279(1):367-78.
Varney, M. A., Jachec, C., Deal, C., Hess, S. D., Daggett, L. P., Skvoretz, R., Urcan, M., Morrison, J. H., Moran, T., Johnson, E. C., & Veliçelebi, G. (1996). Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. The Journal of Pharmacology and Experimental Therapeutics, 279(1), 367-78.
Varney MA, et al. Stable Expression and Characterization of Recombinant Human Heteromeric N-methyl-D-aspartate Receptor Subtypes NMDAR1A/2A and NMDAR1A/2B in Mammalian Cells. J Pharmacol Exp Ther. 1996;279(1):367-78. PubMed PMID: 8859015.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. AU - Varney,M A, AU - Jachec,C, AU - Deal,C, AU - Hess,S D, AU - Daggett,L P, AU - Skvoretz,R, AU - Urcan,M, AU - Morrison,J H, AU - Moran,T, AU - Johnson,E C, AU - Veliçelebi,G, PY - 1996/10/1/pubmed PY - 1996/10/1/medline PY - 1996/10/1/entrez SP - 367 EP - 78 JF - The Journal of pharmacology and experimental therapeutics JO - J Pharmacol Exp Ther VL - 279 IS - 1 N2 - The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/2B are described. In whole-cell electrophysiological recordings, application of NMDA/glycine elicited inward currents at negative holding potentials in human NMDAR1A/2A (hNMDAR1A/2A)- and hNMDAR1A/2B-expressing cells. The current-voltage relationships determined in both cell lines in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heteromeric complexes are formed in these stable cell lines. Stimulation of NMDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressing cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/2A. NMDA/glycine-evoked increases in Ca++ levels were inhibited by CGS 19755, (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These data suggest that cells stably expressing recombinant heteromeric hNMDAR1A/2A and hNMDAR1A/2B represent pharmacologically valid experimental systems to study human NMDA receptors. SN - 0022-3565 UR - https://www.unboundmedicine.com/medline/citation/8859015/Stable_expression_and_characterization_of_recombinant_human_heteromeric_N_methyl_D_aspartate_receptor_subtypes_NMDAR1A/2A_and_NMDAR1A/2B_in_mammalian_cells_ L2 - https://jpet.aspetjournals.org/cgi/pmidlookup?view=long&pmid=8859015 DB - PRIME DP - Unbound Medicine ER -