Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells

M. A. Varney, C. Jachec, C. Deal, S. D. Hess, L. P. Daggett, R. Skvoretz, M. Urcan, John Morrison, T. Moran, E. C. Johnson, G. Veliçelebi

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Abstract

The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N- methyl-o-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 Mg1+ 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 antiNMDAR1A 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)propyl1-phosphonate, MK-801, ketamine and ifenprodil. (±)-3-(2-Carboxypiperazin-4-yl)propyl-l- phosphonate was a 3-fold more potent antagonist at hNMDAR1N/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDARIA/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.

Original languageEnglish (US)
Pages (from-to)367-378
Number of pages12
JournalJournal of Pharmacology and Experimental Therapeutics
Volume279
Issue number1
StatePublished - Oct 1 1996
Externally publishedYes

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N-Methyl-D-Aspartate Receptors
N-Methylaspartate
Cell Line
Glycine
Organophosphonates
selfotel
Pharmacology
Dizocilpine Maleate
Ketamine
Patch-Clamp Techniques
Immunoprecipitation
N-methyl D-aspartate receptor subtype 2A
Glutamic Acid
Complementary DNA
Antibodies
Population

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. / Varney, M. A.; Jachec, C.; Deal, C.; Hess, S. D.; Daggett, L. P.; Skvoretz, R.; Urcan, M.; Morrison, John; Moran, T.; Johnson, E. C.; Veliçelebi, G.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 279, No. 1, 01.10.1996, p. 367-378.

Research output: Contribution to journalArticle

Varney, MA, Jachec, C, Deal, C, Hess, SD, Daggett, LP, Skvoretz, R, Urcan, M, Morrison, J, Moran, T, Johnson, EC & 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', Journal of Pharmacology and Experimental Therapeutics, vol. 279, no. 1, pp. 367-378.
Varney, M. A. ; Jachec, C. ; Deal, C. ; Hess, S. D. ; Daggett, L. P. ; Skvoretz, R. ; Urcan, M. ; Morrison, John ; Moran, T. ; Johnson, E. C. ; Veliçelebi, G. / Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells. In: Journal of Pharmacology and Experimental Therapeutics. 1996 ; Vol. 279, No. 1. pp. 367-378.
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abstract = "The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N- methyl-o-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 Mg1+ 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 antiNMDAR1A 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)propyl1-phosphonate, MK-801, ketamine and ifenprodil. (±)-3-(2-Carboxypiperazin-4-yl)propyl-l- phosphonate was a 3-fold more potent antagonist at hNMDAR1N/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDARIA/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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AU - Jachec, C.

AU - Deal, C.

AU - Hess, S. D.

AU - Daggett, L. P.

AU - Skvoretz, R.

AU - Urcan, M.

AU - Morrison, John

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N2 - The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N- methyl-o-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 Mg1+ 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 antiNMDAR1A 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)propyl1-phosphonate, MK-801, ketamine and ifenprodil. (±)-3-(2-Carboxypiperazin-4-yl)propyl-l- phosphonate was a 3-fold more potent antagonist at hNMDAR1N/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDARIA/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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