Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific

Johannes J. Krupp, Bryce Vissel, Stephen F. Heinemann, Gary Westbrook

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Abstract

Intracellular Ca2+ can reversibly reduce the activity of native N- methyl-D-aspartate (NMDA) receptors in hippocampal neurons, a phenomenon termed Ca2+-dependent inactivation. We examined inactivation in heteromeric NMDA receptors expressed in human embryonic kidney (HEK) 293 cells using whole-cell recording. NR1-1a/2A heteromers showed reversible inactivation that was very similar to native NMDA receptors in cultured hippocampal neurons. Inactivation was dependent on the extracellular Ca2+ concentration and the degree of intracellular Ca2+ buffering. In 2 mM extracellular Ca2+, inactivation resulted in a 46.1 ± 12.6% reduction in the whole-cell current during a 5-sec agonist application. Inactivation of NR1-1a/2A heteromers was unaffected by calcineurin inhibitors, staurosporine, or phalloidin. NR1-1a/2D heteromers also showed a similar degree of inactivation. In contrast, NR1-1a/2B and NR1-1a/2C heteromers showed no significant inactivation. At saturating concentrations of NMDA (1 mM), NR1-1a/2A heteromers also showed Ca- and glycine-independent desensitization, as seen in native hippocampal neurons. Ca2+- and glycine- independent desensitization was less pronounced in NR1-1a/2B heteromers and absent in NR1-1a/2C heteromers. Activation of NR1-1a/2C heteromers triggered intracellular Ca2+ transients similar to NR1-1a/2A heteromers as verified by combined Ca2+ imaging and whole-cell recording. Thus differences in Ca2+ permeability were not responsible for the lack of inactivation in NR1-1a/2C heteromers. Our results show that inactivation of recombinant NMDA receptors requires either the NR2A or NR2D subunit, whereas both inactivation and desensitization were absent in NR2C-containing receptors. The gating of inactivating NMDA receptors is more likely to be influenced by ongoing NMDA receptor activity and Ca2+ transients, perhaps consistent with the prominent expression of NR2A in hippocampus and cerebral cortex.

Original languageEnglish (US)
Pages (from-to)1680-1688
Number of pages9
JournalMolecular Pharmacology
Volume50
Issue number6
StatePublished - Dec 1996

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N-Methyl-D-Aspartate Receptors
Calcium
Patch-Clamp Techniques
Neurons
Glycine
Phalloidine
Staurosporine
N-Methylaspartate
Cerebral Cortex
Permeability
Hippocampus
Kidney

ASJC Scopus subject areas

  • Pharmacology

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Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. / Krupp, Johannes J.; Vissel, Bryce; Heinemann, Stephen F.; Westbrook, Gary.

In: Molecular Pharmacology, Vol. 50, No. 6, 12.1996, p. 1680-1688.

Research output: Contribution to journalArticle

Krupp, Johannes J. ; Vissel, Bryce ; Heinemann, Stephen F. ; Westbrook, Gary. / Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific. In: Molecular Pharmacology. 1996 ; Vol. 50, No. 6. pp. 1680-1688.
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abstract = "Intracellular Ca2+ can reversibly reduce the activity of native N- methyl-D-aspartate (NMDA) receptors in hippocampal neurons, a phenomenon termed Ca2+-dependent inactivation. We examined inactivation in heteromeric NMDA receptors expressed in human embryonic kidney (HEK) 293 cells using whole-cell recording. NR1-1a/2A heteromers showed reversible inactivation that was very similar to native NMDA receptors in cultured hippocampal neurons. Inactivation was dependent on the extracellular Ca2+ concentration and the degree of intracellular Ca2+ buffering. In 2 mM extracellular Ca2+, inactivation resulted in a 46.1 ± 12.6{\%} reduction in the whole-cell current during a 5-sec agonist application. Inactivation of NR1-1a/2A heteromers was unaffected by calcineurin inhibitors, staurosporine, or phalloidin. NR1-1a/2D heteromers also showed a similar degree of inactivation. In contrast, NR1-1a/2B and NR1-1a/2C heteromers showed no significant inactivation. At saturating concentrations of NMDA (1 mM), NR1-1a/2A heteromers also showed Ca- and glycine-independent desensitization, as seen in native hippocampal neurons. Ca2+- and glycine- independent desensitization was less pronounced in NR1-1a/2B heteromers and absent in NR1-1a/2C heteromers. Activation of NR1-1a/2C heteromers triggered intracellular Ca2+ transients similar to NR1-1a/2A heteromers as verified by combined Ca2+ imaging and whole-cell recording. Thus differences in Ca2+ permeability were not responsible for the lack of inactivation in NR1-1a/2C heteromers. Our results show that inactivation of recombinant NMDA receptors requires either the NR2A or NR2D subunit, whereas both inactivation and desensitization were absent in NR2C-containing receptors. The gating of inactivating NMDA receptors is more likely to be influenced by ongoing NMDA receptor activity and Ca2+ transients, perhaps consistent with the prominent expression of NR2A in hippocampus and cerebral cortex.",
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