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Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds.

Publication ,  Journal Article
Blanke, ML; VanDongen, AMJ
Published in: J Biol Chem
August 1, 2008

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

August 1, 2008

Volume

283

Issue

31

Start / End Page

21519 / 21529

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Receptors, N-Methyl-D-Aspartate
  • Receptors, Glutamate
  • Protein Structure, Tertiary
  • Point Mutation
  • Oocytes
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Conformation
  • Glycine
 

Citation

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Blanke, M. L., & VanDongen, A. M. J. (2008). Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds. J Biol Chem, 283(31), 21519–21529. https://doi.org/10.1074/jbc.M709190200
Blanke, Marie L., and Antonius M. J. VanDongen. “Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds.J Biol Chem 283, no. 31 (August 1, 2008): 21519–29. https://doi.org/10.1074/jbc.M709190200.
Blanke ML, VanDongen AMJ. Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds. J Biol Chem. 2008 Aug 1;283(31):21519–29.
Blanke, Marie L., and Antonius M. J. VanDongen. “Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds.J Biol Chem, vol. 283, no. 31, Aug. 2008, pp. 21519–29. Pubmed, doi:10.1074/jbc.M709190200.
Blanke ML, VanDongen AMJ. Constitutive activation of the N-methyl-D-aspartate receptor via cleft-spanning disulfide bonds. J Biol Chem. 2008 Aug 1;283(31):21519–21529.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

August 1, 2008

Volume

283

Issue

31

Start / End Page

21519 / 21529

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Receptors, N-Methyl-D-Aspartate
  • Receptors, Glutamate
  • Protein Structure, Tertiary
  • Point Mutation
  • Oocytes
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Conformation
  • Glycine