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Regulation of NMDA receptor by polyamine and its derivatives

Research Project

Project/Area Number 08044249
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
Research InstitutionChiba University

Principal Investigator

IGARASHI Kazuei  Faculty of Pharmaceutical Sciences, 薬学部, 教授 (60089597)

Co-Investigator(Kenkyū-buntansha) WILLIAMS Keith  University of Pennsylvania, 医学部, 助教授
KASHIWAGI Keiko  Faculty of Pharmaceutical Sciences, 薬学部, 助手 (80169424)
KEITH Willia  ペンシルバニア大学, 医学部, 助教授
Project Period (FY) 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥2,200,000 (Direct Cost: ¥2,200,000)
KeywordsPolyamine / Spermine / NMDA receptor / Antagonist / Site-directed mutagenesis / 部位突然変異
Research Abstract

1. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with pot D,a Polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339 or E342) to neutral amino acids reduced or abolished stimulation by spermine without affecting voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg^<2+>. Residue E342 in NR1 appears to be critical for spermine stimulation. Next, sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutate … More d. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine(D669A), orglutamate (D669E) abolished spermine stimulation. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg^<2+> or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor.
2. The effects of several N-sulfonyl-polyamines, including N^1-dansyl-spermine (N^1-DanSpm) and N^1-(n-octanesulfonyl)-spermine (N^1-OsSpm), were studied at recombinant NMDA receptors expressed in Xenopus oocytes. N^1-DanSpm and N^1-OsSpm inhibited NMDA receptors and were about 1,000-fold more potent than spermine in oocytes voltage-clamped at -70 mV.Block by N^1-DanSpm and N^1-OsSpm was strongly voltage-dependent, being more pronounced at hyperpolarized membrane potentials. Less

Report

(2 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] K.Kashiwagi et al.: "An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons." Mol.Pharmacol.49. 1131-1141 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] S.Sugiyama et al.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Kashiwagi et al.: "Spermidine-preferential uptake system in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Williams et al.: "Activation of N-methyl-D-aspartate receptors by glycine : Role of an aspartate residue in the M3-M4 loop of the NR1 subunit." Mol.Pharmacol.50. 701-708 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] S.Sugiyama et al.: "The 1.8 Å X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protein Science. 5. 1984-1990 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] J.Chao et al.: "N^1-Dansylspermine and N^1-(n-octanesulfonyl)-spermine,novel glutamate receptor antagonists : Block and permeation of N-methyl-D-aspartatereceptors." Mol.Pharmacol.51(in press). (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Kashiwagi et al.: "An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons." Mol.Pharmacol.49. 1131-1141 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] S.Sugiyama et al.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Kashiwagi et al.: "Spermidine-preferential uptake system in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Williams et al.: "Activation of N-methyl-D-aspartate receptors by glycine : Role of an aspartate residue in the M3-M4 loop of the NR1 subunit." Mol.Pharmacol.50. 701-708 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] S.Sugiyama et al.: "The 1.8* X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protein Science. 5. 1984-1990 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] J.Chao et al.: "N^1-Dansylspermine and N^1-(n-octanesulfonyl)-spermine, novel glutamate receptor antagonists : Block and permeation of N-methyl-D-aspartate receptors." Mol.Pharmacol.51 (in press). (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] K.Kashiwagi et al.: "An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons." Mol.Pharmacol.49. 1131-1141 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] S.Sugiyama et al.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] K.Kashiwagi et al.: "Spermidine-preferential uptakesystem in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein." J.Biol.Chem.271. 12205-12208 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] K.Williams et al.: "Activation of N-methyl-D-aspartate receptors by glycine:Role of an aspartate residue in the M3-M4 loop of the NR1 subunit." Mol.Pharmacol.50. 701-708 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] S.Sugiyama et al.: "The 1.8Å X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding." Protein Science. 5. 1984-1990 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] J.Chao et al.: "N^1-Dansylspermine and N^1-(n-octanesulfonyl)-spermine,novel glutamate receptor antagonists: Block and permeation of N-methyl-D-aspartate receptors." Mol.Pharmacol.51(in press). (1997)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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