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Studies on receptor structure-function relationship using molecular biological techniques and GFP molecular visualization

Research Project

Project/Area Number 13672319
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionNational Institute of Health Sciences

Principal Investigator

NAKAZAWA Kenichi  National Institute of Health Sciences Division of Pharmacology Section Leader Research Officer, 薬理部・第二室室長 (00198061)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
KeywordsATP / purinoceptor / ion channel / molecular biology / GFP visularization / mutagenesis / structure-function relationship / Xenopus oocyte / P2X受容体 / GFP分子可視化法 / GFP / 発現系 / 共焦点顕微鏡 / 膜電流解析
Research Abstract

To clarify the relationship between the structure and the function of ATP-activated receptor/channel (P2X receptor), mutations (amino acid substitutions) were introduced by molecular biological techniques. GFP molecular visualization was combined to verify the expression of the mutants. Previous studies had shown that the activity of P2X2 receptor is lost when Gly247 was replaced with alanine. This region is assumed to be topologically similar to the ATP-binding domain of tRNA amino acyl synthetases. In these enzymes, aromatic amino acid residues are believed to contribute to the recognition of the adenine moiety of ATP molecules. Thus, two highly conserved aromatic amino acid residues (Phe240 and Trp256) were replaced with non-aromatic residues. The electrophysiological data obtained with these replacements showed that Trp256 is indispensable for the channel activity. Tyrosine partly compensated for Trp256, but other amino acids including phenylalanine failed. The expression of the non-functional mutants on cell surface was confirmed by the fluorescent images of GFP-connected receptors and the immunoblotting analysis of membrane fractions. The results suggested that the replacement of Trp256 does not affect protein translation or trafficking processes, but affects the structure of the receptor protein in cell membrane. Through these studies, the combination of molecular biological techniques and GFP molecular visualization is valuable for the clarification of the receptor structure and function relationship. In addition, the study with neutral amino acid introduction has shown that the amino acid at the position 333 sterically prevents calcium ion approach to the channel pore. It has been also shown that an intracellular disulfide bond affects the responsiveness to ATP.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Nakazawa, K., Ojima, H., Ohno, Y.: "A highly conserved tryptophane residue indispensable for cloned rat neuronal P2X receptor activation"Neuroscience Letters. 324. 141-144 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K., Sawa, H., Ojima, H., Ishii-Nozawa, R., Takeuchi, K., Ohno, Y.: "Size of side-chain at channel pore mouth affects Ca^<2+> block of P2X_2 receptor"European Journal of Pharmacology. 449. 207-211 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K.: "Encyclopedia of Molecular Medicine (T.Creighton ed.)"John Willey & Sons. 6 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K., Ojima, H., Ohno, Y.: "A highly conserved tryptophane residue indispensable for cloned rat neuronal P2X receptor activation"Neuroscience Letters. 324. 141-144 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K., Sawa,H., Ojima, H., Ishii-Nozawa,R., Takeuchi, K., Ohno, Y.: "Size of side-chain at channel pore mouth affects Ca^<2+> block of P2X_2 receptor"European Journal of Pharmacology. 449. 207-211 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K., T. Creighton ed: "Encyclopedia of Molecular Medicine"Neuroscience Letters, 324, 141-144, 2002. 6 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nakazawa, K., Ojima, H.Ohno, Y.: "A highly conserved tryptophane residue indispensable for cloned rat neuronal P2X receptor activation"Neuroscience Letters. 324. 141-144 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Sato, K., Matsuki, N., Ohno, Y., Nakazawa, K.: "Effects of 17beta-estradiol and xenoestrogens on the neuronal survival in the organotypic hippocampal culture"Neuroendocrinology. 76. 223-234 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nakazawa, K., Sawa, H., Ojima, H., Ishii-Nozawa, R., Takeuchi, K., Ohno, Y.: "Size of side-chain at channel pore mouth affects Ca^<2+> block of P2X_2 receptor"Eur. J. Pharmacol.. 449. 207-211 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Koizumi, S., Saito, Y., Nakazawa, K., Nakajima, K., Sawada, J.I., Kohsaka, S., Illes, P., Inoue K.: "Spatial and temporal aspects of Ca^<2+> signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes"Life Sciences. 72. 431-442 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nakazawa, K., Ohno, Y: "Modulation by estrogens and xenoestrogens of recombinant human neuronal nicotinic receptors"Eur. J. Pharmacol.. 430. 175-183 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 中澤憲一: "ATP受容体の構造と機能"生体の科学. 52. 152-157 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nakazawa, K., Ojima, H., Ohno, Y.: "A highly conserved tryptophane residue indispensable for cloned rat neuronal P2X receptor activation"Neurosci. Lett.. (in press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nakazawa, K.: "Encyclopedia of Molecular Medicine(T.Creighton)"John Wiley & Sons. 6 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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