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Investigation on regulatory mechanisms for smooth muscle receptor-operated Ca^<2+> permeable cation channels using lipid bilayer incorporation of plasma membrane vesicles.

Research Project

Project/Area Number 10670086
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General pharmacology
Research InstitutionKYUSHU UNIVERSITY

Principal Investigator

INOUE Ryuji  Dept.Pharmacol.Grad.Sch.Med.Sci., KYUSHU UNIVERSITY Associate professor, 大学院・医学研究院, 助教授 (30232573)

Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
Keywordsreceptor / cation channel / plasma membrane vesicles / smooth muscle / lipid bilayer reconstitution / 微細再構成形質膜
Research Abstract

We have established the protocol of reconstituting G-protein-coupled receptor activated cation channels into the lipid bilayer, with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca^<2+>-dependent and ATP-sensitive K^+ channels (Toro et al., 1990). Ultracentrifugation of crude plasma membrane vesicles across the five discontinuous sucrose gradients provided a plasma membrane fraction (at 25/30% (w/w) sucrose interface) highly enriched with plasmalemmal proteins. Under Na^+-rich conditions, incorporation of these plasma membrane vesicles into the bilayer produced GTPγS (100μM)-activatable channel activities that are inhibited by GDPβS (1mM), sensitive to Ca^<2+> and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied depending on Na^+ concentration, but not affected by Cl^-. These results strongly indicate that the reconstituted channels activated by GTPγS belong to a class of voltage-dependent, Ca^<2+>-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also strongly point to the usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle in the future.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Inoue,Ryuji: "Nucleotide dependence revealed by flash photolysis of muscarinic cation(I_<cat>)in guinea-pig ileal smooth muscle cells."Jpn.J.Pharmacol.. 79. 47P (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Inoue,Ryuji: "Intracellular ATP slows time-dependent decline of muscarinic cation current in guinea-pig ileal smooth muscle."American Journal of Physiology Cell Physiology. 297. C1307-C1318 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Doira,Naoko: "Reconstitution in lipid bilayer of smooth muscle cation channels activated through a GTP-binding protein."Journal of Smooth Muscle Research. (in press.). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Inoue, Ryuji: "Nucleotide dependence revealed by flash photolysis of muscarinic cation (I_<cat>) in guinea-pig ileal smooth muscle cells."Jpn.J.Pharmacol.. 79. 47P (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Inoue, Ryuji: "Intracellular ATP slows time-dependent decline of muscarinic cation current in guinea-pig ileal smooth muscle."Am.J.Physiol.Cell Physiol.. 297. C307-C1318 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Doira, Naoko: "Reconstitution in lipid bilayer of smooth muscle cation channels activated through a GTP-binding protein."J.Smooth Mus.Res.. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary

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Published: 1998-04-01   Modified: 2016-04-21  

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