• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Molecular elucidation of CaMKII-mediated regulation of vascular receptor-operated Ca^<2+> entry channel TRPC6.

Research Project

Project/Area Number 17590221
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General pharmacology
Research InstitutionFukuoka University

Principal Investigator

INOUE Ryuji  Fukuoka University, School of Medicine, Professor, 医学部, 教授 (30232573)

Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
Keywordsreceptor-operated Ca^<2+> channel / vascular smooth muscle / Ca^<2+> mobilization / TRP protein / calmodulin-dependent kinase / phosphorylation / activation mechanism / amino acid mutation / カルモジュリン依存性キナーゼ / リン酸化
Research Abstract

TRPC6 is one of major TRP isoforms in vascular tissues and acts as a nonselective cation channel associated with vascular tone generation and remodeling. In our recent study, we have found that, in addition to a number of main mechanisms contributing the activation of this channel such as diacylyglycerol and IP3-receptor/calmodulin /phosphatidylinosides interaction, the phosphorylated state of the channel by calmodulin-dependent kinase II (CaMKII) greatly influence the process of activation both before and during receptor stimulation. To elucidate the molecular basis of this regulation, we have performed the following experiments. For this purpose, we performed a mutation analysis of CAMKII consensus motifs on wild-type TRPC6 and its chimera with N-terminal (NT) and transmembrane (TM) domains of TRPC7 (T776).
Search for the CAMKII consensus motif `RXX(S/T)' identified 8 and 7 candidate sequences on the NT or TM regions of TRPC6 and T776, respectively, but not on the C-terminus. Alanine … More substitution in these sequences revealed that only the mutations T487A in wild type TRPC6 and T433A in T776 strongly attenuated Ba^<2+> influx evoked by carbachol (100μM) without affecting their cell-membrane localized expression. The critical importance of T487A for TRPC6 activation was also confirmed by patch clamp experiments, where carbachol-induced current (ITRPC6) was reduced from 9.4±2.3 to 1.2±0.4 (pA/pF, n=5) by alanine substitution. In addition, substitution of T487 with glutamine, which confers a permanently negative charge therefore, caused potentiation of the current and Ba2+ response to carbachol, with prolong deactivation after termination of receptor stimulation. On the other hand, the cell membrane localized expression of TRPC6 protein assessed by its immunofluorescence did not appreciably change by these mutations. Concidering the proposed membrane topology derived from the structural analysis of TRPC1, T487 in TRPC6 and T433 in T776 are likely located on a long intracellular stretch between the second and third TM domains (II- III loop). These results suggest, combined with the requisiteness of a putative calmodulin binding site (CIRB) for channel activation, that close spatial arrangement of CIRB and the II-III loop might allow effective phosphorylation of T487 by CAMKII. This might in turn prime and/or facilitate the TRPC6 channel gating toward 'opening', which might be greatly affected by the charged state of T487 and negatively charged phospholipids in its close vicinity.
In the course of molecular elucidation of CaMKII-mediated regulation, we laso noticed that one of consensus phosphorylation motif T69 is common with that of protein kinase G (PKG), Since PKG is an important target of the ubiquitous physiological vasorelaxant nitric oxide (NO) and also since vasoconstrictor-induced Ca2+ entry is known to be strongly inhibited by activation of NO/cGMP/PKG pathway, we explored the potential role of this system in regulating TRPC6 channel activity. The magnitude of ITRPC6 was greatly inhibited by pretreatment with a NO donor, S-nitrosoacetyl penicillamine (SNAP) (by 70% at 100 μ M) in a voltage-independent manner, but cell-surface expression of TRPC6 protein was not appreciably reduced. Similar extent of inhibition was produced by a membrane-permeable analogue of cGMP 8-bromo cGMP (8-Br-cGMP; 100 g M). Both the inhibitory effects of SNAP and 8-Br-cGMP were nearly abolished by simultaneous administration of the PKG inhibitor KT5823 (10 μ M), PKG-specific inhibitory peptide DT-3 or alanine substitution for T69. These results suggest that PKG-mediated phosphorylation is another powerful mechanism to control TRPC6 channel activity, which may in situ operate as a tonic negative feedback via NO produced in adjacent endothelial cells or migrating inflammatory cells. Less

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (23 results)

All 2007 2006 2005 2004

All Journal Article (23 results)

  • [Journal Article] Membrane stretch-induced activation of a TRPM4-like nonselective cation channel in cerebral artery myocytes.2007

    • Author(s)
      Hiromitsu Morita, Akira Honda, Ryuji Inoue, 他4名
    • Journal Title

      Journal of Pharmacological Sciences 103

      Pages: 417-426

    • NAID

      10024313668

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels.2007

    • Author(s)
      Nakamura Y, Yasukouchi M, Kobayashi S, Uehara K, Honda A, Inoue R, 他2名
    • Journal Title

      Pflugers Archiv Eur J Physiol. 453(4)

      Pages: 455-462

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels.2007

    • Author(s)
      Nakamura, Y., Yasukouchi, M., Kobayashi, S., Uehara, K., Honda, A., Inoue, R., Imanaga, I., Uehara, A..
    • Journal Title

      Pflugers Archiv Eur J Physiol. 453(4)

      Pages: 455-62

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Membrane stretch-induced activation of a TRPM4-like nonselective cation channel in cerebral artery myocytes.2007

    • Author(s)
      Morita, H., Honda, A., Inoue, R., Ito, Y., Abe, K., Nelson, M.T., Brayden, J.E..
    • Journal Title

      Journal of Pharmacological Sciences 103

      Pages: 417-426

    • NAID

      10024313668

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels.2007

    • Author(s)
      Nakamura Y, Yasukouchi M, Kobayashi S, Uehara K, Honda A, Inoue R, Imanaga I, Uehara A.
    • Journal Title

      Pflugers Archiv Eur J Physiol. 453(4)

      Pages: 455-462

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Transient receptor potential C3/6 channels are essential for angiotensin II-induced cardiac hypertrophy.2006

    • Author(s)
      Onohara N, Nishida M, Inoue R, 他6名
    • Journal Title

      EMBO Journal 25(22)

      Pages: 5305-5316

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Nitric oxide activates TRP channels by cysteine S-nitrosylation.2006

    • Author(s)
      Yoshida, T, Inoue, R, 他8名
    • Journal Title

      Nature Chemical Biology 2(11)

      Pages: 596-607

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Transient receptor potential channels in cardiovascular function and disease.2006

    • Author(s)
      Inoue R, Jensen LJ, 他5名
    • Journal Title

      Circulation Research 99

      Pages: 119-131

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] TRP channels : molecular diversity and physiological function.2006

    • Author(s)
      Nishida M, Hara Y, Yoshida T, Inoue R, Mori Y
    • Journal Title

      Microcirculation 13(7)

      Pages: 535-550

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Depletion of intracellular Ca^<2+> store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells.2006

    • Author(s)
      Yoshida, I., Monji, A., Tashiro, K., Nakamura, K., Inoue, R., Kanba, S.
    • Journal Title

      Neurochemistry International 48

      Pages: 696-702

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Nitric oxide activates TRP channels by cysteine S-nitrosylation.2006

    • Author(s)
      Yoshida, T., Inoue, R., Morii, T., Hara, Y., Takahashi, N., Yamamoto, S., Tominaga, M., Shimizu, S., Sato, Y., Mori, Y.
    • Journal Title

      Nature Chemical Biology 2(11)

      Pages: 596-607

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Transient receptor potential C3/6 channels are essential for angiotensin II-induced cardiac hypertrophy.2006

    • Author(s)
      Onohara, N., Nishida, M., Inoue, R., Kobayashi, H., Sumimoto, H., Sato, Y., Mori, Y., Nagao, T., Kurose, H.
    • Journal Title

      EMBO Journal 25(22)

      Pages: 5305-5316

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Transient receptor potential channels in cardiovascular function and disease.2006

    • Author(s)
      Inoue, R., Jensen, L.J., Shi, J., Morita, H., Nishida, M., Honda, A., Ito, Y.
    • Journal Title

      Circulation Research 99

      Pages: 119-131

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Depletion of intracellular Ca^<2+> store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells.2006

    • Author(s)
      Yoshida, I., Monji, A., Tashiro, K., Nakamura, K., Inoue, R., Kanba, S
    • Journal Title

      Neurochemistry International 48

      Pages: 696-702

    • Related Report
      2006 Annual Research Report
  • [Journal Article] TRP channels : molecular diversity and physiological function.2006

    • Author(s)
      Nishida M, Hara Y, Yoshida T, Inoue R, Mori Y.
    • Journal Title

      Microcirculation 13(7)

      Pages: 535-550

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Depletion of intracellular Ca^<2+> store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells.2006

    • Author(s)
      Yoshida, I., Monji, A., Tashiro, K., Nakamura, K., Inoue, R., Kanba, S.
    • Journal Title

      Neurochemistry International (In press)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] G α _<12/13>-and reactive oxygen species-dependent activation of c-Jun NH_2-terminal kinase and p38 MAPK by angiotensin receptor stimulation in rat neonatal cardiomyocytes.2005

    • Author(s)
      Nishida, M., Tanabe, S., Maruyama, Y., Nagamatsu, Y., Takagahara, S., Turner, J.H., Kozasa, T., Kobayashi, H., Sato, Y., Kawanishi, T., Inoue, R., Nagao, T., Kurose, H.
    • Journal Title

      Journal of Biological Chemistry 280(18)

      Pages: 18434-18441

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] G α _<12/13>-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.2005

    • Author(s)
      Fujii, T., Onohara, N., Maruyama, Y, Tanabe, S., Kobayashi, H., Fukutomi, M., Nagamatsu, Y., Nishihara, N., Inoue, R., Sumimoto, H., Shibasaki, F., Nagao, T., Nishida, M., Kurose, H.
    • Journal Title

      Journal of Biological Chemistry 280(24)

      Pages: 23041-23047

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] TRP channels as a newly emerging non-voltage-gated Ca^<2+> entry channel superfamily.2005

    • Author(s)
      Inoue, R.
    • Journal Title

      Current Pharmaceutical Design 11 (15)

      Pages: 1899-1914

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Gα_<12/13>-and reactive oxygen species-dependent activation of c-Jun NH_2-terminal kinase and p38 MAPK by angiotensin receptor stimulation in rat neonatal cardiomyocytes.2005

    • Author(s)
      Nishida, M., Tanabe, S., Maruyama, Y., Nagamatsu, Y., Takagahara, S., Turner, J.H., Kozasa, T., Kobayashi, H., Sato, Y., Kawanishi, T., Inoue, R., Nagao, T., Kurose, H
    • Journal Title

      Journal of Biological Chemistry 280(18)

      Pages: 18434-18441

    • Related Report
      2005 Annual Research Report
  • [Journal Article] α_<12/13>-mediated production of reactive oxygen species is critical for angiotensin receptor-induced NFAT activation in cardiac fibroblasts.2005

    • Author(s)
      Fujii, T., Onohara, N., Maruyama, Y, Tanabe, S., Kobayashi, H., Fukutomi, M., Nagamatsu, Y, Nishihara, N., Inoue, R, Sumimoto, H, Shibasaki, F, Nagao, T., Nishida, M., Kurose, H
    • Journal Title

      Journal of Biological Chemistry 280(24)

      Pages: 23041-23047

    • Related Report
      2005 Annual Research Report
  • [Journal Article] TRP channels as a newly emerging non-voltage-gated Ca^<2+> entry channel superfamily.:,2005

    • Author(s)
      Inoue R
    • Journal Title

      Current Pharmaceutical Design 11(15)

      Pages: 1899-1914

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells.2004

    • Author(s)
      Shi, J., Mori, E., Mori, Y., Mori, M., Li, J., Ito Y., Inoue, R.
    • Journal Title

      Journal of Physiology 561(2)

      Pages: 415-432

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary

URL: 

Published: 2005-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi