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Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference

  • Akihiro Mizutani
  • Katsuhiro Kawaai
  • Chihiro Hisatsune
  • Hideaki Ando
  • Takayuki Michikawa
  • Katsuhiko MikoshibaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 645)

Abstract

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca2+ release channels localized on intracellular Ca2+ stores and play a role in the generation of complex patterns of intracellular Ca2+ signals. We show herein experimental protocols for the identification of associating proteins of IP3R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP3R isoforms and their associating proteins using RNA interference. These methods will provide clues to understand the exact nature of how the signaling complex contributes to the generation of spatio-temporal patterns of intracellular Ca2+ signals.

Key words

IP3 Ca2+ Affinity chromatography GST-fusion protein siRNA Ca2+ oscillation IRBIT ERp44 

Notes

Acknowledgments

This work was supported by grants from the Education, Culture, Sports, and Technology of Japan.

References

  1. 1.
    Bezprozvanny, I. (2005) The inositol 1,4,5-trisphosphate receptors. Cell Calcium 38, 261–272.CrossRefPubMedGoogle Scholar
  2. 2.
    Foskett, J. K., White, C., Cheung, K. H. and Mak, D. O. (2007) Inositol trisphosphate receptor Ca2+ release channels. Physiol. Rev. 87, 593–658.CrossRefPubMedGoogle Scholar
  3. 3.
    Mikoshiba, K. (2007) IP3 receptor/Ca2+ channel: from discovery to new signaling concepts. J. Neurochem. 102, 1426–1446.CrossRefPubMedGoogle Scholar
  4. 4.
    Berridge, M. J. (1993) Inositol trisphosphate and calcium signalling. Nature 361, 315–325.CrossRefPubMedGoogle Scholar
  5. 5.
    Iwai, M., Tateishi, Y., Hattori, M., Mizutani, A., Nakamura, T., Futatsugi, A., Inoue, T., Furuichi, T., Michikawa, T. and Mikoshiba, K. (2005) Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant. J. Biol. Chem. 280, 10305–10317.CrossRefPubMedGoogle Scholar
  6. 6.
    Tu, H., Wang, Z., Nosyreva, E., De Smedt, H. and Bezprozvanny, I. (2005) Functional characterization of mammalian inositol 1,4,5-trisphosphate receptor isoforms. Biophys. J. 88, 1046–1055.CrossRefPubMedGoogle Scholar
  7. 7.
    Newton, C. L., Mignery, G. A. and Sudhof, T. C. (1994) Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J. Biol. Chem. 269, 28613–28619.PubMedGoogle Scholar
  8. 8.
    Choe, C. U. and Ehrlich, B. E. (2006) The inositol 1,4,5-trisphosphate receptor (IP3R) and its regulators: sometimes good and sometimes bad teamwork. Sci. STKE 2006, re15.CrossRefPubMedGoogle Scholar
  9. 9.
    Ando, H., Mizutani, A., Matsu-ura, T. and Mikoshiba, K. (2003) IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor. J. Biol. Chem. 278, 10602–10612.CrossRefPubMedGoogle Scholar
  10. 10.
    Higo, T., Hattori, M., Nakamura, T., Natsume, T., Michikawa, T. and Mikoshiba, K. (2005) Subtype-specific and ER lumenal environment-dependent regulation of inositol 1,4,5-trisphosphate receptor type 1 by ERp44. Cell 120, 85–98.CrossRefPubMedGoogle Scholar
  11. 11.
    Ando, H., Mizutani, A., Kiefer, H., Tsuzurugi, D., Michikawa, T. and Mikoshiba, K. (2006) IRBIT suppresses IP3 receptor activity by competing with IP3 for the common binding site on the IP3 receptor. Mol. Cell 22, 795–806.CrossRefPubMedGoogle Scholar
  12. 12.
    Hattori, M., Suzuki, A. Z., Higo, T., Miyauchi, H., Michikawa, T., Nakamura, T., Inoue, T. and Mikoshiba, K. (2004) Distinct roles of inositol 1,4,5-trisphosphate receptor types 1 and 3 in Ca2+ signaling. J. Biol. Chem. 279, 11967–11975.CrossRefPubMedGoogle Scholar
  13. 13.
    Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. and Mikoshiba, K. (1989) Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature 342, 32–38.CrossRefPubMedGoogle Scholar
  14. 14.
    Dykxhoorn, D. M. and Lieberman, J. (2005) The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Ann. Rev. Med. 56, 401–423.CrossRefPubMedGoogle Scholar
  15. 15.
    Scherer, L. J. and Rossi, J. J. (2003) Approaches for the sequence-specific knockdown of mRNA. Nat. Biotechnol. 21, 1457–1465.CrossRefPubMedGoogle Scholar
  16. 16.
    Kawaai, K., Ishida, S., Matsu-ura, T., Kuroda, Y., Ogawa, N., Tashiro, T., Enomoto, M., Hisatsune, C. and Mikoshiba, K. (2008) Optimization of transfection conditions for gene expression, siRNA knock-down, and live cell imaging using FuGene HD Transfection reagent. Biochemica 280, 21–24.Google Scholar
  17. 17.
    Mendes, C. C., Gomes, D. A., Thompson, M., Souto, N. C., Goes, T. S., Goes, A. M., Rodrigues, M. A., Gomez, M. V., Nathanson, M. H. and Leite, M. F. (2005) The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria. J. Biol. Chem. 280, 40892–40900.CrossRefPubMedGoogle Scholar
  18. 18.
    Galeotti, N., Quattrone, A., Vivoli, E., Bartolini, A. and Ghelardini, C. (2007) Knockdown of the type 2 and 3 inositol 1,4,5-trisphosphate receptors suppresses muscarinic antinociception in mice. Neuroscience 149, 409–420.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Akihiro Mizutani
    • 1
  • Katsuhiro Kawaai
    • 1
    • 2
  • Chihiro Hisatsune
    • 1
  • Hideaki Ando
    • 3
  • Takayuki Michikawa
    • 1
    • 4
  • Katsuhiko Mikoshiba
    • 1
    • 4
    Email author
  1. 1.Laboratory for Developmental NeurobiologyBrain Science Institute, RIKENSaitamaJapan
  2. 2.Department of Chemistry and Biological ScienceAoyama-Gakuin UniversityKanagawaJapan
  3. 3.Oregon Stem Cell CenterOregon Health & Sciences UniversityPortlandUSA
  4. 4.Calcium Oscillation Project, International Cooperative Research Project-Solution Oriented Research for Science and TechnologyJapan Science and Technology AgencySaitamaJapan

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