Skip to main content
SpringerLink
Log in

Rapid functional assays of intracellular Ca2+ channels

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

Abstract

Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs. A low-affinity Ca2+ indicator (mag-fluo-4) trapped within the ER of permeabilized cells is shown to report changes in luminal free Ca2+ concentration reliably. An automated fluorescence plate reader allows rapid measurement of Ca2+ release from intracellular stores mediated by IP3R. The method can be readily adapted to other cell types or to the analysis of other intracellular Ca2+ channels. This protocol can be completed in 2–3 h.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: FlexStation assays of ER Ca2+ content.
Figure 2: Concentration-dependent release of Ca2+ by IP3 and AdA.
Figure 3: FlexStation analyses of HEK cells.
Figure 4: FlexStation analyses of mutant IP3R expressed in DT40 cells.

Similar content being viewed by others

References

  1. Laude, A.J. et al. Rapid functional assays of recombinant IP3 receptors. Cell Calcium 38, 45–51 (2005).

    Article  CAS  Google Scholar 

  2. Monteith, G.R. & Bird, G.S. Techniques: high-throughput measurement of intracellular Ca2+ — back to basics. Trends Pharmacol. Sci. 26, 218–223 (2005).

    Article  CAS  Google Scholar 

  3. Tsien, R.Y. Fluorescent probes of cell signalling. Annu. Rev. Neurosci. 12, 227–253 (1989).

    Article  CAS  Google Scholar 

  4. Tsien, R.Y. Imagining imaging's future. Nat. Rev. Mol. Cell Biol. 4, SS16–SS21 (2003).

    Google Scholar 

  5. Johnston, P.A. & Johnston, P.A. Cellular platforms for HTS: three case studies. Drug Discov. Today 7, 353–363 (2002).

    Article  CAS  Google Scholar 

  6. Williams, D.A., Fogarty, K.E., Tsien, R.Y. & Fay, F.S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using fura-2. Nature 318, 558–561 (1985).

    Article  CAS  Google Scholar 

  7. Hofer, A.M. & Machen, T.E. Technique for in situ measurement of calcium in intracellular inositol 1,4,5-trisphosphate-sensitive stores using the fluorescent indicator mag-fura-2. Proc. Natl. Acad. Sci. USA 90, 2598–2602 (1993).

    Article  CAS  Google Scholar 

  8. Combettes, L., Cheek, T.R. & Taylor, C.W. Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization. EMBO J. 15, 2086–2093 (1996).

    Article  CAS  Google Scholar 

  9. Miyakawa, T. et al. Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J. 17, 1303–1308 (1999).

    Article  Google Scholar 

  10. Bygrave, F.L. & Benedetti, A. What is the concentration of calcium ions in the endoplasmic reticulum? Cell Calcium 19, 547–551 (1996).

    Article  CAS  Google Scholar 

  11. Hofer, A.M. & Machen, T.E. Direct measurement of free Ca2+ in organelles of gastric epithelial cells. Am. J. Physiol. 267, G442–G451 (1994).

    CAS  PubMed  Google Scholar 

  12. Simpson, A.W. Fluorescent measurement of [Ca2+]c: basic practical considerations. Methods Mol. Biol. 312, 3–36 (2006).

    PubMed  Google Scholar 

  13. Invitrogen. Molecular Probes Handbook — A Guide to Fluorescent Probes and Labelling Technologies Tenth Edn. 〈http://probes.invitrogen.com/handbook/〉.

  14. Taylor, C.W. in Calcium Signaling (ed. Putney, J.W.) p.p. 111–130 (CRC Press, Boca Raton, Florida, USA, 2000).

    Google Scholar 

  15. Winding, P. & Berchtold, M.W. The chicken B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Meth. 249, 1–16 (2001).

    Article  CAS  Google Scholar 

  16. Suguwara, H., Kurosaki, M., Takata, M. & Kurosaki, T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 16, 3078–3088 (1997).

    Article  Google Scholar 

  17. Thomas, D. et al. A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28, 213–223 (2000).

    Article  CAS  Google Scholar 

  18. Patel, S. & Taylor, C.W. Quantal responses to inositol 1,4,5-trisphosphate are not a consequence of Ca2+ regulation of inositol 1,4,5-trisphosphate receptors. Biochem. J. 312, 789–794 (1995).

    Article  CAS  Google Scholar 

  19. Correa, V. et al. Structural determinants of adenophostin A activity at inositol trisphosphate receptors. Mol. Pharmacol. 59, 1206–1215 (2001).

    Article  CAS  Google Scholar 

  20. Van Rossum, D.B. et al. Agonist-induced Ca2+ entry determined by inositol 1,4,5-trisphosphate recognition. Proc. Natl. Acad. Sci. USA 101, 2323–2327 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Wellcome Trust, and the Biotechnology and Biological Sciences Research Council (BBSRC). We thank S. Lummis (Department of Biochemistry, Cambridge University, UK) for access to her FlexStation.

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

    Stephen C Tovey, Yi Sun & Colin W Taylor

Authors
  1. Stephen C Tovey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Colin W Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Colin W Taylor.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tovey, S., Sun, Y. & Taylor, C. Rapid functional assays of intracellular Ca2+ channels. Nat Protoc 1, 259–263 (2006). https://doi.org/10.1038/nprot.2006.40

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.40

  • Springer Nature Limited

This article is cited by

Search

Navigation