Skip to main content

Advertisement

SpringerLink
Log in

FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera

  • Letter
  • Published:

From Nature Biotechnology

View current issue Submit your manuscript

Abstract

When and where proteins associate is a central question in many biomolecular studies. Förster resonance energy transfer (FRET) measurements can be used to address this question when the interacting proteins are labeled with appropriate donor and acceptor fluorophores. We describe an improved method to determine FRET efficiency that uses a mode-locked laser, a confocal microscope and a streak camera. We applied this method to study the association of α and β1 subunits of the human cardiac sodium channel. The subunits were tagged with the cyan and yellow variants of the green fluorescent protein (GFP) and expressed in human embryonic kidney (HEK293) cells. Pronounced FRET between the channel subunits in the endoplasmic reticulum (ER) suggested that the subunits associate before they reach the plasma membrane. The described method allows simultaneous measurement of donor and acceptor fluorescence decays and provides an intrinsically validated estimate of FRET efficiency.

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: Experimental setup.
Figure 2: Streak images, fluorescence spectra and fluorescence decays.
Figure 3: Energy migration analysis.

Similar content being viewed by others

References

  1. Catterall, W.A. Cellular and molecular biology of voltage-gated sodium channels. Physiol. Rev. 72, S15–S48 (1992).

    Article  CAS  Google Scholar 

  2. Fozzard, H.A. & Hanck, D.A. Structure and function of voltage-dependent sodium channels: comparison of brain II and cardiac isoforms. Physiol. Rev. 72, 887–926 (1996).

    Article  Google Scholar 

  3. Nuss, H.B., Chiamvimonvat, N., Perez-Garcia, M.T., Tomaselli, G.F. & Marban, E. Functional association of the β1 subunit with human cardiac (hH1) and rat skeletal muscle sodium channel a subunits expressed in Xenopus oocytes. J. Gen. Physiol. 106, 1171–1191 (1995).

    Article  CAS  Google Scholar 

  4. Qu, Y. et al. Modulation of cardiac Na+ channel expression in Xenopus oocytes by β1 subunits. J. Biol. Chem. 270, 25696–25701 (1995).

    Article  CAS  Google Scholar 

  5. Zimmer, T. et al. Functional expression of GFP-linked human heart sodium channel (hH1) and subcellular localization of the α subunit in HEK293 cells and dog cardiac myocytes. J. Membr. Biol. 186, 1–12 (2002).

    Article  CAS  Google Scholar 

  6. Zimmer, T., Biskup, C., Bollensdorff, C. & Benndorf, K. The β1 subunit but not the β2 subunit colocalizes with the human heart Na+ channel (hH1) already within the endoplasmic reticulum. J. Membr. Biol. 186, 13–21 (2002).

    Article  CAS  Google Scholar 

  7. Hink, M.A., Visser N.V., Borst, J.W., van Hoek, A. & Visser, A.J.W.G. Practical use of corrected fluorescence excitation and emission spectra of fluorescence proteins in Förster resonance energy transfer (FRET) studies. J. Fluoresc. 13, 185–188 (2003).

    Article  CAS  Google Scholar 

  8. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  9. Malhotra, J.D. et al. Characterization of sodium channel α and β subunits in rat and mouse cardiac myocytes. Circulation 103, 1303–1310 (2001).

    Article  CAS  Google Scholar 

  10. Kusumi, A. et al. Development of a streak-camera based time resolved microscope fluorimeter and its application to studies of membrane fusion in single cells. Biochemistry 30, 6517–6527 (1991).

    Article  CAS  Google Scholar 

  11. Xu, X. et al. Detection of programmed cell death using fluorescence energy transfer. Nucleic Acids Res. 26, 2034–2035 (1998).

    Article  CAS  Google Scholar 

  12. Clegg, R.M. Fluorescence resonance energy transfer. in Fluorescence Imaging Spectroscopy and Microscopy. (eds. Wang, X.F. & Herman, B.) 179–252 (John Wiley, New York, 1996).

    Google Scholar 

  13. Patterson, G.H., Piston, D.W. & Barisas, B.G. Förster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank U. Meisel, G. Möhler, U. Simon, G. Watzinger, G. Weiss and R. Wolleschensky (Carl Zeiss Jena GmbH) for their help in adapting the LSM to our needs. We also appreciate the support of U. Denzer and G. Rousseau (Hamamatsu Photonics Germany) for the installation of the streak camera setup. We are grateful to K. Schoknecht, A. Kolchmeier, S. Bernhardt, G. Ditze and A. Hertel for excellent technical assistance.

Author information

Authors and Affiliations

  1. Friedrich-Schiller-University Jena, Institute of Physiology II, Teichgraben 8, Jena, 07740, Germany

    Christoph Biskup, Thomas Zimmer & Klaus Benndorf

Authors
  1. Christoph Biskup
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Zimmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Klaus Benndorf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Christoph Biskup or Klaus Benndorf.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Biskup, C., Zimmer, T. & Benndorf, K. FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera. Nat Biotechnol 22, 220–224 (2004). https://doi.org/10.1038/nbt935

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt935

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation