Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai (original) (raw)

Nature volume 443, pages 226–229 (2006)Cite this article

Abstract

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule)1,2 and Orai3,4,5. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion1,6. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells3. The overexpression of Stim and Orai together markedly increases CRAC current5,7,8,9. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1–S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241 (2005)
    Article CAS Google Scholar
  2. Roos, J. et al. STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445 (2005)
    Article CAS Google Scholar
  3. Feske, S. et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179–185 (2006)
    Article ADS CAS Google Scholar
  4. Vig, M. et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312, 1220–1223 (2006)
    Article ADS CAS Google Scholar
  5. Zhang, S. L. et al. Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity. Proc. Natl Acad. Sci. USA 103, 9357–9362 (2006)
    Article ADS CAS Google Scholar
  6. Zhang, S. L. et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437, 902–905 (2005)
    Article ADS CAS Google Scholar
  7. Mercer, J. C. et al. Large store-operated calcium-selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1. J. Biol. Chem. doi:10.1074/jbc.M604589200 (published online 28 June 2006)
  8. Peinelt, C. et al. Amplification of CRAC current by STIM1 and CRACM1 (Orai1). Nature Cell Biol. 8, 771–773 (2006)
    Article CAS Google Scholar
  9. Soboloff, J. et al. Orai1 and STIM reconstitute store-operated calcium channel function. J. Biol. Chem. 281, 20661–20665 (2006)
    Article CAS Google Scholar
  10. Yeromin, A. V., Roos, J., Stauderman, K. A. & Cahalan, M. D. A store-operated calcium channel in Drosophila S2 cells. J. Gen. Physiol. 123, 167–182 (2004)
    Article CAS Google Scholar
  11. Almers, W. & McCleskey, E. W. Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. J. Physiol. (Lond.) 353, 585–608 (1984)
    Article CAS Google Scholar
  12. Hess, P. & Tsien, R. W. Mechanism of ion permeation through calcium channels. Nature 309, 453–456 (1984)
    Article ADS CAS Google Scholar
  13. Hoth, M. & Penner, R. Calcium release-activated calcium current in rat mast cells. J. Physiol. (Lond.) 465, 359–386 (1993)
    Article CAS Google Scholar
  14. Lepple-Wienhues, A. & Cahalan, M. D. Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells. Biophys. J. 71, 787–794 (1996)
    Article CAS Google Scholar
  15. Zweifach, A. & Lewis, R. S. Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes. J. Gen. Physiol. 107, 597–610 (1996)
    Article CAS Google Scholar
  16. Kozak, J. A., Kerschbaum, H. H. & Cahalan, M. D. Distinct properties of CRAC and MIC channels in RBL cells. J. Gen. Physiol. 120, 221–235 (2002)
    Article Google Scholar
  17. Bakowski, D. & Parekh, A. B. Monovalent cation permeability and Ca2+ block of the store-operated Ca2+ current ICRAC in rat basophilic leukemia cells. Pflugers Arch. 443, 892–902 (2002)
    Article CAS Google Scholar
  18. Prakriya, M. & Lewis, R. S. Potentiation and inhibition of Ca2+ release-activated Ca2+ channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors. J. Physiol. (Lond.) 536, 3–19 (2001)
    Article CAS Google Scholar
  19. Ellinor, P. T., Yang, J., Sather, W. A., Zhang, J. F. & Tsien, R. W. Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. Neuron 15, 1121–1132 (1995)
    Article CAS Google Scholar
  20. Prakriya, M. & Lewis, R. S. Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels. J. Gen. Physiol. 119, 487–507 (2002)
    Article CAS Google Scholar
  21. Zweifach, A. & Lewis, R. S. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc. Natl Acad. Sci. USA 90, 6295–6299 (1993)
    Article ADS CAS Google Scholar
  22. Voets, T., Janssens, A., Droogmans, G. & Nilius, B. Outer pore architecture of a Ca2+-selective TRP channel. J. Biol. Chem. 279, 15223–15230 (2004)
    Article CAS Google Scholar
  23. Prakriya, M. et al. Orai1 is an essential pore subunit of the CRAC channel. Nature advance online publication, doi:10.1038/nature05122 (20 August 2006)

Download references

Acknowledgements

We thank L. Forrest for assistance in cell culture and G. Chandy for use of molecular reagents in his laboratory. This work was supported by a grant from the National Institutes of Health (M.D.C.), by a fellowship from the George E. Hewitt Foundation (S.L.Z.), and by a Scientist Development Grant from the American Heart Association (Y.Y.).Author Contributions A.V.Y. was responsible for all patch-clamp experiments and analysis. S.L.Z. was responsible for all molecular biology and biochemistry experiments, with the assistance of W.J. Y.Y. and O.S. performed RT–PCR. M.D.C. provided advice and overall direction, and supervised project planning and execution.

Author information

Author notes

  1. Andriy V. Yeromin and Shenyuan L. Zhang: *These authors contributed equally to this work

Authors and Affiliations

  1. Department of Physiology & Biophysics and Center for Immunology, University of California, Irvine, California, 92697, USA
    Andriy V. Yeromin, Shenyuan L. Zhang, Weihua Jiang, Ying Yu, Olga Safrina & Michael D. Cahalan
  2. Biophysics and Center for Immunology, University of California,
    Andriy V. Yeromin, Shenyuan L. Zhang, Weihua Jiang, Ying Yu, Olga Safrina & Michael D. Cahalan

Authors

  1. Andriy V. Yeromin
    You can also search for this author inPubMed Google Scholar
  2. Shenyuan L. Zhang
    You can also search for this author inPubMed Google Scholar
  3. Weihua Jiang
    You can also search for this author inPubMed Google Scholar
  4. Ying Yu
    You can also search for this author inPubMed Google Scholar
  5. Olga Safrina
    You can also search for this author inPubMed Google Scholar
  6. Michael D. Cahalan
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toMichael D. Cahalan.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–3. (PDF 146 kb)

Supplementary Notes

This file contains Supplementary Methods, Supplementary Table 1 and Supplementary Figure Legends. (DOC 42 kb)

Rights and permissions

About this article

Cite this article

Yeromin, A., Zhang, S., Jiang, W. et al. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.Nature 443, 226–229 (2006). https://doi.org/10.1038/nature05108

Download citation

Editorial Summary

Calcium pump

Two groups report the molecular identification of the long-sought CRAC channel as a plasma membrane protein known variously as olf186-F, Orai and CRACM1. The CRAC channel is of fundamental importance to Ca2+ signalling mechanisms in cell biology.