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Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

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.

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Figure 1: Orai interacts with Stim and generates increased store-operated currents in S2 cells.
Figure 2: Mutation E180D of Orai alters ion selectivity of the CRAC current.
Figure 3: Monovalent current in the absence of divalent ions exhibits altered ion selectivity in the Orai(E180D) mutant.
Figure 4: Charge-neutralizing mutations in the S1–S2 loop.

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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.

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  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

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  1. Andriy V. Yeromin
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Correspondence to Michael D. Cahalan.

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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

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