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STIM1 gates the store-operated calcium channel ORAI1 in vitro

Nature Structural & Molecular Biology volume 17, pages 112–116 (2010)Cite this article

Abstract

Store-operated Ca2+ entry through the plasma membrane Ca2+ release–activated Ca2+ (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca2+ flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.

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Figure 1: Recombinant ORAI1 and STIM1 proteins used in the present study.
Figure 2: STIM1 cytoplasmic fragments interact with ORAI1 assembled in yeast membranes and with two cytoplasmic fragments of ORAI1.
Figure 3: STIM1 triggers ORAI1-dependent Ca2+ efflux from membrane vesicles of S. cerevisiae.
Figure 4: STIM1 activates ORAI1 channels in membrane vesicles from S. cerevisiae.

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Acknowledgements

We are grateful to H. Li for guidance on protein expression and purification. We thank R. Rao for S. cerevisiae strain NY17 and for advice on membrane protein expression in that sec6-4 strain, R.Y. Tsien for plasmids encoding the calcium sensors D3cpV and D4cpV, J. Cregg for advice on protein expression in P. pastoris, the Department of Neurobiology, Harvard Medical School, for use of their spectrofluorimeter and T. Rapoport for access to SEC-MALLS instrumentation. This work was supported by US National Institutes of Health grants AI40127, GM075256 and AI084167 (to A.R. and P.G.H.) and by an Irvington Fellowship from the Cancer Research Institute and a Postdoctoral Fellowship from the Leukemia and Lymphoma Society (to Y.Z.).

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  1. Yubin Zhou and Paul Meraner: These authors contributed equally to this work.

Authors and Affiliations

  1. Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, Massachusetts, USA

    Yubin Zhou, Paul Meraner, Hyoung T Kwon, Danya Machnes, Masatsugu Oh-hora, Yun Huang, Antonio Stura, Anjana Rao & Patrick G Hogan

  2. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Yubin Zhou, Paul Meraner, Masatsugu Oh-hora, Yun Huang & Anjana Rao

  3. Department of Cell Biology and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Jochen Zimmer

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Contributions

P.G.H. set overall goals for the project and coordinated the work; Y.Z., P.M., A.R. and P.G.H. designed experiments and wrote the manuscript; Y.Z. prepared and characterized the STIM1 reagents, measured STIM-ORAI interactions and conducted the assays using mammalian cells; P.M. prepared and characterized sec6-4 ORAI1 vesicles; Y.Z. and P.M. conducted the in vitro Ca2+ flux assays; D.M. and H.T.K. developed the P. pastoris membrane-flotation assay; M.O., with Y.Z., carried out reconstitution and Ca2+-imaging experiments using STIM1−/− T cells; J.Z., with Y.Z., carried out SEC-MALLS analyses; Y.H., with Y.Z., contributed confocal microscopy; A.S. helped P.M. with construction of S. cerevisiae expression plasmids.

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Correspondence to Patrick G Hogan.

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Competing interests

A.R. and P.G.H. are founders and scientific advisors to CalciMedica, Inc., a biotechnology company developing treatments for autoimmune and inflammatory diseases.

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Zhou, Y., Meraner, P., Kwon, H. et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat Struct Mol Biol 17, 112–116 (2010). https://doi.org/10.1038/nsmb.1724

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