Store-operated Ca 2+ entry through the plasma membrane Ca 2+ release-activated Ca 2+ (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 Ca 2+ 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.Ca 2+ influx through the CRAC channel in mammalian T cells and mast cells is essential for transcriptional responses and other effector responses to physiological stimuli [1][2][3][4] . Gating of the CRAC channel is a classical instance of store-operated Ca 2+ entry, where an initial release of Ca 2+ from internal cellular stores, by depleting the stores, triggers sustained Ca 2+ signaling due to opening of plasma membrane Ca 2+ channels [5][6][7] . RNA interference (RNAi) screens and determination of the genetic basis of a human severe combined immunodeficiency (SCID) syndrome have identified two proteins required for CRAC channel function: STIM1, a protein anchored in the endoplasmic reticulum (ER) that senses depletion of ER Ca 2+ stores [8][9][10] , andCorrespondence should be addressed to P.G.H. (hogan@idi.harvard.edu). 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Structural & Molecular Biology website. AUTHOR CONTRIBUTIONSP.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 Ca 2+ flux assays; D.M. and H.T.K. developed the P. pastoris membrane-flotation assay; M.O., with Y.Z., carried out reconstitution and Ca 2+ -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. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/nsmb/.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. NIH Public Access RESULTS Expression of recombinant ORAI1 and STIM...

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