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J. Biol. Chem., Vol. 282, Issue 22, 16232-16243, June 1, 2007

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Biochemical and Functional Characterization of Orai Proteins^*

From the Department of Pathology, Harvard Medical School, and the CBR Institute for Biomedical Research, Boston, Massachusetts 02115

Stimulation of immune cells triggers Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca²⁺ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca²⁺ release-activated Ca²⁺ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca²⁺ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca²⁺ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

Received for publication, October 12, 2006 , and in revised form, January 11, 2007.

^* This work was supported in part by National Institutes of Health (NIH) Grants GM075256 and AI40127 (to A. R.) and NIH Grant AI066128 and grants from the Charles H. Hood and March of Dimes Foundations (to S. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ Both authors contributed equally to this work.

² Supported by a Ford Foundation predoctoral fellowship. A fellow of the Ryan Foundation.

³ Recipient of a fellowship from Uehara Memorial Foundation.

⁴ To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, The CBR Institute for Biomedical Research, Rm. 152, Warren Alpert Bldg., 200 Longwood Ave., Boston MA 02115. Tel.: 617-278-3260; Fax: 617-278-3280; E-mail: arao{at}cbr.med.harvard.edu.

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