1. Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611, USA
2. Harvard Medical School and the CBR Institute for Biomedical Research, 200 Longwood Avenue, Boston, Massachusetts 02115, USA
3. *These authors contributed equally to this work

Correspondence to: Anjana Rao² Correspondence and requests for materials should be addressed to A.R. (Email: arao@cbr.med.harvard.edu).

Abstract

Stimulation of immune cells causes depletion of Ca²⁺ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca²⁺ entry through store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels, an essential signal for lymphocyte activation and proliferation^{1, 2}. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca²⁺ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane^{3, 4, 5}. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca²⁺ entry downstream of STIM^{6, 7, 8}. Combined overexpression of Orai and Stim in Drosophila cells⁸, or Orai1 and STIM1 in mammalian cells^{9, 10, 11}, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca²⁺ influx, increase current carried by monovalent cations, and render the channel permeable to Cs⁺. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.

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