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Orai1 is an essential pore subunit of the CRAC channel

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 proliferation1,2. 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 membrane3,4,5. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM6,7,8. Combined overexpression of Orai and Stim in Drosophila cells8, or Orai1 and STIM1 in mammalian cells9,10,11, 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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Figure 1: Orai1 is a four-transmembrane protein expressed at the cell surface and with its N and C termini located in the cytoplasm.
Figure 2: The conserved glutamate residues in transmembrane segments TM1 and TM3 have an important role in store-operated Ca 2+ influx.
Figure 3: E106D and E190Q mutations in the transmembrane segments of human Orai1 alter the ion selectivity of CRAC channels.
Figure 4: E106D and E190Q mutations of human Orai1 alter the Cs + selectivity of CRAC channels.

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Acknowledgements

This work was supported by grants from the National Institutes of Health to A.R. and S.F.; from the Charles H. Hood Foundation to S.F.; and a Scientist Development Award from the American Heart Association to M.P.

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Correspondence to Anjana Rao.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Figure Legends and Supplementary Methods. (DOC 68 kb)

Supplementary Figure 1

Orai1 is a plasma membrane protein with intracellular N- and C-termini. (PDF 1505 kb)

Supplementary Figure 2

Alignment of protein sequences of Drosophila Orai and its three mammalian homologues Orai1, Orai2 and Orai3. (PPT 29 kb)

Supplementary Figure 3

Expression of mutants in S2 cells and selective depletion of endogenous dOrai by dsRNA treatment. (PPT 73 kb)

Supplementary Figure 4

Evolutionarily conserved E106 in Orai1 is required for Orai1-mediated Ca2+ influx. (PPT 836 kb)

Supplementary Figure 5

Orai1 mutant proteins are expressed in the plasma membrane. (PPT 6137 kb)

Supplementary Figure 6

Ca2+ influx in SCID T cells expressing glutamate mutants of Orai1 is sensitive to inhibition by La3+. (PPT 61 kb)

Supplementary Figure 7

Leak-corrected current in control cells is negligible. (PPT 22 kb)

Supplementary Figure 8

Concentration dependence of the block of monovalent ion current by Ca2+. (PPT 18 kb)

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Prakriya, M., Feske, S., Gwack, Y. et al. Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 230–233 (2006). https://doi.org/10.1038/nature05122

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