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Gated regulation of CRAC channel ion selectivity by STIM1

Abstract

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states, typically without changes in ion selectivity1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, stromal interaction molecule 1 (STIM1), remains unknown. CRAC channels are distinguished by a very high Ca2+ selectivity and are instrumental in generating sustained intracellular calcium concentration elevations that are necessary for gene expression and effector function in many eukaryotic cells2. Here we probe the central features of the STIM1 gating mechanism in the human CRAC channel protein, ORAI1, and identify V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produce constitutively active CRAC channels that are open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels are not Ca2+-selective, their Ca2+ selectivity is dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity is also seen in wild-type ORAI1 channels by increasing the number of STIM1 activation domains that are directly tethered to ORAI1 channels, or by increasing the relative expression of full-length STIM1. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels but rather a tuneable feature that is bestowed on otherwise non-selective ORAI1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism in which permeation and gating are closely coupled.

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Figure 1: State-dependent accessibility of pore-lining residues localizes the activation gate to the extracellular TM1 region.
Figure 2: Mutations at V102 cause STIM1-independent constitutive ORAI1 activation.
Figure 3: STIM1 regulates ion selectivity of constitutively active V102C ORAI1 channels.
Figure 4: STIM1 dose-dependently modulates the ion selectivity of ORAI1 channels.

References

  1. Olcese, R. And yet it moves: conformational states of the Ca2+ channel pore. J. Gen. Physiol. 129, 457–459 (2007)

    CAS  Article  Google Scholar 

  2. Hogan, P. G., Lewis, R. S. & Rao, A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533 (2010)

    CAS  Article  Google Scholar 

  3. Mignen, O., Thompson, J. L. & Shuttleworth, T. J. Orai1 subunit stoichiometry of the mammalian CRAC channel pore. J. Physiol. 586, 419–425 (2008)

    CAS  Article  Google Scholar 

  4. Penna, A. et al. The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456, 116–120 (2008)

    ADS  CAS  Article  Google Scholar 

  5. Madl, J. et al. Resting-state Orai1 diffuses as homotetramer in the plasma membrane of live mammalian cells. J. Biol. Chem. 285, 41135–41142 (2010)

    CAS  Article  Google Scholar 

  6. McNally, B. A., Yamashita, M., Engh, A. & Prakriya, M. Structural determinants of ion permeation in CRAC channels. Proc. Natl Acad. Sci. USA 106, 22516–22521 (2009)

    ADS  CAS  Article  Google Scholar 

  7. Zhou, Y., Ramachandran, S., Oh-Hora, M., Rao, A. & Hogan, P. G. Pore architecture of the ORAI1 store-operated calcium channel. Proc. Natl Acad. Sci. USA 107, 4896–4901 (2010)

    ADS  CAS  Article  Google Scholar 

  8. Karlin, A. & Akabas, M. H. Substituted-cysteine accessibility method. Methods Enzymol. 293, 123–145 (1998)

    CAS  Article  Google Scholar 

  9. Prakriya, M. & Lewis, R. S. Regulation of CRAC channel activity by recruitment of silent channels to a high open-probability gating mode. J. Gen. Physiol. 128, 373–386 (2006)

    CAS  Article  Google Scholar 

  10. Yamashita, M., Navarro-Borelly, L., McNally, B. A. & Prakriya, M. Orai1 mutations alter ion permeation and Ca2+-dependent inactivation of CRAC channels: evidence for coupling of permeation and gating. J. Gen. Physiol. 130, 525–540 (2007)

    CAS  Article  Google Scholar 

  11. Prakriya, M. et al. Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 230–233 (2006)

    ADS  CAS  Article  Google Scholar 

  12. Yeromin, A. V. et al. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443, 226–229 (2006)

    ADS  CAS  Article  Google Scholar 

  13. Vig, M. et al. CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr. Biol. 16, 2073–2079 (2006)

    CAS  Article  Google Scholar 

  14. Mullins, F. M., Park, C. Y., Dolmetsch, R. E. & Lewis, R. S. STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels. Proc. Natl Acad. Sci. USA 106, 15495–15500 (2009)

    ADS  CAS  Article  Google Scholar 

  15. Scrimgeour, N., Litjens, T., Ma, L., Barritt, G. J. & Rychkov, G. Y. Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins. J. Physiol. 587, 2903–2913 (2009)

    CAS  Article  Google Scholar 

  16. Derler, I. et al. A Ca2+ release-activated Ca2+ (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2+-dependent inactivation of ORAI1 channels. J. Biol. Chem. 284, 24933–24938 (2009)

    CAS  Article  Google Scholar 

  17. Feske, S. et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179–185 (2006)

    ADS  CAS  Article  Google Scholar 

  18. Doyle, D. A. Structural changes during ion channel gating. Trends Neurosci. 27, 298–302 (2004)

    CAS  Article  Google Scholar 

  19. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003)

    ADS  CAS  Article  Google Scholar 

  20. Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T. & Rees, D. C. Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282, 2220–2226 (1998)

    ADS  CAS  Article  Google Scholar 

  21. Navarro-Borelly, L. et al. STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy. J. Physiol. 586, 5383–5401 (2008)

    CAS  Article  Google Scholar 

  22. Hoover, P. J. & Lewis, R. S. Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1). Proc. Natl Acad. Sci. USA 108, 13299–13304 (2011)

    ADS  CAS  Article  Google Scholar 

  23. Li, Z. et al. Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits. Cell Res. 21, 305–315 (2011)

    ADS  CAS  Article  Google Scholar 

  24. Yellen, G. The voltage-gated potassium channels and their relatives. Nature 419, 35–42 (2002)

    ADS  CAS  Article  Google Scholar 

  25. Peinelt, C., Lis, A., Beck, A., Fleig, A. & Penner, R. 2-Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1-dependent gating of CRAC channels. J. Physiol. 586, 3061–3073 (2008)

    CAS  Article  Google Scholar 

  26. Zhang, S. L. et al. Store-dependent and -independent modes regulating Ca2+ release-activated Ca2+ channel activity of human Orai1 and Orai3. J. Biol. Chem. 283, 17662–17671 (2008)

    CAS  Article  Google Scholar 

  27. Schindl, R. et al. 2-aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size. J. Biol. Chem. 283, 20261–20267 (2008)

    CAS  Article  Google Scholar 

  28. Yamashita, M., Somasundaram, A. & Prakriya, M. Competitive modulation of CRAC channel gating by STIM1 and 2-aminoethyldiphenyl borate (2-APB). J. Biol. Chem. 286, 9429–9442 (2011)

    CAS  Article  Google Scholar 

  29. Feng, M. et al. Store-independent activation of Orai1 by SPCA2 in mammary tumors. Cell 143, 84–98 (2010)

    CAS  Article  Google Scholar 

  30. Radzicka, A., Pedersen, L. & Wolfenden, R. Influences of solvent water on protein folding: free energies of solvation of cis and trans peptides are nearly identical. Biochemistry 27, 4538–4541 (1988)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank C. Lingle, R. Lewis, K. Swartz, J. Sack, A. Gross, L. Tirado-Lee and T. Hornell for discussions and comments on the manuscript. This work was supported by NIH grant NS057499 to M.P. An American Heart Association predoctoral fellowship supported A.S.

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B.A.M. generated, expressed and functionally characterized the ORAI mutants by patch-clamp electrophysiology with help from M.Y. The Ca2+ imaging, FRET imaging, TIRF microscopy and western blot analysis of ORAI1 mutants were performed by A.S. B.A.M, A.S. and M.P. contributed to the writing and editing of the paper.

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Correspondence to Murali Prakriya.

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The authors declare no competing financial interests.

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McNally, B., Somasundaram, A., Yamashita, M. et al. Gated regulation of CRAC channel ion selectivity by STIM1. Nature 482, 241–245 (2012). https://doi.org/10.1038/nature10752

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