Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Sep 14;443(7108):226-9.
doi: 10.1038/nature05108. Epub 2006 Aug 20.

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Andriy V Yeromin  1 , Shenyuan L Zhang, Weihua Jiang, Ying Yu, Olga Safrina, Michael D Cahalan
Affiliations

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Andriy V Yeromin et al. Nature. .

Abstract

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Orai interacts with Stim and generates increased store-operated currents in S2 cells
a, Representative co-immunoprecipitation of Stim and Orai (n = 3). Input: total cell lysate, showing an equal amount of samples prepared for immunoprecipitation. Cells were transfected with HA-olf186-F (Orai) and Stim-V5-His (Stim) constructs, as indicated. M refers to molecular weight markers (kDa). b, Activation of CRAC current (at −130 mV) by three methods: IP3 (10 μM IP3 added to high-Ca2+ pipette solution; current density −11.4 ± 3.1 pA pF−1, n = 5); thapsigargin (high-Ca2+ pipette solution and thapsigargin added to bath; −7.6 ± 2.5 pA pF−1, n = 2); or passive store-depletion (Ca2+-free pipette solution; −28.7 ± 1.8 pA pF−1, n = 27). All three methods produced substantially greater CRAC current density than passive store depletion in control cells (−2.5 ± 0.4 pA pF−1, n = 18).
Figure 2
Figure 2. Mutation E180D of Orai alters ion selectivity of the CRAC current
a, Time courses of inward current at −130 mV and outward current at 90 mV in representative cells overexpressing wild-type Orai (WT, black) or Orai(E180D) mutant (red). Ca2+-free internal solution. b, I–V curves at times indicated in a. c, I–V curves of wild-type Orai-induced current in Ca2 solution containing 160 mM Na+ and 2 mM Ca2+ and in choline external solution with 1.1 mM Na+ and 2 mM Ca2+. (Complete solution recipes are indicated in Supplementary Table 1.) d, Representative I–V curves for Orai(E180D) with the same solutions as c (n = 3). e, Divalent cation selectivity of wild-type Orai CRAC current. I–V curves are normalized to current values at −130 mV in Ca2+ solution. Test solutions contained 20 mM test divalent and 124 mM Na+. External solutions for e and f are labelled according to colour. f, I–V curves (not leak-subtracted) for Orai(E180D) with the same divalent cations, normalized to currents at 90 mV in Ca2+ solution.
Figure 3
Figure 3. Monovalent current in the absence of divalent ions exhibits altered ion selectivity in the Orai(E180D) mutant
a, Time course of inward current in a cell expressing wild-type Orai. Bars indicate external solution exchange. b, Same as a but for Orai(E180D). c, I–V curves for wild-type Orai at times indicated in a. d, I–V curves for Orai(E180D) at times indicated in b. e, I–V curves of Orai(E180D)-induced current in the presence of varying external [Ca2+]. f, [Ca2+] dependence of Orai(E180D) CRAC current at −130 (squares) and 90 mV (circles), scaled to the current in divalent-free solution. Ca2+-dependent block (mean ± s.e.m.) was fitted by the function y = 1/(1 + [Ca2+]/IC50), where IC50 is the calculated half-blocking Ca2+ concentration for inward current at −130 mV (48 ± 13 μM; n = 13, 8, 4, 6 and 2 cells for 20, 2, 0.2, 0.02 and 0.002 mM free external [Ca2+], respectively) and outward current at 90 mV (2.05 ± 0.78 mM; n = 13, 8, 4, 5 and 3 cells for 20, 2, 0.2, 0.02 and 0.002 mM free external [Ca2+], respectively).
Figure 4
Figure 4. Charge-neutralizing mutations in the S1–S2 loop
a, I–V curves in a cell expressing the Orai(E180A) mutant (without Stim) compared to control. b, I–V curves for Orai(D184A), Orai(D186A) and Orai(N188A) mutants. c, CRAC current density in transfected S2 cells. Each point represents the maximal CRAC current density (pA pF−1) in a single cell, plotted as absolute values (from left to right): GFP-transfected control; Orai(E180A) (P = 1.7 × 10−4 relative to control); Orai(E180A) plus Stim (P = 8.2 × 10−3); wild-type Orai plus Stim; Orai(E180D) plus Stim, inward current; Orai(D184A) plus Stim; Orai(D186A) plus Stim; Orai(N188A) plus Stim; and Orai(E180D) plus Stim, outward current. d, Suppression of CRAC current by 5 nM Gd3+ in wild-type Orai and the D184A, E180D and D186A Orai mutants. Bars indicate time of Gd3+ application; dashed lines indicate the zero-current level.
See this image and copyright information in PMC

Similar articles

  • Orai1 is an essential pore subunit of the CRAC channel.
    Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG. Prakriya M, et al. Nature. 2006 Sep 14;443(7108):230-3. doi: 10.1038/nature05122. Epub 2006 Aug 20. Nature. 2006. PMID: 16921383
  • The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.
    Penna A, Demuro A, Yeromin AV, Zhang SL, Safrina O, Parker I, Cahalan MD. Penna A, et al. Nature. 2008 Nov 6;456(7218):116-20. doi: 10.1038/nature07338. Epub 2008 Sep 28. Nature. 2008. PMID: 18820677 Free PMC article.
  • Store-Independent Orai Channels Regulated by STIM.
    Zhang X, Gueguinou M, Trebak M. Zhang X, et al. In: Kozak JA, Putney JW Jr, editors. Calcium Entry Channels in Non-Excitable Cells. Boca Raton (FL): CRC Press/Taylor & Francis; 2018. Chapter 11. In: Kozak JA, Putney JW Jr, editors. Calcium Entry Channels in Non-Excitable Cells. Boca Raton (FL): CRC Press/Taylor & Francis; 2018. Chapter 11. PMID: 30299650 Free Books & Documents. Review.
  • Studies of Structure-Function and Subunit Composition of Orai/STIM Channel.
    Fahrner M, Schindl R, Romanin C. Fahrner M, et al. In: Kozak JA, Putney JW Jr, editors. Calcium Entry Channels in Non-Excitable Cells. Boca Raton (FL): CRC Press/Taylor & Francis; 2018. Chapter 2. In: Kozak JA, Putney JW Jr, editors. Calcium Entry Channels in Non-Excitable Cells. Boca Raton (FL): CRC Press/Taylor & Francis; 2018. Chapter 2. PMID: 30299645 Free Books & Documents. Review.
  • Biochemical and functional characterization of Orai proteins.
    Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Oh-hora M, Neems DS, Hogan PG, Rao A. Gwack Y, et al. J Biol Chem. 2007 Jun 1;282(22):16232-43. doi: 10.1074/jbc.M609630200. Epub 2007 Feb 9. J Biol Chem. 2007. PMID: 17293345
See all similar articles

Cited by

See all "Cited by" articles

References

    1. Liou J, et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol. 2005;15:1235–1241. - PMC - PubMed
    1. Roos J, et al. STIM1, an essential and conserved component of store-operated Ca2+ channel function. J Cell Biol. 2005;169:435–445. - PMC - PubMed
    1. Feske S, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441:179–185. - PubMed
    1. Vig M, et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry. Science. 2006;312:1220–1223. - PMC - PubMed
    1. Zhang SL, et al. Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity. Proc Natl Acad Sci USA. 2006;103:9357–9362. - PMC - PubMed

Publication types

MeSH terms