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. 2013 Apr 19;288(16):11263-72.
doi: 10.1074/jbc.M113.450254. Epub 2013 Feb 27.

Differential Roles of the C and N Termini of Orai1 Protein in Interacting With Stromal Interaction Molecule 1 (STIM1) for Ca2+ Release-Activated Ca2+ (CRAC) Channel Activation

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Free PMC article

Differential Roles of the C and N Termini of Orai1 Protein in Interacting With Stromal Interaction Molecule 1 (STIM1) for Ca2+ Release-Activated Ca2+ (CRAC) Channel Activation

Hongying Zheng et al. J Biol Chem. .
Free PMC article

Abstract

The entry of extracellular Ca(2+), which is mediated by Ca(2+) release-activated Ca(2+) (CRAC) channels, is essential for T cell activation and the normal functioning of other immune cells. Although the molecular components of CRAC channels, the Orai1 pore-forming subunit and the STIM1-activating subunit have been recently identified, the gating mechanism by which Orai1 channels conduct Ca(2+) entry upon Orai1-STIM1 interaction following Ca(2+) store release remains elusive. Herein, we show that C-terminal truncations or point mutations prevented Orai1 from binding to STIM1 and subsequent channel opening. In contrast, an Orai1 mutant with an N-terminal truncation interacted with but failed to be activated by STIM1. Moreover, Orai1 channels with C-terminal disruption, but not N-terminal truncation, could be gated by fused functional domains of STIM1. Interestingly, the channel activities of Orai1 mutants carrying either an N-terminal or a C-terminal truncation were restored by a methionine mutation at the putative gating hinge, the conserved Gly-98 site in the first transmembrane segment (TM1) of Orai1. Collectively, these results support a stepwise gating mechanism of STIM1-operated Orai1 channels; the initial binding between STIM1 and the C terminus of Orai1 docks STIM1 onto the N terminus of Orai1 to initiate conformational changes of the pore-lining TM1 helix of Orai1, leading to the opening of the channel.

Figures

FIGURE 1.
FIGURE 1.
Schematic diagram of the Orai1 mutants with truncations or point mutations described in the present study.
FIGURE 2.
FIGURE 2.
Both intracellular termini of Orai1 are indispensable for STIM1-mediated channel activation. A, confocal images of HEK293A cells transfected with mCherry-CAD (red) alone or in combination with full-length eGFP-Orai1-E106A (106A, 301 residues, green) or truncated eGFP-Orai1-E106A: ΔC1 (1–276), ΔC2 (1–266), or ΔN2 (81–301) are presented. The CAD of STIM1 was not recruited to the peripheral regions of the PM by the Orai1-E106A-ΔC1 or ΔC2 mutants. Scale bar, 10 μm. B and C, representative intracellular free Ca2+ traces ([Ca2+]i, mean ± S.E.) show TG-triggered store-operated Ca2+ entry in HEK293A cells transfected with (i) STIM1 + eGFP (n = 14 cells); (ii) STIM1 + eGFP-Orai1 (positive control; n = 15); STIM1 + eGFP-Orai1 mutants: (iii) E106A (negative control; n = 14); (iv) L273S (n = 15); (v) ΔC1 (n = 10); (vi) ΔC2 (n = 9); (vii) ΔN1 (n = 5); and (viii) ΔN2 (n = 6), respectively. TG (2 μm) was used to deplete the Ca2+ store. Top bars and vertical lines on the x axis indicate the time of solution exchange (supplemental Table S1). D, averaged peak [Ca2+]i values coupled to TG-dependent Ca2+ influx for the nine groups of cells (from left to right, n = 56, 88, 24, 15, 15, 15, 16, 42, and 38 cells) represented in B and C. N/A, not available.
FIGURE 3.
FIGURE 3.
Tethering the functional domains of STIM1 to Orai1 bypasses the requirement for the C terminus, but not the N terminus, of Orai1 for channel activation. A and B, representative averaged [Ca2+]i traces from HEK293A cells expressing eGFP (n = 14), full-length (FL) Orai1-SS-eGFP (n = 3), Orai1-E106A-SS-eGFP (n = 15), Orai1-L273S-SS-eGFP (n = 10), Orai1-ΔC1-SS-eGFP (n = 8), or Orai1-ΔN2-SS-eGFP (n = 16) are shown. Solution exchanges are indicated. Disruption of the N terminus (ΔN2), but not the C terminus (L273S or ΔC1), of Orai1-SS-eGFP abolished spontaneous Ca2+ entry. C, the summary of averaged resting [Ca2+]i levels from cells expressing eGFP (37 cells) or Orai1-SS-eGFP proteins: full length (22 cells), E106A (74 cells), L273S (29 cells), ΔC1 (26 cells), ΔN1 (18 cells), ΔN2 (69 cells), ΔN1-ΔC1 (20 cells), and ΔN2-ΔC1 (77 cells). D, representative time course of inward Orai1-SS-eGFP currents, measured at −110 mV, is shown. E, corresponding I-V curves are presented for the time points indicated in D. Ch: choline. F, Orai1-ΔN2-SS-eGFP is mainly expressed in the peripheral regions of the PM. Scale bar, 10 μm. G and H, representative time course and I-V relationship of Orai1-ΔN2-SS-eGFP currents are shown. Traces in E and H are leak-subtracted; the observed residual current after 10 μm Gd3+ (CRAC channel blocker) treatment was considered as the leak. I, break-in inward current densities (picoamperes/picofarads (pA/pF)) were averaged from cells transfected with wild-type Orai1-SS-eGFP (full-length, 9 cells), Orai1-E106A-SS-eGFP (7 cells), Orai1-L273S-SS-eGFP (4 cells), Orai1-ΔC1-SS-eGFP (7 cells), Orai1-ΔN2-SS-eGFP (10 cells), or Orai1-ΔN2-ΔC1-SS-eGFP (7 cells). *, p < 0.01 in comparison with full-length Orai1-SS-eGFP.
FIGURE 4.
FIGURE 4.
Characterization of the Orai1-G98X mutants. A, prediction of TM helices in human Orai1 proteins. B, probabilities of the original glycine residue and the individual amino acid substitutions at the Gly-98 site of Orai1 to form transmembrane helices. C and D, prediction of TM helices in Orai1-G98D (C) and Orai1-G98M (D). E and F, representative [Ca2+]i recordings show STIM1-independent spontaneous Ca2+ entry in HEK cells overexpressing Orai1-G98L (n = 8), Orai1-G98F (n = 9), Orai1-G98E (n = 3), or Orai1-G98Y (n = 12), but not in cells overexpressing Orai1-G98R (n = 9). 2-APB, 2-aminoethyl diphenylborinate. G, bar graph summarizes the averaged resting [Ca2+]i levels of cells expressing WT GFP-Orai1 or various Orai1-G98X mutants without the co-expression of STIM1 (from left to right, n = 58, 7, 11, 13, 14, 15, 15, 22, 28, 3, 2, 3, 2, 57, 3, 8, 27, 15, 8, and 16 cells). *, p < 0.01 in comparison with WT GFP-Orai1.
FIGURE 5.
FIGURE 5.
G98M is a novel gain-of-function Orai1 mutation. A and B, representative [Ca2+]i responses from HEK293A cells expressing wild-type Orai1 (A, Orai1-WT, n = 5) or the Orai1-G98M mutant (B, n = 10) are presented. Gd3+-sensitive, spontaneous Ca2+ entry through Orai1-G98M channels is shown. C and D, representative time course and corresponding I-V curves of STIM1-independent, nonselective cationic currents mediated by Orai1-G98M channels are presented. The red trace in D shows currents from a representative control cell expressing WT eGFP-Orai1 alone, which was recorded under the same conditions as G98M (the black trace). Ch: choline. E and F, fluorescent images (green + differential interference contrast) of HEK cells expressing eGFP-Orai1-G98M (E) or eGFP-Orai1-G98D (F) indicate that the Orai1-G98M proteins are less toxic to host cells than the Orai1-G98D proteins. Scale bar, 10 μm.
FIGURE 6.
FIGURE 6.
Drosophila Orai-G170M channels spontaneously conduct Ca2+ influx in HEK293A cells. A, sequence alignment of the TM1 regions of the human Orai1 and Drosophila Orai proteins. B, resting [Ca2+]i levels in HEK cells co-expressing maxGFP and Drosophila Orai (n = 15) or in cells co-expressing maxGFP and dOrai-G170M (n = 5) are shown. C, averaged resting [Ca2+]i levels of cells overexpressing maxGFP plus WT dOrai (25 cells) or dOrai-G170M (27 cells) are summarized. D, representative I-V curves from cells expressing dOrai (the red trace) or dOrai-G170M (the black trace) are shown. E, comparison of the normalized break-in inward currents (−110 mV) through WT (7 cells) and dOrai-G170M mutant channels (10 cells). *, p < 0.01. pA/pF, picoamperes/picofarads.
FIGURE 7.
FIGURE 7.
G98M restores the channel activity of Orai1 truncated at the N or C terminus. A and B, averaged cytosolic Ca2+ concentrations from HEK cells expressing: (A) Orai1-ΔC2 (n = 11) or Orai1-G98M-ΔC2 (n = 8) or (B) Orai1-ΔN2 (n = 8) or G98M-ΔN2 (n = 7). C, the bar graph presents the resting [Ca2+]i levels in cells transfected with five pairs of Orai1 cDNA constructs, with or without the G98M mutation: L273S (31 and 59 cells), ΔC1 (27 and 46 cells), ΔC2 (25 and 70 cells), ΔN1 (22 and 28 cells), and ΔN2 (36 and 46 cells), respectively. D and E, representative store-independent currents conducted by exogenous Orai1-ΔC2, G98M-ΔC2, Orai1-ΔN2, or G98M-ΔN2 channels without the co-expression of STIM1 are shown. F, comparison of the normalized break-in current amplitude at −110 mV (picoamperes/picofarads (pA/pF)) from the wild type and a series of mutated Orai1 channels to the Orai1-G98M channels with corresponding mutations: full-length (FL, 13 and 19 cells), L273S (6 and 5 cells), ΔC2 (6 and 19 cells), and ΔN2 (6 and 7 cells), respectively. All current traces in this figure are leak-subtracted; the currents after 10 μm Gd3+ treatment were considered as the leak. *, p < 0.01.
FIGURE 8.
FIGURE 8.
CAD does not co-localize with the Orai1-G98M-ΔC1 or ΔC2 mutants. Confocal images show the expression of mCherry-CAD (red) alone or in combination with full-length eGFP-Orai1-G98M, eGFP-Orai1-G98M-ΔC1, or ΔC2 (green) in HEK cells. Scale bar, 10 μm. N.A., not available.
FIGURE 9.
FIGURE 9.
G98M restores the channel activity of Orai1 truncated at both the N and the C termini. A, representative [Ca2+]i traces recorded from cells transfected with WT Orai1-ΔN2-ΔC2 (n = 10) or Orai1-G98M-ΔN2-ΔC2 (n = 8) are shown. B, summary of resting [Ca2+]i levels of HEK cells expressing WT Orai1-ΔN1-ΔC2 or Orai1-ΔN2-ΔC2 (red columns, 17 and 65 cells, respectively) or the corresponding Orai1-G98M truncations (black columns, 25 and 52 cells, respectively). C, overexpression of Orai1-G98M-ΔN2-ΔC2 elicits a preactivated current (the black trace), whereas Orai1-ΔN2-ΔC2 channels carry a negligible spontaneous current (the red trace). D, averaged current densities (−110 mV, break-in) from cells overexpressing WT Orai1-ΔN2-ΔC2 (n = 5) or Orai1-G98M-ΔN2-ΔC2 (n = 6) are presented. *, p < 0.01. pA/pF, picoamperes/picofarads.
FIGURE 10.
FIGURE 10.
Schematic gating model for Orai1 channels upon STIM1 activation. A, two subunits of an Orai1 channel are shown for clarity. The Glu-106, Gly-98, and Arg-91 sites are indicated. Prior to store depletion, STIM1 molecules are not engaged with Orai1, and the channel remains closed. SAM, sterile α-motif. B, the three sequential steps of STIM1-operated channel activation are highlighted in green. Upon Ca2+ release from the ER, the C terminus of Orai1 directly binds to STIM1 (Step 1) and docks STIM1 onto the N terminus of Orai1 (Step 2), which leads to the conformational change of the pore-lining TM1 of Orai1 (Step 3) to gate the channel for Ca2+ entry. This model does not imply a particular stoichiometry between STIM1 and Orai1 in this gating process.

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