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. 2011 Oct 25;108(43):17838-43.
doi: 10.1073/pnas.1114821108. Epub 2011 Oct 10.

Mutations in Orai1 Transmembrane Segment 1 Cause STIM1-independent Activation of Orai1 Channels at Glycine 98 and Channel Closure at Arginine 91

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Mutations in Orai1 Transmembrane Segment 1 Cause STIM1-independent Activation of Orai1 Channels at Glycine 98 and Channel Closure at Arginine 91

Shenyuan L Zhang et al. Proc Natl Acad Sci U S A. .
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Abstract

Stim and Orai proteins comprise the molecular machinery of Ca(2+) release-activated Ca(2+) (CRAC) channels. As an approach toward understanding the gating of Orai1 channels, we investigated effects of selected mutations at two conserved sites in the first transmembrane segment (TM1): arginine 91 located near the cytosolic end of TM1 and glycine 98 near the middle of TM1. Orai1 R91C, when coexpressed with STIM1, was activated normally by Ca(2+)-store depletion. Treatment with diamide, a thiol-oxidizing agent, induced formation of disulfide bonds between R91C residues in adjacent Orai1 subunits and rapidly blocked STIM1-operated Ca(2+) current. Diamide-induced blocking was reversed by disulfide bond-reducing agents. These results indicate that R91 forms a very narrow part of the conducting pore at the cytosolic side. Alanine replacement at G98 prevented STIM1-induced channel activity. Interestingly, mutation to aspartate (G98D) or proline (G98P) caused constitutive channel activation in a STIM1-independent manner. Both Orai1 G98 mutants formed a nonselective Ca(2+)-permeable conductance that was relatively resistant to block by Gd(3+). The double mutant R91W/G98D was also constitutively active, overcoming the normal inhibition of channel activity by tryptophan at the 91 position found in some patients with severe combined immunodeficiency (SCID), and the double mutant R91C/G98D was resistant to diamide block. These data suggest that the channel pore is widened and ion selectivity is altered by mutations at the G98 site that may perturb α-helical structure. We propose distinct functional roles for G98 as a gating hinge and R91 as part of the physical gate at the narrow inner mouth of the channel.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Diamide treatment induces R91C to form homo-disulfide bonds. (A) [Ca2+]i responses (mean ± SE) before and following store depletion by addition of 2 μM TG to HEK cells transfected with STIM1 + Myc-Orai1 (positive control; n = 5 cells), STIM1 + R91W Myc-Orai1 (n = 8 cells), STIM1 + C-less Myc-Orai1 (n = 4 cells), and STIM1 + R91C Myc-Orai1 (n = 10 cells). Data shown are representative of at least three experiments with >15 cells for each condition. Vertical lines indicate the time of solution exchange. Ca0 indicates zero free Ca2+; Ca2 indicates 2 mM Ca2+ (Table S1). (B) Representative Western blotting (n = 4) of Myc-tagged WT Orai1 and R91C Orai1 from cells treated without or with 10 mM diamide; total cell lysates were boiled either with or without DTT (100 mM, 10 min at 70 °C). MW refers to molecular weight markers (kDa). (C) Representative Western blotting of Myc-tagged C-less Orai1 and C-less R91C Orai1 from cells treated with or without diamide and DTT. (D) Representative anti-GFP Western blotting of cell lysates with overexpressed GFP-Orai1 and R91C GFP-Orai1 proteins.
Fig. 2.
Fig. 2.
R91C Orai1 channels are blocked by oxidation treatment. (A and B) [Ca2+]i responses (±SE) before and following store depletion by addition of 2 μM TG. Effect of diamide (Dia) (1 mM) and following DTT (2 mM) application on Ca2+ influx in cells cotransfected with STIM1 and either WT Orai1 or R91C Orai1 (n = 5 and 8 cells, respectively). (C and D) Pretreatment with diamide (1 mM) has no effect on WT Orai1 but prevents Ca2+ influx through R91C Orai1 (n = 7 cells for each). DTT restores Ca2+ influx through R91C channels. (E) Representative result for block of R91C Orai1 CRAC current by diamide treatment (10 mM, n = 13 cells); recovery upon BMS treatment (5 mM, n = 6 cells). Traces are leak-subtracted; the current at break-in was considered as the leak. (F) Corresponding I-V curves for the time points indicated in E.
Fig. 3.
Fig. 3.
G98 mutants form spontaneously activated Orai1 channels. (A) Resting [Ca2+]i in cells expressing WT Orai1 without STIM1 (n = 8 cells) or in cells expressing G98D Orai1 (n = 7 cells). Representative data are shown; average values include 46 ± 4 nM, 41 cells for WT Orai1; 260 ± 28 nM, 81 cells for G98D; P < 0.0001 for WT compared with G98D; P < 0.0001 for Ca2 compared with Ca0 in 44 cells transfected with G98D. 2-APB application (50 μM). (B) Resting [Ca2+]i in cells expressing G98P Orai1 (n = 8 cells); 190 ± 42 nM, 14 cells for G98P; P < 0.0001 for WT compared with G98P; P = 0.0032 for Ca2 compared with Ca0 in 14 G98P cells. Gd3+ application (10 μM). (C) Time course of inward and outward G98D Orai1 currents, measured at −120 and +100 mV, respectively. Currents are representative of six experiments. ChoI, choline. (D) Corresponding I-V relationships. Numbered traces and colors correspond to times indicated in C. (E and F) Time course and I-V relationships of G98P Orai1 currents. Currents are representative of four experiments.
Fig. 4.
Fig. 4.
Dominance of G98D mutation. (A) G98D restores channel activity of R91W Orai1. Expression of R91W/G98D Orai1 results in preactivated cationic current. (B) Corresponding I-V curves for conditions indicated in A. (C and D) Properties of nonselective R91C/G98D Orai1 current: cationic permeability (C) and corresponding I-V relationships (D). (E) Diamide application does not suppress R91C/G98D current (n = 3 cells). (F) Corresponding I-V curves for the time points indicated in E.
Fig. 5.
Fig. 5.
Schematic models of Orai1/CRAC channel gating. Two of the pore-lining Orai1 TM1 segments are shown, with E106, G98, and R91 highlighted from outside to inside. (A) E106 sites near the outside control Ca2+ selectivity. The open pore has a diameter <3.3 Å, the diameter of an impermeant Cs+ ion. Near the inside, the R91 sites prevent ion flow in the closed state of the channel. Located in the middle of TM1, the G98 sites permit a conformational change that opens the channel upon store depletion followed by STIM1–Orai1 interaction. (B) R91W Orai1, irreversibly closed by a greasy tryptophan plug and hence a “dead” channel. (C) Spontaneously open G98D Orai1, with a dilated pore allowing nonselective currents (leaky) to permeate. (D) G98D/R91W double mutant, spontaneously open and leaky, allowing monovalent cation permeation despite tryptophan at position 91.

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