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A Calcium-Accumulating Region, CAR, in the Channel Orai1 Enhances Ca(2+) Permeation and SOCE-induced Gene Transcription

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A Calcium-Accumulating Region, CAR, in the Channel Orai1 Enhances Ca(2+) Permeation and SOCE-induced Gene Transcription

Irene Frischauf et al. Sci Signal.

Abstract

The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.

Conflict of interest statement

Competing financial interests: All authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Molecular dynamics simulations identifies CAR in extracellular loop1 of human Orai1.
(A) Atomic 3D model of human Orai1 (E62 – G297), including the loop segments that were not present in the Drosophila Orai crystal structure on which this human Orai1 model is based. The panel shows a side view of a modelled hexameric structure with each monomer individually colored. (B) Side view of a representative snapshot of a molecular dynamics simulation of the Orai1 pore showing Ca2+ binding (yellow balls) to the selectivity filter (Glu106 sidechain shown) and the loop1 segments (Asp110 sidechain shown). (C and E) Top view of a representative snapshot of a molecular dynamics simulation of Orai1 and Orai1-D110A simulation. (D) One-dimensional density number of ion concentration through the Orai1 channel (black) and Orai1-D110A (red) from the intracellular to the extracellular side (in Z-direction). The dotted lines mark the membrane headgroup regions and the peak between 7 and 8 nm corresponds to the selectivity filter.
Figure 2
Figure 2. CAR promotes Ca2+ permeation through the Orai1 channel.
(A) Whole-cell patch-clamp experiments show time course of currents mediated by mCherry-tagged STIM1 coexpressed with wild-type or mutant eYFP-tagged Orai1 as indicated in HEK 293 cells (n = 9 -10). Currents were only measured for cells positive for mCherry-tagged STIM1 and wild-type or mutant eYFP-tagged Orai1. Current activation by endoplasmic reticulum store-depletion was mediated by 20 mM EGTA in the patch pipette and stepwise current increases were recorded at 0.3 mM, 1 mM and 10 mM Ca2+ concentration with Na+-containing (A, C) or Na+-free (B) solution. Representative current-voltage relationship with properties of Ca2+-selective currents are shown for wt-Orai1 and Orai1-D110A (A,B). (B) Whole-cell patch-clamp experiments show time course of currents mediated mCherry-tagged STIM1 coexpressed with wild-type or mutant eYFP-tagged Orai1 as indicated in HEK 293 cells (n = 7 – 10). Store depletion was achieved and currents were recorded as in (A) with increasing Ca2+ concentrations in a Na+-free solution. Orai1 and Orai1-D110A maximum currents at various extracellular Ca2+ concentrations were compared for statistical significance by t-test. In a Na+-containing solution (A), currents are significantly different (p < 0.05) in a 1 mM Ca2+ solution. In a Na+-free extracellular solution (B) currents were significantly different (p < 0.05) in a 0.3, 0.5, 1, and 2 mM Ca2+ solution. (C) Similar time-course for indicated mutants as in (A,B). Currents of Orai1-D112A or Orai1-D114A were compared by t-test for significance and are not significantly different to those of wild-type Orai1.
Figure 3
Figure 3. CAR promotes Ca2+ permeation through the Orai2 channel.
(A) Whole-cell patch-clamp experiments show time course of currents mediated mCherry-tagged STIM1 coexpressed with wild-type or mutant eYFP-tagged Orai2 as indicated in HEK cells exposed to 20 mM EGTA in the patch pipette and then stepwise increase in Ca2+ concentration with Na+-containing solution (n = 8 – 9). Wild-type Orai2 and mutant Orai2 were analyzed by t-test for statistical significance for their maximum currents at various extracellular Ca2+ concentrations. Their currents are significantly different (p < 0.05) in a 1, 10, and 110 mM Ca2+ solution. (B, C) Representative current-voltage relationship of Orai2- or Orai2-E84Q-mediated SOCE currents for experiments shown in (A).
Figure 4
Figure 4. Activation of the Orai1 CAR mutant results in decreased SOCE-induced cytosolic Ca2+ concentrations and NFAT signaling.
(A) Time course of cytosolic Ca2+ concentration measured by Fura-2 microscopy for HEK 293 cells coexpressing mCherry-tagged STIM1 and eYFP-tagged wild-type or mutant Orai1, or mock-transfected cells, or and unstimulated cells that were not exposed to thapsigargin (TG). In Ca2+-free extracellular solution, ER stores were depleted with thapsigargin (TG) at 3 min and 0.3 mM Ca2+ was added at 12 min (n = 12 - 25). STIM + Orai1 data were compared to STI + Orai-D110A at XX by t-test and determined significantly different (p <0.05) upon addition of extracellular Ca2+. (B) Representative images of GFP-NFAT localization in mock-transfected HEK cells or HEK cells coexpressing Cherry-tagged STIM1 and the indicated eYFP-tagged Orai1 before (upper panel) or 30 minutes after exposure to 1 μM TG (lower panel) in a 0.3 mM Ca2+-containing bath solution. (C) Time course showing the ratio of nucleus to cytosolic GFP-NFAT fluorescence intensity for cells coexpressing GFP-NFAT with the indicated transfected proteins. ER store depletion was induced by 1 μM TG at 5 minutes in a Ca2+-free solution and 0.3 mM Ca2+ was added at 10 minutes (n = 5 – 12). The ratios for NFAT + STIM1 + Orai1 were compared to those for NFAT + STIM1 + Orai1-D110A using a t-test at XX and determined significantly different (p < 0.05) in a 0.3 mM Ca2+ solution (D) Shown are quantitative analysis and representative images of activation of an NFAT-controlled reporter gene (NFAT reporter) in RBL mast cells expressing RFP under the control of an NFAT-regulated promoter, CFP-tagged STIM1, and eYFP-tagged wild-type or mutant Orai1 that were exposed to 100 nM TG for 3.5 hours in 0.3 mM or 1.8 mM Ca2+-containing medium. Representative images are shown on the left, the percent of cells positive for RFP fluorescence are shown in the middle (n = 34 – 86 cells), and the intensity of RFP fluorescence is shown on the right (n = 34 – 86 cells). Data were analyzed by t-test for statistical significance (p < 0.05) as indicated by the stars. (E) Representative HEK 293 cells show CFP-tagged STIM1 cluster formation with eYFP-tagged Orai1 or eYFP-tagged Orai1-D110A upon store depletion with 1 μM TG and presence of 2mM Ca2+ solution. R-factor as a measure of the linear correlation between STIM1 and Orai1, as well as STIM1 and Orai1-D110A before and after store depletion by TG (n = 28 – 39). (F) The NFAT reporter gene, CFP-tagged STIM1 and eYFP-tagged Orai1 or eYFP-tagged Orai1-D110A coexpressed in WM3734 melanoma cells were treated with 100 nM thapsigargin for one hour in a 0.4 mM Ca2+-containing media. Intensity of NFAT driven RFP expression was determined in those cells that exhibited STIM1 and Orai1 or Orai1-D110A expression 24h hours after thapsigargin treatment. Data were analyzed by t-test for statistical significance (p < 0.05) and determined that Orai1-D110A intensity is significantly decreased compared to wild-type Orai1.
Figure 5
Figure 5. Loop3 residues form electrostatic interactions with CAR in Orai1
(A) Representative snapshot of the intermolecular (left panel) and intramolecular (right panel) electrostatic interaction of Arg210 with Asp112 predicted by molecular dynamics simulation. Potential Asp112-Arg210 contacts were counted based on a distance ≤ 4.5 Å between the carboxyl carbon of loop1 aspartic acid and the guanidino carbon of loop3 arginine as shown in the time-courses (left inset graph for intermolecular interaction, and right inset graph for intramolecular interaction). The time-dependent number of intramolecular and intermolecular contacts between Asp112 in loop1 and Arg210 in loop3 for a single Orai1 channel within 100 ns is shown in the central inset. (B) Selected single cysteine mutants in Orai1 loop1 (L109C, D110C, A111C, D112C, H113C, D114C, P117C, L110C) are shown as sticks in a 3D homology model of Orai1. (C) Percentage of dimerization for cysteine mutants upon CuP treatment were calculated. Data are shown as the average + SEM (n = 4 -8). (D) Structural modelling of the loop3 domain in Orai1 yielded a random coil region, highlighting the residues (P201, L202, K203, K204, R210, P211, K214, P216, S218, A221, S225, T230, P231, I237, T240), which were individually engineered to cysteines. (E) Percentage of dimerization for cysteine mutants upon CuP treatment were calculated. Data are shown as the average + SEM (n = 4 – 8).
Figure 6
Figure 6. Disruption of CAR through forced dimerization between loop1 and loop3 inhibits SOCE.
(A) Oligomerization of the indicated Orai1 single and double cysteine mutants overexpressed in HEK 293 cells were detected by SDS PAGE (12%, left; 8% right) after incubation with 1mM CuP (upper panel) or after the addition of 5 mM BMS (lower panel). (B) Whole-cell patch-clamp experiments show time course of currents mediated by Cherry-tagged STIM1 coexpressed with wild-type eYFP-tagged Orai1 or Orai1 mutant as indicated in HEK cells exposed to 20 mM EGTA in the patch pipette and a 10 mM Ca2+-containing solution, and upon maximum current activation, BMS (5 mM) was added (n = 8 - 11). Analysis by t-test, with p < 0.05 for statistical significance, indicated that Orai1-D112C maximum currents are significantly different from those of Orai1-D112C-R210C in the absence but not in the presence of BMS. (C) Current-voltage relationship of the store-operated activation of Orai1-D112C-R210C and upon BMS stimulation from a representative experiment from (B). (D) The BMS-dependent increase in currents mediated by wild-type (wt) eYFP-tagged Orai1 and the indicated eYFP-tagged loop3 cysteine mutants upon maximum store-dependent activation. All Orai1 mutants, except P201C, yielded a store-dependent current when coexpressed with STIM1 in HEK 293 cells exposed to 20 mM EGTA in the patch pipette and a 10 mM Ca2+-containing solution. Data are shown as the mean + SEM. (E) The BMS-dependent increase in currents mediated by wild-type (wt) eYFP-tagged Orai1 and the indicated YFP-tagged loop1 cysteine mutants upon maximum store-dependent activation under the same conditions as in (D). (F) Whole-cell patch-clamp experiments show time course of currents mediated by Cherry-tagged STIM1 coexpressed with wild-type eYFP-tagged Orai1 or Orai1 mutant in a 1mM Ca2+ containing bath solution (n= 7-11 cells). Maximum currents of Orai1-D112C and Orai1-D112C-R210C were analyzed by t-test before and after addition of BMS for statistical significance, and are significantly different (p < 0.05) before addition of BMS. (G) Comparison of relative BMS-dependent stimulation in currents mediated by Cherry-tagged STIM1 and eYFP-tagged Orai1-D112C-R210C coexpressed in HEK 293 cells in the presence of the indicated extracellular solutions (n = 6 - 11). Data were analyzed by t-test for statistical significance (p < 0.05) of maximum currents upon BMS treatment, determining that 1mM and 10mM Ca2+ currents are significantly different compared to sodium based currents.
Figure 7
Figure 7. Ca2+ and loop3 compete for binding to CAR.
(A) Top view of a representative snapshot of the molecular dynamics simulation of Orai1-R210A-K214A. Ca2+ ions are represented as yellow balls, Na+ as orange balls. (B) One-dimensional density number of ion concentration through the Orai1 channel (black) and the double mutant (left) or the single mutants (right) from the intracellular to the extracellular side (in Z-direction). Dotted lines mark the membrane head group regions and the peak between 7 and 8 nm corresponds to the selectivity filter. (C) Whole-cell patch-clamp experiments show time course of currents mediated by Cherry-tagged STIM1 and YFP-tagged wild-type or mutant Orai1 expressed in HEK 293 cells exposed to 20 mM EGTA in the patch pipette and then exposed to the indicated Ca2+ solutions (n = 10 – 14). Maximum currents were analyzed by t-test for statistical significance; Orai1 and Orai1-R210A-K214A are significantly different (p < 0.05) in a 0.3 mM and 1 mM Ca2+ solution. Orai1 and Orai1-R210A maximum currents are significantly different in a 0.3 mM Ca2+ solution.

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