Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states

Abstract

We applied single-molecule photobleaching to investigate the stoichiometry of human Orai1 and Orai3 channels tagged with eGFP and expressed in mammalian cells. Orai1 was detected predominantly as dimers under resting conditions and as tetramers when coexpressed with C-STIM1 to activate Ca(2+) influx. Orai1 was also found to be tetrameric when coexpressed with STIM1 and evaluated following fixation. We show that fixation rapidly causes release of Ca(2+), redistribution of STIM1 to the plasma membrane, and STIM1/Orai1 puncta formation, and may cause the channel to be in the activated state. Consistent with this possibility, Orai1 was found predominantly as a dimer when coexpressed with STIM1 in living cells under resting conditions. We further show that Orai3, like Orai1, is dimeric under resting conditions and is predominantly tetrameric when activated by C-STIM1. Interestingly, a dimeric Orai3 stoichiometry was found both before and during application of 2-aminoethyldiphenyl borate (2-APB) to activate a nonselective cation conductance in its STIM1-independent mode. We conclude that the human Orai1 and Orai3 channels undergo a dimer-to-tetramer transition to form a Ca(2+)-selective pore during store-operated activation and that Orai3 forms a dimeric nonselective cation pore upon activation by 2-APB.

Fig. 1.

Functional expression of Orai1 and Orai3. (A) Fura-2 ratiometric Ca²⁺ measurements in response to removal and readdition of extracellular Ca²⁺, followed by application of 50 μM of 2-APB. Cells were transfected with eGFP–Orai1 alone (light gray), a 1:5 ratio of eGFP–Orai1/C-STIM1 (black), or eGFP–Orai1 E106A (a dominant-negative nonconducting pore mutant) together with C-STIM1 (dark gray). Measurements were made 3 to 5 h after transfection. Traces show means and SEM from one representative experiment of six experiments; each experiment typically included >50 cells. (B) Initial basal cytosolic [Ca²⁺] levels when bathed in solution containing 2 mM Ca²⁺ (Left), and the increase in cytosolic [Ca²⁺] resulting from readmission of 2 mM Ca²⁺ after bathing in zero [Ca²⁺] solution (Right). Error bars indicate SEM from six experiments; *P < 0.05 compared with mock-transfected cells (open bars). (C) Fura-2 Ca²⁺ measurements in eGFP–Orai3-transfected cells in response to removal and readdition of extracellular Ca²⁺, followed by application of 50 μM of 2-APB. Black trace is from cells transfected with eGFP–Orai3 (WT) together with C-STIM1, gray trace from cells transfected with eGFP–Orai3 alone, light gray trace from cells transfected with an eGFP-tagged dominant-negative mutant (E81Q, EQ) of Orai3, and dark gray trace from cells transfected with eGFP–Orai3 E81Q together with C-STIM1. (D) Analysis of Ca²⁺ measurements from experiments similar to C, showing resting [Ca²⁺] levels, peak [Ca²⁺] levels following Ca²⁺ readdition, and peak [Ca²⁺] levels following application of 2-APB. Error bars indicate 1 SEM from 6 to 12 experiments.

Fig. 2.

Subunit stoichiometry of Orai1 at rest and when activated by coexpression of C-STIM1. (A and B) Representative traces showing single-spot bleaching steps in fixed HEK cells expressing, respectively, eGFP–Orai1 alone, or eGFP–Orai1 plus C-STIM1. Arrows indicate visually identified bleaching steps. Fluorescence is expressed in arbitrary units (AU). (C) Histograms showing percentages of spots showing one, two, three, or four bleaching steps in fixed cells expressing eGFP–Orai1 alone (open bars; 146 spots, 14 cells) or eGFP–Orai1 plus C-STIM1 (filled bars; 237 spots, 14 cells). Comparison of the bleaching step distributions for eGFP–Orai1 with and without C-STIM1 yielded P < 0.0001 (χ² test).

Fig. 3.

Fixation evokes release of intracellular Ca²⁺, and in cells expressing STIM1 results in redistribution of Orai1 and STIM1 and shifts bleaching patterns of eGFP–Orai1 from primarily dimeric to tetrameric. (A) Histograms showing percentages of spots showing one, two, three, or four bleaching steps in fixed HEK cells expressing Orai1–eGFP alone (open bars; 185 spots, 16 cells), in fixed cells expressing eGFP–Orai1 + full-length STIM1 (gray bars; 114 spots, 13 cells); and in living cells expressing eGFP–Orai1 + full-length STIM1 (black bars; 90 spots, 7 cells), or mCherry–Orai1 + full-length STIM1 (hatched bars; 137 spots, 11 cells). Comparison of bleaching step distributions for eGFP–Orai1 + STIM1-expressing cells before and after fixation yielded P < 0.0001 (χ² test). (B) Fura-2 Ca²⁺ measurements in HEK cells expressing eGFP–Orai1 + STIM1 in the absence of extracellular Ca²⁺. Arrow indicates application times of 5 μM ionomycin (filled circles), 1 μM thapsigargin (open circles), and 4% PFA (triangles). (C–F) PFA fixation triggers redistribution of YFP–STIM1 and eGFP–Orai1. (C) A single cell expressing Orai1 + YFP–STIM1 before PFA application. (D) The same cell, imaged after treatment with Ca²⁺-free PBS solution containing 4% PFA (pH 7.4) at 23 °C for 5 min, and then washed with fresh PBS for 30 min. (E and F) Corresponding images before and after PFA treatment of a cell expressing eGFP–Orai1 + STIM1. The brightness and contrast settings in D and F were adjusted compared with the prefixation images in C and E to compensate for fluorescence quenching induced by PFA.

Fig. 4.

Subunit stoichiometry of eGFP–Orai3 alone and when activated by C-STIM1 or by 2-APB. (A–C) Representative traces showing bleaching steps from experiment in fixed cells expressing: (A) eGFP–Orai3 alone, (B) eGFP–Orai3 + C-STIM1, or (C) eGFP–Orai3 alone following application of 100 μM of 2-ABP. Arrows indicate visually identified bleaching steps. (D) Histograms showing percentages of spots showing one, two, three, or four bleaching steps in HEK cells expressing: eGFP–Orai3 alone (white bars, 287 spots, 19 cells), eGFP–Orai3 + C-STIM1 (gray bars; 257 spots, 16 cells), or eGFP–Orai3 following application of 100 μM of 2-APB (black bars; 235 spots, 23 cells). Comparison of bleaching step distributions for eGFP–Orai3 and eGFP–Orai3 +2-APB gave no significant difference (P = 0.127; χ² test).