The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Abstract

Orai channels mediate store-operated Ca²⁺ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca²⁺ entry. Moreover, each Orai1 concatemer mediated Ca²⁺ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca²⁺ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca²⁺ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.

Keywords: Ca2+ signaling; Crac channels; Orai channels; Orai1; STIM1; calcium; calcium channel; cell signaling; concatemer; ion channel; signal transduction; store-operated channel; stromal interaction molecule 1 (STIM1).

FIGURE 1.

Orai1 concatemers localize to the plasma membrane and exist in there with molecular masses corresponding to those predicted by their sequence. A, deconvolved fluorescence images of HEK-O1^koS1⁺ cells expressing tdTomato-tagged Orai1 monomer, dimer, trimer, tetramer, pentamer, or hexamer constructs revealing their plasma membrane localization. B, Western blot of surface-isolated proteins isolated from the HEK-O1^koS1⁺ cells expressing tdTomato-tagged Orai1 monomers, dimers, trimers, tetramers, pentamers, or hexamers shown in A. Cells were biotinylated, and surface proteins were isolated with NeutrAvidin resin, eluted with DTT-containing sample buffer, and assessed by Western analysis with Orai1 antibody. The calculated molecular masses for tdTomato-linked Orai1 concatemers are: 91.2 kDa (monomer); 127.1 kDa (dimer); 163 kDa (trimer); 198.9 kDa (tetramer); 234.8 kDa (pentamer); 270.7 kDa (hexamer). C, comparison of the surface (PM) expression of Orai1 hexamer with that expressed elsewhere in the cell. Western blotting analysis used an Orai1 antibody to detect surface-biotinylated (left) and non-biotinylated (right) Orai1 protein derived from HEK-O1^koS1⁺ cells expressing tdTomato-tagged Orai1 hexamer. Following biotinylation of cells, lysates were applied to NeutrAvidin resin. The flow-through lane is the fraction that was not retained on NeutrAvidin resin. The surface protein lane is the fraction eluted with sample buffer/DTT after binding to NeutrAvidin resin. Details are given under “Experimental Procedures.”

FIGURE 2.

Orai1 dimeric, trimeric, tetrameric, pentameric, and hexameric concatemers mediate store-operated Ca²⁺ entry and CRAC current indistinguishable from that mediated by Orai1 monomer. tdTomato-tagged Orai1 concatemer constructs were transiently expressed in HEK-O1^koS1⁺ cells. A, fura-2 ratiometric Ca²⁺ add-back measurements for cells transfected with the concatemers shown. Ca²⁺ stores were released upon the addition of 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). Traces (means ± S.E.) are representative of three independent experiments. B, summary scatter plots with means ± S.D. for peak Ca²⁺ entry of all individual cells recorded in three independent experiments with the Orai1 concatemer constructs shown in A. C, representative I/V plots from whole-cell recordings from HEK-O1^koS1⁺ cells transiently expressing each of the tdTomato-tagged concatenated Orai1 constructs shown in A. D, summary statistics of the reversal potential measurements for each Orai1 concatemer construct described in C. Scatter plots are means ± S.D. for all the cells recorded in three independent experiments.

FIGURE 3.

Orai1 pore-inactive E106A subunit substitution in concatenated Orai dimers and tetramers. A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 dimer constructs shown. Ca²⁺ stores were released upon the addition of 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). Traces are for wild-type Orai1 dimer (OO, red), dimer with Orai1 E106A substitution at the first (N-terminal) position (XO, green), second (C-terminal) position (OX, blue), E106A substitution at both positions (XX, orange), or untransfected control cells (black). B, fura-2 ratiometric Ca²⁺ responses as in A with cells expressing similar levels of tdTomato-tagged Orai1 tetrameric concatemers, including the all-wild-type Orai1 tetramer (OOOO, red), Orai1 tetramer with the first subunit pair E106A-mutated (XXOO, blue), Orai1 tetramer with the second subunit pair E106A-mutated (OOXX, green), or untransfected control cells (black). C, representative I/V plots from whole-cell recordings from HEK-O1^koS1⁺ cells transiently expressing each of the tdTomato-tagged concatenated Orai1 tetramer constructs shown in B. Summary scatter plot (means ± S.D.) of reversal potential measurements taken from multiple I/V curves for the OOOO and OOXX constructs, as shown. Current for the XXOO construct was essentially zero. D, fura-2 ratiometric Ca²⁺ responses as in B with cells expressing similar levels of tetrameric concatemers, but instead, all tagged with tdTomato at the N terminus. Traces are for the all-wild-type Orai1 tetramer (OOOO, red), Orai1 tetramer with the first subunit pair E106A-mutated (XXOO, blue), Orai1 tetramer with the second subunit pair E106A-mutated (OOXX, green), or untransfected control cells (black). For each of the results shown in A, B, and D, traces (means ± S.E.) are representative of three independent experiments. In each case, summary scatter plots with means ± S.D. of the percentage of peak Ca²⁺ entry relative to wild type peak Ca²⁺ entry are shown for all the cells derived from three independent experiments.

FIGURE 4.

The Orai1 tetrameric and hexameric concatemers differ in the effects of substituting Orai1 pore-inactive E106A subunits at the first or second positions. A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar fluorescence levels of the C-terminal tdTomato-tagged Orai1 tetramers shown. Ca²⁺ stores were released upon the addition of 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). Left, traces are for wild-type Orai1 tetramer (OOOO; red) or Orai1 tetramer substituted with E106A at the first position (XOOO, orange). Middle, traces are for wild-type Orai1 tetramer (OOOO, red) or Orai1 tetramer substituted with E106A at the second position (OXOO, purple). Untransfected control cells are shown for each experiment (black). Traces (means ± S.E.) are representative of three independent experiments. Right, summary scatter plots of peak Ca²⁺ entry relative to wild type; results are means ± S.D. of all cells derived from three independent experiments. B, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca²⁺ measurements were as in A. Left, traces are for wild-type Orai hexamer (OOOOOO, red) or Orai1 hexamer substituted with E106A at the first position (XOOOOO, turquoise). Right, traces are for wild-type Orai1 hexamer (OOOOOO, red) or Orai1 hexamer substituted with E106A at the second position (OXOOOO, brown). Untransfected control cells are shown for each experiment (black). Traces (means ± S.E.) are representative of three independent experiments. C, summary scatter plots of peak Ca²⁺ entry relative to wild type; results are means ± S.D. of all cells from three independent experiments. The summary results for the XO and OX dimers from Fig. 3A are included for comparison.

FIGURE 5.

Substitution of pore-inactive E106A subunits in the Orai1 hexamer reveals that all the subunits in the hexamer contribute to channel function. A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar fluorescence levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca²⁺ stores were released upon the addition of 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). In each case, traces for wild-type Orai1 hexamer (OOOOOO, red) are compared with traces for Orai1 hexamer substituted with E106A at the third (OOXOOO, orange), fourth (OOOXOO, gray), fifth (OOOOXO, blue), or sixth position (OOOOOX, purple). Untransfected control cells are also shown (black). B, Ca²⁺ add-back measurements were as in A using hexamers substituted with pairs of E106A residues. In each case, traces for wild-type Orai1 hexamer (OOOOOO, red) are compared with traces for Orai1 hexamer substituted with two E106A subunits at the first and second (XXOOOO, purple), third and fourth (OOXXOO, green), or fifth and sixth position (OOOOXX, blue). Untransfected control cells are also shown (black). Traces (means ± S.E.) are representative of three independent experiments. Note that for the OOOOXO data (A, third panel), the wild-type trace (OOOOOO) is the same as that shown for the OXOOOO data (Fig. 4B, right panel) because the data were obtained in the same experiment. In each case, summary scatter plots of peak Ca²⁺ entry relative to wild type are shown; results are means ± S.D. of all cells from three independent experiments.

FIGURE 6.

The function of the Orai1 tetramer or Orai1 hexamer is substantially reduced by co-expression of pore-inactive Orai1 E106A monomer, and this effect is independent of any STIM1 binding of the monomer. A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 tetramer (left) or hexamer (right), either with (blue traces) or without (red traces) co-expression of Orai1 E106A monomer. Ca²⁺ stores were released with 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). In each case, traces (means ± S.E.) are representative of all cells in two independent experiments. B, summary scatter plots of peak Ca²⁺ entry in the presence of E106A monomer when compared with Ca²⁺ entry without monomer; results are means ± S.D. of three independent experiments represented in A. C, exactly the same experimental design as in A except that the monomer used is the double mutant, Orai1 E106A/L273D, which is devoid of any ability to bind STIM1. Traces (means ± S.E.) are representative of three independent experiments. D, summary scatter plots of peak Ca²⁺ entry in the presence of E106A monomer when compared with Ca²⁺ entry without monomer; results are means ± S.D. of all cells in two independent experiments represented in C. Note that the levels of expression of tdTomato-tagged Orai1 tetramer or hexamer were not affected by Orai1 E106A monomer expression, and in each experiment, the level of tdTomato fluorescence, with or without monomer expression, was the same.

FIGURE 7.

Model of the possible assembly of concatenated Orai1 constructs to form functional hexameric channels. The model depicts the ability of concatenated Orai1 dimers (A) or tetramers (B) to form functional hexameric channels through the insertion of three N-terminal dimers into a hexameric trimer-of-dimers complex. The concatenated Orai1 hexamer is distinct from the dimer and tetramer in being able to form a hexameric ring (C) in which all six of the subunits contribute to the function of the channel. The data suggest that the hexamer can additionally form a trimer-of-dimers complex similar to that formed by the tetramer. Details of the evidence and logic favoring formation of these functional assemblies are given under “Results and Discussion.” 36-aa linker, 36-amino acid linker.

FIGURE 8.

The position and number of pore-inactive E106A subunits substituted in the Orai1 hexamer reveal profound differences in channel function likely reflecting different combinations of assembly as described under “Results and Discussion.” A, fura-2 ratiometric Ca²⁺ add-back measurements for HEK-O1^koS1⁺ cells expressing similar levels of the C-terminal tdTomato-tagged Orai1 hexamers shown. Ca²⁺ stores were released with 2.5 μm ionomycin in Ca²⁺-free medium followed by the addition of 1 mm Ca²⁺ (arrows). The trace for wild-type Orai1 hexamer (OOOOOO, red) is compared with that for Orai1 hexamer substituted with E106A at the first three positions (XXXOOO, orange). B, Ca²⁺ add-back measurements as in A using Orai1 hexamer substituted with three E106A residues in the second, fourth, and sixth positions (OXOXOX, olive). Traces (means ± S.E.) are representative of three independent experiments. C, summary scatter plots of peak Ca²⁺ entry when compared with wild type; results are means ± S.D. of all cells in three independent experiments represented in A or B. D, Ca²⁺ add-back measurements as in A using Orai1 hexamer substituted with three E106A residues in the last three positions (OOOXXX, cyan). E, Ca²⁺ add-back measurements as in A using Orai1 hexamer substituted with four E106A residues in the last four positions (OOXXXX; green). Traces (means ± S.E.) are representative of three independent experiments. F, summary scatter plots of peak Ca²⁺ entry when compared with wild type; results are means ± S.D. of all cells in three independent experiments represented in D or E.