Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Abstract

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca²⁺ influx and Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule)^1,2 and Orai^3–5. Stim probably functions as a sensor of luminal Ca²⁺ content and triggers activation of CRAC channels in the surface membrane after Ca²⁺ store depletion^1,6. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca²⁺ influx and CRAC channel activity in patient T cells³. The overexpression of Stim and Orai together markedly increases CRAC current^5,7–9. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca²⁺ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1–S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca²⁺-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd³⁺. These results indicate that Orai itself forms the Ca²⁺-selectivity filter of the CRAC channel.

Orai and Orai1 possess four hydrophobic stretches that are predicted to span the membrane. On the basis of strategies used previously for several different ion channels, we made point mutations to investigate the most conserved loop between putative transmembrane segments (Supplementary Fig. 1a) and examined properties of ion selectivity, current–voltage rectification and block that are intimately associated with a pore-forming subunit. Wild-type or mutant Orai proteins were overexpressed together with wild-type Stim in S2 cells; messenger RNA and protein expression were verified by RT–PCR and western blotting (Fig. 1a and Supplementary Fig. 1b). In addition, by co-immunoprecipitation of the epitope-tagged wild-type proteins, we evaluated whether Stim and Orai are associated with each other before and after Ca²⁺ store depletion. When cells were cultured without stimulation, only limited interaction was seen. However, Ca²⁺ store depletion triggered by thapsigargin, which inhibits the SERCA (sarco/endoplasmic reticulum Ca²⁺-ATPase) re-uptake pump, markedly increased Stim–Orai protein interaction when either protein was used to pull down the other by co-immunoprecipitation (Fig. 1a). This result shows that both transfected proteins are expressed and, moreover, that they interact, either directly or as part of a complex, after Ca²⁺ store depletion to initiate CRAC channel activation. During whole-cell recording on the co-transfected cells, dialysis of a Ca²⁺ chelator (BAPTA) into the cytoplasm activated a very large Ca²⁺-selective inward current, as shown previously⁵. Using a pipette solution that maintains normal Ca²⁺ store content to prevent spontaneous channel opening, we confirmed that addition of inositol-1,4,5-trisphosphate (IP₃) inside or thapsigargin (TG) outside resulted in greatly augmented CRAC current (Fig. 1b).

Figure 1. Orai interacts with Stim and generates increased store-operated currents in S2 cells.

a, Representative co-immunoprecipitation of Stim and Orai (n = 3). Input: total cell lysate, showing an equal amount of samples prepared for immunoprecipitation. Cells were transfected with HA-olf186-F (Orai) and Stim-V5-His (Stim) constructs, as indicated. M refers to molecular weight markers (kDa). b, Activation of CRAC current (at −130 mV) by three methods: IP₃ (10 μM IP₃ added to high-Ca²⁺ pipette solution; current density −11.4 ± 3.1 pA pF⁻¹, n = 5); thapsigargin (high-Ca²⁺ pipette solution and thapsigargin added to bath; −7.6 ± 2.5 pA pF⁻¹, n = 2); or passive store-depletion (Ca²⁺-free pipette solution; −28.7 ± 1.8 pA pF⁻¹, n = 27). All three methods produced substantially greater CRAC current density than passive store depletion in control cells (−2.5 ± 0.4 pA pF⁻¹, n = 18).

Expression of an Orai mutant with a glutamate-to-aspartate mutation at position 180 (Orai(E180D)), again together with wild-type Stim, generated a current that developed with the normal kinetics of CRAC current during passive Ca²⁺ store depletion, but also exhibited marked alterations in ion selectivity and rectification. In contrast to the normal inward CRAC current, Orai(E180D) expression produced a very large outward current and a smaller inward current that developed in parallel (Fig. 2a). Current–voltage (I–V) curves of the Orai(E180D)-induced current revealed an outwardly rectifying shape with a reversal potential of 6.0 ± 0.4 mV (n = 14 cells), in contrast to the characteristic inwardly rectifying I–V curves with a reversal potential >50 mV of native or wild-type Orai-induced CRAC current (Fig. 2b).

Figure 2. Mutation E180D of Orai alters ion selectivity of the CRAC current.

a, Time courses of inward current at −130 mV and outward current at 90 mV in representative cells overexpressing wild-type Orai (WT, black) or Orai(E180D) mutant (red). Ca²⁺-free internal solution. b, I–V curves at times indicated in a. c, I–V curves of wild-type Orai-induced current in Ca2 solution containing 160 mM Na⁺ and 2 mM Ca²⁺ and in choline external solution with 1.1 mM Na⁺ and 2 mM Ca²⁺. (Complete solution recipes are indicated in Supplementary Table 1.) d, Representative I–V curves for Orai(E180D) with the same solutions as c (n = 3). e, Divalent cation selectivity of wild-type Orai CRAC current. I–V curves are normalized to current values at −130 mV in Ca²⁺ solution. Test solutions contained 20 mM test divalent and 124 mM Na⁺. External solutions for e and f are labelled according to colour. f, I–V curves (not leak-subtracted) for Orai(E180D) with the same divalent cations, normalized to currents at 90 mV in Ca²⁺ solution.

In a series of ion-substitution experiments, we evaluated the ion selectivity properties of current induced by Orai(E180D). Substitution of Na⁺ by choline while maintaining constant Ca²⁺ in the external solution—an experimental procedure that has little effect on native CRAC current¹⁰ or CRAC current with overexpressed wild-type Orai (Fig. 2c)—reduced the inward current and shifted the reversal potential to hyperpolarized potentials (Fig. 2d), indicating that the Orai(E180D)-induced inward current is carried predominantly by Na⁺ rather than by Ca²⁺. Next, different divalent cation species in the external solution were tested, resulting in a I–V pattern of wild-type Orai-induced CRAC current similar to native CRAC current described previously¹⁰. Ca²⁺, Sr²⁺ and Ba²⁺(20 mM in each case, while maintaining high external Na⁺) resulted in increased inward current (Fig. 2e), indicating that each of these divalent cations is permeant, whereas Mg²⁺ is relatively impermeant. In contrast, representative I–V curves of Orai(E180D)-induced current (shown in Fig. 2f with the same colour code for comparison with Fig. 2e) reveal an entirely different pattern. A tenfold increase of Ca²⁺ concentration ([Ca²⁺]) in the external solution significantly decreased the outward current and did little to the small inward current. Substitution of 2 mM external Ca²⁺ by 20 mM Sr²⁺ had little effect; Mg²⁺ increased the current; and Ba²⁺ significantly increased the inward current while decreasing the outward current (Supplementary Fig. 2a). These results indicate that divalent cations block the outward current (carried by Cs⁺) in the potency sequence Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺, and the inward current (carried by Na⁺) in the potency sequence Ca²⁺ ≈ Sr²⁺ > Mg²⁺ > Ba²⁺. Together, these ion-substitution experiments indicate that the Orai(E180D) mutation transforms the CRAC channel from being selective for divalent cations to being a monovalent-selective channel that selects against, and indeed is blocked by, divalent cations.

CRAC channels share with voltage-gated Ca²⁺ channels the property of monovalent ion permeation when external divalent ions are removed^11–15. Upon withdrawal of external divalent cations, Na⁺ carries significant current through CRAC channels, whereas Cs⁺ is far less permeant^10,14,16,17. The monovalent CRAC current normally declines within 10 s, a process termed depotentiation¹⁵ that is due to removal of external Ca²⁺. To check whether the Orai(E180D) mutation alters monovalent CRAC current, both wild-type- and Orai(E180D)-induced CRAC currents were recorded in divalent-free Na⁺ and Cs⁺ test solutions. Three clear differences can be discerned (Fig. 3). First, the inward Na⁺ current was much larger in the Orai(E180D) mutant and did not depotentiate to the same extent as either wild-type Orai (Fig. 3a, b) or native CRAC current in S2 cells¹⁰. Second, in contrast to the relatively small Cs⁺ current density in wild-type Orai (−1.6 ± 0.6 pA pF⁻¹, n = 4), Orai(E180D)-induced Cs⁺ current density was much larger (−191 ± 46 pA pF⁻¹, n = 4), similar in amplitude to the Na⁺ current (−238 ± 43 pA pF⁻¹, n = 9), and the large Cs⁺ current did not depotentiate (Fig. 3b). Third, the measured reversal potential in Na⁺external solution was 7.9 ± 0.3 mV (n = 9) for Orai(E180D), whereas it was 52.4 ± 0.9 mV (n = 5) for wild-type Orai (Fig. 3c, d). Calculated from reversal potentials, the average permeability ratio P_Na/P_Cs changed from 8.0 for wild-type Orai to 1.4 as a result of the E180D mutation, indicating an increase in pore diameter or altered electrostatic interactions with cations. I–V curves for Orai(E180D)-induced current changed shape with varying external [Ca²⁺] (Fig. 3e), revealing voltage-dependent block (Fig. 3f) that causes outward rectification in physiological saline. This examination of ion selectivity properties shows that a conservative mutation of glutamate to aspartate at position 180 of Orai transforms the CRAC channel from being Ca²⁺-selective and inwardly rectifying to one that conducts Na⁺ or Cs⁺ and is outwardly rectifying due to voltage-dependent Ca²⁺ block.

Figure 3. Monovalent current in the absence of divalent ions exhibits altered ion selectivity in the Orai(E180D) mutant.

a, Time course of inward current in a cell expressing wild-type Orai. Bars indicate external solution exchange. b, Same as a but for Orai(E180D). c, I–V curves for wild-type Orai at times indicated in a. d, I–V curves for Orai(E180D) at times indicated in b. e, I–V curves of Orai(E180D)-induced current in the presence of varying external [Ca²⁺]. f, [Ca²⁺] dependence of Orai(E180D) CRAC current at −130 (squares) and 90 mV (circles), scaled to the current in divalent-free solution. Ca²⁺-dependent block (mean ± s.e.m.) was fitted by the function y = 1/(1 + [Ca²⁺]/IC₅₀), where IC₅₀ is the calculated half-blocking Ca²⁺ concentration for inward current at −130 mV (48 ± 13 μM; n = 13, 8, 4, 6 and 2 cells for 20, 2, 0.2, 0.02 and 0.002 mM free external [Ca²⁺], respectively) and outward current at 90 mV (2.05 ± 0.78 mM; n = 13, 8, 4, 5 and 3 cells for 20, 2, 0.2, 0.02 and 0.002 mM free external [Ca²⁺], respectively).

Expression of the charge-neutralizing Orai(E180A) mutant completely abolished native CRAC current (Fig. 4a), indicating that alanine-containing subunits prevent ion conduction in hetero-multimeric CRAC channels by a dominant-negative action. In contrast, expression of Orai(D184A), Orai(D186A) or Orai(N188A) mutants with Stim resulted in increased CRAC current with unaltered I–V shape (Fig. 4b) and normal monovalent Na⁺ current that exhibited depotentiation and low Cs⁺ permeability (data not shown). Figure 4c summarizes the effect of each Orai mutant tested on CRAC current density. Expressed by itself or together with Stim, only Orai(E180A) potently inhibited native CRAC current. The inward current density and I–V shape in cells expressing Orai(D184A), Orai(D186A) and Orai(N188A) mutants did not differ significantly from current observed in cells expressing wild-type Orai. Orai(E180D) was unique in producing very large outward currents as a result of altered ion selectivity.

Figure 4. Charge-neutralizing mutations in the S1–S2 loop.

a, I–V curves in a cell expressing the Orai(E180A) mutant (without Stim) compared to control. b, I–V curves for Orai(D184A), Orai(D186A) and Orai(N188A) mutants. c, CRAC current density in transfected S2 cells. Each point represents the maximal CRAC current density (pA pF⁻¹) in a single cell, plotted as absolute values (from left to right): GFP-transfected control; Orai(E180A) (P = 1.7 × 10⁻⁴ relative to control); Orai(E180A) plus Stim (P = 8.2 × 10⁻³); wild-type Orai plus Stim; Orai(E180D) plus Stim, inward current; Orai(D184A) plus Stim; Orai(D186A) plus Stim; Orai(N188A) plus Stim; and Orai(E180D) plus Stim, outward current. d, Suppression of CRAC current by 5 nM Gd³⁺ in wild-type Orai and the D184A, E180D and D186A Orai mutants. Bars indicate time of Gd³⁺ application; dashed lines indicate the zero-current level.

Gd³⁺ very effectively suppresses native and wild-type Orai-induced CRAC current⁵. In addition to ion selectivity properties, block by Gd³⁺ was evaluated in all mutants (Fig. 4d). Two charge-neutralizing mutations, aspartate to alanine at positions 184 and 186, and, to a lesser degree, mutation of asparagine to alanine at position 188, significantly reduced the potency of Gd³⁺ block (Fig. 4d and Supplementary Fig. 2b). As shown previously⁵, wild-type Orai CRAC current was potentiated by 2-aminoethyldiphenyl borate (2-PB) at a low concentration (5 μM) and inhibited at a high (20 μM) concentration (Supplementary Fig. 3a), similar to effects of 2-APB on native CRAC current in Jurkat and in S2 cells^10,18. The effects of 2-APB on Orai(E180D)-induced CRAC current were complex but still showed potentiation and inhibition (Supplementary Fig. 3b). The time course of Orai(E180D) outward CRAC current showed an immediate decrease preceding potentiation by 5 μM 2-APB, and an immediate increase preceding inhibition during 20 μM application (n = 3). The pharmacological analysis provides support for the conclusion that the S1–S2 loop is involved in ion selectivity and block by Gd³⁺, and may contribute to the complex effects of 2-APB on CRAC current.

Our results demonstrate that thapsigargin-triggered store depletion dynamically strengthens an interaction between Stim and Orai, supporting a model for CRAC channel activation in which Stim serves as the Ca²⁺ sensor to detect store depletion and as the messenger to activate CRAC channels in the plasma membrane. More importantly, we conclude that Orai is a bona fide ion channel, based on the following: (1) RNA-interference-mediated knockdown of Orai expression suppresses thapsigargin-dependent Ca²⁺ influx and CRAC channel activity; (2) overexpression of Orai with or without Stim augments CRAC currents that exhibit biophysical properties identical to native CRAC current; and (3) mutations of negatively charged residues within the putative pore region of Orai significantly alter ion selectivity, current rectification and affinity to a charged channel blocker without altering channel activation kinetics. The marked alteration of these properties by a targeted point mutation provides definitive evidence that Orai embodies the pore-forming subunit of the CRAC channel.

The consensus sequence within the S1–S2 loop, 179VEVQLDxD186, contains the critical glutamate (bold) shown here to control ion selectivity properties of the CRAC channel, and two aspartates (underlined) that may help to attract Gd³⁺ (and Ca²⁺) towards the pore. It is not similar to pore sequences found in other channels. Unlike the pore regions of voltage-gated Ca²⁺ (CaV) channels, which contain a relatively long loop and a ring of critical glutamates from different domains that form a high-affinity Ca²⁺-binding site¹⁹, the putative pore sequence of Orai is very short, and the key residue for ion selectivity (E180) is adjacent to the putative S1 segment. Nevertheless, because withdrawal of external divalent ions reveals permeability to monovalent cations in both CaV^11,12 and CRAC channels^10,14,16,17, it is possible that the CRAC channel ion-selectivity filter is also formed by a ring of glutamates and that the mechanism of Ca²⁺ permeation is similar, although the single-channel conductance and maximum permeant ion size of the CRAC channel selectivity filter are smaller than that of the CaV channel^10,17,20,21. Negatively charged side chains also contribute to Ca²⁺ selectivity of TRPV6; in this instance, aspartate (at position 541) is proposed to coordinate with Ca²⁺ ions and line the selectivity filter in a ring structure formed by four subunits²². The CRAC channel may be a multimer that includes several identical Orai subunits, as a non-conducting pore mutant (E180A) exerts a strong dominant-negative action on native CRAC current. Biochemical approaches and cysteine-scanning mutagenesis should be useful to elucidate better the unique pore architecture of the CRAC channel.

Note added in proof: While in proof, our attention was drawn to another paper reporting complementary findings regarding the control of ion selectivity in the CRAC channel²³. In our study, the critical glutamate residue is 180 in the Orai protein encoded by the Invitrogen S2 cell complementary DNA (GenBank accession number DQ503470, as described⁵). The corresponding residue is 178 in the Drosophila genome database (accession number AY071273), and 106 in the human Orai1 homologue (accession number BC015369).

Methods

Molecular cloning and mutagenesis

Generation of pAc5.1/EGFP, pAc5.1/D-STIM, pAc5.1/D-STIM-V5-His and pAc5.1/olf186-F (GenBank accession number DQ503470) were as described⁵. Orai pore mutants were made by exchanging the corresponding codons (E180A, GAG to GCG; E180D, GAG to GAC; D184A, GAT to GCT; D186A, GAT to GCT; N188A, AAT to GCT; Supplementary Fig. 1) using the QuikChange site-directed mutagenesis kit (Stratagene). The pAc5.1/HA-olf186-F clone was made by adding the haemagglutinin tag via PCR and re-cloned into the XhoI and NotI sites of pAc5.1/V5-His B expression vector. Resulting clones were confirmed by sequencing. Description of the primers and conditions for cloning are available upon request. Both HA-olf186-F and Stim-V5-His were verified for normal function by whole-cell recording.

Whole-cell recording

Cells transfected (Amaxa) with Orai and Stim constructs were identified by fluorescence of co-transfected GFP. Patch-clamp experiments were performed at room temperature in the standard whole-cell recording, using conditions similar to those reported previously¹⁰. The membrane capacitance of S2 cells selected for recording was 11.6 ± 0.4 pF (n = 131 cells). Membrane potentials were corrected for a liquid junction potential of 10 mV between the pipette and bath solution. The series resistance (2–7 MΩ) was not compensated. The membrane potential was held at −10 mV, and 220-ms voltage ramps from −130 to 90 mV alternating with 220-ms pulses to −130 mV were delivered every 2 s. Only cells with high input resistance (>2 GΩ) were selected for recording. External and pipette solution recipes are listed in Supplementary Table 1. Analysed data are presented as mean ± s.e.m.

Co-immunoprecipitation

Two days after transfection, 5 × 10⁶ cells were treated with either Ringer's solution or zero-Ca²⁺ Ringer's solution plus 1 μM thapsigargin using solutions described⁶ for 15 min at room temperature. Cells transfected with Stim-V5-His only or HA-olf186-F only were used as controls. Cell extracts were then prepared using RIPA lysis buffer (Upstate) according to the manufacturer's instructions. The extracts were pre-cleared with protein A/G beads (Pierce) and protein concentration was determined, using the Pierce BCA Protein Reagent Kit. The cell lysate was then diluted to approximately 1 mg ml⁻¹ total protein with PBS and mixed with either anti-HA monoclonal antibodies (1 μg per 100 μg total protein, Santa Cruz) or anti-V5 monoclonal antibodies (1 μg per 100 μg total protein, Invitrogen) for 4 h at 4 °C. Equal amounts of samples were mixed with either normal mouse IgG or vehicle as negative controls. Protein A/G beads were subsequently added (1 μl per 1 μg IgG) to rock overnight at 4 °C. Proteins were eluted by boiling in 4× sample buffer (Invitrogen). Samples were resolved by SDS–PAGE and analysed by standard western blotting techniques. Anti-HA monoclonal antibodies (Santa Cruz) were used at a dilution of 1:200. Anti-V5 monoclonal antibodies (Invitrogen) were used at a dilution of 1:2,500. Proteins were detected by developing with the SuperSignal (Pierce) detection system. Samples treated with normal mouse IgG or vehicle showed no detectable signal for both Stim-V5-His and HA-olf186-F (data not shown), indicating that the immunoprecipitation and co-immunoprecipitation are specific.