Regulation of Orai1/STIM1 mediated ICRAC by intracellular pH

Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai1, are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Previously it has been shown that the amplitude of CRAC current (ICRAC) strongly depends on extracellular and intracellular pH. Here we investigate the intracellular pH (pHi) dependence of ICRAC mediated by Orai1 and STIM1ectopically expressed in HEK293 cells. The results indicate that pHi affects not only the amplitude of the current, but also Ca2+ dependent gating of CRAC channels. Intracellular acidification changes the kinetics of ICRAC, introducing prominent re-activation component in the currents recorded in response to voltage steps to strongly negative potentials. ICRAC with similar kinetics can be observed at normal pHi if the expression levels of Orai1 are increased, relative to the expression levels of STIM1. Mutations in the STIM1 inactivation domain significantly diminish the dependence of ICRAC kinetics on pHi, but have no effect on pHi dependence of ICRAC amplitude, implying that more than one mechanism is involved in CRAC channel regulation by intracellular pH.

that pH i affects STIM1/Orai1 interaction 13 . Furthermore, mutation of His155 in the intracellular loop of Orai1 to phenylalanine (H155F) abolishes the effect of alkalinisation on I CRAC and diminishes I CRAC inhibition caused by acidification of pH i 10 . The Orai1 region containing H155 has previously been implicated in fast Ca 2+ -dependent inactivation (FCDI) of I CRAC , and therefore may be involved in STIM1/Orai1 interactions 14 .
In this work, we investigated pH i dependence of I CRAC mediated by WT Orai1 and WT or mutated STIM1 ectopically expressed in HEK293 cells at two Orai1:STIM1 expression ratios. Using cells expressing WT Orai1 and WT STIM1 we confirmed that I CRAC amplitude strongly depends on pH i and showed that intracellular acidification introduces a strong re-activation component in I CRAC traces recorded in response to voltage steps between −80 mV and −140 mV. (In this study, term "re-activation" is used exclusively in relation to slow increase of I CRAC amplitude during voltage steps from 0 mV to potentials between −80 and −140 mV, and is opposite of FCDI.) As shown previously, this I CRAC re-activation could also be observed at normal pH i , but only in the cells that were transfected with higher amounts of Orai1 cDNA, relative to STIM1 9,15,16 . To investigate whether there is any overlap between the mechanisms that regulate dependence of I CRAC on pH i and pH o , we used E106D Orai1 mutant. Glutamate 106 in the selectivity centre of Orai1 pore was previously shown to mediate I CRAC dependence on pH o 9 . In further search for the potential protonation sites that may be responsible for I CRAC dependence on pH i , we evaluated EE482/483AA and DD475/476AA double mutations within STIM1 inactivation domain (ID STIM ). Considering that ID STIM is highly negatively charged and is indispensable for Ca 2+ dependent inactivation of I CRAC 17,18 , it is logical to hypothesise that it is involved in pH i sensitivity of CRAC channel.

Results
Intracellular pH affects CRAC channel gating. To investigate I CRAC dependence on pH i , the currents were recorded using pipette solutions with pH i adjusted to 6.3, 7.3 or 8.3. Previously it has been shown that the current amplitude, fast Ca 2+ dependent inactivation (FCDI), re-activation, potentiation by 2-APB, and selectivity of CRAC channels for divalent cations strongly depend on the relative amounts of Orai1 and STIM1 proteins in the cell 15,16 . It is possible that other properties of I CRAC , including pH dependence, are also influenced by the Orai1:STIM1 expression ratios. Therefore, we investigated the effects of pH i on I CRAC at two transfection conditions. To achieve different expression ratios, HEK293T cells were transfected with Orai1-and STIM1-containing plasmids at either 1:4 or 1:1 molar ratios. For both transfection conditions, the amplitude of I CRAC exhibited strong dependence on pH i . I CRAC was smaller at pH i 6.3 and larger at pH i 8.3, compared to pH i 7.3 (Fig. 1a). Consistent with previous publications, at pH i 7.3 and 6.3 cells transfected with higher relative amount of STIM1 (1 Orai1: 4 STIM1 ratio) produced larger I CRAC , compared to cells transfected with equal amounts of STIM1 and Orai1 (1 Orai1: 1 STIM1 ratio; Fig. 1a). However, the effect of the relative expression ratio on the current amplitude, at least within the employed transfection range (1:4 and 1:1), was absent when pH i was raised to 8.3 (Fig. 1a). Majority of cells at pH i 7.3 produced I CRAC with noticeable FCDI at potentials between −80 and −140 mV when transfected with 1 Orai1: 4 STIM1 ratio (Fig. 1b,i). Some re-activation was also evident with longer pulses (Fig. 1b,ii). It was observed that raising pH i to 8.3 resulted in elimination of visible signs of the re-activation component, even with longer pulses. However, the extent of FCDI of the currents recorded in response to 200 ms pulses at pH i 8.3 was also reduced, compared to pH i 7.3, and the time course of inactivation was significantly slower (c.f. Fig. 1ci and bi). In contrast, lowering pH i to 6.3 produced I CRAC with pronounced re-activation at potentials between −80 and −140 mV and no visible FCDI (Fig. 1d).
To compare I CRAC Ca 2+ dependent gating (FCDI and re-activation) under different conditions and a range of membrane potentials, we used the amplitudes of tail-currents obtained at −100 mV after voltage steps between −140 and +80 mV, normalised to the amplitude of the tail current after a step to +80 mV (see Methods) 9 . The resulting data were used to construct apparent P o curves 9 . I CRAC that exhibited FCDI and little or no re-activation produced apparent P o data that could be fitted with a standard Boltzmann equation (eq. 1), whereas I CRAC with pronounced re-activation exhibited bell-shaped P o curves which could not be fitted with a single Boltzmann function ( Fig. 2a). At the 1Orai1:4STIM1 transfection ratio FCDI was more pronounced at pH i 7.3 than at pH i 8.3. At pH i 6.3, the P o curve was bell-shaped with a maximum at −20 mV, which was expected, considering the presence of re-activation. However, despite the apparent absence of FCDI in current traces recorded at pH i 6.3 (Fig. 1d), the FCDI was still present, and the extent of it, relative to the maximum P o , was similar to that of I CRAC recorded at pH i 7.3 (Fig. 2a).
Larger I CRAC amplitude at alkaline pH i is due to pH dependence of EGTA. Phenomenologically, the effect of intracellular acidification on the I CRAC kinetics and P o (Figs 1 and 2) was similar to the effect of increasing Orai1 expression relative to STIM1 16 . I CRAC recorded at pH i 7.3 in the cells transfected with 1Orai1:1STIM1 ratio showed strong re-activation during voltage steps from 0 mV to −120 mV and produced bell-shaped P o curve, which looked similar to the P o curve obtained at pH i 6.3 with 1 Orai1: 4 STIM1 transfection ratio (c.f. Fig. 2a and b). Lowering pH i to 6.3 in cells transfected with 1 Orai1: 1 STIM1 ratio further increased the re-activation (Fig. 2c). In contrast, rising pH i to 8.3 virtually eliminated current re-activation (Fig. 2c). The apparent P o curves obtained at pH i 8.3 in cells transfected with 1:1 and 1:4 Orai1:STIM1 ratios were almost identical between two transfection conditions (Fig. 2d). The observed changes in the kinetics and the extent of I CRAC FCDI induced by raising pH i to 8.3 (Fig. 1c) are similar to those caused by replacing EGTA with BAPTA at pH i 7.3 9,19,20 . Due to its' ability to bind Ca 2+ faster than EGTA, BAPTA is believed to reduce Ca 2+ concentration at the intracellular mouth of CRAC channels, thus slowing down and reducing FCDI 19,20 . The apparent P o curve obtained at pH i 7.3 using cells transfected with 1 Orai1: 4 STIM1 ratio and BAPTA as Ca 2+ buffer, was virtually identical to P o curves obtained at pH i 8.3 and EGTA in the pipette solution (Fig. 2d). Using BAPTA in the internal solution instead of EGTA with pH i 6.3 also decreased I CRAC re-activation at negative potentials and therefore reduced positive apparent P o (Fig. 2d).  To investigate whether intracellular Ca 2+ buffer contributes to the dependence of I CRAC on pH i , we used extracellular application of 30 mM NH 4 Cl, which is known to alkalinise pH i 13,21 . Application of NH 4 Cl to the bath, when EGTA was used as Ca 2+ buffer in the pipette solution, drastically increased the I CRAC amplitude (Fig. 3a,b) and caused inhibition of both I CRAC FCDI and re-activation ( Fig. 3c), in agreement with the results obtained using pipette solution with EGTA and pH i 8.3 (Figs 1a and 2a,c). In contrast, application of NH 4 Cl when BAPTA was used in the pipette solution instead of EGTA, had very little effect on I CRAC amplitude (Fig. 3a,b).

Is there any overlap between mechanisms regulating I CRAC dependence on pH o and pH i ?
Previous investigations have shown that the amplitude of native I CRAC in different cell types and I CRAC mediated by heterologously expressed Orai1 and STIM1 strongly depends on extracellular pH [8][9][10]12 . Superficially, the dependence of I CRAC amplitude on pH o looks similar to its dependence on pH i 8-10, 12 . However, possible reasons for similarities between pH i and pH o effects on I CRAC have not been yet considered. Could changing pH o affect pH i in patch clamping experiments? To investigate this question, we used cells transfected with Orai1 and STIM1 at 1:1 molar ratio, which showed a very pronounced re-activation at negative potentials (Fig. 4a). Raising pH i to 8.3 eliminates I CRAC re-activation at negative potentials (Fig. 2b), and if raising pH o results in a rise of pH i , one would expect a reduction of current re-activation. The results show that increasing pH o from 7.4 to 8.3 does not reduce I CRAC re-activation and has no effect on the P o curve (Fig. 4). Therefore, it can be safely concluded that pH i in these patch clamping experiments is not affected by changes in pH o . One of the main residues responsible for I CRAC dependence on pH o is Glu 106 in the Orai1 pore 9 . Could pH i affect protonation of Glu 106 in the Orai1 pore? Despite the observation that I CRAC kinetics is unaffected by pH o , it is possible that I CRAC amplitude dependence on pH i and pH o is mediated by the same protonatable site in the pore. To investigate this possibility, we used an E106D Orai1 mutant. Previous studies have shown that the E106D Orai1 differs from WT Orai1 in several respects 9 . Firstly, it is less selective for Ca 2+ and supports a significant Na + conductance. Secondly, while E106D-mediated I CRAC exhibits strong inactivation at negative potentials that looks similar to FCDI of WT I CRAC (Fig. 5a, cf. Fig. 1b), it has a different underlying mechanism. The inactivation of E106D-mediated I CRAC during steps to negative potentials is caused by Ca 2+ block of Na + permeation through the pore; it does not require interaction with ID STIM , and it is not affected by BAPTA or Orai1:STIM1 transfection ratios 9 . Finally, and importantly for this investigation, the Ca 2+ dependent block of Na + permeation through the E106D pore is strongly pH o dependent, whereas the peak amplitude of I CRAC mediated by E106D Orai1 is not influenced by pH o 9 . Changing the pipette solution pH revealed that the amplitude of E106D-mediated I CRAC was pH i -dependent -the current was strongly inhibited by pH i 6.3 and enhanced by pH i 8.3, similarly to WT I CRAC (Fig. 5c, cf. Fig. 1a). E106D-mediated I CRAC recorded in the absence of Na + in the bath solution, when Ca 2+ was the only permeating cation, also showed pH i dependence of the amplitude similar to that of WT I CRAC (Fig. 5d). However, the kinetics and the extent of Ca 2+ dependent block of Na + permeation through E106D Orai1 was not appreciably affected by pH i (Fig. 5b, cf. Figs 5a and 1d). If Asp 106 could be protonated from the intracellular side at low pH i , one would expect the changes in E106D-mediated I CRAC to be similar to those induced by low which was not the case. These results demonstrate that pH i and pH o affect I CRAC through different mechanisms, and that Glu 106 which is located in the Orai1 pore does not mediate the pH i -dependence of I CRAC amplitude.
The effects of mutations in STIM inactivation domain on I CRAC dependence on pH i . One of the domains within STIM1/Orai1 complex critically important for FCDI is located on STIM1 between residues 470 and 491 (ID STIM ), C-terminal to CRAC activation domain (CAD) 17,18 . Thus, neutralisation of Aspartate and Glutamate residues within a cluster of 7 negatively charged amino acids (475DDVDDMDEE483) in ID STIM results in drastic changes in I CRAC FCDI 18 . It is possible that protonation/deprotonation of some of these residues contribute to I CRAC dependence on pH i . To investigate this possibility, we investigated pH i dependence of two double mutants of STIM1, DD475/476AA, which produced I CRAC with diminished FCDI (Fig. 6a,c), and EE482/483AA, which produced I CRAC with enhanced FCDI (Fig. 6b,c) 17,18 . Using pipette solutions with pH adjusted to 6.3, 7.3 or 8.3 we found that the amplitude of I CRAC mediated by each of these STIM1 mutants co-expressed with WT Orai1 exhibited dependence on pH i similar to that of WT I CRAC (Fig. 6d; cf. Fig. 1a). Next, we investigated the effects of DD475/476AA and EE482/483AA STIM1 mutations on the dependence of I CRAC kinetics on pH i . At the transfection ratio of 1 Orai1: 4 STIM1 the apparent P o for I CRAC mediated by DD475/476AA-STIM1 mutant showed a weaker dependence on pH i , compared to WT I CRAC (Fig. 7a; cf. Fig. 2a). Although pH i 8.3 reduced the re-activation component (Fig. 7a), as it did in WT I CRAC (Fig. 2a), pH i 6.2 failed to induce a significant change in the apparent P o of the Orai1/ DD475/476AA-STIM1 mediated current (Fig. 7a). Changing the transfection ratio to 1 Orai1: 1 DD475/476AA-STIM1 did not affect the apparent P o , or its dependence on pH i (Fig. 7b). However, we were unable to obtain the apparent P o curve at pH i 6.3 as the amplitude of the current was too small for a reliable extraction of the data. I CRAC mediated by Orai1 and EE482/483AA-STIM1 mutant also exhibited a weaker dependence of the kinetics on pH i , compared to WT CRAC (Fig. 7c,d). At the transfection ratio 1 Orai1: 4 EE482/483AA-STIM1, lowering pH i to 6.3 introduced a small re-activation component to the current (Fig. 7c). This can be seen on P o curve as deviation from simple Boltzmann distribution, whereas increasing pH i to 8.3 slightly reduced the extent of FCDI at negative potentials (Fig. 7c). At the transfection ratio 1:1, the changes in FCDI and re-activation induced by changes in pH i were more pronounced than at the ratio 1:4 (Fig. 7d, cf. Fig. 7c), however, these changes were significantly smaller than those induced by pH i changes in the WT I CRAC (Fig. 2a,c; cf. Fig. 7c,d). Overall, DD475/476AA and EE482/483AA STIM1 double mutations significantly diminished the dependence of I CRAC FCDI on pH i and the relative Orai1/STIM1 expression ratio, without affecting pH i dependence of I CRAC amplitude.
One of the distinctive properties of Orai1/STIM1 mediated I CRAC is inhibition by high (over 100 µM) and potentiation by low (below 10 µM) concentrations of 2-APB, whereas application of intermediate concentrations of 2-APB (10-50 µM) cause transient potentiation of I CRAC followed by inhibition 22 . Previously we have shown that the extent of I CRAC potentiation by 2-APB depends on the relative expression levels of STIM1 and Orai1 16 . The higher the expression of Orai1, relative to STIM1, the stronger the potentiation 16 . Here we investigated whether potentiation of I CRAC amplitude by 50 µM 2-APB is affected by pH i . The amplitude of I CRAC in cells transfected with WT STIM1 and Orai1 at 4:1 ratio increased more than 4-fold at acidic pH i of 6.3, but only 1.3-fold when pH i was raised to 8.3, compared to a potentiation of 2.5-fold at pH 7.3 (Fig. 8a). Despite the lack of pH i effect on FCDI and the apparent P o of I CRAC mediated by Orai1/EE482/483AA-STIM1, the dependence of 2-APB mediated potentiation of this mutant on pH i remained unchanged, compared to WT I CRAC (Fig. 8b).

Discussion
The key findings of this paper can be summarised as follows -(i) pH i regulates both, the amplitude of I CRAC and Ca 2+ dependent gating of CRAC channels; (ii) increase in I CRAC amplitude in response to alkaline pH i in the presence of EGTA in the pipette solution is a result of pH dependence of the Ca 2+ buffering properties of EGTA, not the CRAC channel itself; (iii) Glutamate 106 in the selectivity centre of Orai1 pore, which mediates I CRAC dependence on pH o , does not contribute to I CRAC dependence on pH i ; (iv) negatively charged residues in ID STIM domain play a role in pH i regulation of CRAC channel gating kinetics but not the amplitude of I CRAC . These data suggest that several mechanisms contribute to I CRAC regulation by pH i . It has been shown previously that increasing the amounts of Orai1 relative to STIM1 results in a smaller I CRAC that exhibits re-activation at negative potentials which masks FCDI 15,16 . The results presented here show that intracellular acidification has an effect on I CRAC similar to that of increasing the relative amounts of Orai1 (or decreasing the relative amounts of STIM1). Comparable changes in I CRAC kinetics and amplitude caused by intracellular acidification and increased Orai1:STIM1 ratio suggest that low pH i reduces the affinity of STIM1 binding to Orai1, likely due to protonation of specific residues, which is equivalent to a reduction of available STIM1. This notion is supported by previous observations that acidification of cytoplasm due to hypoxia reduces FRET between Orai1and STIM1 and inhibits I CRAC 13 . In the study of Mancarella et al. (2012) the effect of hypoxia on I CRAC could be mimicked by application of extracellular propionate, which lowers pH i , and reversed by application of NH 4 Cl, which raises pH i 13 . Intracellular acidification was shown to reduce FRET between STIM1-YFP and Orai1-CFP, but no change was observed in STIM1/Orai1 co-localisation in puncta 13 . These results suggested that pH i affects STIM1/Orai1 functional coupling leading to channel opening, but not the interactions that trap STIM1 and Orai1 in puncta 13 . The pH dependent changes in I CRAC kinetics reported here also point to the conclusion that intracellular acidification disrupts STIM1/Orai1 functional interactions.  (d) ID STIM mutants I CRAC amplitude was measured at −100 mV from the responses to 100 ms voltage ramps from −120 to 120 mV, at indicated pH i . HEK293T cells were transfected with Orai1 and DD475/6AA STIM1 or EE482/3AA STIM1 plasmids at 1:4 ratio.
Inhibition of I CRAC by low pH i has been demonstrated previously in several publications 8,10,12 . They all agree that I CRAC , both endogenous and mediated by ectopically expressed Orai1 and STIM1, is inhibited by approximately 70-90% at pH i of around 6, compared to pH i 7.3 8, 10, 12 . In contrast, the effects of alkalinisation of pH i above 7.3 on I CRAC are inconsistent between different studies 8,10,12 . The results of the present work suggest that the reason for the discrepancy is likely to be due to the type of intracellular Ca 2+ buffer used. Studies employing BAPTA in the pipette did not find much increase in I CRAC amplitude at higher pH i , whereas studies that used EGTA reported a significant potentiation of I CRAC amplitude by alkalinisation 8, 10, 12 . Calculations using Maxchelator (http://maxchelator.stanford.edu/) indicate that Ca 2+ buffering capacity of EGTA is highly pH dependent, and raising pH by one unit increases EGTA binding affinity to Ca 2+ two orders in magnitude, changing K d from 1.28 × 10 −7 M at pH 7.3 to 1.4 × 10 −9 M at pH 8.3, whereas pH dependence of Ca 2+ buffering by BAPTA is weak.
The observations reported here which show strong increase in I CRAC amplitude in response to NH 4 Cl application to the bath when EGTA is used in the pipette, and virtual absence of such effect when intracellular Ca 2+ is buffered with BAPTA, suggest that the Ca 2+ binding properties of EGTA play a significant part in I CRAC pH i dependence in the presence of EGTA, particularly, when pH i rises above 7.5. The increase in I CRAC amplitude at alkaline pH i is likely to be due to stronger and faster Ca 2+ binding by EGTA, rather than increase in pH i per se. pH dependence of EGTA Ca 2+ binding properties creates unwanted complications for the interpretation of the experimental results. However, many physiological intracellular Ca 2+ buffers are likely to exhibit strong pH dependence, similarly to EGTA 23,24 . This is supported by the observations that intracellular alkalinisation induced by application of NH 4 Cl to the bath in Ca 2+ imaging experiments, when cells have endogenous intracellular Ca 2+ buffering, potentiates store-operated Ca 2+ entry in platelets and HT-29 cells 21,25 . Therefore, results obtained using EGTA, rather than BAPTA, may have more physiological relevance. Much bigger amplitude of I CRAC activated by IP 3 in the presence of BATPA in the pipette solution, compared to EGTA, was noticed very early on 19. However, the reason for this difference remains poorly understood.
The only residue that has been implicated in I CRAC dependence on pH i so far is His 155 in Orai1 10 . H155F mutation in Orai1 was shown to abolish the increase of I CRAC amplitude in response to intracellular alkalinisation, but I CRAC mediated by H155F-Orai1 was still inhibited by about 60% at low pH i 10 , which implies that His 155 is unlikely to be the only site that mediates I CRAC regulation by pH i . Data presented in this work indicate that Glut 106 in the Orai1 selectivity centre, which can be protonated from the extracellular side 9 , does not contribute to pH i dependence at all, which also suggests that Orai1 pore is not permeable to protons. Presence of seven negatively charged residues within ID STIM and the fact that neutralisation of three of them, D476, D478, and D479, significantly reduced the FCDI, similarly to acidic pH i , made ID STIM a good candidate for the pH i sensor of CRAC channel 18 . The results presented here indicate that ID STIM is not involved in pH i dependence of the I CRAC amplitude, but mutations in ID STIM affect pH i regulation of I CRAC Ca 2+ dependent gating. The kinetics of I CRAC mediated by Orai1/EE482/483AA-STIM1 or Orai1/DD475/476AA-STIM1 was not appreciably affected by either acidic, or alkaline pH i . It is unlikely, however, that protonation/deprotonation of negatively charged resides in ID STIM is responsible for the changes in I CRAC kinetics induced by the changes in pH i . Neutralisation of aspartates 482 and 483 increases FCDI, so protonation of these aspartates alone cannot be responsible for reduced FCDI at acidic pH i . It has been shown previously that neutralization of these aspartates together with glutamates in ID STIM reduce FCDI 18 , i.e. the effect of neutralisation of glutamates overcomes the effect of neutralisation of aspartates. This suggests that if glutamates in the ID STIM were protonated at acidic pH i , Orai1/EE482/483AA-STIM1 would display dependence of kinetics on pH i similar to that of WT I CRAC . However, this was not the case, which excludes ID STIM as a direct pH i sensor.
Interestingly, EE482/483AA-STIM1 significantly diminished the dependence of I CRAC kinetics not only on pH i , but also on the relative expression levels of STIM1 and Orai1. This could've been a result of saturating levels of expression of the mutant STIM1, compared to Orai1. If the expression levels of STIM1 are very high, moderate changes in the affinity of STIM1 biding to Orai1 due to changes in pH i , or moderate increase in Orai1 expression, are unlikely to have an appreciable effect on I CRAC kinetics. However, when the amounts of STIM1 are close to saturating, 2-APB does not potentiate I CRAC 16 . Application of 2-APB to Orai1 EE482/483AA-STIM1 mediated I CRAC caused the same level of potentiation as in WT I CRAC at all intracellular pH tested. This indicates that expression levels of mutant STIM1 were not different from that of WT STIM1, and that pH i affected functional coupling of Orai1 with mutant STIM1 in the same way it has affected it's functional coupling with WT STIM1. The lack of the dependence of Orai1 EE482/483AA-STIM1 I CRAC kinetics on the Orai1:STIM1 relative expression ratio and pH i suggests that the minimum number of this mutant STIM1 peptides which is needed to open Orai1 pore, is sufficient to support fully functional I CRAC FCDI.
In conclusion, the results presented here support the hypothesis that I CRAC inhibition by intracellular acidification is caused by disruption of functional coupling of STIM1 and Orai1, whereas the increase in I CRAC amplitude at alkaline pH i in the presence of EGTA is mainly due to increased Ca 2+ buffering capacity of EGTA. Negatively charged ID STIM is not a direct pH i sensor, but mutations neutralising negative charges in ID STIM affect pH i dependence of I CRAC kinetics by changing the interaction between STIM1 and Orai1.

Methods
Cell culture and transfections. HEK-293T cells [human embryonic kidney-293 cells expressing the large T antigen of SV40 (simian virus 40)] (A.T.C.C. CRL 11268) were cultured at 37 °C in 5% (v/v) CO 2 in air in DMEM (Dulbecco's modified Eagle's medium) supplemented with 100 μM nonessential amino acids, 2 mM L-glutamine and 10% fetal bovine serum 9,16 . To co-express WT Orai1 with WT STIM1 or double STIM1 mutants (EE482/483AA and DD475/476AA), cells seeded on glass cover slips were transfected using Polyfect (Qiagen) . The pH i dependence of I CRAC potentiation by 2-APB. The Y-axis represents the ratio between the amplitudes of I CRAC recorded immediately after and before application of 50 µM 2-APB to the bath. The I CRAC amplitude was measured at −100 mV from the responses to 100 ms voltage ramps from −120 to 120 mV, applied every 2 seconds. HEK 293 T cells were transfected with WT STIM1 and Orai1 (a) or EE482/3AA STIM1and Orai1(b) at 4:1 ratio. pH of the pipette solution is indicated below the bars.
Patch clamping. Whole-cell patch clamping was performed at room temperature (23 °C) using a computer based patch-clamp amplifier (EPC-9, HEKA Elektronik) and PULSE software (HEKA Elektronik) as previously described 9,16 . The control bath solution contained 140 mM NaCl, 4 mM CsCl, 10 mM CaCl 2 , 2 mM MgCl 2 and 10 mM HEPES adjusted to pH 7.4 with NaOH. Depletion of intracellular Ca 2+ stores was achieved using 20 μM Ins(3,4,5)P3 (Sigma) added to an internal solution containing 130 mM caesium glutamate, 10 mM CsCl, 5 mM MgCl 2 , 1 mM MgATP, 10 mM EGTA and either 10 mM MES adjusted to pH 6.3 with NaOH, or 10 mM HEPES adjusted to pH 7.3 or 8.3 with NaOH. Patch pipettes were pulled from borosilicate glass and fire polished to give a pipette resistance between 2 and 4 MΩ. Series resistance did not exceed 15 MΩ and was 50-70% compensated. Traces obtained before activation of I CRAC , or after its inhibition with 10 µM La 3+ were used for leakage subtraction.
Data analysis. To obtain apparent (relative) open probability (P o ) curves of CRAC channels, instantaneous tail currents recorded in response to voltage steps to −100 mV after test pulses between −140 and 80 mV, applied every 5 s in 20 mV increments, were normalised to the amplitude of the instantaneous tail current recorded after test pulse to 80 mV and plotted against corresponding test pulse voltage 9 . The length of the test pulses was set to 150 ms to make sure that both gating processes of I CRAC -inactivation and re-activation are captured in one protocol. Were possible, the data points were fitted with the Boltzmann distribution with an offset of the form: where P min is an offset, V is the membrane potential, V 1/2 is the half-maximal activation potential (V 1/2 corresponds to the inflexion point of the P o curve) and k is the slope factor. However, in many cases apparent P o data could not be fitted with Boltzmann distribution and the data points were fitted with a smooth curve using cubic spline procedure in Prizm 6 software.