To better understand the molecular mechanisms underlying F99 gate opening, we began our studies by carefully mapping the movements of F99 in molecular dynamics (MD) simulations of gain-of-function (GOF) and loss-of-function (LOF) dOrai mutants (Figure 2). Although there are now several available structures of activating mutants (Hou et al., 2018; Liu et al., 2019), we performed simulations using the 3.35 Å crystal structure of Drosophila melanogaster Orai ([PDB ID:4HKR]; Hou et al., 2012) because it remains the most complete, highest-resolution structure and has been utilized in previous MD studies (Bonhenry et al., 2019; Dong et al., 2019). Further, given the high sequence identity between dOrai and human Orai1 (hOrai1) within the transmembrane domains, channel phenotypes identified using MD simulations in dOrai are likely to be translatable to electrophysiological results in hOrai1.

For our simulations, we took advantage of mutations at a well-studied gating locus, H134 on TM2 (equivalent to dOrai H206), to model inactive, partially active, or fully active channels. The histidine side-chain of H134 faces the non-pore-lining surface of TM1 and has been suggested to stabilize the closed channel state by acting as a steric brake at the TM1-TM2/3 ring interface (Yeung et al., 2018; Figure 2—figure supplement 1A). Previous studies have described several interesting GOF or LOF mutations at hOrai1 H134 that either inhibit or constitutively activate Orai1 gating (Frischauf et al., 2017; Yeung et al., 2018). Compared to WT channels, the LOF mutant hOrai1 H134Y (dOrai H206Y) conducts small, non-selective Orai1 currents when co-expressed with STIM1, reflecting a defect in STIM1-mediated gating (Yeung et al., 2018). A second mutant, hOrai1 H134Q (dOrai H206Q), is modestly active at rest but can be further gated by STIM1. By contrast, a third mutant, hOrai1 H134C (dOrai H206C), is one of the most strongly active variants, displaying similar properties as STIM1-gated channels even without STIM1, including inward-rectifying, Ca²⁺-selective currents and displacement of the F99 gate (Bulla et al., 2019; Frischauf et al., 2017; Yeung et al., 2018). The channel activities of these mutants, as measured by electrophysiology (Figure 2—figure supplement 1B), correlate well with metrics such as the extent of pore helix rotation, outer pore dilation, and pore hydration observed in MD simulations (Figure 2—figure supplement 2B; [see also Yeung et al., 2018]).

Within these dOrai H206 simulations, we looked for conformational changes in the TM1-TM3 hydrophobic cluster that correlated with pore opening for potential clues on the molecular mechanism of channel gate opening. The time trajectories of dOrai across all variants showed that most of the residues in the hydrophobic stack display spatial fluctuations of small amplitude, consistent with their role as a rigid relay from TM3 to TM1. However, intriguingly, in simulations of the active variants H206Q/C (equivalent to hOrai1 H134Q/C), the M173 (hOrai1 M101) side-chain was uniquely flexible and reversibly sampled rotamers that deviated from the crystal structure (Figure 2C,D, Figure 2—video 2). We observed that the flexibility of M173 allowed it to interact with the hydrophobic gate residue, F171 (hOrai1 F99) of neighboring subunits (Figure 2C,D). Specifically, the M173-F171 distance often reached 4–6 Å (Figure 2E), sufficiently close for direct contact between the two side-chains. This finding is notable as sulfur-aromatic interactions involving Met and Phe residues are known to play distinctive roles in protein function, often by stabilizing protein structures (Valley et al., 2012; Weber and Warren, 2019). Because the M173-F171 interaction was preferentially seen only in the activating mutants and not in WT or LOF mutants (Figure 2A–D, Figure 2—videos 1 and 2), we considered the possibility that this interaction may promote the opening of the channel gate in the activating mutants.

To investigate this hypothesis, we examined how interactions between M173 and F171 correlated with metrics of channel activity in WT, H206Y, H206Q, and H206C channels (Figure 2G–K). Previous studies have concluded that sulfur-aromatic interactions occur at distances less than 7 Å, with a peak distance of ≈ 5 Å (Gómez-Tamayo et al., 2016; Valley et al., 2012). Therefore, we quantified the interactions between M173 (hOrai1 M101) with the hydrophobic gate F171 (hOrai1 F99) as well as with its normal ‘resting state’ partner in the hydrophobic stack, F259 (hOrai1 F187 in TM3), by measuring distances between the sulfur atom on M173 and the center of mass of the aromatic rings of F171 and F259 (Figure 2E,F). This analysis showed that the distance between M173 and F259 oscillated between 4 and 7 Å, with an average value of 5.46 ± 0.04 Å (WT), 5.38 ± 0.05 Å (H206Y), 5.65 ± 0.06 Å (H206Q), and 5.65 ± 0.04 Å (H206C). These values indicate that the interaction of the M173 side-chain with F259 within the TM1-TM3 hydrophobic stack is relatively stable and maintained across open and closed variants (Figure 2F).

By contrast, the distance between M173 and F171 showed much larger variability and depended on channel activity. In closed WT and LOF H206Y (hOrai1 H134Y) channels, there was a broad distribution of F171-M173 distances from 5 Å to 10 Å (Figure 2A,B,E). In the activated channels H206Q and H206C, however, the distribution was markedly shifted toward shorter distances, with a peak between 4 and 7 Å, centered at 6.6 ± 0.1 Å for H206Q and 6.4 ± 0.2 Å for H206C (Figure 2C–E). These distances imply the presence of a stable sulfur-aromatic interaction between M173 and F171 in the open variants. Importantly, the M173-F171 interaction distance was correlated with several metrics of channel activation, including pore helix rotation, pore hydration, and pore diameter across all dOrai variants (Figure 2G–K), with shorter interaction distances most strongly associated with F171 rotation. The increased magnitude of counterclockwise angular rotations of F171 C_α in the activated mutants also correlated well with those of M173, suggesting that as the channel opens, M173 rotates along with F171 to enable it to contact F171 on the adjacent helix (Figure 2—figure supplement 2D). Together, these results suggest that whereas M173 interacts with F259 in both open and closed channels, it interacts with F171 only in open states. This pattern of M173 interactions seen in the closed and open mutants suggests that M173 (hOrai1 M101) acts as a bridge between F259 (hOrai1 F187) and F171 (hOrai1 F99) in the adjacent protomer to help stabilize the rotated F171 gate in the open state. The energetic contribution of the M173-F171 interaction for Orai gating will be analyzed in the Discussion section.

While the Met-Phe interactions described above may be mediated by side-chain fluctuations, rigid body motions of the TM1 backbone may also contribute to these interactions. To assess this possibility, we quantified the distances between the C_β of M173 and the center of the F171 and F259 rings in the different H206 mutants (Figure 2—figure supplement 2E,F). Compared to the sulfur atom distances, C_β measurements are likely to be driven more by pore helix rotation because the M173 C_β is closer to the center of the TM1 helix. In WT and H206Y channels, M173 is predominantly facing TM3 as part of the TM1-TM3 hydrophobic clamp, so that C_β is closer to F259 and farther away from F171. However, in the activated states of the H206Q/C channels, the TM1 rotation angle distribution displayed an additional peak corresponding to distances closer to F171 and farther from F259 (Figure 2—figure supplement 2E,F). Interestingly, analysis of the M173 C_β-F259 distances revealed clear differences between closed (WT and H206Y) and open (H206C and H206Q) channel states (Figure 2—figure supplement 2F). This result suggests that the TM1 helix is sufficiently mobile to display conformational alterations at the M173 C_β position between open and closed states. By contrast, as already indicated above, no differences were seen in the distance measured from the sulfur atom to the F259 ring (Figure 2F), indicating that the M173 side-chain exhibits sufficient flexibility and that the sulfur atom is intrinsically drawn toward the F259 ring.

The finding that M173-F171 (hOrai1 M101-F99) interactions are augmented in MD simulations of active channel states led us to consider whether artificially forcing an interaction between M101 and F99 in hOrai1 via a metal-ion bridge can directly activate the channel. Metal-ion bridges in double cysteine mutants have been exploited in many studies of ion channel gating to probe the conformational changes underlying gating and to stabilize channels in specific states (Holmgren et al., 1996; Li et al., 2010; Loussouarn et al., 2001; McNally et al., 2012; Puljung and Zagotta, 2011; Rulísek and Vondrásek, 1998). We therefore introduced cysteines at F99 and M101 in hOrai1 and examined the effects of applying the thiol-reactive divalent ion Cd²⁺ in F99C/M101C Orai1 channels overexpressed in HEK293-H cells without STIM1 (Figure 3A).

F99C/M101C channels are partially open at baseline, producing non-selective CRAC currents (V_rev = −5.4 ± 2.7 mV) in line with previous results indicating that the F99C mutation disrupts the hydrophobic barrier in the pore (Yamashita et al., 2017). Strikingly, Cd²⁺ administration dramatically enhanced this F99C/M101C current with a slow time course over tens of seconds (Figure 3C,E,F). Over the 40 s application of 5 µM Cd²⁺, the non-selective F99C/M101C Orai1 current increased 12-fold over the baseline current (Figure 3C,D). Washout of Cd²⁺ by 20 mM Ca²⁺ Ringer’s solution caused an additional rapid increase in the current (Figure 3C,E,F). Previous reports have shown that application of Cd²⁺ to the single F99C mutant results in strong blockade of Orai1 currents when F99C residues are oriented in a pore-facing configuration (McNally et al., 2009; Yamashita et al., 2017). Thus, we reasoned that the rapid current increase in the F99C/M101C double mutant following washout of Cd²⁺ arises from the removal of Cd²⁺ block by the permeating Ca²⁺ ions. The Cd²⁺-mediated potentiation was stable and could only be reversed by reducing agent bis(2-mercaptoethylsulfone) (BMS) (Figure 3C,E,F), indicating that the trapping of Cd²⁺ between the introduced cysteine residues is destabilized only by directly disrupting the coordinating thiol groups.

WT and single cysteine mutants F99C and M101C did not exhibit Cd²⁺-dependent current enhancement (McNally et al., 2009), indicating that cysteines at both positions are required for the potentiation effect. Further, when STIM1 was co-expressed, the extent of Cd²⁺-induced current potentiation was dramatically reduced (Figure 3D). Because STIM1 stabilizes F99 is an activated state with F99 facing away from the pore and toward M101, we reasoned that the additional gating induced by a Cd²⁺ bridge between F99C and M101C is diminished in the STIM1-bound state of Orai1. However, one caveat to this interpretation is that STIM1 does not activate the F99C/M101C channels strongly (I = −1.49 pA/pF at baseline versus −3.17 pA/pF following STIM1 binding). Thus, we cannot formally out that the smaller degree of STIM1-activated Cd²⁺ potentiation of F99C/M101C channels is due to unknown indirect effects such as stabilization of the channel in an intermediate state where the Cd²⁺-binding site is obscured. Nevertheless, the robust potentiation of F99C/M101C activity in STIM1-free channels is strong indication of the importance of M101-F99 interaction for pore opening.

Increasing the Cd²⁺ dose from 5 µM to 500 µM dramatically increased the extent of channel potentiation in F99C/M101C channels, up to 150 times the baseline amplitude at 300 μM Cd²⁺ (Figure 3E,G). However, even at 500 µM Cd²⁺ potentiation did not reach saturation. Further increases could not be quantified because Cd²⁺ precipitated out of the 20 mM Ca²⁺ Ringer’s solution at higher concentrations. In order to distinguish whether the Cd²⁺ potentiation site is close to the pore or away from it, we varied the permeant ion concentration, reasoning that a site located in or close to the pore would be highly sensitive to the concentration of permeant ions. Specifically, current potentiation is expected to decline at higher concentrations of permeant Ca²⁺ ions due to a knockoff effect of Cd²⁺ by Ca²⁺ ions. As predicted for a Ca²⁺-permeable channel, F99C/M101C channels conducted more baseline current when the external Ca²⁺ concentration was raised from 20 to 110 mM Ca²⁺ (Figure 3F). However, when applied in the 110 mM Ca²⁺ solution, Cd²⁺ induced significantly less current potentiation than that in 20 mM Ca²⁺ solution (Figure 3F,G), indicating that raising the permeant ion concentration diminishes Cd²⁺-induced gating. Based on this result, we conclude that Ca²⁺ flux in the pore competes with Cd²⁺ binding at its potentiation site and thus the Cd²⁺ binding site is located close to the pore where it is influenced by permeant ions.

To test whether the gating induced by Cd²⁺ is also seen with other thiol-reactive probes, we tested the effects of Zn²⁺, a slightly smaller metal ion that, like Cd²⁺, can also coordinate with thiol groups (Yellen et al., 1994). Administration of Zn²⁺ also elicited potentiation of Orai1 current (Figure 3—figure supplement 1), suggesting that the potentiation effect is not unique to Cd²⁺ but arises due to bridging of F99C to M101C via a thiol-reactive divalent ion. These results are consistent with the hypothesis that the M101-F99 interaction stabilizes the open state.

If stabilizing a M101-F99 interaction augments channel activation, does facilitating the resting state interaction between M101 and F187 (as seen in the closed channel crystal structure) favor the closed state? We tested this idea using the hOrai1 M101C/F187C mutant. We have previously shown that Orai1 F187C is a Ca²⁺-selective, constitutively active mutant (Yeung et al., 2018). Similarly, the M101C/F187C double cysteine mutant was also constitutively active and highly Ca²⁺ selective (Figure 4B). Strikingly, when 5 μM Cd²⁺ was applied to M101C/F187C, the current was almost completely inhibited (Figure 4B). This current decrease was uniform across the voltage ramp (V_rev = 41.0 ± 6.3 mV without Cd²⁺ and V_rev = 35.8 ± 8.9 mV with 5 µM Cd²⁺; p=0.65), which suggests that it arises from channel inhibition rather than pore block. In addition, the reversibility of the effect by BMS is consistent with that of a cysteine-mediated mechanism (Figure 4B). The rapid Cd²⁺-induced inhibition was only seen in the double Cys mutants. Neither the single F187C nor double M101A/F187C and M101C/H134S mutants showed any effect with Cd²⁺ (Figure 4C), indicating that cysteines are required at both F187 and M101 for inducing channel closure.

In contrast to the strong dependence on the permeant ion concentration seen for the Cd²⁺-mediated potentiation of F99C/M101C channels, Cd²⁺-induced channel closure in the M101C/F187C mutant was largely insensitive to the permeant ion concentration. Specifically, Cd²⁺ blockade in 110 mM Ca²⁺ Ringer’s was not notably different than that seen in 20 mM Ca²⁺ solution (Figure 4E,F), suggesting that the inhibition site is likely not in the pore. This conclusion is also supported by the strikingly different Cd²⁺ sensitivities of Cd²⁺ inhibition on M101C/F187C channels versus potentiation in F99C/M101C channels. Whereas inhibition in M101C/F187C channels quickly reached saturation with increasing Cd²⁺ with an apparent K_d of ~0.3 μM (Figure 4F), potentiation in the F99C/M101C channels did not reach saturation even at 500 µM Cd²⁺ (Figure 3G). The latter effect is likely due to a knockoff effect of the permeant Ca²⁺ ions on Cd²⁺ occupancy at the potentiation site between F99C and M101C. Application of Zn²⁺ also induced channel closure in M101C/F187C channels, suggesting that the inhibition effect is generalizable to other thiol-reactive metals (Figure 3—figure supplement 1). We conclude that stabilizing the resting interaction between M101 and F187 evokes pore closure, presumably by releasing the F99 gate into its pore-facing configuration (Figure 4A).

If the sulfur-aromatic interaction is critical for gate opening, then mutations of M101 to other amino acids that cannot support sulfur-aromatic interactions should disrupt channel function. Consistent with this hypothesis, nearly every substitution that we tested, including M101G/A/S/T/C/V/L/I, yielded LOF channels that lost gating by STIM1 (Figure 5A,B). Notably, mutations of M101 to Leu or Ile, which have comparable or even greater hydrophobicity as the native Met, also produced channels that could not be activated by STIM1 (Figure 5A,B). The plasma membrane fluorescence of Orai1-YFP was similar across the mutants, indicating that reduced channel expression is not the cause of the smaller currents. Moreover, with the exception of some polar and charged substitutions (M101Q/N/D/K/E), no difference was seen in co-localization of the YFP-tagged Orai1 mutants with CFP-CAD nor in E-FRET between the two proteins (Figure 5—figure supplement 1), indicating that loss of gating was not due to gross changes in the ability of the Orai1 variants to interact with STIM1. Introduction of the M101L mutation into the constitutively active H134S and constitutively conducting V102C mutants also attenuated currents in these mutants (Figure 5—figure supplement 2). This latter result in V102C is not entirely unexpected since previous studies have indicated that the V102C mutant also shows greater spontaneous counter-clockwise fluctuations of the F99 gate region compared to WT channels, consistent with models showing that gate opening and pore hydration are intimately coupled in CRAC channels (Yamashita et al., 2017; Yeung et al., 2018).

The one exception to the LOF effect induced by mutations at M101 was the Phe mutant, which surprisingly produced a GOF phenotype. M101F was not only normally gated by STIM1 (Figure 5A), but was also modestly active and Ca²⁺-selective (V_rev = 44.9 ± 5.7 mV) at rest in the absence of STIM1 (Figure 5C). Interestingly, the M101F substitution was able to confer channel activity to LOF mutant H134W (Figure 5—figure supplement 2) which cannot be gated by STIM1 (Yeung et al., 2018), suggesting that the effect of M101F is directly on the F99 gate. As will be discussed further below, we postulate that the constitutive activity of M101F likely arises from interactions of the introduced Phe side-chain with F99 and F187 created by the introduced aromatic ring at M101. Collectively, these M101 mutants demonstrate that hydrophobicity alone is not enough for channel function and reaffirm the conclusion that the sulfur-containing Met residue at position 101 mediates a specialized function in Orai1 gating in addition to its role in the hydrophobic cluster.

In contrast to the LOF Orai1 phenotypes of mutations at M101, mutation of F187 to smaller residues including Gly, Ala, Ser, and Cys produced GOF channels with inwardly rectifying CRAC channel-like current-voltage relationships. Large and hydrophobic F187 substitutions including Tyr and Trp, on the other hand, remained closed in the absence of STIM1 (Figure 5—figure supplement 3). Based on these results, we conclude that the large, hydrophobic side-chain of F187 on TM3 is required for stabilizing the closed state of the channel. However, because M101C channels are not constitutively open, we surmise that F187 may prevent spontaneous pore opening through mechanisms beyond its interaction with M101, potentially involving other residues of the TM1-TM3 hydrophobic stack. Without additional these interactions, the pore transitions into a Ca²⁺-selective state that is similar to the one evoked by STIM1 binding.

The experimental results showing that loss of the sulfur-aromatic gate latch compromises channel gating led us to hypothesize that M101 LOF mutations stabilize the F99 gate in the closed pore-facing orientation, resulting in diminished pore hydration at the channel gate. To address this hypothesis, we performed MD simulations of dOrai M173L (LOF hOrai1 M101L) and dOrai M173F (GOF hOrai1 M101F) to investigate the effects of the mutations on pore hydration and orientation of the F99 gate. These simulations revealed that the M173L channel displayed significantly smaller spontaneous counter-clockwise pore helix rotations than the WT channel (Figure 6D), with the angular position of F171 shifted by 10 ± 1° for M173L channels and 17 ± 1° for WT channels as measured with respect to the crystallographic orientation of the F171 side-chain in our coordinate system (30.6°, [PDB ID:4HKR]; Hou et al., 2012). The decrease in pore helix rotation of M173L was accompanied by markedly lower hydration of the hydrophobic stretch of the pore (Figure 6B). By contrast, M173F channels, which are modestly active in the absence of STIM1 (Figure 5C), showed a statistically significant increase in the extent of pore helix rotation relative to WT (Figure 6D) and a similar level of pore hydration as WT channels (Figure 6B).

Examination of the simulation trajectories showed that the introduced Leu, which is both shorter and more hydrophobic than the native Met, spends the majority of the simulation time in the hydrophobic cluster at the TM1-TM3 interface and away from F171, suggesting that it cannot interact with F171 to facilitate pore opening (Figure 6—video 1). Quantification of the M173L-F171 distance as measured from the C_β atom of M173 indicated a subtle but clear shift towards larger distances in this variant. This shift was accompanied by an increase in the interaction propensity between M173L and F259 (Figure 6E,F and Figure 6—video 1). By contrast, in simulations of M173F channels, we observed that the Phe side-chain, which is shorter and less flexible than the native Met at position 173, results in a bridge between F171 and the TM3 residue F259, causing F171 to be displaced away from the pore and increasing pore helix rotation (Figure 6—video 2). We conclude that the M173L mutation stabilizes F99 in a pore-facing configuration, and that the resulting decrease in pore hydration relative to WT channels in the hydrophobic stretch contributes to the LOF phenotype of the M101L mutant.

HEK293-H cells (Thermo Fisher Scientific) were maintained in suspension at 37°C with 5% CO₂ in CD293 medium supplemented with 4 mM GlutaMAX (Invitrogen). The HEK293 cell line is a permanent line established from primary embryonic human kidney and transformed with sheared human adenovirus type 5 DNA. The E1A adenovirus gene is expressed in these cells to optimize protein production. HEK293-H cells were cloned from the original 293 cell line and adapted to CD293 serum-free medium for growth in suspension. Cell line identity has been authenticated by ThermoFisher Scientific, and cells were tested negative for mycoplasma by qPCR detection assay. For imaging and electrophysiology, cells were plated onto poly-L-lysine coated coverslips one day before transfection and grown in a medium containing 44% DMEM (Corning), 44% Ham’s F12 (Corning), 10% fetal bovine serum (HyClone), 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin.

The Orai1 mutants employed for electrophysiology were engineered into a pEYFP-N1 vector (Clontech) to produce C-terminally tagged Orai1-YFP proteins (Navarro-Borelly et al., 2008). mCherry-STIM1 and CFP-CAD were kind gifts of Dr. R. Lewis (Stanford University, USA). All mutants were generated by the QuikChange Mutagenesis Kit (Agilent Technologies) and the mutations were confirmed by DNA sequencing. For electrophysiology, the indicated Orai1 constructs were transfected into HEK293-H cells either alone (200 ng DNA per coverslip) or together with STIM1 (100 ng Orai1 and 500 ng STIM1 DNA per coverslip). For FRET and confocal microscopy experiments, cells were transfected with Orai1-YFP alone (200 ng DNA per coverslip) or with CFP-CAD constructs (100 ng each per coverslip). All transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific) 24–48 hr prior to electrophysiology or imaging experiments.

The standard extracellular Ringer’s solution used for electrophysiological experiments contained 130 mM NaCl, 4.5 mM KCl, 20 mM CaCl₂, 10 mM tetraethylammonium chloride (TEA-Cl), 10 mM D-glucose, and 5 mM HEPES (pH 7.4 with NaOH). For the FRET and confocal imaging studies, the Ringer’s solution contained 2 mM CaCl₂ and 150 mM NaCl with the other components as above. The 110 mM Ca²⁺ solution contained 110 mM CaCl₂, 10 mM D-glucose, and 5 mM HEPES (pH 7.4 with NaOH). The DVF Ringer’s solution contained 150 mM NaCl, 10 mM HEDTA, 1 mM EDTA, 10 mM TEA-Cl and 5 mM HEPES (pH 7.4). The internal solution contained: 135 mM Cs aspartate, 8 mM MgCl₂, 8 mM Cs-BAPTA, and 10 mM HEPES (pH 7.2 with CsOH).

Currents were recorded in the standard whole-cell configuration at room temperature on an Axopatch 200B amplifier (Molecular Devices) interfaced to an ITC-18 input/output board (Instrutech). Routines developed by R. S. Lewis (Stanford) on the Igor Pro software (Wavemetrics) were employed for stimulation, data acquisition and analysis. Data are corrected for the liquid junction potential of the pipette solution relative to Ringer’s in the bath (−10 mV). The holding potential was +30 mV. The standard voltage stimulus consisted of a 100 ms step to –100 mV followed by a 100 ms ramp from –100 to +100 mV applied at 1 s intervals. I_CRAC was typically activated by passive depletion of ER Ca²⁺ stores by intracellular dialysis of 8 mM BAPTA. All currents were acquired at 5 kHz and low pass filtered with a 1 kHz Bessel filter built into the amplifier. All data were corrected for leak currents collected in 100–200 µM LaCl₃.

Analysis of current amplitudes was typically performed by measuring the peak currents during the −100 mV pulse. Specific mutants were categorized as gain-of-function if their currents exceeded 2 pA/pF, which is more than ten times the current density of WT Orai1 without STIM1. Reversal potentials were measured from the average of several leak-subtracted sweeps in each cell. Fractional inhibition of current was quantified as: Inhibition=(1-I_b/I_Ctrl), where I_b is the Orai1 current in the presence of Cd²⁺, and I_Ctrl is the Orai1 current prior to application of the blocker (Cd²⁺).

HEK293-H cells transfected with Orai1-YFP and CFP-CAD DNA constructs were imaged using wide-field epifluorescence microscopy on an IX71 inverted microscope (Olympus, Center Valley, PA). Cells were imaged with a 60X oil immersion objective (UPlanApo NA 1.40), a 175 W Xenon arc lamp (Sutter, Novatao, CA), and excitation and emission filter wheels (Sutter, Novato, CA). At each time point, three sets of images (CFP, YFP, and FRET) were captured on a cooled EM-CCD camera (Hamamatsu, Bridgewater, NJ) using optical filters specific for the three images as previously described. Image acquisition and analysis was performed with SlideBook software (Imaging Innovations Inc, Denver, CO). Images were captured at exposures of 100–500 ms with 1 × 1 binning. Lamp output was attenuated to 25% by a 0.6 ND filter in the light path to minimize photobleaching. All experiments were performed at room temperature.

FRET analysis was performed as previously described (Navarro-Borelly et al., 2008). The microscope-specific bleed-through constants (a = 0.12; b = 0.008; c = 0.002 and d = 0.33) were determined from cells expressing cytosolic CFP or YFP alone. The apparent FRET efficiency was calculated from background-subtracted images using the formalism (Zal and Gascoigne, 2004):EFRET=FcFc+GIDDwhere F_c = I_DA aIAA - dI_DD.

I_DD, I_AA, and I_DA refer to the background subtracted CFP, YFP, and FRET images, respectively. The instrument dependent G factor had the value 1.85 ± 0.1. E-FRET analysis was restricted to cells with YFP/CFP ratios in the range of 2–6 to ensure that E-FRET was compared across identical acceptor to donor ratios, and measurements were restricted to regions of interest drawn at the plasma membrane.

HEK293-H cells expressing various Orai1-YFP mutants and CFP-CAD were imaged on an Andor XDI Revolution spinning-disk confocal microscope equipped with a 100X oil immersion objective. Cells were maintained at 37°C with 5% CO₂. Fluorophores were excited with 445 nm (CFP) and 515 nm (YFP) laser diodes with the intensity of laser light attenuated to 15–40% for CFP and 5–30% for YFP. Images were obtained at 512 × 512 pixels at an exposure of 200–500 ms per frame and a slice thickness of 0.8 μm. An average of four frames was used for each image. Images analysis was performed using NIH ImageJ software (NIH, Bethesda, MD).

Molecular models were constructed using the crystal structure of the Drosophila melanogaster Orai1 channel (4HKR) (Hou et al., 2012). Missing residues of the M1-M2 loop (amino acids 181 to 190) and the M2-M3 loop (amino acids 220 to 235) were modeled de novo using MODELLER (Fiser and Sali, 2003). System preparation was performed using CHARMM-GUI membrane builder (Jo et al., 2007). The C terminus was truncated at residue 329 for all chains and the N and C terminus were acetylated and amidated, respectively. The protein was embedded within a hydrated 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer with 150 mM NaCl to obtain a hexagonal cell with box vectors 104.2 × 104.2×126.5 Å. Pore waters were not modeled. The simulation cell consisted of ~112K atoms. Single point mutations were made using CHARMM-GUI to create the H206Y, H206Q, H206C, M173L, and M173F systems. The CHARMM36 force field was used for protein (Best et al., 2012; MacKerell et al., 1998), ions, and lipids (Klauda et al., 2010) along with the TIP3P water model (Jorgensen et al., 1983).

All simulations were performed using GROMACS 2016.3 (Murtola et al., 2015) without modification to the CHARMM-GUI output parameters (with the exception of additional equilibration steps and extended production simulation length). Lennard-Jones interactions were cut off at 1.2 nm and a force-based switching function with a range of 1.0 nm was used. Electrostatic interactions were calculated using particle-mesh Ewald (Darden et al., 1993; Essmann et al., 1995) with a real-space cut-off distance of 1.2 nm. Nonbonded interactions were calculated using Verlet neighbor lists (Páll and Hess, 2013; Verlet, 1967). All simulations were performed at constant temperature (323.15 K) and pressure (one atm) using the Nosé-Hoover thermostat (Hoover, 1985; Nosé, 1984) with temperature coupling of 1.0 ps and the Parrinello-Rahman barostat (Nosé and Klein, 1983; Parrinello and Rahman, 1980) with a time constant of 5.0 ps, respectively. All hydrogen bonds were constrained using the LINCS algorithm (Hess, 2008). The integration time step was two fs. All systems followed the standard energy minimization and six-step equilibration procedure of CHARMM-GUI (Jo et al., 2007), followed by two successive 10 ns protein-restrained simulations conducted in the NPT ensemble. In these equilibration steps, position restraints were applied to main chain backbone atoms and then Cα atoms, with restraint strength of 1000 kJ mol⁻¹ nm⁻². Twenty simulation repeats were created for WT, H206Q/C, ten simulation repeats were created for H206Y, and thirty repeats were created for M173L/F systems with randomized initial velocities for production simulations. Production simulations were conducted for 400–500 ns for all simulation repeats for an aggregate total of 73.6 µs.

Prior to analysis, all simulation frames were aligned such that the principal axis formed by TM1 helix C_α atoms were aligned to the box vector z. Analysis was performed on all simulation frames spaced at 1.0 ns after removing the first 100 ns of data from each simulation repeat. All axial coordinates were measured with respect to the center of mass of the pore helix C_α atoms (residues 141 to 174). Axial histograms of water oxygen atoms, Na⁺, and Cl^-, was computed within a cylinder of radius of 10 Å centered at the pore center of mass. Error bars were computed using the standard error of mean over all simulation repeats. Pearson correlation coefficients were computed using observables measured at all data points for all simulation repeats, with all subunit specific properties averaged across all six subunits. Potential mean force plots in Figure 7B were generated from the M173-F171 distance distributions, where the energy change is denoted as W(d)–W(0) and the equilibrium constant is represented by p(d)/p(d₀):Wd-W0=-RTlnp(d)p(d0)where p(d) is the probability distribution of the distance d between M173 and F171, d₀ is an arbitrary reference separation, R is the universal gas constant, and W(d) is the potential of mean force (PMF) or free energy at d.