A Quartet of Native Orai Channel Isoforms Orchestrates Graded NFAT Activation and Transcription

Reviewer #1 (Public review):

Summary:

This study quantifies the ability of the four isoforms of the calcium-release calcium-activated (CRAC) channel Orai to mediate calcium entry and transcriptional responses. By genetically invalidating each isoform and by separately re-expressing them in Orai-deficient human embryonic kidney cells, the authors show that the rates of calcium entry across the four native Orai calcium channel isoforms (Orai1α/β, Orai2, Orai3) correlate with the degree of NFAT activation. They further show that the two alternatively translated isoforms Orai1α and Orai1β are interchangeable, as their expression in Orai1-deficient primary mouse T cells induces identical cytokine responses and transcriptional programmes, and that individuals bearing frameshift mutations causing a loss of the Orai1α isoform do not exhibit immune, muscular, or dermatologic features and have preserved T cells' Ca2+ and transcriptional responses.

Strengths:

The data are of high quality, relying on clean cell line and mouse knockout models to link calcium entry rates directly to NFAT activation, and human data from homozygous and heterozygous frameshift mutation carriers confirm that Orai1α and Orai1β are functionally redundant in vivo.

Weaknesses:

An acknowledged limitation is that some conclusions depend on transient overexpression experiments. The authors should explicitly address whether Orai1α could possess non-redundant functions under unique physiological environments not captured by these assays.

Reviewer #2 (Public review):

Summary:

The authors aim to assess the differential role of Orai isoforms for mediating SOCE and NFAT1 and/or NFAT4 nuclear translocation primarily in the context of T cells. For this purpose, they have used genetically modified cells and appropriate human genetic conditions. All isoforms were expressed individually (with deletions of other isoforms) at roughly equivalent levels so that data across isoforms could be compared unambiguously. Their experiments convincingly identify Orai1 as the main driver of SOCE and NFAT1/4 translocation in the HEK293 cell line and in primary T cells. The two Orai1 isoforms appear functionally equivalent, and loss of the longer isoform (Orai1α) is compensated in humans by the presence of the shorter isoform (Orai1β).

Strengths:

Overall, their judicious use of appropriate knockouts, mutants and expression constructs allows for unambiguous interpretation of data regarding the key role of Orai1α and Orai1β in T cell physiology. They also demonstrate a role for Orai3 in driving SOCE in a breast cancer cell line.

Weaknesses:

The main shortcoming of this manuscript is its inability to place the findings in a context that would be of interest to a broader audience. It would be helpful to provide a metanalysis from existing public databases (human and/or murine) of the known expression in various cell types and tissues of Orai1α, Orai1β, Orai2 and Orai3. This could suggest possible roles for each Orai isoform in tissues other than immune cells. Further, the physiological relevance of the two NFAT isoforms studied, NFAT1 and NFAT4, needs some elaboration, both in the context of T cells and other tissues.

Reviewer #3 (Public review):

Summary:

The work by Abdelnaby et al. investigated the different Orai isoforms, and also especially Orai1 alpha and beta for NFAT 1 and NFAT 4 translocation, but also its impact in genetically modified T-cells. This is a very well-performed work that manages to monitor NFAT nuclear/cytosol ratio even in a time-dependent manner. Overall, this study has been very well performed, is clearly written and discusses the literature very accurately.

Strengths:

For monitoring NFAT translocation, the authors have managed to observe this pattern even in a time-dependent manner. The authors may like to describe how this analysis was done with more details, as I believe some software assistance was needed to distinguish the nuclear to cytosolic region for a huge number of cells. Clearly, this analysis provides a very clear picture of the time course of translocation and allows for a precise statistical analysis.

Remarkably, NFAT1 and NFAT4 translocation was not substantially different for Orai1 alpha- and Orai1 beta-mediated Ca2+ signals. This has also been observed previously by Zhang et al. for NFAT1 from the same laboratory. Clearly, this is in contrast to the work of reference 38 (Kar et al.). All these publications have been performed with a huge amount of data, and I believe that clearly stating this difference in results from previous work is an additional important aspect of this manuscript.

In line with the similar NFAT translocation efficiency of Orai1 alpha- or beta-containing cells, the authors carefully evaluated differentially expressed genes in CD4+ T cells. They did not find a substantial difference in these cells that were transduced with Orai1 alpha or beta - clear orthogonal evidence for a conserved function of Orai1 alpha and beta for transcriptional activation.

Weaknesses:

This reviewer identified no obvious weaknesses in this work.

Reviewer #4 (Public review):

Summary

Orai1, Orai2 and Orai3 are the pore-forming subunits of CRAC channels, and Orai1 itself exists as two N-terminally distinct translational isoforms, Orai1α and Orai1β. Despite well-documented differences in the biophysical properties of these four proteins (Ca²⁺-dependent inactivation, expression pattern, evolutionary conservation), it has been unclear whether these differences translate into distinct transcriptional outputs through the Ca²⁺-calcineurin-NFAT axis, or whether NFAT responses are simply scaled by the amount of Ca²⁺ that each channel variant lets through. The authors address this using HEK293 cell lines engineered by CRISPR/Cas9 to retain only a single native Orai homologue (OraiDKO lines) or none at all (Orai-TKO, reconstituted at near-native levels with a weak TK promoter), and extend their findings to primary murine CD4⁺ T cells lacking endogenous Orai1, as well as to human primary T cells and population genetic datasets from individuals carrying naturally occurring loss-offunction alleles that selectively eliminate Orai1α while sparing Orai1β. Across these systems, the authors report a consistent rank order of NFAT1/NFAT4 activation (Orai1β {greater than or equal to} Orai1α >> Orai2 > Orai3) that mirrors the rank order of SOCE amplitude, and show that a fast Ca²⁺ chelator (BAPTA) but not a slow one (EGTA) blocks NFAT1 activation, consistent with a requirement for local, channel-proximal Ca²⁺ signals rather than global cytosolic Ca²⁺ elevation. Human individuals homozygous for Orai1αselective null alleles are clinically unaffected and show normal or even enhanced SOCE/NFAT responses, contrasting sharply with the severe CRAC channelopathy phenotype produced by mutations affecting residues shared by both isoforms. Together, the data support a model in which the graded strength of SOCE, rather than isoform-specific coupling machinery, is the principal determinant of NFAT activation and downstream gene expression.

Strengths

The central strength of this study is its genetic strategy. Rather than relying on overexpression of individual Orai/STIM constructs in a background where multiple endogenous Orai paralogues are still present (a common confound in this field), the authors generated HEK293 clones that retain only one native Orai isoform, and separately reconstituted Orai-TKO cells with individual isoforms at nearendogenous expression levels using a weak TK promoter, validated by immunofluorescence and Western blotting. This design substantially reduces the risk that observed differences reflect artifacts of Orai/STIM stoichiometry rather than genuine isoform-intrinsic properties, a concern the authors explicitly raise and address (citing prior work showing that Orai/STIM overexpression itself can alter regulator sensitivity).

The multi-tier validation strategy is unusually thorough for this type of mechanistic question. The same qualitative conclusion, that NFAT activation scales with SOCE magnitude rather than isoform identity, is supported independently by (i) engineered HEK293 lines under both maximal (thapsigargin) and physiological (carbachol) stimulation, (ii) an orthogonal cellular system (MCF7 breast cancer cells natively dominated by Orai3) in which a "weak" channel is shown to support robust NFAT1 activation when sufficiently abundant, (iii) reconstitution of Orai1-deficient primary murine CD4⁺ T cells with matched, near endogenous levels of Orai1α or Orai1β, assessed by SOCE, endogenous NFAT1 localization (ImageStream), cytokine production, and genome-wide RNA-seq, and (iv) human population genetics across four independent cohorts (UK Biobank, gnomAD, Qatar Biobank, All of Us) combined with direct cellular phenotyping of human T cells from individuals with defined Orai1α genotypes. The convergence of cell line, mouse, and human genetic data on a single coherent model considerably strengthens confidence in the conclusions beyond what any single approach could provide.

The chelator experiment (Figure 4) is a clean, well-controlled test of local versus global Ca²⁺ signaling, using the classical BAPTA/EGTA differential kinetic-buffering logic, and the result (BAPTA-sensitive, EGTA-insensitive NFAT1 activation for both isoforms) is internally consistent and clearly presented.

The RNA-seq analysis (Figure 6) is a valuable addition, showing near-identical global transcriptional programs driven by Orai1α and Orai1β (log2 fold-change correlation R ≈ 0.92) and demonstrating that the larger number of nominal differentially expressed genes for Orai1α is attributable to statistical thresholding rather than a qualitatively distinct transcriptional signature. This substantially strengthens the claim that isoform identity has little independent influence on the transcriptional output of SOCE beyond its effect on Ca²⁺ influx magnitude.

Finally, the human genetic component is a genuine strength that elevates the physiological relevance of the paper considerably. Directly testing individuals who are natural "knockouts" for one Orai1 isoform but not the other is a powerful complement to the reductionist cell biology, and the finding that Orai1αnull carriers are clinically well and immunologically not compromised (indeed showing modestly enhanced SOCE/NFAT signaling) provides an unusually direct refutation of a specific published mechanistic model (the AKAP79-Orai1α N-terminus coupling hypothesis).

Weaknesses

Some tension remains between the HEK293/human T cell data, where Orai1β is moderately but consistently more efficient than Orai1α at driving SOCE and NFAT activation, and the murine primary CD4⁺ T cell data, where the two isoforms are functionally indistinguishable. The authors offer a plausible explanation, that retroviral expression levels in T cells were high enough to mask subtle isoform differences, and they partially address this by gating on Amtlow (lower-expressing) cells.

The TK-promoter reconstitution system, while a clear improvement over CMV-driven overexpression and validated as achieving comparable expression across isoforms, still involves ectopic expression in an Orai-null background rather than truly endogenous expression from the native locus. Describing this system as achieving "near-native" levels is reasonable given the Western blot validation shown in the supplement, but readers would benefit from the manuscript being explicit that this is a reconstitution model rather than unperturbed endogenous expression, and from a brief discussion of what residual differences (in, for example, membrane trafficking, promoter-driven transcript stability, or subtle stoichiometric mismatch with STIM) could still confound isoform comparisons.

Physiological cells normally co-express Orai1α and Orai1β at varying ratios (as the authors themselves show in Figure S2 across T helper subsets), and it is not established whether heteromeric Orai1α/Orai1β channels (which have been reported by others to form) behave as a simple functional average of the two homomeric channels or display emergent properties. The current study's conclusions rest entirely on isoform-pure systems, and this leaves open whether the "SOCE magnitude" model generalizes cleanly to the mixed populations of channels present in most native cells.

Several of the human genetic subgroup analyses involve modest sample sizes. The number of homozygous carriers of the two Orai1α-specific truncating variants varies substantially by cohort (for example, only 2 and 0 homozygotes identified in QBB), and the UK Biobank clinical phenotype comparison is based on 24 mutation carriers versus 984 controls. While the overall pattern (absence of enrichment for CRAC channelopathy associated diagnostic codes) is reassuring and consistent across the core infection, ectodermal, and myopathy categories, the confidence intervals in these comparisons are necessarily wide, and the absence of statistically significant differences in a modestly sized cohort should be interpreted as consistent with, rather than definitive proof of, phenotypic equivalence. Likewise, the direct cellular characterization of NFAT translocation and SOCE in human T cells from wild-type, heterozygous, and homozygous Orai1α-null Qatar Genome Project donors (Figure 8) is based on a single individual per genotype, which, although understandable given the rarity of the relevant genotype, limits the ability to distinguish genotype effects from inter-individual variability.

The manuscript proposes that NFAT activation is governed principally by the magnitude of local, channel-proximal Ca²⁺ signals rather than isoform-specific decoding machinery, but does not fully address whether isoform-specific differences in Ca²⁺-dependent inactivation kinetics (which shape the temporal profile, not just the amplitude, of the Ca²⁺ signal) could themselves constitute a form of isoform-specific "coding" that is conceptually distinct from, but difficult to fully disentangle from, simple amplitude scaling. A brief discussion of how the SOCE magnitude and Ca²⁺ signal kinetics/duration are related, and whether they can be cleanly separated in this experimental system, would help readers judge the boundaries of the magnitude-based model.