Feedback modulation of orai1alpha and orai1beta protein content mediated by STIM proteins

Received: 18 May 2024 | Accepted: 20 September 2024 DOI: 10.1002/jcp.31450 RESEARCH ARTICLE Feedback modulation of Orai1αand Orai1βprotein content mediated by STIM proteins Joel Nieto‐Felipe 1 |Alvaro Macias‐Díaz 1 |Vanesa Jimenez‐Velarde 1 | Jose J. Lopez 1 |Gines M. Salido 1 |Tarik Smani 2,3 |Isaac Jardin 1 | Juan A. Rosado 1 1 Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers (IMPB), University of Extremadura, Caceres, Spain 2 Group of Cardiovascular Pathophysiology, Institute of Biomedicine of Seville, University Hospital of Virgen del Rocío/University of Seville/CSIC, Seville, Spain 3 Department of Medical Physiology and Biophysics, Faculty of Medicine, University of Seville, Seville, Spain Correspondence Juan A. Rosado and Isaac Jardin, Department of Physiology (Cellular Physiology Research Group), Institute of Molecular Pathology Biomarkers (IMPB), University of Extremadura, 10003‐Caceres, Spain. Email: [email protected] and [email protected] Funding information Agencia Estatal de Investigación; Junta de Extremadura Abstract Store‐operated Ca 2+ entry is a mechanism controlled by the filling state of the intracellular Ca 2+ stores, predominantly the endoplasmic reticulum (ER), where ER‐ resident proteins STIM1 and STIM2 orchestrate the activation of Orai channels in the plasma membrane, and Orai1 playing a predominant role. Two forms of Orai1, Orai1αand Orai1β, have been identified, which arises the question whether they are equally regulated by STIM proteins. We demonstrate that STIM1 preferentially activates Orai1αover STIM2, yet both STIM proteins similarly activate Orai1β. Under resting conditions, there is a pronounced association between STIM2 and Orai1α. STIM1 and STIM2 are also shown to influence the protein levels of the Orai1 variants, independently of Ca 2+ influx, via lysosomal degradation. Interestingly, Orai1α and Orai1βappear to selectively regulate the protein level of STIM1, but not STIM2. These observations offer crucial insights into the regulatory dynamics between STIM proteins and Orai1 variants, enhancing our understanding of the intricate processes that fine‐tune intracellular Ca 2+ signaling. KEYWORDS Orai1α, Orai1β, STIM1, STIM2, store‐operated Ca 2+ entry 1|INTRODUCTION The tetra‐pass plasma membrane (PM) protein Orai1 belongs to a highly selective Ca 2+ channel family, ubiquitously conserved throughout the animal kingdom, that are known for their vital role in the finely modulated spatio‐temporal signaling of Ca 2+ across the PM. Working in conjunction with its mammalian paralogues, Orai2 and Orai3, Orai1 is instrumental in facilitating store‐operated calcium entry (SOCE), a fundamental process essential for agonist‐induced Ca 2+ mobilization. This mobilization regulates various cellular functions, including gene expression, cell proliferation, differentiation, and the immune response in both electrically excitable and non‐excitable cells (Emrich et al., 2021; Kar et al., 2021;Zhang et al., 2019). SOCE is initiated by the release of Ca 2+ from intracellular stores, primarily the endoplasmic reticulum (ER). This reduction in the luminal Ca 2+ concentration is detected by stromal interaction J Cell Physiol. 2024;e31450. wileyonlinelibrary.com/journal/jcp | 1of22 https://doi.org/10.1002/jcp.31450 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2024 The Author(s). Journal of Cellular Physiology published by Wiley Periodicals LLC. Joel N. Felipe and Alvaro Macias‐Díaz are contributed equally to this work. Tarik Smani and Juan A. Rosado are co‐senior authors. molecule proteins, STIM1 and STIM2, a type of integral single‐pass ER proteins that possess highly conserved luminal EF‐hand motifs, which have the ability to bind Ca 2+ , enabling STIM proteins to effectively detect and monitor stored Ca 2+ levels within the ER (Roos et al., 2005). STIM1 and STIM2 exhibit distinct characteristics and biological roles. STIM1 primarily functions as the principal stimulator of Orai/CRAC channels, whereas STIM2, with less affinity for Ca 2+ ions, acts as a comparatively slower and less potent activator of Orai channels, and it is considered to be involved in maintaining stable levels of Ca 2+ in both, the cytosol and ER, under resting conditions (Brandman et al., 2007). Upon ER depletion, STIM proteins activate, oligomerize, and translocate to regions close to the PM. There, they associate with and initiate the activation of Orai channels through a specific cytosolic region located within the STIM C‐terminus (Muik et al., 2009; Park et al., 2009; Yuan et al., 2009). This region, known as Orai1 activating small fragment (OASF) (Muik et al., 2009), STIM1‐ Orai1 activating region (SOAR) (Yuan et al., 2009) or CRAC activation domain (CAD) (Park et al., 2009), includes two coiled‐coil structures, highly conserved among family members, containing a homomerization domain, required for coupling to Orai1, and a regulatory domain, which modulates the extent of coupling to Orai1 (Muik et al., 2009). Subsequently, the activation of Orai channels leads to a significant influx of Ca 2+ from the extracellular environment (Prakriya et al., 2006; Zhang et al., 2005). Currently, there are no reported cases of alternative splicing for Orai1 mRNA; however, alternative initiation of translation has been observed in mammalian cells, giving rise to two Orai1 isoforms. The canonical full‐length Orai1 protein, designated as Orai1α, comprises 301 amino acids, whereas a shorter variant, termed Orai1β, originates from an alternative initiation of translation commencing at either methionine 64 or methionine 71. This variant consequently lacks the N‐terminal 63 or 70 amino acids found in the full‐length Orai1 protein (Fukushima et al., 2012; Putney, 2013). While both variants of Orai1 demonstrate similar efficacy in supporting the highly Ca 2+ ‐ selective and store‐operated I CRAC currents (Desai et al., 2015), notable functional and biophysical distinctions exist between Orai1α and Orai1β. Functionally, Orai1α, in conjunction with STIM1 and TRPC1, mediates the less Ca 2+ ‐selective store‐operated current I SOC (Cheng et al., 2011; Jardin et al., 2008; Kim et al., 2009) and the arachidonate‐regulated current I ARC (Desai et al., 2015). In contrast, Orai1βdoes not participate in I ARC , and its involvement in I SOC is celltype‐dependent (Sanchez‐Collado et al., 2022). Furthermore, Orai1α is essential for the NF‐κB transcription factor activation, while Orai1β does not exhibit this capability (Nieto‐Felipe et al., 2023). Biophysically, Orai1αhas been shown to be more susceptible to rapid Ca 2+ ‐ dependent inactivation compared to Orai1β(Desai et al., 2015). However, the interaction patterns between STIM1 or STIM2 and Orai1 variants, as well as their effectiveness in activating Orai1αand Orai1β, have not been studied to date. In a resting cell, Orai1 channels are present in two distinct cellular regions, the PM, which accounts for approximately 40%, and a subplasmalemal compartment (Hodeify et al., 2015). Upon cell stimulation, intracellular Ca 2+ store depletion leads to an accumulation of Orai1 proteins in the PM, in a STIM1‐dependent mechanism known as Orai1 “trafficking trap”that reduces Orai1 recycling and compartmentalization, thereby supporting and sustaining SOCE (Hodeify et al., 2015). As we have previously reported, this mechanism requires the participation of SNAP‐25 (Woodard et al., 2008). We have recently demonstrated that while both Orai1 variants constitutively translocates to the PM in resting cells, Orai1βrequires Orai1αto adequately translocate to the PM upon store depletion and support SOCE (Jardin et al., 2023). Furthermore, Orai1 undergoes rapid and dynamic recycling at the PM via endocytosis, a process modulated by the chaperonin‐containing T‐complex protein 1 (CCT) (Hodeify et al., 2018). However, it is still unknown whether both variants of Orai1 are subjected to the same mechanism. The objective of this study is to investigate the effectiveness of STIM1 and STIM2 in activating Ca 2+ signals through Orai1αand Orai1β and to characterize the role of STIM proteins in the turnover of Orai1α and Orai1β.Herewereporttwoimportantaspectsoftheregulationof Orai1 variants. Firstly, while STIM1 is a stronger activator of Orai1α compared to STIM2, both isoforms activate Orai1β‐dependent Ca 2+ signals to a similar extent. Secondly, our results indicate that the expression of STIM proteins can modulate the protein content of Orai1α and Orai1β. This suggests that the regulation of SOCE may involve a feedback mechanism where the extent of Ca 2+ influx through these channels depends not only on their intrinsic properties but also on the number and location of available channels in the PM. Therefore, our study provides valuable insights into the activation of Orai1αand Orai1βby STIM1 and STIM2, as well as the differential regulation of their expression. These findings contribute to a better understanding of the complex mechanisms underlying Ca 2+ signalingandtheroleofOrai1variantsin cellular functions. 2|MATERIALS AND METHODS 2.1 |Reagents and antibodies Fura‐2 acetoxymethyl ester (fura‐2/AM) was from Molecular Probes. High‐glucose Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, trypsin, penicillin/streptomycin, Trizma base, streptavidinconjugated agarose beads, rabbit polyclonal anti‐STIM2 antibody (catalog number STIM2‐201AP, epitope: amino acids 600‐650 of human STIM2), mouse monoclonal anti‐PMCA antibody (clone 5F10; catalog number MA3‐914, epitope: amino acids 724–783 of human PMCA), Clean‐Blot™ IP detection reagent, SuperSignal ® West Dura extended duration substrate reagent, sulfo‐NHS‐LC biotin, Live/Dead viability/cytotoxicity kit and Pierce™BCA protein assay kit were purchased from ThermoFisher Scientific (Waltham). Complete EDTA‐free protease inhibitor cocktail tablets were from Roche Diagnostics GmbH. DharmaFECT kb transfection reagent was obtained from Horizon Discovery. Thapsigargin (TG), carbachol (CCh), protein A agarose beads, cycloheximide, HEPES (4‐(2‐ Hydroxyethyl)piperazine‐1‐ethanesulfonic acid), EGTA (ethylene glycol‐ bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid), EDTA (ethylenedinitri lotetraacetic acid), bovine serum albumin (BSA), sodium azide, dimethyl‐ BAPTA, sodium ascorbate, bafilomycin A1, MG132, rabbit polyclonal anti‐ 2of22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Orai1 antibody (catalog number O8264, epitope: amino acids 288‐301 of human Orai1) and rabbit polyclonal anti‐β‐actin antibody (catalog number A2066, epitope: amino acids 365‐375 of human β‐actin) were obtained from MilliporeSigma (Burlington). Mouse monoclonal Anti‐GOK/STIM1 antibody (Clone 44/GOK; catalog number 610954, epitope: amino acids: 25–139 of human STIM1) was purchased from BD Biosciences. Horseradish peroxidase‐conjugated goat anti‐mouse immunoglobulin G (IgG) antibody and goat anti‐rabbit IgG antibody were from Jackson laboratories. Thymidine kinase (TK)‐and CMV‐promoter pEGFP‐N1‐Orai1α‐ eGFP and pEGFP‐N1‐Orai1β‐eGFP plasmids were kindly provided by Mohamed Trebak (Department of Pharmacology and Chemical Biology, University of Pittsburgh). CMV‐promoter STIM1‐D76A, STIM1‐YFP, STIM2‐YFP, STIM1‐mCherry and STIM2‐mCherry were a gift from Christoph Romanin (Institute of Biophysics, Johannes Kepler University Linz). pEX‐CMV‐SP‐YFP‐STIM2(15‐746) EF hand D80A was a gift from Tobias Meyer (Addgene plasmid # 18864; http://n2t.net/addgene: 18864; RRID:Addgene_18864). STIM1‐OASF and STIM2‐OASF were kinkly provided by Rainer Schindl (Division of Biophysics, Medical University of Graz, Austria). N‐glycosidase F (PNGaseF) from Elizabethkingia miricola was from New England Biolabs. All other reagents were of an analytical grade. 2.2 |Cell culture and transfections CRISPR‐generated STIM1/2 double‐knockout HEK293 cells (DKO) and parental HEK‐293 cells were kindly provided by Mohamed Trebak and cultured at 37°C with a 5% CO 2 in high‐glucose DMEM supplemented with 10% (v/v) fetal bovine serum and 100 U/mL penicillin and streptomycin, as described previously (Albarran et al., 2016). Cells used throughout the study are free of mycoplasma. For transient transfections, cells were grown to 60% to 80% confluency and transfected with TK‐or CMV‐promoter Orai1α‐EGFP and Orai1β‐EGFP or CMV‐promoter STIM1‐YFP, STIM2‐YFP or STIM2‐D80A‐YFP, depending on the experimental conditions, using DharmaFECT kb transfection reagent and were used 48 h after transfection. Unless otherwise stated, expression plasmids were used at the Orai1:STIM ratio of 1:2 to maintain Orai1‐ STIM stoichiometry as previously established (Zhou et al., 2015). When cells were transfected with a combination of STIM1 and STIM2 the Orai:STIM1:STIM2 ratio was 1:1:1 to maintain stoichiometry as mentioned above. Cell viability after transfection throughout the study, estimated using the Live/Dead viability/cytotoxicity kit, was in the range between 92% and 95%. For Western blot analysis and immunoprecipitation, cells (2×10 6 )wereplatedin100‐mm petri dish and cultured for 48 h, while, for Ca 2+ imaging and confocal analysis cells (4 × 10 5 ) were seeded in a 35‐mm six‐well multidish. 2.3 |Immunoprecipitation and Western blot analysis Immunoprecipitation and Western blot analysis were performed as described previously (Sanchez‐Collado et al., 2019). Briefly, cells cultured on 100‐mm petri dish (2 × 10 6 cells) were stimulated with 2μM TG or with vehicle and subsequently lysed with ice‐cold NP‐40 buffer pH 8 containing 137 mM NaCl, 20 mM Tris, 2 mM EDTA, 10% glycerol, 1% Nonidet P‐40, 1 mM Na 3 VO 4 , and complete EDTA‐free protease inhibitor tablets. Cell lysates (1 mL) were immunoprecipitated by incubation with 2 µg of anti‐Orai1 antibody and 50 µL of protein A‐agarose overnight at 4°C on a rotary platform. Cell lysates and immunoprecipitates were resolved by 10% SDS‐PAGE and separated proteins were electrophoretically transferred onto nitrocellulose membranes for subsequent probing. Blots were incubated overnight with 10% (w/v) BSA in Tris‐buffered saline with 0.1% Tween‐20 (TBST) to block residual protein binding sites. Immunodetection of STIM1, STIM2, Orai1 or β‐actin was achieved by incubation for 1 h with anti‐STIM1 antibody diluted 1:500 in TBST, 1 h with anti‐STIM2 antibody diluted 1:1000 in TBST, 1 h with anti‐ Orai1 antibody diluted 1:1000 in TBST or 45 min with anti‐β‐actin antibody diluted 1:2000 inTBST. The primary antibody was removed, and blots were washed six times for 5 min each with TBST. To detect the primary antibody, blots were incubated for 1 h with horseradish peroxidase‐conjugated goat anti‐mouse IgG antibody, horseradish peroxidase‐conjugated goat anti‐rabbit IgG antibody diluted 1:10000 in TBST, or Clean‐Blot™IP Detection Reagent diluted 1:250 in TBST, and then exposed to enhanced chemiluminiscence reagents for 5 min. The antibody binding was assessed with a ChemiDoc Imaging System (Bio‐Rad, Madrid, Spain) and the density of bands was measured using ImageJ software v.1.8.0_172 (NIH). Data were normalized to the amount of protein recovered by the antibody used for the immunoprecipitation or to β‐actin from the same gel. 2.4 |Determination of cytosolic free‐Ca 2+ concentration ([Ca 2+ ] I ) Cells were loaded with fura‐2byincubationwith5μMfura‐2/AM for 30 min at 37°C. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti2) with an image acquisition and analysis system for videomicroscopy (NIS‐Elements Imaging Software v.5.02.00, Nikon). Cells were continuously superfused at room temperature with HEPES‐buffered saline (HBS) containing (in mM) 125 NaCl, 5 KCl, 1 MgCl 2 , 5 glucose, and 25 HEPES, pH 7.4, supplemented with 0.1% (w/v) BSA. Cells were examined at 40× magnification (Nikon CFI S FLUOR 40× Oil) and were alternatively excited with light from a xenon lamp passed through a high‐speed monochromator Optoscan ELE 450 (Cairn Research) at 340/380 nm. Fluorescence emission at 510 nm was detected using a cooled digital sCMOS camera PCO Panda 4.2 (Excelitas PCOGmbH)andrecordedusingNIS‐Elements AR software (Nikon). Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data were presented as ΔF 340 /F 380 as described previously (Sanchez‐ Collado et al., 2022). CCh‐evoked Ca 2+ mobilization was estimated as the area under the curve measured as the integral of the rise in fura‐2flu- orescence ratio 10 min after the addition of the agonist and taking a sample every second. TG‐evoked Ca 2+ entry was estimated as the area NIETO‐FELIPE ET AL. | 3of22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License under the curve measured as the integral of the rise in fura‐2fluores- cence ratio 2½ min after the addition of TG and taking a sample every second. 2.5 |Analysis of Ca 2+ oscillations The analysis of Ca 2+ oscillations was performed as described previously (Yoast et al., 2020). All Ca 2+ traces obtained in the Ca 2+ ‐imaging experiments were plotted using GraphPad Prism v.8.4.3 (GraphPad Software). The number of oscillations per 20 min were counted by the function signal. find_peaks from the SciPy 1.8.1 library for Python 3.9. Then, cells were classified in two groups and a percentage of each group of cells was calculated for each individual coverslip. The first group, oscillating cells, includes cells showing regenerative oscillations after CCh stimulation, where each oscillation returns to baseline before the start of the next oscillation. The second group, non‐oscillating cells, includes cells showing a sustained or transient cytosolic Ca 2+ signal for at least 3 min after stimulation. 2.6 |Confocal microscopy analysis Cells were seeded in 35‐mm six‐well multidish and transfected with the indicated plasmids. To determine subcellular location of proteins and Förster resonance energy transfer (FRET) cells were imaged 48 h post transfection using a confocal microscope (LSM900, Zeiss) with 63× oil immersion objective, using an image acquisition and analysis system for video microscopy (ZEN Software, Zeiss). For FRET analysis, appropriate cross‐talk calibration factors were determined for each construct on each raw of FRET experiments. After threshold determination and background subtraction, the corrected FRET (Eapp) was calculated on a pixel‐to‐pixel basis with a custom‐made software integrated in FIJI ImageJ 1.54 f according to the method published previously (Singh et al., 2006). To assess the expression of Orai1 variants in the presence or absence of STIM proteins, background subtraction and thresholding techniques were applied. The NaN Background mathematical process was used to eliminate noise. Subsequently, seven to 10 random locations, measuring 2.5 µm 2 each, were selected for fluorescence measurement using custom software integrated into FIJI ImageJ 1.54 f. 2.7 |Biotinylation of surface proteins Biotinylation of surface proteins was performed as described previously (Jardin et al., 2023). Briefly, cells were washed three times with phosphate‐buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH 2 PO 4 ,8mMNa 2 HPO 4 •2H 2 O, pH 7,4), subsequently resuspended in biotinylation buffer (PBS, pH 8.0) and surface labeled with 1 mg/mL sulfo‐ NHS‐LC biotin at room temperature. Labeling was stopped 1 h after reaction with PBS, pH 8.0, supplemented with 100 µM Tris and washed 2 times in supplemented PBS, pH 8.0. Biotinylated cells were subsequently lysed with ice‐cold NP‐40 buffer, pH 8.0, containing 137 mM of NaCl, 20mMofTris,2mMofEDTA,10%glycerol,1%NonidetP‐40, 1 mM of Na 3 VO 4 , and complete EDTA‐free protease inhibitor tablets and protein lysates were incubated with streptavidin‐conjugated agarose beads overnight at 4°C on a rocking platform. Biotinylated proteins bound to streptavidin‐conjugated agarose beads were isolated by centrifugation andwashed3timesinNP40buffer.Biotinylated fraction was loaded and separated in 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and analyzed by Western blot using anti‐PMCA antibody, as control. 2.8 |Statistical analysis All data are presented as the mean ± standard error of mean (SEM). Analysis of statistical significance was performed using GraphPad Prism v.8.4.3 (GraphPad Software). Kruskal–Wallis test combined with Dunn´s post hoc test were used to compare the different experimental groups. For comparison between two groups, the Mann–Whitney U test was used. All data with p< 0.05 was deemed significant; “ns”= nonsignificant. 3|RESULTS 3.1 |Efficacy of STIM1 and STIM2 activating Ca 2+ signals through Orai1αand Orai1β To investigate the functional interaction of Orai1 variants and STIM proteins we have explored thapsigargin (TG)‐induced SOCE in STIM1/2 double‐knockout HEK‐293 cells (DKO cells) cotransfected with CMV‐ driven STIM1‐YFP or STIM2‐YFP together with Orai1α‐eGFP or Orai1β‐ eGFP. The expression plasmids were used at the Orai1:STIM ratio of 1:2 to maintain Orai1‐STIM stoichiometry as previously reported (Yang et al., 2012). Forty‐eight hours after transfection of STIM1‐YFP or STIM2‐YFP, a single protein band with molecular weight around 105 and 130 kDa were observed (Figure 1f). Furthermore, 48 h after transfection of Orai1α‐eGFP or Orai1β‐eGFP several bands over 60 and 50 kDa, respectively, were observed, due to Orai1αand Orai1βglycosylation, as previously reported (Fukushima et al., 2012;Sanchez‐Collado et al., 2022). In the absence of extracellular Ca 2+ ,treatmentofuntransfected (wild type (WT)) HEK‐293 cells with 2 µM TG, an inhibitor of the sarco/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA), induced a transient increase in the fura‐2 fluorescence ratio due to Ca 2+ release from the intracellular Ca 2+ stores. Subsequent addition of Ca 2+ to the extracellular medium leads to a rise in the fura‐2 fluorescence ratio that is indicative of Ca 2+ influx (Figure 1a).AsshowninFigure1f, DKO cells do not express STIM proteins and show impaired SOCE (Figure 1a)despite Ca 2+ release from the intracellular stores was similar to that in WT cells. Cotransfection with Orai1αor Orai1βin combination with STIM1 rescued SOCE in DKO cells (Figure 1b–e) resulting in an extent of Ca 2+ influx that exceeded that in WT cells (probably due to the overexpression of the Orai1 variants and STIM1); however, in DKO cells, Cotransfection of STIM2 with Orai1αwaslesseffectivethanSTIM1(Figure1b,d,e). 4of22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Meanwhile, the efficiency of STIM2 stimulating Ca 2+ entry through Orai1βwas similar to that of STIM1 (Figure 1c–e). Finally, co‐expression of STIM1 and STIM2 with the Orai1 variants generated SOCE with similar extent as STIM1 alone (Figure 1b–e). These findings indicate that STIM2 is less effective inducing SOCE by Orai1αbut exhibit a similar efficacy to STIM1 activating Orai1β.Figure1f shows the expression of the target proteins under the different experimental conditions. As expected, Orai1 wasdetectedasmultiplebandscorrespondingtoOrai1αand the short form, Orai1β, and to different posttranslational modifications, including N‐linked glycosylation and phosphorylation, among others (Fukushima et al., 2012;Sanchez‐Collado et al., 2019). Quantification of the protein expression for Figure 1f is shown in Supporting Information S1: Figure S1a–d. Unexpectedly, DKO cells exhibit a significantly smaller endogenous expression of total Orai1, as well as Orai1αand Orai1β (Supporting Information S1: Figures S1a and S2a) and expression of STIM proteins further attenuates the endogenous expression of Orai1 (Supporting Information S1: Figure S1a).DatashowninSupporting Information S1: Figure S1c,d, reveal the Orai1 variant:STIM1:STIM2 expression ratio of 1:1:1 vs the Orai1 variant:STIM1 or Orai1 variant:STIM2 ratio of 1:2 as mentioned in Material and Methods. Next, we explored the role of STIM1 and STIM2 on carbachol (CCh)‐induced Ca 2+ mobilization in STIM1/2 DKO HEK‐293 cells expressing either Orai1αor Orai1β. In the presence of 1.8 mM extracellular Ca 2+ , CCh (10 µM) elicited Ca 2+ mobilization. Traces from five representative cells are depicted in Figure 2a–f. About 99% of WT HEK‐293 cells responded to CCh with regenerative Ca 2+ oscillations (Figure 2a). In DKO cells, CCh induced a transient rise in the fura‐2 fluorescence ratio due to Ca 2+ release from the intracellular stores and did not respond with Ca 2+ oscillations (Figure 2b). The magnitude of Ca 2+ mobilization induced by CCh was significantly FIGURE 1 Ca 2+ entry through the Orai1 variants induced by STIM1 and STIM2. (a) Fura‐2‐loaded wild type HEK‐293 cells (WT) and STIM1, 2‐DKO HEK‐293 cells (DKO) were suspended in a Ca 2+ ‐free (100 µM EGTA) HBS and then stimulated with 2 µM TG followed by reintroduction of external Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ entry. (b, c) DKO HEK‐239 cells were cotransfected with CMV‐driven plasmids for either Orai1α‐eGFP (O1α; b) or Orai1β‐eGFP (O1β; c) and either STIM1‐YFP (S1), STIM2‐YFP (S2) or both (S1 + S2) plasmids, as described. Forty‐eight hours later cells were loaded with fura‐2 and suspended in a Ca 2+ ‐free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. (d, e) quantification of TG‐evoked Ca 2+ release (d) and entry (e) determined as described in materials and methods. Bar graphs are represented as mean ± SEM and expressed as fold change over control (WT HEK‐293 cells). From left to right, n= 224, 112, 50, 46, 37, 41, 41 and 23; n values correspond to individual cells. Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). * p< 0.05, ** p< 0.01, ***p< 0.001 and ****p< 0.0001 as compared to WT HEK‐293 cells. $$ p< 0.01 and $$$$ p< 0.0001 as compared to DKO HEK‐293 cells. ϕ p< 0.05 as compared to DKO cells transfected with STIM1 expressing plasmid. (f) WT HEK‐293 cells (lane 1) and DKO cells either transfected with empty vector or with CMV‐driven Orai1αor Orai1βin combination with either STIM1, STIM2 or both plasmids (lanes 2‐9) were lysed and then subjected to 10% SDSPAGE and Western blot analysis with the anti‐STIM2, anti‐STIM1 or anti‐Orai1 (C‐terminal) antibody, as described in material and methods. Membranes were reprobed with the anti‐β‐actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Blots are representative of four separate experiments. DKO, double‐knockout, HBS, HEPES‐ buffered saline; SEM, standard error of mean; WT, wild type. NIETO‐FELIPE ET AL. | 5of22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 2 CCh‐evoked Ca 2+ mobilization in cells expressing the Orai1 variants in combination with STIM1 or STIM2. (a‐f) Representative Ca 2+ mobilization in response to 10 µM CCh measured using fura‐2inwildtypeHEK‐293 cells (WT), STIM1,2‐DKO HEK‐293 cells (DKO) and DKO cells cotransfected with CMV‐driven plasmids for either Orai1α‐eGFP (O1α)orOrai1β‐eGFP (O1β)andeitherSTIM1‐YFP (S1), STIM2‐YFP (S2) or both (S1 + S2) plasmids, as described. Cells were superfused with HBS containing 2 mM Ca 2+ and stimulated with 10 µM CCh at 1 min (indicated by arrow). Representative traces from five cells/condition were chosen to represent the datasets. (g) Quantification of Ca 2+ mobilization for all the conditions from a to f estimated in all the cells. (for g, from left to right, n= 110, 141, 67, 34, 89 and 70; n‐values correspond to individual cells). Scatter plots are representedasmean±SEMandexpressedasfoldchangeovercontrol(WTHEK‐293 cells). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). ****p<0.0001 ascomparedto WTHEK‐293 cells. $$ p<0.01, $$$ p<0.001 and $$$$ p<0.0001ascompared to DKO cells. ϕ p< 0.05 as compared to DKO cells transfected with STIM1 expressing plasmid. CCh, carbachol; DKO, double‐knockout, HBS, HEPES‐buffered saline; SEM, standard error of mean; WT, wild type. 6of22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License greater in WT, which reveals the role of STIM proteins in CChinduced Ca 2+ signals (Figure 2g;p< 0.0001). When DKO cells were cotransfected with CMV‐driven Orai1αor Orai1βin combination with STIM1 or STIM2 expression plasmid most cells responded to CCh with a rather sustained increase in the fura‐2 fluorescence ratio and Ca 2+ mobilization was rescued (Figure 2c–g). We noticed that the Ca 2+ mobilization induced by CCh in DKO cells expressing Orai1α was greater in the presence of STIM1 than of STIM2 (Figure 2g; p< 0.0001). Despite we did not detect any significant difference between STIM1 and STIM2 in cells expressing Orai1β(Figure 2g), our findings further indicate that STIM1 is more effective than STIM2 mediating CCh‐induced Ca 2+ mobilization. We realized that, using expression plasmids with CMV promoter for Orai1 variants and STIM proteins, cells did not respond to CCh with regenerative Ca 2+ oscillations, mostly due to the overexpression of the target proteins (Sanchez‐Collado et al., 2022). Hence, we repeated these experimental protocols using plasmids with thymidine kinase (TK) promoter, which yield a protein expression level significantly smaller than plasmids with the CMV promoter (Nieto‐Felipe et al., 2023). DKO cells were transfected with shOrai1 to reduce the expression of native Orai1 (Figure 3). Cotransfection with TK‐driven Orai1αor Orai1βwith STIM1 rescued SOCE in DKO cells (Figure 3a–e); however, as observed with CMV‐driven plasmids, cotransfection of STIM2 with Orai1αwas less effective than STIM1 (Figure 3b,d,e;p< 0.0001), while the Ca 2+ signal generated in cells expressing Orai1βwas similar in the presence of STIM1 or STIM2 (Figure 3c,d,f). These findings further indicate that STIM2 is less effective inducing SOCE by Orai1α, but not by Orai1β, as mentioned FIGURE 3 Ca 2+ entry through the Orai1 variants induced by STIM1 and STIM2 using thymidine kinase (TK)‐driven plasmids. (a) Fura‐2‐loaded wild type HEK‐293 cells (WT) and STIM1,2‐DKO HEK‐293 cells (DKO) were suspended in a Ca 2+ ‐free HBS (100 µM EGTA) and then stimulated with 2 µM TG followed by reintroduction of external Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ entry. (b, c) DKO cells were cotransfected with shOrai1 alone or in combination with TK‐driven plasmids for either Orai1α‐eGFP (O1α;b)orOrai1β‐eGFP (O1β;c)andeitherSTIM1‐GFP (S1) or STIM2‐GFP (S2) plasmids. Forty‐8 h later cells were loaded with fura‐2 and suspended in a Ca 2+ ‐free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. (d, e) quantification of TG‐evoked Ca 2+ release (d) and entry (e) determined as described in materials and methods. Bar graphs are represented as mean ± SEM and expressed as fold change over control (WT HEK‐293 cells). From left to right, n= 203, 133, 105, 82, 70 and 109; n values correspond to individual cells. Data were statistically analyzed using Kruskal–Wallistestwithmultiplecomparisons(Dunn´stest).**p< 0.01 and ****p<0.0001as comparedtoWTHEK‐293 cells. $$$ p<0.001 and p< 0.0001 as compared to DKO cells. ϕϕϕ p< 0.001 and ϕϕϕϕ p< 0.0001 as compared to DKO cells transfected with STIM1 expressing plasmid. (f) WT HEK‐293 cells (lane 1) and DKO cells (lanes 2–9) were transfected with empty vector (lanes 1 and 2), or with shOrai1 alone (lane 3) or in combination with TK‐driven Orai1αor Orai1βand either STIM1, STIM2 or both plasmids (lanes 4–9), as described, and then were lysed and subjected to 10% SDS‐PAGE and Western blot analysis with the anti‐STIM2, anti‐STIM1 or anti‐Orai1 (C‐terminal) antibody, as described in material and methods. Membranes were reprobed with the anti‐β‐actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. Blots are representative of four separate experiments. DKO, double‐knockout, HBS, HEPES‐buffered saline; SEM, standard error of mean; WT, wild type. NIETO‐FELIPE ET AL. | 7of22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 4 (See caption on next page). 8of22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License above using CMV‐driven plasmids. Figure 3f shows the expression of the target proteins under the different experimental conditions. Quantification of the protein expression for Figure 1f is shown in Supporting Information S1: Figure S1e–h, and the effect of shOrai1 on native Orai1 expression in DKO cells is more clearly observed in Supporting Information S1: Figure S2b. Furthermore, we analyzed the functional role of STIM1 and STIM2 in CCh‐evoked Ca 2+ oscillations using TK‐driven plasmids. In the presence of 1.8 mM extracellular Ca 2+ , WT HEK‐293 cells responded to CCh with regenerative Ca 2+ oscillations (Figure 4a,h,i), with an average of 10.0 ± 0.4 oscillations/20 min (Figure 4j). In DKO cells, where SOCE is impaired, only 22% of the cells responded with Ca 2+ oscillations after stimulation with CCh, with an average of 3.2 ± 0.2 oscillations/20 min (Figure 4b,h). The remaining cells responded with transient Ca 2+ signals because of Ca 2+ release from the intracellular stores. As a result, the magnitude of Ca 2+ mobilization induced by CCh was significantly greater in WT, which further confirms the role of STIM proteins in CCh‐induced Ca 2+ signals (Figure 4g;p< 0.0001). When DKO cells were cotransfected with TK‐ driven Orai1αor Orai1βin combination with STIM1 or STIM2 expression plasmid, the percentage of cells that responded with regenerative Ca 2+ oscillations enhanced to 72% and 54% (for Orai1α+ STIM1 and Orai1α+ STIM2, respectively) and 58% and 69% (for Orai1β+ STIM1 and Orai1β+ STIM2, respectively) (Figure 4h). Only the combination of the expression of Orai1α+ STIM1 in DKO cells resulted in a percentage of oscillating cells that was not statistically different from WT cells (Figure 4h;p< 0.0001). Interestingly, we have not found significant differences in the percentage of oscillating cells among the DKO cells transfected with any of the combinations tested of Orai1 variants and STIM isoforms (Figure 4h). No combination completely rescued the number of oscillations observed in WT cells (Figure 4j;p< 0.01). Co‐expression of Orai1α and STIM1 in DKO cells completely rescued CCh‐induced Ca 2+ mobilization (Figure 4g), meanwhile co‐expression of Orai1βwith STIM1 or STIM2 only partially rescued CCh‐induced Ca 2+ mobilization, and expression of Orai1αand STIM2 was unable to rescue Ca 2+ mobilization induced by CCh in DKO cells as compared to WT cells (Figure 4g;p< 0.0001) thus resulting in a Ca 2+ response that is significantly smaller than that generated by co‐expression of Orai1αand STIM1 (Figure 4g;p< 0.0001). Altogether, our results indicate that STIM1 is more efficient that STIM2 generating Orai1α‐dependent TG‐and CCh‐evoked Ca 2+ signals. By contrast, STIM1 and STIM2 exhibit a similar efficiency activating Ca 2+ signals generated by Orai1β. 3.2 |Interaction of Orai1 variants with STIM1 and STIM2 STIM proteins associate with and activate Orai channels to mediate SOCE. Here we have used confocal Förster Resonance Energy Transfer (FRET) to examine the interaction of STIM1 and STIM2 with Orai1 variants in living HEK293 cells. Co‐expression of CMV‐driven C‐terminally tagged Orai1α‐eGFP (Figure 5a,c)orC‐terminally tagged Orai1β‐eGFP (Figure 5b,d) together with CMV‐driven C‐terminally tagged STIM1‐mCherry (Figure 5a,b) or STIM2‐mCherry (Figure 5c,d), revealed substantial PM (Orai1α‐eGFP and Orai1β‐eGFP) or intracellular localization (STIM1‐mCherry and STIM2‐mCherry). A mean nFRET value of 0.106 ± 0.002, 0.095 ± 0.003, 0.159 ± 0.009 and 0.074 ± 0.001 suggests a direct coupling of STIM1‐Orai1α, STIM1‐ Orai1β, STIM2‐Orai1αand STIM2‐Orai1β, respectively, while the cell is in a quiescent state. One of the functions attributed to STIM2 is its ability to activate Orai1 to counteract minor leakage from the ER (Ahmad et al., 2022). It is noteworthy that, in a quiescent cell, the interaction between STIM2 and Orai1αis significantly more pronounced compared to the other studied conditions (Figure 5a–d, control, and 5E control; p< 0.0001). As expected, stimulation with 2 µM TG for 1 min in the presence of 1.8 mM extracellular Ca 2+ resulted in a significant increase in the mean nFRET value in all tested combinations, STIM1‐Orai1α: 0.378 ± 0.030; STIM1‐Orai1β: 0.334 ± 0.037; STIM2‐Orai1α: 0.269 ± 0.018; and STIM2‐Orai1β: 0.114 ± 0.002. Our data show a strong association between STIM1 and both Orai1 variants or STIM2 and Orai1α(Figure 5a–c, TG, and Figure 5e, TG; p< 0.0001), while a weaker association between STIM2 and Orai1βupon TG stimulation was observed (Figure 5d, TG, and Figure 5e, TG; p< 0.05). We have further investigated the interaction of Orai1 variants with STIM1 and STIM2 by looking for coimmunoprecipitation between the target proteins from cell lysates. Immunoprecipitation and subsequent SDS‐PAGE and Western blot analysis were FIGURE 4 CCh‐evoked Ca 2+ oscillations in cells expressing the Orai1 variants in combination with STIM1 or STIM2. (a‐f) Representative Ca 2+ mobilization in response to 10 µM CCh measured using fura‐2 in wild type HEK‐293 cells (WT), STIM1,2‐DKO HEK‐293 cells (DKO) and DKO cells cotransfected with either shOrai1 and thymidine kinase (TK)‐driven Orai1α‐eGFP (O1α) or Orai1β‐eGFP (O1β) and either STIM1‐GFP (S1) or STIM2‐GFP (S2) plasmids, as described. Cells were superfused with HBS containing 2 mM Ca 2+ and stimulated with 10 µM CCh at 1 min (indicated by arrow). Representative traces from five cells/condition were chosen to represent the datasets. (g) Quantification of Ca 2+ mobilization for all the conditions from a to f estimated in all the cells. (for g, from left to right, n= 187, 286, 91, 61, 110 and 126; n‐values correspond to individual cells). (h–j) Quantification of the percentage of oscillating cells (h), non‐oscillating cells (i) and total oscillations/cell in 20 min (j) for data presented in a–f (for h to j, from left to right, n= 8, 8, 6, 5, 5 and 6; n values correspond to independent experiments). Scatter plots are represented as mean ± SEM and expressed as fold change over control (WT HEK‐293 cells). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). *p< 0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001 as compared to WT HEK‐293 cells. $ p< 0.05, and p< 0.0001 as compared to DKO cells. ϕϕϕϕ p< 0.0001 as compared to DKO cells transfected with STIM1 expressing plasmid. CCh, carbachol; DKO, double‐knockout, HBS, HEPES‐buffered saline; SEM, standard error of mean; WT, wild type. NIETO‐FELIPE ET AL. | 9of22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License proteins. To address this question, we expressed STIM1 or STIM2 in STIM1/2 DKO cells alone or in combination with Orai1αand Orai1βand analyzed the protein content of the STIM proteins. Our results indicate that when STIM1 was co‐expressed with Orai1αor Orai1βits protein content was significantly reduced (Figure 12a;p< 0.0001). By contrast, the protein content of STIM2 was not found to be affected by expression of the Orai1 variants (Figure 12b). These findings suggest that STIM1 turnover is promoted by Orai1αand Orai1βwhile STIM2 escapes from this regulatory mechanism. 4|DISCUSSION It is well established that mammalian STIM proteins, STIM1 and STIM2, differ in their properties and biological functions. STIM1 is the main activator of Orai/CRAC channels while STIM2, with lower affinity to Ca 2+ , has been proposed as a slower and weaker Orai activator that stabilizes basal cytosolic and ER Ca 2+ levels (Brandman et al., 2007). In agreement with the previous observations (Brandman et al., 2007; Soboloff et al., 2012) here we show that STIM1 is a stronger activator that STIM2 triggering Orai1α‐dependent Ca 2+ signals. By contrast, we provide for the first time evidence supporting that STIM1 and STIM2 exhibit a similar strength activating Ca 2+ signals generated by Orai1βdespite the weaker association of STIM2 with Orai1βcompared to STIM1. This finding might be attributed to a stronger activation of Orai1βby STIM2; however, the constitutively active mutants of STIM1 (STIM1D76A) and STIM2 (STIM2D80A), as well as STIM1‐OASF and STIM2‐OASF, induce a similar activation of Ca 2+ influx through Orai1β, which suggests that both STIM isoforms are similar activators of Orai1β. Native CRAC channels have been reported to consist of heteromers of the three Orai isoforms (Yoast et al., 2020), and previous studies have shown that STIM2 exhibits basal interaction with Orai1 and Orai2, a process that might regulate basal cytosolic and ER Ca 2+ levels and SOCE upon stimulation with low agonist concentrations (Emrich et al., 2021). Concerning the Orai1 variants, our FRET analysis demonstrates a significantly enhanced basal interaction between STIM2 and Orai1α,ascompared to Orai1β, a difference that has not been observed between STIM1 and the Orai1 forms. This observation might suggest that Orai1αwould play a relevant role in the modulation of resting cytosolic and ER Ca 2+ levels, although further studies are required to test this hypothesis. FIGURE 9 Effect of STIM1 and STIM2 expression on the Orai1αand Orai1βprotein level. (a) HEK‐293 cells were transfected with empty vector (Ctrl), CMV‐driven STIM1‐YFP or STIM2‐YFP expression plasmids or with shSTIM1 or esiSTIM2, as indicated. Forty‐eight hours later cells lysed. Cell lysates were treated with PNGaseF and then subjected to 10% SDS‐PAGE and Western blot analysis using anti‐STIM1, anti‐STIM2 or anti‐Orai1 C‐terminal antibody. Membranes were reprobed with anti β‐actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. These results are representative of six separate experiments. (b–e) Quantification of Orai1α, Orai1β, STIM1 and STIM2 expression under the different experimental conditions normalized to the β‐actin expression. Data are represented as mean ± SEM and expressed as fold change (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). *p< 0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001 as compared to the protein expression in WT cells. SEM, standard error of mean; WT, wild type. 16 of 22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License The canonical role of STIM proteins is to communicate the filling state of the intracellular Ca 2+ stores to the Orai channels in the PM to mediate SOCE. Modulation of Ca 2+ entry through Orai channels is essential to generate physiological Ca 2+ signals and maintain intracellular Ca 2+ homeostasis. A variety of regulatory mechanisms have been reported, including intrinsic mechanisms such as fast Ca 2+ ‐ dependent inactivation (FCDI) of Orai channels (Hoth & Penner, 1993) and, based on the different sensitivity of the three Orai isoforms to FCDI, fine‐tuning of Ca 2+ entry by Orai heteromerization (Yoast et al., 2020). Other SOCE regulatory mechanisms require the participation of regulatory proteins and/or organelles such as slow Ca 2+ ‐dependent inactivation (Jha et al., 2013; Parekh, 2017). Here we provide evidence for a role of STIM1 and STIM2 in the turnover of Orai1αand Orai1βthat might serve as an additional feedback modulation of SOCE. We base this statement on different findings: First, in STIM1/2 DKO cells, expression of STIM1 and STIM2 significantly attenuated the protein content and PM location of Orai1αand Orai1β. This difference cannot be attributed to interference between the expression plasmids as we did not detect changes in the expression of the Orai1 variants upon co‐expression of Orai1αwith Orai1βor with other cellular proteins using the same CMV‐driven plasmids and experimental procedure (Jardin et al., 2023). Concerning the PM expression of the Orai1 variants, the expression of STIM proteins did not alter the increase in the PM location of Orai1 observed upon Ca 2+ store depletion relative to control, which confirms that STIM proteins modulate the protein content of Orai1 but not its functionality. Our previous studies using Orai1 knockout HEK‐293 cells have reported that while at resting conditions there is a pool of both Orai1 variants in the PM, upon Ca 2+ store depletion there is a detectable PM enrichment of Orai1αwhile Orai1βbehaves as a defective protein that requires the presence of Orai1αto accumulate in the PM (Jardin et al., 2023). In the present FIGURE 10 STIM1 and STIM2 regulate lysosomal degradation of Orai1αand Orai1β. STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven Orai1α‐eGFP (a, c, e) or Orai1β‐eGFP (b, d, f) in combination with either STIM1‐YFP, STIM2‐YFP or both plasmids, as indicated. Forty‐eight hours later cells were either treated with cycloheximide (CHX; 10 µM) or the vehicle for 7 h and lysed (a, b) or were treated with cycloheximide (10 µM) alone or in combination with 10 µM MG132 for 7 h and lysed (c, d) or were treated with cycloheximide (10 µM) alone or in combination with 1 µM bafilomycin A1 (BafA1) for 7 h and lysed (e, f). Cell lysates were analyzed by SDS‐PAGE and Western blot analysis using anti‐STIM1, anti‐STIM2 or anti‐Orai1 C‐terminal antibody. Membranes were reprobed with anti β‐actin antibody. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. These results are representative of six separate experiments. Bar graphs show quantification of Orai1αand Orai1βexpression under the different experimental conditions normalized to the β‐actin expression. Data are represented as mean ± SEM and expressed as fold change (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). *p< 0.05, **p< 0.01, ***p< 0.001 and ****p< 0.0001 as compared to the Orai expression in the absence of STIM proteins. DKO, double‐knockout; SEM, standard error of mean. NIETO‐FELIPE ET AL. | 17 of 22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License study we have detected an increase in the surface expression of transiently expressed Orai1βafter stimulation with TG in the absence of cell transfection with Orai1α, which we attribute to the presence of native Orai1αin these cells. Second, the extent of the drop in Orai1 variant protein content depends on the amount of STIM expressed as observed when DKO cells were cotransfected with STIM and Orai1 variants expression plasmids at 1:1 and 1:2 ratios. Third, the STIM protein content modulates the expression of native Orai1α and Orai1βas demonstrated when WT HEK‐293 cells were transfected with STIM1 or STIM2 expression plasmid or, alternatively, with shRNA/siRNA specific for each STIM protein. Forth, STIM‐ induced Orai1αand Orai1βturnover is likely to involve interaction FIGURE 11 (See caption on next page). 18 of 22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License between both proteins as demonstrated by the enhanced drop in the Orai1 protein level induced by the constitutively active mutants STIM1‐D76A and STIM2‐D80A or the STIM1‐OASF region. To our knowledge this is the first evidence for a role of STIM proteins in the regulation of Orai1 variant content, and, in this regulatory function, the role of both STIM proteins is indistinguishable. The content of a given protein depends on the rate of synthesis and degradation. By using CHX, which blocks protein synthesis by impairing translation elongation (Schneider‐Poetsch et al., 2010)wehavefound that STIM proteins do not alter the synthesis of the Orai1 variants, as the percentage of inhibition of their expression in the presence of STIMs is similar when protein synthesis is allowed or impaired. Similarly, inhibition of ubiquitin‐proteasomal degradation has negligible effects, if any, on the Orai1 protein content. By contrast, impairment of the lysosomal proton gradient using bafilomycin A1 completely reversed the effect mediated by expression of STIM1 and STIM2, which demonstrates that STIM proteins modulate the protein level of Orai1αand Orai1βby endolysosomal degradation. Our findings are consistent with a previous study that revealed the role of lysosomal degradationinOrai1proteinturnover (Yeh et al., 2020). Furthermore, we have confirmed that the STIM1 OASF region mimics the degradation of Orai1 caused by STIM1, which indicates that OASF is sufficient for this process and strongly suggests that Orai1 degradation requires OASF‐Orai1 interaction. Nevertheless, Orai1 degradation does not depend on Ca 2+ influx through the channel, as expression of STIM proteins induces degradation of Orai1αbearing the pore‐dead E106Q mutation. We have found that the protein content of STIM1 is modulated by the expression of the Orai1 variants, which might suggest that both proteins recycle simultaneously. Interestingly, we have not detected any significant drop in the STIM2 protein content after co‐expression with the Orai1 variants, thus, indicating that STIM2 recycles by a mechanism that is independent of Orai1. As for Orai1, STIM1 and STIM2 recycling has been previously reported to be mediated by autophagy/lysosomal degradation and is independent of the proteasome (Keil et al., 2010; Serwach & Gruszczynska‐Biegala, 2020). However, the half‐life of STIM2 ( >24 h) (Rana et al., 2015) has been reported to be significantly greater than that of STIM1 (19 h) (Saitoh et al., 2011). The results presented here, demonstrating that STIM2 recycling is independent of Orai1 interaction, provides an explanation for the greater stability of STIM2 and are consistent with a role for STIM2 in the maintenance of basal Ca 2+ levels. Therefore, our results provide evidence that support two important aspects of the regulation of Orai1 variants function. On the one hand, while STIM1 is a stronger activator of Orai1αthan STIM2, both STIM proteins exhibit a similar strength activating Orai1β‐dependent Ca 2+ signals. Furthermore, our results provide evidence supporting that STIM proteins play a relevant role in Orai1α and Orai1βrecycling, which might serve as an additional feedback regulatory mechanism of SOCE as the extent of Ca 2+ influx through Orai1αand Orai1βis expected to depend not only on the intrinsic properties of the channels itself, such as Ca 2+ ‐dependent inactivation, but also on the number of available channels and its PM location. AUTHOR CONTRIBUTIONS Joel N. Felipe: Formal analysis; investigation; methodology. Alvaro Macias‐Díaz: Formal analysis; investigation; methodology. Vanesa Jimenez‐Velarde: Investigation. Jose J. Lopez: Methodology; writing—review and editing. Gines M. Salido: Writing—review and editing. Tarik Smani: Conceptualization; funding acquisition; supervision; writing—review and editing. Isaac Jardin: Conceptualization; data curation; formal analysis; investigation; methodology; supervision; validation; writing—original draft; writing—review and editing. Juan A. Rosado: Conceptualization; data curation; funding acquisition; project administration; resources; supervision; validation; writing—original draft; writing—review and editing. All the authors approved the final edited version. The degree of contribution to the FIGURE 11 Degradation of Orai1αand Orai1βby STIMs does not require Ca 2+ influx through the channel. (a) STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven Orai1α‐eGFP or a dominant negative Orai1αmutant (Orai1αdn), in combination with either STIM1‐YFP, STIM2‐YFP or the STIM1 OASF fragment, as indicated. Forty‐eight hours later cells were lysed, and cell lysates were analyzed by SDS‐PAGE and Western blot analysis using anti‐STIM1, anti‐STIM2 or anti‐Orai1 C‐terminal antibody. Membranes were reprobed with anti β‐actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. These results are representative of six separate experiments. Bar graphs show quantification of Orai1αand Orai1αdn expression under the different experimental conditions normalized to the β‐actin expression. Data are represented as mean ± SEM and expressed as fold change (experimental/ control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). **p< 0.01 and ****p< 0.0001 as compared to the expression of Orai1αor Orai1αdn in the absence of STIM proteins and fragments. (b) STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven Orai1αor a dominant negative Orai1αmutant (Orai1αdn), in combination with STIM1, as indicated. Fortyeight hours later cells were loaded with fura‐2 and suspended in a Ca 2+ ‐free HBS (100 µM EGTA added) and then stimulated with TG (2 µM) followed by the addition of extracellular Ca 2+ (final concentration 1.8 mM) to initiate Ca 2+ influx. Bar graphs represent quantification of TG‐ evoked Ca 2+ release and entry determined as described in materials and methods. Data are presented as mean ± SEM and expressed as fold change over control (cells expressing Orai1αand STIM1). Data were statistically analyzed using Mann–Whitney U test. ****p< 0.0001 as compared to cells expressing Orai1αand STIM1. (c) STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven STIM1 OASF fragment. Forty‐eight hours later GFP fluorescence was detected using an LSM900 confocal microscope. The images show representative confocal images of the STIM1 OASF region. The scale bar represents 100 μm. (d) STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven Orai1αor a dominant negative Orai1αmutant (Orai1αdn), in combination with STIM1 or the STIM1 OASF fragment, as indicated. Forty‐eight hours later cells were loaded with fura‐2 and suspended in a Ca 2+ ‐free HBS (100 µM EGTA added). Ca 2+ was added to the extracellular medium at a final concentration of 1.8 mM to initiate Ca 2+ influx. DKO, double‐knockout, SEM, standard error of mean. NIETO‐FELIPE ET AL. | 19 of 22 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License design, to performance of the experiments, and to manuscript writing were used to assign authorship order among co‐first authors. ACKNOWLEDGMENTS We thank Sandra Alvarado for assistance in some experiments and María Barriga for technical assistance. This research is part of the projects PID2022‐136279NB‐C21 and PID2022‐136279NB‐ C22 funded by MICIU/AEI/10.13039/501100011033/ and by “ERDF A way of making Europe”, and Junta de Extremadura‐ Fondo Europeo de Desarrollo Regional (FEDER; Grant IB20007 and GR18061) to JAR and TS. JN‐F and AM are supported by a contract from AEI/Spanish Ministry of Science and Innovation and INVESTIGO, respectively. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. FIGURE 12 STIM1 stability is modulated by Orai1αand Orai1β.STIM1,2‐DKO HEK‐293 cells were cotransfected with CMV‐driven STIM1‐YFP (a) or STIM2‐YFP (b) in combination with Orai1α‐eGFP or Orai1β‐eGFP, with a relative Orai1:STIM expression ratio of 1:2. Forty‐eight hours later cells were lysed and cell lysates were analyzed by SDS‐PAGE and Western blot analysis using anti‐STIM1 or anti‐STIM2 antibody. Membranes were probed with anti‐Orai1 C‐terminal antibody or anti β‐actin antibody. Molecular masses indicated on the right were determined using molecular‐mass markers run in the same gel. These results are representative of 6 separate experiments. Bar graphs represent the quantification of STIM1 and STIM2 expression under the different experimental conditions normalized to the β‐actin expression. Data are represented as mean ± SEM and expressed as fold increase (experimental/control). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ****p<0.0001ascomparedto the STIM1 expression in the absence of Orai1 variants. DKO, double‐knockout,SEM,standarderrorofmean. 20 of 22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License ETHICS STATEMENT Experimental procedures were approved by the local ethical committee (University of Extremadura and Extremadura Health Service). 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(2024). Feedback modulation of Orai1αand Orai1βprotein content mediated by STIM proteins. Journal of Cellular Physiology, e31450. https://doi.org/10.1002/jcp.31450 22 of 22 | NIETO‐FELIPE ET AL. 10974652, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/jcp.31450 by Readcube (Labtiva Inc.), Wiley Online Library on [11/10/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License