Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

Calumenin is a multiple EF-hand Ca²⁺-binding protein localized in the sarcoplasmic reticulum (SR) with C-terminal SR retention signal HDEF. Recently, we showed evidence that calumenin interacts with SERCA2 in rat cardiac SR (Sahoo, S. K., and Kim, D. H. (2008) Mol. Cells 26, 265–269). The present study was undertaken to further characterize the association of calumenin with SERCA2 in mouse heart by various gene manipulation approaches. Immunocytochemical analysis showed that calumenin and SERCA2 were partially co-localized in HL-1 cells. Knockdown (KD) of calumenin was conducted in HL-1 cells and 80% reduction of calumenin did not induce any expressional changes of other Ca²⁺-cycling proteins. But it enhanced Ca²⁺ transient amplitude and showed shortened time to reach peak and decreased time to reach 50% of baseline. Oxalate-supported Ca²⁺ uptake showed increased Ca²⁺ sensitivity of SERCA2 in calumenin KD HL-1 cells. Calumenin and SERCA2 interaction was significantly lower in the presence of thapsigargin, vanadate, or ATP, as compared with 1.3 μm Ca²⁺, suggesting that the interaction is favored in the E1 state of SERCA2. A glutathione S-transferase-pulldown assay of calumenin deletion fragments and SERCA2 luminal domains suggested that regions of 132–222 amino acids of calumenin and 853–892 amino acids of SERCA2-L4 are the major binding partners. On the basis of our in vitro binding data and available information on three-dimensional structure of Ca²⁺-ATPases, a molecular model was proposed for the interaction between calumenin and SERCA2. Taken together, the present results suggest that calumenin is a novel regulator of SERCA2, and its expressional changes are tightly coupled with Ca²⁺-cycling of cardiomyocytes.     

Pleiotropic roles of calumenin (calu-1), a calcium-binding ER luminal protein, in Caenorhabditis elegans

Calumenin is a Ca²⁺ binding protein localizing at the lumen of the endoplasmic reticulum (ER). Although it has been implicated in various diseases, the in vivo functions of calumenin are largely unknown. Here, we report that calumenin has pleiotropic roles in muscle and cuticle function in Caenorhabditis elegans. Mutant analysis revealed that the calu-1 is required for regulating fertility, locomotion and body size. In addition, calu-1 is important for two behaviors, defecation and pharyngeal pumping, consistent with its ability to bind Ca²⁺. The genetic analysis further suggested the possibility that calu-1 regulates the pharyngeal pumping together with the inositol 1,4,5-triphosphate (IP₃) receptor encoded by itr-1. Taken together, our data suggest that calumenin is important for calcium signaling pathways in C. elegans.     

The rapidly expanding CREC protein family: members,localization, function,and role in disease

Although many aspects of the physiological and pathophysiological mechanisms remain unknown, recent advances in our knowledge suggest that the CREC proteins are promising disease biomarkers or targets for therapeutic intervention in a variety of diseases. The CREC family of low affinity, Ca²⁺‐binding, multiple EF‐hand proteins are encoded by five genes, RCN1, RCN2, RCN3, SDF4, and CALU, resulting in reticulocalbin, ER Ca²⁺‐binding protein of 55 kDa (ERC‐55), reticulocalbin‐3, Ca²⁺‐binding protein of 45 kDa (Cab45), and calumenin. Alternative splicing increases the number of gene products. The proteins are localized in the cytosol, in various parts of the secretory pathway, secreted to the extracellular space or localized on the cell surface. The emerging functions appear to be highly diverse. The proteins interact with several different ligands. Rather well‐described functions are attached to calumenin with the inhibition of several proteins in the endoplasmic or sarcoplasmic reticulum membrane, the vitamin K₁ 2,3‐epoxide reductase, the γ‐carboxylase, the ryanodine receptor, and the Ca²⁺‐transporting ATPase. Other functions concern participation in the secretory process, chaperone activity, signal transduction as well as participation in a large variety of disease processes.     

Calumenin has a role in the alleviation of ER stress in neonatal rat cardiomyocytes

Disturbance of endoplasmic reticulum (ER) homeostasis causes ER stress (ERS), and triggers the unfolded protein response (UPR) that consequently reduces accumulation of unfolded proteins by increasing the quantity of ER chaperones. Calumenin, a Ca²⁺-binding protein with multiple EF hand motifs, which is located in the ER/SR, is highly expressed during the early developmental stage of the heart, similar to other ER-resident chaperones. The aim of this study was to investigate the functional role of calumenin during ERS in the heart. Like other chaperones (e.g., GRP94 and GRP78), calumenin expression was highly upregulated during ERS induced by 10 μg/ml tunicamycin, but attenuated in the presence of 500 μM PBA, the chemical chaperone in neonatal rat ventricular cardiomyocytes (NRVCs). Upon 7.5-fold overexpression of calumenin using a recombinant adenovirus system, the expression levels of ERS markers (GRP78, p-PERK, and p-elF2α) and ER-initiated apoptosis markers (CHOP and p-JNK) were reduced, whereas the survival protein BCL-2 was upregulated during ERS compared to the control. Evaluation of cell viability by TUNEL assay showed that apoptosis was also significantly reduced by calumenin overexpression in ERS-induced cells. Taken together, our results suggest that calumenin plays an essential role in the alleviation of ERS in neonatal rat cardiomyocytes.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

The myristoylation of guanylate cyclase-activating protein-2 causes an increase in thermodynamic stability in the presence but not in the absence of Ca²⁺

Guanylate cyclase activating protein‐2 (GCAP‐2) is a Ca²⁺‐binding protein of the neuronal calcium sensor (NCS) family. Ca²⁺‐free GCAP‐2 activates the retinal rod outer segment guanylate cyclases ROS‐GC1 and 2. Native GCAP‐2 is N‐terminally myristoylated. Detailed structural information on the Ca²⁺‐dependent conformational switch of GCAP‐2 is missing so far, as no atomic resolution structures of the Ca²⁺‐free state have been determined. The role of the myristoyl moiety remains poorly understood. Available functional data is incompatible with a Ca²⁺‐myristoyl switch as observed in the prototype NCS protein, recoverin. For the homologous GCAP‐1, a Ca²⁺‐independent sequestration of the myristoyl moiety inside the proteins structure has been proposed. In this article, we compare the thermodynamic stabilities of myristoylated and non‐myristoylated GCAP‐2 in their Ca²⁺‐bound and Ca²⁺‐free forms, respectively, to gain information on the nature of the Ca²⁺‐dependent conformational switch of the protein and shed some light on the role of its myristoyl group. In the absence of Ca²⁺, the stability of the myristoylated and non‐myristoylated forms was indistinguishable. Ca²⁺ exerted a stabilizing effect on both forms of the protein, which was significantly stronger for myr GCAP‐2. The stability data were corroborated by dye binding experiments performed to probe the solvent‐accessible hydrophobic surface of the protein. Our results strongly suggest that the myristoyl moiety is permanently solvent‐exposed in Ca²⁺‐free GCAP‐2, whereas it interacts with a hydrophobic part of the protein's structure in the Ca²⁺‐bound state.     

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca²⁺ leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca²⁺ leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic β-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca²⁺ imaging to monitor the effects on ER Ca²⁺ leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca²⁺ leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca²⁺ leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic β-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.     

ATP increases within the lumen of the endoplasmic reticulum upon intracellular Ca2+ release

Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca²⁺ and redox insensitive, which makes it possible to monitor Ca²⁺-coupled ER ATP dynamics specifically. The approach uncovers a cell type–specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca²⁺ release is coupled to an increase of ATP within the ER. The Ca²⁺-coupled ER ATP increase is independent of the mode of Ca²⁺ mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca²⁺ depletion of the organelle. Our data highlight a novel Ca²⁺-controlled process that supplies the ER with additional energy upon cell stimulation.     

Electron Paramagnetic Resonance Spectroscopy of Nitroxide-Labeled Calmodulin

Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca²⁺)₄-form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2–3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca²⁺ to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca²⁺ binding. The midpoint of the Ca²⁺-mediated transition determined by EPR occurred at a higher Ca²⁺ concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca²⁺)₄-CaM], contains a compact intermediate corresponding to [(Ca²⁺)₂-CaM], and the present results suggest that the spin probes are reporting on Ca²⁺ binding to the last two sites in the N-terminal domain, i.e. for the [(Ca²⁺)₂-CaM] → [(Ca²⁺)₄-CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca²⁺-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

When worlds collide: IP3 receptors and the ERAD pathway

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca²⁺ store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP₃) receptors are activated, since in addition to acting as release channels for stored ER Ca²⁺, IP₃ receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP₃ receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP₃ receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP₃ receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP₃ receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca²⁺ stores to IP₃ and may protect cells against deleterious effects of over-activation of Ca²⁺ signaling pathways.     

Auxiliary Ca2+ binding sites can influence the structure of CIB1

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca²⁺ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K_d, 10⁻²M) Ca²⁺ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca²⁺ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca²⁺ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca²⁺ concentrations.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca²⁺ leak using synthetic Ca²⁺ indicators that report changes in both the cytoplasmic ([Ca²⁺]_i) and the luminal ER ([Ca²⁺]_ER) Ca²⁺ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca²⁺ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca²⁺]_ER but did not decrease the ER Ca²⁺ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP₃R and only Orai3 channel supported the 2-APB-induced ER Ca²⁺ leak, while Orai1 and Orai2 inhibited this type of ER Ca²⁺ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca²⁺ leak but did not modify the ER Ca²⁺ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca²⁺]_i response after TG but only when the ER store had been overloaded with Ca²⁺ by eliminating the acidic internal Ca²⁺ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca²⁺ leak but forms an ER Ca²⁺ leak channel that is limiting the overloading with Ca²⁺ of the ER store.     

Bcl-2 Modulates Endoplasmic Reticulum and Mitochondrial Calcium Stores in PC12 Cells

Endoplasmic reticulum (ER) and mitochondria are intracellular organelles and their interactions are directly involved in different processes such as Ca²⁺ signaling in cell survival and death mechanisms. Bcl-2 is an anti-apoptotic protein intrinsically related to ER and mitochondria, modulating Ca²⁺ content in these organelles. We investigated the effects of Bcl-2 overexpression on ER and mitochondrial Ca²⁺ dynamics in PC12 cells. Bcl-2 overexpressing and control cells were loaded with Fura 2/AM and stimulated with different drugs. Results showed that in Bcl-2 cells, ACh induced a lower Ca²⁺ response compared to control. Ca²⁺ release induced by TG was decreased in Bcl-2 cells, however, it was greater in Caff induced Ca²⁺ rise. In addition, FCCP induced a higher Ca²⁺ release in Bcl-2 cells. These results suggest that Bcl-2 overexpression modulate the ER Ca²⁺ pools differently and the release of ER Ca²⁺ may increase mitochondrial Ca²⁺ accumulation. These alterations of intracellular Ca²⁺ stores are important mechanisms for the control of Ca²⁺ signaling.     

Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

Ca²⁺ (calcium) homoeostasis and signalling rely on physical contacts between Ca²⁺ sensors in the ER (endoplasmic reticulum) and Ca²⁺ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca²⁺ sensors oligomerize upon Ca²⁺ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca²⁺ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca²⁺ have been studied but responses towards elevated cytosolic Ca²⁺ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P₂-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P₂, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca²⁺ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca²⁺ and PI(4,5)P₂ concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca²⁺ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.