Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca²⁺ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

STIM1 regulates store-operated Ca entry in oocytes

The single transmembrane-spanning Ca²⁺-binding protein, STIM1, has been proposed to function as a Ca²⁺ sensor that links the endoplasmic reticulum to the activation of store-operated Ca²⁺ channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca²⁺ entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca²⁺ stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca²⁺ stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca²⁺ influx after store depletion and subsequent Ca²⁺ add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca²⁺ channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca²⁺ levels in the store-depleted oocytes after Ca²⁺ add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca²⁺ store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca²⁺ entry.     

Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca²⁺ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca²⁺ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca²⁺ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca²⁺ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca²⁺ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRAS^G13D), and have shown that reduced Ca²⁺ release from the ER and mitochondrial Ca²⁺ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca²⁺ Entry (SOCE) and its underlying current, I_CRAC are under the influence of KRAS^G13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca²⁺ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRAS^G13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRAS^G13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca²⁺ entry downstream of KRAS^G13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis.     

Mechanosensitive Ca2+ permeant cation channels in human prostate tumor cells

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca²⁺-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd³⁺ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca²⁺]_i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca²⁺ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca²⁺ influx to a small Ca²⁺-activated K⁺ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca²⁺ signaling that may play a role in tumor progression.     

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca²⁺ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca²⁺ increase generated by an interplay between ER-Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca²⁺ depletion and binds Orai1 to activate Ca²⁺ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca²⁺ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca²⁺ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca²⁺ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca²⁺ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca²⁺ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca²⁺ influx.     

Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.     

Ion channels and anti-cancer immunity

The outcome of a malignant disease depends on the efficacy of the immune system to destroy cancer cells. Key steps in this process, for example the generation of a proper Ca²⁺ signal induced by recognition of a specific antigen, are regulated by various ion channel including voltage-gated Kv1.3 and Ca²⁺-activated KCa3.1 K⁺ channels, and the interplay between Orai and STIM to produce the Ca²⁺-release-activated Ca²⁺ (CRAC) current required for T-cell proliferation and function. Understanding the immune cell subset-specific expression of ion channels along with their particular function in a given cell type, and the role of cancer tissue-dependent factors in the regulation of operation of these ion channels are emerging questions to be addressed in the fight against cancer disease. Answering these questions might lead to a better understanding of the immunosuppression phenomenon in cancer tissue and the development of drugs aimed at skewing the distribution of immune cell types towards killing of the tumour cells.     

STIM1 Regulates Platelet-Derived Growth Factor-Induced Migration and Ca2+ Influx in Human Airway Smooth Muscle Cells

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca²⁺ sensor that activates Orai1, the Ca²⁺ channel responsible for store-operated Ca²⁺ entry (SOCE). We investigated the role of STIM1 in [Ca²⁺]_i and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca²⁺-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca²⁺]_i followed by sustained [Ca²⁺]_i elevation. Sustained increases in [Ca²⁺]_i due to PDGF-BB were significantly inhibited by a Ca²⁺ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca²⁺]_i or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca²⁺ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.     

A Novel Native Store-operated Calcium Channel Encoded by Orai3: SELECTIVE REQUIREMENT OF Orai3 VERSUS Orai1 IN ESTROGEN RECEPTOR-POSITIVE VERSUS ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS*

Store-operated calcium (Ca²⁺) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca²⁺ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca²⁺ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ current (I_CRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca²⁺ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I_CRAC in estrogen receptor-positive (ER⁺) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER⁻) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER⁺ breast cancer cells represent the first example where the native SOCE pathway and I_CRAC are mediated by Orai3. Future studies implicating Orai3 in ER⁺ breast cancer progression might establish Orai3 as a selective target in therapy of ER⁺ breast tumors.     

Lysosomal cysteine protease cathepsin S is involved in cancer cell motility by regulating store-operated Ca2+ entry

Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca²⁺-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca²⁺ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.     

Involvement of STIM1 in the proteinase‐activated receptor 1‐mediated Ca2+ influx in vascular endothelial cells

Thrombin increases the cytosolic Ca²⁺ concentrations and induces NO production by activating proteinase‐activated receptor 1 (PAR₁) in vascular endothelial cells. The store‐operated Ca²⁺ influx is a major Ca²⁺ influx pathway in non‐excitable cells including endothelial cells and it has been reported to play a role in the thrombin‐induced Ca²⁺ signaling in endothelial cells. Recent studies have identified stromal interaction molecule 1 (STIM1) to function as a sensor of the store site Ca²⁺ content, thereby regulating the store‐operated Ca²⁺ influx. However, the functional role of STIM1 in the thrombin‐induced Ca²⁺ influx and NO production in endothelial cells still remains to be elucidated. Fura‐2 and diaminorhodamine‐4M fluorometry was utilized to evaluate the thrombin‐induced changes in cytosolic Ca²⁺ concentrations and NO production, respectively, in porcine aortic endothelial cells transfected with small interfering RNA (siRNA) targeted to STIM1. STIM1‐targeted siRNA suppressed the STIM1 expression and the thapsigargin‐induced Ca²⁺ influx. The degree of suppression of the STIM1 expression correlated well to the degree of suppression of the Ca²⁺ influx. The knockdown of STIM1 was associated with a substantial inhibition of the Ca²⁺ influx and a partial reduction of the NO production induced by thrombin. The thrombin‐induced Ca²⁺ influx exhibited the similar sensitivity toward the Ca²⁺ influx inhibitors to that seen with the thapsigargin‐induced Ca²⁺ influx. The present study provides the first evidence that STIM1 plays a critical role in the PAR₁‐mediated Ca²⁺ influx and Ca²⁺‐dependent component of the NO production in endothelial cells. J. Cell. Biochem. 108: 499–507, 2009. © 2009 Wiley‐Liss, Inc.     

Store-independent Orai1-mediated Ca2+ entry and cancer

Ca²⁺ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca²⁺ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca²⁺ channels. Orai1 mediates the highly Ca²⁺-selective Ca²⁺ release-activated current (I_CRAC) and participates in the less Ca²⁺-selective store-operated current (I_SOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca²⁺ influx mechanisms, including the arachidonate-regulated Ca²⁺ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca²⁺ influx processes activated by the secretory pathway Ca²⁺-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca²⁺-activated K⁺ channel 3 (SK3) or the voltage-dependent K_v10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca²⁺ influx activation through Orai1 channels and their role in the development of different cancer features.     

Tunicamycin desensitizes store-operated Ca entry to ATP and mitochondrial potential

Tunicamycin effect on thapsigargin-induced store-operated calcium entry was investigated. Ca²⁺ influx was stimulated by 50% upon exposure of Jurkat cells to tunicamycin. Moreover, tunicamycin efficiently prevented the inhibition of store-operated calcium entry caused by dissipation of mitochondrial membrane potential. Protective action of tunicamycin on store-operated Ca²⁺ entry was also partially preserved in Jurkat cells depleted of ATP, while Ca²⁺ entry into ATP-deprived cells grown in tunicamycin-free medium was almost completely inhibited. Tunicamycin-evoked changes in cellular Ca²⁺ fluxes coincided with decreased glycosylation of STIM1 protein. Although the latter observation is correlative and needs additional confirmation it may suggest that deglycosylation of STIM1 protein deprives store-operated calcium entry system of an important regulatory mechanism. This study suggests a novel mechanism of modulation of the activity of store-operated calcium channels in lymphoidal cells.     

Plasma Membrane Localized GCaMP-MS4A12 by Orai1 Co-Expression Shows Thapsigargin- and Ca2+-Dependent Fluorescence Increases

Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca²⁺ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca²⁺ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca²⁺ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca²⁺ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca²⁺. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.     

Intrinsic Disorder Mediates Cooperative Signal Transduction in STIM1

Intrinsically disordered domains have been reported to play important roles in signal transduction networks by introducing cooperativity into protein–protein interactions. Unlike intrinsically disordered domains that become ordered upon binding, the EF-SAM domain in the stromal interaction molecule (STIM) 1 is distinct in that it is ordered in the monomeric state and partially unfolded in its oligomeric state, with the population of the two states depending on the local Ca^2 + concentration. The oligomerization of STIM1, which triggers extracellular Ca^2 + influx, exhibits cooperativity with respect to the local endoplasmic reticulum Ca^2 + concentration. Although the physiological importance of the oligomerization reaction is well established, the mechanism of the observed cooperativity is not known. Here, we examine the response of the STIM1 EF-SAM domain to changes in Ca^2 + concentration using mathematical modeling based on in vitro experiments. We find that the EF-SAM domain partially unfolds and dimerizes cooperatively with respect to Ca^2 + concentration, with Hill coefficients and half-maximal activation concentrations very close to the values observed in vivo for STIM1 redistribution and extracellular Ca^2 + influx. Our mathematical model of the dimerization reaction agrees quantitatively with our analytical ultracentrifugation-based measurements and previously published free energies of unfolding. A simple interpretation of these results is that Ca^2 + loss effectively acts as a denaturant, enabling cooperative dimerization and robust signal transduction. We present a structural model of the Ca^2 +-unbound EF-SAM domain that is consistent with a wide range of evidence, including resistance to proteolytic cleavage of the putative dimerization portion.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

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.