Role of the STIM1 C-terminal Domain in STIM1 Clustering

Store-operated Ca²⁺ entry (SOCE) represents a ubiquitous Ca²⁺ influx pathway activated by the filling state of intracellular Ca²⁺ stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca²⁺ sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca²⁺ stores are full, these domains are inactive to prevent constitutive Ca²⁺ entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca²⁺ stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.     

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺ entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺ buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺ imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.     

Lipid rafts are essential for the regulation of SOCE by plasma membrane resident STIM1 in human platelets

STIM1 is a transmembrane protein essential for the activation of store-operated Ca²⁺ entry (SOCE), a major Ca²⁺ influx mechanism. STIM1 is either located in the endoplasmic reticulum, communicating the Ca²⁺ concentration in the stores to plasma membrane channels or in the plasma membrane, where it might sense the extracellular Ca²⁺ concentration. Plasma membrane-located STIM1 has been reported to mediate the SOCE sensitivity to extracellular Ca²⁺ through its interaction with Orai1. Here we show that plasma membrane lipid raft domains are essential for the regulation of SOCE by extracellular Ca²⁺. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn²⁺ entry, which was inhibited by increasing concentrations of extracellular Ca²⁺. Platelet treatment with methyl-β-cyclodextrin, which removes cholesterol and disrupts the lipid raft domains, impaired the inactivation of Ca²⁺ entry induced by extracellular Ca²⁺. Methyl-β-cyclodextrin also abolished translocation of STIM1 to the plasma membrane stimulated by treatment with TG and prevented TG-evoked co-immunoprecipitation between plasma membrane-located STIM1 and the Ca²⁺ permeable channel Orai1. These findings suggest that lipid raft domains are essential for the inactivation of SOCE by extracellular Ca²⁺ mediated by the interaction between plasma membrane-located STIM1 and Orai1.     

A Reciprocal Shift in Transient Receptor Potential Channel 1 (TRPC1) and Stromal Interaction Molecule 2 (STIM2) Contributes to Ca2+ Remodeling and Cancer Hallmarks in Colorectal Carcinoma Cells

We have investigated the molecular basis of intracellular Ca²⁺ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca²⁺ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca²⁺ levels, agonist-induced Ca²⁺ increases, store-operated Ca²⁺ entry (SOCE), and store-operated currents (I_SOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca²⁺ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca²⁺ release-activated Ca²⁺ currents (I_CRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced I_CRAC plus a nonselective I_SOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca²⁺ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca²⁺ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca²⁺ remodeling and tumor features in colon cancer.     

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.     

Potent functional uncoupling between STIM1 and Orai1 by dimeric 2-aminodiphenyl borinate analogs

The coupling of ER Ca²⁺-sensing STIM proteins and PM Orai Ca²⁺ entry channels generates “store-operated” Ca²⁺ signals crucial in controlling responses in many cell types. The dimeric derivative of 2-aminoethoxydiphenyl borinate (2-APB), DPB162-AE, blocks functional coupling between STIM1 and Orai1 with an IC₅₀ (200 nM) 100-fold lower than 2-APB. Unlike 2-APB, DPB162-AE does not affect L-type or TRPC channels or Ca²⁺ pumps at maximal STIM1–Orai1 blocking levels. DPB162-AE blocks STIM1-induced Orai1 or Orai2, but does not block Orai3 or STIM2-mediated effects. We narrowed the DPB162-AE site of action to the STIM–Orai activating region (SOAR) of STIM1. DPB162-AE does not prevent the SOAR–Orai1 interaction but potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores SOAR–Orai binding but only slowly restores Orai1 channel-mediated Ca²⁺ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1–Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.     

Knockdown of STIM1 inhibits 6-hydroxydopamine-induced oxidative stress through attenuating calcium-dependent ER stress and mitochondrial dysfunction in undifferentiated PC12 cells

Abstract

Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca²⁺ signaling systems and are considered to be important players in regulating neuronal Ca²⁺ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undifferentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca²⁺-dependent manner. Downregulation of STIM1 expression by specific small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown significantly attenuated 6-OHDA-induced intracellular Ca²⁺ overload by inhibiting endogenous store-operated calcium entry (SOCE). The effect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca²⁺ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca²⁺ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca²⁺ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the first evidence that inhibition of STIM1-meditated intracellular Ca²⁺ dyshomeostasis protects undifferentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD.     

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.     

Overexpression of Orai1 and STIM1 proteins alters regulation of store-operated Ca2+ entry by endogenous mediators

Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca²⁺ entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca²⁺-independent phospholipase A₂ β (iPLA₂β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA₂β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA₂β but not STIM1. These data confirm the role of iPLA₂β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.     

Unraveling STIM2 function

The discovery of molecular players in capacitative calcium (Ca²⁺) entry, also referred to as store-operated Ca²⁺ entry (SOCE), supposed a great advance in the knowledge of cellular mechanisms of Ca²⁺ entry, which are essential for a broad range of cellular functions. The identification of STIM1 and STIM2 proteins as the sensors of Ca²⁺ stored in the endoplasmic reticulum unraveled the mechanism by which depletion of intracellular Ca²⁺ stores is communicated to store-operated Ca²⁺ channels located in the plasma membrane, triggering the activation of SOCE and intracellular Ca²⁺-dependent signaling cascades. Initial studies suggested a dominant function of STIM1 in SOCE and SOCE-dependent cellular functions compared to STIM2, especially those that participate in immune responses. Consequently, most of the subsequent studies focused on STIM1. However, during the last years, STIM2 has been demonstrated to play a more relevant and complex function than initially reported, being even important to sustain normal life in mice. These studies have led to reconsider the role of STIM2 in SOCE and its relevance in cellular physiology. This review is intended to summarize and provide an overview of the current data available about this exciting isoform, STIM2, and its actual position together with STIM1 in the mechanism of SOCE.     

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.     

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.     

Constitutive Activation of the Calcium Sensor STIM1 Causes Tubular-Aggregate Myopathy

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca²⁺ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca²⁺-binding EF hands. Ca²⁺ stores are refilled through a process called store-operated Ca²⁺ entry (SOCE). Upon Ca²⁺-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca²⁺ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca²⁺ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca²⁺ level in TAM cells and a dysregulation of intracellular Ca²⁺ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.