STIM and ORAI proteins in the nervous system

Stromal interaction molecules (STIM) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM proteins which, in turn, bind and open calcium channels in the plasma membrane formed by the proteins ORAI1, ORAI2, and ORAI3. The resulting store-operated calcium entry (SOCE), mostly controlled by the principal components STIM1 and ORAI1, has been particularly characterized in immune cells. In the nervous system, all STIM and ORAI homologs are expressed. This review summarizes current knowledge on distribution and function of STIM and ORAI proteins in central neurons and glial cells, i.e. astrocytes and microglia. STIM2 is required for SOCE in hippocampal synapses and cortical neurons, whereas STIM1 controls calcium store replenishment in cerebellar Purkinje neurons. In microglia, STIM1, STIM2, and ORAI1 regulate migration and phagocytosis. The isoforms ORAI2 and ORAI3 are candidates for SOCE channels in neurons and astrocytes, respectively. Due to the role of SOCE in neuronal and glial calcium homeostasis, dysfunction of STIM and ORAI proteins may have consequences for the development of neurodegenerative disorders, such as Alzheimer''s disease.     

STIM and ORAI proteins in the nervous system

Stromal interaction molecules (STIM) 1 and 2 are sensors of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM proteins which, in turn, bind and open calcium channels in the plasma membrane formed by the proteins ORAI1, ORAI2, and ORAI3. The resulting store-operated calcium entry (SOCE), mostly controlled by the principal components STIM1 and ORAI1, has been particularly characterized in immune cells. In the nervous system, all STIM and ORAI homologs are expressed. This review summarizes current knowledge on distribution and function of STIM and ORAI proteins in central neurons and glial cells, i.e. astrocytes and microglia. STIM2 is required for SOCE in hippocampal synapses and cortical neurons, whereas STIM1 controls calcium store replenishment in cerebellar Purkinje neurons. In microglia, STIM1, STIM2, and ORAI1 regulate migration and phagocytosis. The isoforms ORAI2 and ORAI3 are candidates for SOCE channels in neurons and astrocytes, respectively. Due to the role of SOCE in neuronal and glial calcium homeostasis, dysfunction of STIM and ORAI proteins may have consequences for the development of neurodegenerative disorders, such as Alzheimer's disease.     

STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage‐dependent calcium (Ca_V) channels. A second cold‐activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co‐expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store‐operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold‐induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.     

STIM1 is necessary for store-operated calcium entry in turning growth cones

J. Neurochem. (2012) 122, 1155-1166. ABSTRACT: Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.     

Activation and regulation of store‐operated calcium entry

The process of store-operated Ca²⁺ entry (SOCE), whereby Ca²⁺ influx across the plasma membrane is activated in response to depletion of intracellular Ca²⁺ stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca²⁺ sensors within the ER that, upon Ca²⁺ store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1

Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca²⁺ sensor and activator of Orai1 channels. In resting cells with replete Ca²⁺ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca²⁺ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca²⁺ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

Store-operated calcium entry remains fully functional in aged mouse skeletal muscle despite a decline in STIM1 protein expression

Store-operated Ca(2+) entry (SOCE) is a robust mechanism in skeletal muscle, supported by abundant STIM1 and Orai1 in the junctional membranes. The precise role of SOCE in skeletal muscle Ca(2+) homeostasis and excitation-contraction coupling remains to be defined. Regardless, it remains important to determine whether the function and capacity of SOCE changes in aged skeletal muscle. We identified an approximate 40% decline in the expression of the integral SOCE protein, stromal interacting molecule 1 (STIM1), but no such decline in its coupling partner, Orai1, in muscle fibers from aged mice. To determine whether this changed aspects of SOCE functionality in skeletal muscle in aged mice, Ca(2+) in the cytoplasm and t-system were continuously and simultaneously imaged on a confocal microscope during sarcoplasmic reticulum Ca(2+) release and compared to experiments under identical conditions using muscle fibers from young mice. Normal activation, deactivation, Ca(2+) influx, and spatiotemporal characteristics of SOCE were found to persist in skeletal muscle from aged mice. Thus, SOCE remains a robust mechanism in aged skeletal muscle despite the decline in STIM1 protein expression, suggesting STIM1 is in excess in young skeletal muscle.     

Coiled-Coil Formation Conveys a STIM1 Signal from ER Lumen to Cytoplasm

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STIM1-Ca2+ signaling in coronary sinus cardiomyocytes contributes to interatrial conduction

Pacemaker action potentials emerge from the sinoatrial node (SAN) and rapidly propagate through the atria to the AV node via preferential conduction pathways, including one associated with the coronary sinus. However, few distinguishing features of these tracts are known. Identifying specific molecular markers to distinguish among these conduction pathways will have important implications for understanding atrial conduction and atrial arrhythmogenesis. Using a Stim1 reporter mouse, we discovered stromal interaction molecule 1 (STIM1)-expressing coronary sinus cardiomyocytes (CSC)s in a tract from the SAN to the coronary sinus. Our studies here establish that STIM1 is a molecular marker of CSCs and we propose a role for STIM1-CSCs in interatrial conduction. Deletion of Stim1 from the CSCs slowed interatrial conduction and increased susceptibility to atrial arrhythmias. Store-operated Ca²⁺ currents (I_soc) in response to Ca²⁺ store depletion were markedly reduced in CSCs and their action potentials showed electrical remodeling. Our studies identify STIM1 as a molecular marker for a coronary sinus interatrial conduction pathway. We propose a role for SOCE in Ca²⁺ signaling of CSCs and implicate STIM1 in atrial arrhythmogenesis.     

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.     

STIM和Orai在钙库操纵的钙内流途径及心脑血管疾病中的作用

钙库操纵的钙内流(SOCE)是调节钙离子(Ca2+)内流进入细胞最普遍的一种途径,它的通道称为钙库操纵的钙内流通道(SOC)。SOC存在于大多数非兴奋细胞和部分兴奋细胞上,近年来确定,STIM和Orai是组成SOC的两种主要蛋白质。本文就近年来对SOCE途径的机制,STIM和Orai不同亚型的结构、功能及在心脑血管疾病中的作用作一综述。     

Orai1 and STIM1 mediate SOCE and contribute to apoptotic resistance of pancreatic adenocarcinoma

The store-operated calcium channels (SOCs) represent one of the major calcium-entry pathways in non-excitable cells. SOCs and in particular their major components ORAI1 and STIM1 have been shown to be implicated in a number of physiological and pathological processes such as apoptosis, proliferation and invasion. Here we demonstrate that ORAI1 and STIM1 mediate store-operated calcium entry (SOCE) in pancreatic adenocarcinoma cell lines. We show that both ORAI1 and STIM1 play pro-survival anti-apoptotic role in pancreatic adenocarcinoma cell lines, as siRNA-mediated knockdown of ORAI1 and/or STIM1 increases apoptosis induced by chemotherapy drugs 5-fluorouracil (5-FU) or gemcitabine. We also demonstrate that both 5-FU and gemcitabine treatments increase SOCE in Panc1 pancreatic adenocarcinoma cell line via upregulation of ORAI1 and STIM1. Altogether our results reveal the novel calcium-dependent mechanism of action of the chemotherapy drugs 5-FU and gemcitabine and emphasize the anti-apoptotic role of ORAI1 and STIM1 in pancreatic adenocarcinoma cells. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.