The family of stromal interaction molecules (STIM) includes two widely expressed single-pass endoplasmic reticulum (ER) transmembrane proteins and additional splice variants that act as precise ER-luminal Ca2+ sensors. STIM proteins mainly function as one of the two essential components of the so-called Ca2+ release-activated Ca2+ (CRAC) channel. The second CRAC channel component is constituted by pore-forming Orai proteins in the plasma membrane. STIM and Orai physically interact with each other to enable CRAC channel opening, which is a critical prerequisite for various downstream signalling pathways such as gene transcription or proliferation. Their activation commonly requires the emptying of the intracellular ER Ca2+ store. Using their Ca2+ sensing capabilities, STIM proteins confer this Ca2+ content-dependent signal to Orai, thereby linking Ca2+ store depletion to CRAC channel opening. Here we review the conformational dynamics occurring along the entire STIM protein upon store depletion, involving the transition from the quiescent, compactly folded structure into an active, extended state, modulation by a variety of accessory components in the cell as well as the impairment of individual steps of the STIM activation cascade associated with disease.
- MeSH
- Calcium Release Activated Calcium Channels * MeSH
- Membrane Proteins metabolism MeSH
- ORAI1 Protein MeSH
- Stromal Interaction Molecule 1 metabolism MeSH
- Stromal Interaction Molecules * metabolism MeSH
- Calcium metabolism MeSH
- Calcium Signaling physiology MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.
- MeSH
- Amino Acids metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Calcium Release Activated Calcium Channels * genetics MeSH
- Membrane Proteins * metabolism MeSH
- Mutation MeSH
- Mice MeSH
- ORAI1 Protein metabolism MeSH
- Stromal Interaction Molecule 1 genetics MeSH
- Calcium metabolism MeSH
- Calcium Channels metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Phosphatidylcholines chemistry metabolism MeSH
- Ion Channel Gating genetics MeSH
- Genetic Vectors chemistry metabolism MeSH
- HEK293 Cells MeSH
- Protein Interaction Domains and Motifs MeSH
- Protein Conformation, alpha-Helical MeSH
- Protein Conformation, beta-Strand MeSH
- Humans MeSH
- Liposomes chemistry metabolism MeSH
- Luminescent Proteins genetics metabolism MeSH
- Patch-Clamp Techniques MeSH
- Mutation MeSH
- Neoplasm Proteins chemistry genetics metabolism MeSH
- ORAI1 Protein chemistry genetics metabolism MeSH
- Stromal Interaction Molecule 1 chemistry genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Genes, Reporter MeSH
- Molecular Dynamics Simulation MeSH
- Amino Acid Substitution MeSH
- Calcium metabolism MeSH
- Calcium Signaling * MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.
Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.
- MeSH
- Lymphocyte Activation MeSH
- Cell Membrane metabolism MeSH
- Drosophila melanogaster MeSH
- Ion Channel Gating MeSH
- HEK293 Cells MeSH
- Protein Conformation MeSH
- Humans MeSH
- Membrane Proteins metabolism MeSH
- Mutation MeSH
- Peptide Hydrolases chemistry MeSH
- ORAI1 Protein chemistry MeSH
- Serine Endopeptidases metabolism MeSH
- Signal Transduction MeSH
- Stochastic Processes MeSH
- Calcium metabolism MeSH
- Calcium Signaling physiology MeSH
- Calcium Channels chemistry MeSH
- Protein Binding MeSH
- Computational Biology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
- MeSH
- Erythrocytes, Abnormal MeSH
- Dyslexia genetics MeSH
- HEK293 Cells MeSH
- Ichthyosis genetics MeSH
- Calcium Release Activated Calcium Channels metabolism MeSH
- Cloning, Molecular MeSH
- Nucleic Acid Conformation MeSH
- Humans MeSH
- Magnetic Resonance Spectroscopy MeSH
- Patch-Clamp Techniques MeSH
- Migraine Disorders genetics MeSH
- Miosis genetics MeSH
- Models, Molecular MeSH
- Mutation genetics MeSH
- Neoplasm Proteins genetics MeSH
- ORAI1 Protein genetics MeSH
- Stromal Interaction Molecule 1 genetics MeSH
- Spleen abnormalities MeSH
- Muscle Fatigue genetics MeSH
- Blood Platelet Disorders genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Store-operated calcium entry (SOCE) is one of regulatory mechanisms which regulates Ca2+ cycling in the heart. SOCE alterations in pathological conditions contribute to progression of heart failure and cardiac hypertrophy by multiple signaling pathways such as Cn/NFAT and CaMKII/MEF2. Several components mediating SOCE have been identified, such as STIM and Orai. Different isoforms of both Orai and STIM have been detected in animal studies, exhibiting distinct functional properties. This study is focused on the analysis of STIM and Orai isoforms expression in the end-stage human failing myocardium. Left ventricle samples isolated from 43 explanted hearts from patients undergoing heart transplant and from 5 healthy donor hearts were used to determine the mRNA levels of Orai1, Orai2 and Orai3, STIM1, STIM2 and STIM2.1 by qRT-PCR. The expression was further analyzed for connection with gender, related co-morbidities, pathoetiology, clinical data and biochemical parameters. We show that Orai1 expression is decreased by 30 % in failing myocardium, even though we detected no significant changes in expression of Orai2 or Orai3. Interestingly, this decrease in Orai1 was gender-specific and was present only in men, with no change in women. The ratio Orai1/Orai3 was significantly lower in males as well. The novel STIM2.1 isoform was detected both in healthy and failing human myocardium. In the end-stage heart failure, the expression of STIM2.1 was significantly decreased. The lower ratio of STIM2.1/STIM2 in failing hearts indicates a switch from SOCE-inhibiting STIM2.1 isoform to stimulatory STIM2.2. STIM1 mRNA levels were not significantly changed. These observed alterations in Orai and STIM expression were independent of functional heart parameters, clinical or biochemical patient characteristics. These results provide detailed insight into the alterations of SOCE regulation in human failing myocardium. Gender-specific change in Orai1 expression might represent a possible mechanism of cardioprotective effects of estrogens. The switch from STIM2.1 to STIM2.2 indicates an amplification of SOCE and could contribute to the hypertrophy development in the filing heart.
- MeSH
- Adult MeSH
- Calcium Release Activated Calcium Channels metabolism MeSH
- Middle Aged MeSH
- Humans MeSH
- Myocardium metabolism MeSH
- Sex Characteristics MeSH
- Protein Isoforms metabolism MeSH
- Stromal Interaction Molecules metabolism MeSH
- Ventricular Remodeling MeSH
- Heart Failure metabolism MeSH
- Case-Control Studies MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.
- MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Neoplasm Proteins chemistry genetics metabolism MeSH
- ORAI1 Protein chemistry genetics metabolism MeSH
- Stromal Interaction Molecule 1 chemistry genetics metabolism MeSH
- Protein Domains MeSH
- Protein Structure, Secondary MeSH
- Calcium Channels chemistry genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.
- MeSH
- Chromatography, Affinity MeSH
- Dynamic Light Scattering MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Isotope Labeling MeSH
- Nitrogen Isotopes chemistry isolation & purification MeSH
- Carbon Isotopes chemistry isolation & purification MeSH
- Humans MeSH
- Neoplasm Proteins chemistry isolation & purification MeSH
- Nuclear Magnetic Resonance, Biomolecular MeSH
- Stromal Interaction Molecule 1 chemistry isolation & purification MeSH
- Protein Domains MeSH
- Recombinant Proteins chemistry isolation & purification MeSH
- Solubility MeSH
- Protein Folding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current. Orai1, which represents the subunit forming the CRAC channel complex, is activated by the ER resident Ca2+ sensor STIM1. The genetically inherited Stormorken syndrome disease has been associated with the STIM1 single point R304W mutant. The resulting constitutive activation of Orai1 mainly involves the CRAC-activating domain CAD/SOAR of STIM1, the exposure of which is regulated by the molecular interplay between three cytosolic STIM1 coiled-coil (CC) domains. Here we present a dual mechanism by which STIM1 R304W attains the pathophysiological, constitutive activity eliciting the Stormorken syndrome. The R304W mutation induces a helical elongation within the CC1 domain, which together with an increased CC1 homomerization, destabilize the resting state of STIM1. This culminates, even in the absence of store depletion, in structural extension and CAD/SOAR exposure of STIM1 R304W leading to constitutive CRAC channel activation and Stormorken disease.
- MeSH
- Erythrocytes, Abnormal metabolism pathology MeSH
- Bacterial Proteins genetics metabolism MeSH
- Point Mutation * MeSH
- Dyslexia genetics metabolism pathology MeSH
- Gene Expression MeSH
- HEK293 Cells MeSH
- Ichthyosis genetics metabolism pathology MeSH
- Protein Interaction Domains and Motifs MeSH
- Ion Transport MeSH
- Protein Conformation, alpha-Helical MeSH
- Humans MeSH
- Luminescent Proteins genetics metabolism MeSH
- Patch-Clamp Techniques MeSH
- Migraine Disorders genetics metabolism pathology MeSH
- Miosis genetics metabolism pathology MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- Neoplasm Proteins chemistry genetics metabolism MeSH
- ORAI1 Protein chemistry genetics metabolism MeSH
- Stromal Interaction Molecule 1 chemistry genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Genes, Reporter MeSH
- Amino Acid Sequence MeSH
- Spleen abnormalities metabolism pathology MeSH
- Amino Acid Substitution MeSH
- Muscle Fatigue genetics MeSH
- Blood Platelet Disorders genetics metabolism pathology MeSH
- Calcium chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
