Molecular mechanisms of STIM/Orai communication Derler, Isabella; Jardin, Isaac; Romanin, Christoph
American Journal of Physiology: Cell Physiology,
04/2016, Volume:
310, Issue:
8
Journal Article
Peer reviewed
Open access
Ca(2+)entry into the cell via store-operated Ca(2+)release-activated Ca(2+)(CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, ...secretion, and cell death. These store-operated Ca(2+)channels open after depletion of intracellular Ca(2+)stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca(2+)sensor, while Orai forms a highly Ca(2+)-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca(2+)permeation into the cell.
Ca
ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell ...is the Ca
release-activated Ca
(CRAC) channel. It consists of the Ca
sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca
ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the "classical" Ca
ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca
store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca
permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The Orai Pore Opening Mechanism Tiffner, Adéla; Maltan, Lena; Weiß, Sarah ...
International journal of molecular sciences,
01/2021, Volume:
22, Issue:
2
Journal Article
Peer reviewed
Open access
Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca
concentrations. The primary source of Ca
entry into the cell is mediated by the Ca
...release-activated Ca
(CRAC) channel. Its action is stimulated in response to internal Ca
store depletion. The fundamental constituents of CRAC channels are the Ca
sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca
-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1-Orai1 machinery.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cancer represents a major health burden worldwide. Several molecular targets have been discovered alongside treatments with positive clinical outcomes. However, the reoccurrence of cancer due to ...therapy resistance remains the primary cause of mortality. Endeavors in pinpointing new markers as molecular targets in cancer therapy are highly desired. The significance of the co-regulation of Ca
-permeating and Ca
-regulated ion channels in cancer cell development, proliferation, and migration make them promising molecular targets in cancer therapy. In particular, the co-regulation of the Orai1 and SK3 channels has been well-studied in breast and colon cancer cells, where it finally leads to an invasion-metastasis cascade. Nevertheless, many questions remain unanswered, such as which key molecular components determine and regulate their interplay. To provide a solid foundation for a better understanding of this ion channel co-regulation in cancer, we first shed light on the physiological role of Ca
and how this ion is linked to carcinogenesis. Then, we highlight the structure/function relationship of Orai1 and SK3, both individually and in concert, their role in the development of different types of cancer, and aspects that are not yet known in this context.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Ca2+ ions play a variety of roles in the human body as well as within a single cell. Cellular Ca2+ signal transduction processes are governed by Ca2+ sensing and Ca2+ transporting proteins. In this ...review, we discuss the Ca2+ and the Ca2+-sensing ion channels with particular focus on the structure-function relationship of the Ca2+ release-activated Ca2+ (CRAC) ion channel, the Ca2+-activated K+ (KCa2+) ion channels, and their modulation via other cellular components. Moreover, we highlight their roles in healthy signaling processes as well as in disease with a special focus on cancer. As KCa2+ channels are activated via elevations of intracellular Ca2+ levels, we summarize the current knowledge on the action mechanisms of the interplay of CRAC and KCa2+ ion channels and their role in cancer cell development.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In immune cells, generation of sustained Ca2+ levels is mediated by the Ca2+ release-activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 ...(STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca2+ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233–450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50–74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca2+ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233–420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233–485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca
2+
sensor in the endoplasmic reticulum (ER), and Orai1, the Ca
2+
ion channel in the plasma membrane. Ca
2+
store ...depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners.
Graphic abstract
Legend: Upon intracellular Ca
2+
store depletion of the endoplasmic reticulum (ER), Ca
2+
dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca
2+
influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca
2+
-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca
2+
store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca
2+
store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up ...avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor–ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Abstract As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the ...identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC50 of approximately 4 μM and exhibited a substantially slower rate of onset than the typical pore blocker La3+ , together with almost no current recovery upon wash-out over 4 min. For the less Ca2+ -selective Orai1 E106D pore mutant, ICRAC inhibition was significantly reduced. FRET experiments indicated that neither STIM1–STIM1 oligomerization nor STIM1–Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the ...calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca2+ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca2+ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca2+ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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