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The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ 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 Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-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 Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ 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).

The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the 1H, 13C, 15N chemical shift assignments of the CC3 domain. This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form. Supplementary Information The online version contains supplementary material available at 10.1007/s12104-021-10042-7.

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.

Store-operated calcium entry (SOCE) through Orai ion channels is an intracellular signaling pathway that is initiated by ligand-induced depletion of calcium from the endoplasmic reticulum (ER) store. The molecular link between SOCE and ER store depletion is thereby provided by a distinct class of single pass ER transmembrane proteins known as stromal interaction molecules (STIM). STIM proteins are equipped with a precise N-terminal calcium sensing domain that enables them to react to changes of the ER luminal calcium concentration. Additionally, a C-terminal coiled-coil domain permits relaying of signals to Orai ion channels via direct physical interaction. In this review, we provide a brief introduction to STIM proteins with a focus on structure and function and give an overview of recent developments in the field of STIM research.

Natural product derived analogues were surveyed, and an oxygenated analog was identified as a potent and selective TRPV6 inhibitor, with high microsomal stability and low off-target effects., Transient receptor potential vanilloid 6 (TRPV6) is a calcium channel implicated in multifactorial diseases and overexpressed in numerous cancers. We recently reported the phenyl-cyclohexyl-piperazine cis-22a as the first submicromolar TRPV6 inhibitor. This inhibitor showed a seven-fold selectivity against the closely related calcium channel TRPV5 and no activity on store-operated calcium channels (SOC), but very significant off-target effects and low microsomal stability. Here, we surveyed analogues incorporating structural features of the natural product capsaicin and identified 3OG, a new oxygenated analog with similar potency against TRPV6 (IC50 = 0.082 ± 0.004 μM) and ion channel selectivity, but with high microsomal stability and very low off-target effects. This natural product-inspired inhibitor does not exhibit any non-specific toxicity effects on various cell lines and is proposed as a new tool compound to test pharmacological inhibition of TRPV6 mediated calcium flux in disease models.

The family of stromal interaction molecules (STIMs), comprising the homologs STIM1 and STIM2 with their different isoforms, has an intricate role in cellular calcium (Ca

The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in 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 (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established 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 is required to allow STIM1 coupling and Orai1 pore opening.Graphical Abstract

STIM1, an ER-located Ca2+ sensor, activates Orai1 channels upon Ca2+-storedepletion. Prior to this, STIM1 undergoes a sequence of conformational changes, which cannot be controlled individually with high spatiotemporal resolution. Ma et al. [1] used the power of optogenetic engineering to transfer light-sensitivity to STIM1 and precisely characterize individual STIM1 activation steps.

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.

Within this work, a fluorescence microscopy approach for the investigation of the distribution of polymer additives in polypropylene is presented. The fluorescent whitening agent 2,5-bis-(5-tert-butyl-benzoxazol-2-yl)-thiophene was used as a model compound representing other groups of polymer additives. So far, methods reported in the literature such as UV and IR microscopy offer a high spatial resolution, however, suffer from poor sensitivities, thus not allowing them to analyze samples with low additive concentrations typically used in engineering materials. Using the fluorescence microscopy technique, it was shown that independent from the applied concentrations (0.1–1.7 wt%), additives are distributed on a spherulitic scale with the majority being found at the spherulite boundary and only traces in the center. Furthermore, it could be demonstrated that the additive distribution is affected not only by the spherulite sizes but also by the cooling rate of the polymer melt leading to more or less...

Small-molecule piperazine derivatives mimic inactivation by calmodulin to selectively and potently block TRPV6 channels., Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo–electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.

The stromal interaction molecule 1 (STIM1) has two important functions, Ca

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell activation and the adaptive immune response. This entry occurs via the prototypic Ca

A new photochromic ligand enables high precision control over native TRPC channels., Lipid-gated TRPC channels are highly expressed in cardiovascular and neuronal tissues. Exerting precise pharmacological control over their activity in native cells is expected to serve as a basis for the development of novel therapies. Here we report on a new photopharmacological tool that enables manipulation of TRPC3 channels by light, in a manner independent of lipid metabolism and with higher temporal precision than lipid photopharmacology. Using the azobenzene photoswitch moiety, we modified GSK1702934A to generate light-controlled TRPC agonists. We obtained one light-sensitive molecule (OptoBI-1) that allows us to exert efficient, light-mediated control over TRPC3 activity and the associated cellular Ca2+ signaling. OptoBI-1 enabled high-precision, temporal control of TRPC3-linked cell functions such as neuronal firing and endothelial Ca2+ transients. With these findings, we introduce a novel photopharmacological strategy to control native TRPC conductances.

[Image: see text] Herein we report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a number of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, we identified a compound switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC(50) ≫ 10 μM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradiation at λ = 365 nm (Z/E = 3:1, IC(50) = 1.7 ± 0.4 μM), thereby allowing precise spatiotemporal control of inhibition. This new tool compound should be useful to deepen our understanding of TRPV6.

A primary calcium (Ca

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.

The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.

A primary Ca

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

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.

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.

Calcium (Ca2+) is an essential second messenger required for diverse signaling processes in immune cells. Ca2+ release-activated Ca2+ (CRAC) channels represent one main Ca2+ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum, and the Ca2+ ion channel Orai in the plasma membrane. After Ca2+ store depletion, STIM1 and Orai couple to each other, allowing Ca2+ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca2+ selectivity, an increase in current density when switching from a Ca2+-containing solution to a divalent-free Na+ one, and fast Ca2+-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.

Among all known second messengers in eukaryotic cells, Ca2+ is one of the most versatile and is involved in a multitude of physiological and cellular processes including cell proliferation, growth, gene expression, muscle contraction, and exocytosis/secretion [1,2]. To act as an intracellular signal molecule, Ca2+ has to enter the cell at specific physiological/cellular situations and time points. One major pathway that allows Ca2+ entry into the cells involves the Ca2+ release-activated Ca2+ (CRAC) channels, which belong to the group of store-operated channels (SOC) [3–14]. In the beginning of the CRAC/SOC channel analysis, these channels were studied and characterized using mainly cells of the immune system, that is, T-lymphocytes and mast cells [9,10,14,15]. Finally, in 2005–2006, the major key players forming the functional CRAC channel complex were identified [16–27]: first, the stromal interaction molecule (STIM), which represents the Ca2+ sensor in the endoplasmic reticulum (ER), and second, Orai, which is located in the plasma membrane (PM) and builds the ion-conducting transmembrane (TM) protein complex. Feske and colleagues [16] had studied a defect in CRAC channel function linked to one form of hereditary severe combined immune deficiency (SCID) syndrome, which allowed the identification of the Orai1 (also initially termed CRACM1) channel protein and its mutated form (Orai1 R91W) in SCID patients. By successfully employing and combining a modified linkage analysis with single-nucleotide polymorphism arrays and a Drosophila RNA interference screen, light was shed on the gene and protein that forms the Ca2+ conducting CRAC channel [16]. Furthermore, the search for homologous proteins using a sequence database research revealed Orai1, Orai2, and Orai3 in higher vertebrates. The three members of the Orai protein family have been analyzed with bioinformatics methods showing that they represent TM proteins with 4 PM spanning domains connected by one intracellular and two extracellular loops and cytosolic N-and C-termini [16,20,28,29]. Several research groups have concentrated on the electrophysiological examination and characterization of Orai proteins revealing the typical high Ca2+ selectivity and low single-channel conductance, concluding that these proteins unequivocally represent the pore-forming entity of the CRAC channel.The CRAC channel-activating protein—stromal interaction molecule (STIM)—has been presented and published by Liou et al. as well as Roos et al. in 2005 [18,19]. Screening about 2300 signaling proteins in Drosophila S2 cells and HeLa cells using an RNA interference-based gene knockdown approach, 2 homologous proteins highly involved in ER store depletion-mediated Ca2+ influx were elucidated—STIM1 and STIM2. These proteins serve as ER-resident Ca2+ sensors, which closely communicate with the CRAC channels upon Ca2+ depletion of the ER [18,19]. Both STIM1 and STIM2 are single-pass TM proteins with the N-terminus in the ER lumen and the larger C-terminal part facing the cytosol. The ER luminal part, which functions as a Ca2+ sensor of [Ca2+]ER, contains, among other parts, a Ca2+-sensing EF hand followed by the α-helical TM domain. The larger cytosolic part of STIM is responsible for coupling to and activation of Orai channels [6,30–33]. Confocal microscopy images reveal an intracellular tubular distribution of STIM1 under resting conditions with full ER Ca2+ stores; however, a small percentage of STIM1 has also been detected in the PM [34]. Lowering the ER-intraluminal Ca2+ concentration represents the initial trigger for STIM1 activation. In the course of store depletion, Ca2+ is released from the STIM1 EF hand domain followed by STIM1 homomerization and translocation to the cell periphery into the so-called ER-PM junctions—regions where the ER membrane is in tight proximity to the plasma membrane. Low [Ca2+]ER finally leads to the formation of oligomeric STIM1 clusters/punctae in these microdomains where Orai channels localize as well. This physical coupling of STIM1 to Orai channels therefore induces Ca2+ influx linked to specific downstream signaling and ER store refilling [30,35–39]. Besides the activation of CRAC channels, STIM1 has been shown to play a role in arachidonate as well as leukotriene C4-stimulated Ca2+ channels (see Chapter 11) as well as TRP channel regulation [40].After the initial characterization of STIM and Orai with limited structural knowledge based on bioinformatics predictions, in 2012, the crystal structures of cytosolic fragments of STIM1 and full-length Orai were reported, allowing new and more focused studies of STIM1 and Orai related to their intra- and intermolecular interactions [41].

Sirolimus (rapamycin) is used in drug-eluting stent strategies and proved clearly superior in this application compared with other immunomodulators such as pimecrolimus. The molecular basis of this action of sirolimus in the vascular system is still incompletely understood. Measurements of cell proliferation in human coronary artery smooth muscle cells (hCASM) demonstrated a higher antiproliferative activity of sirolimus compared with pimecrolimus. Although sirolimus lacks inhibitory effects on calcineurin, nuclear factor of activated T-cell activation in hCASM was suppressed to a similar extent by both drugs at 10 μM. Sirolimus, but not pimecrolimus, inhibited agonist-induced and store-operated Ca2+ entry as well as cAMP response element binding protein (CREB) phosphorylation in human arterial smooth muscle, suggesting the existence of an as-yet unrecognized inhibitory effect of sirolimus on Ca2+ signaling and Ca2+-dependent gene transcription. Electrophysiological experiments revealed that only sirolimus but not pimecrolimus significantly blocked the classical stromal interaction molecule/Orai-mediated, store-operated Ca2+ current reconstituted in human embryonic kidney cells (HEK293). A link between Orai function and proliferation was confirmed by dominant-negative knockout of Orai in hCASM. Analysis of the effects of sirolimus on cell proliferation and CREB activation in an in vitro model of arterial intervention using human aorta corroborated the ability of sirolimus to suppress stent implantation-induced CREB activation in human arteries. We suggest inhibition of store-operated Ca2+ entry based on Orai channels and the resulting suppression of Ca2+ transcription coupling as a key mechanism underlying the antiproliferative activity of sirolimus in human arteries. This mechanism of action is specific for sirolimus and not a general feature of drugs interacting with FK506-binding proteins.

Ca(2+) influx via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels represents a main signaling pathway for T-cell activation as well as mast-cell degranulation. The ER-located Ca(2+)-sensor, STIM1 and the Ca(2+)-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. Their identification, but even more the recent structural resolution of both proteins by X-ray crystallography has substantially advanced the understanding of the activation mechanism of CRAC channels. In this review, we provide a detailed description of the STIM1/Orai1 signaling pathway thereby focusing on the critical domains mediating both, intra- as well as intermolecular interactions and on the ion permeation pathway. Based on the results of functional studies as well as the recently published crystal structures, we portray a mechanistic view of the steps in the CRAC channel signaling cascade ranging from STIM1 oligomerization over STIM1-Orai1 coupling to the ultimate Orai1 channel activation and permeation.

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 IC(50) of approximately 4μM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) 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.

Store-operated Ca2+ entry (SOCE) pathways - Libros de Medicina - Cardiologia general - 155,95

A primary Ca2+ entry pathway in non-excitable cells is established by the Ca2+ release-activated Ca2+ channels. Their two limiting molecular components include the Ca2+-sensor protein STIM1 located in the endoplasmic reticulum and the Orai channel in the plasma membrane. STIM1 senses the luminal Ca2+ content, and store depletion induces its oligomerization into puncta-like structures, thereby triggering coupling to as well as activation of Orai channels. A C-terminal STIM1 domain is assumed to couple to both C- and N-terminal, cytosolic strands of Orai, accomplishing gating of the channel. Here we highlight the inter- and intramolecular steps of the STIM1-Orai signaling cascade together with critical sites of the pore structure that accomplishes Ca2+ permeation.

Ca(2+) influx via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels represents a main signalling pathway for a variety of cell functions, including T-cell activation as well as mast-cell degranulation. Depletion of [Ca(2+)]ER results in activation of Ca(2+) channels within the plasmamembrane that mediate sustained Ca(2+) influx which is required for refilling Ca(2+) stores and down-stream Ca(2+) signalling. The CRAC channel is the best characterized store-operated channel (SOC) with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel have been defined. The ER - located Ca(2+)-sensor, STIM1 and the Ca(2+)-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca(2+)]ER and activates the CRAC channel upon store depletion by direct binding to Orai1 in the plasmamembrane. The identification of STIM1 and Orai1 and recently the structural resolution of both proteins by X-ray crystallography and nuclear magnetic resonance substantiated many findings from structure-function studies which has substantially improved the understanding of CRAC channel activation. Within this review, we summarize the functional and structural mechanisms of CRAC channel regulation, present a detailed overview of the STIM1/Orai1 signalling pathway where we focus on the critical domains mediating interactions and on the ion permeation pathway. We portray a mechanistic view of the steps in the dynamics of CRAC channel signalling ranging from STIM1 oligomerization over STIM1-Orai1 coupling to CRAC channel activation and permeation.

Calcium is an important second messenger involved in many cellular processes such as cell proliferation, T-cell activation, muscle contraction, egg fertilization, or apoptosis. Calcium entry into non-excitable cells is mainly carried by store-operated Ca2+ release-activated Ca2+ (CRAC) channels, whereby Orai was found to be the calcium channel in the plasma membrane and the stromal interaction molecule (STIM) to act as a calcium sensor in the endoplasmatic reticulum (ER), and as an activator of Orai channels. A reduction of Ca2+ in the ER causes STIM to oligomerize, enabling its accumulation at the ER-plasma membrane junctions where it binds directly to Orai to open the Ca2+-channel. Compared with activation, much less is known about the mechanisms underlying Ca2+-dependent inactivation (CDI) processes. It has been proposed that binding of calmodulin (CaM) to a highly conserved N-terminal segment of Orai1 adjacent to its first transmembrane helix is critical for CDI. Hereby it is assumed that CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation. However, the molecular basis of these interactions remains unclear. In this study, the interaction between calmodulin and the N-terminal fragment of Orai1 and its homologue Orai3 was studied by Single Molecule Recognition Force Spectroscopy, Surface Plasmon Resonance, and Microscale Thermophoresis. Both Orai fragments showed a calcium-dependent and highly specific interaction with calmodulin, with slightly longer bond lifetimes and lower dissociation rate constants for Orai3 than for Orai1. Our data support a switch between a 1:1 and a 1:2 binding stoichiometry between CaM and Orai1/3. The latter occurs at low CaM concentration, has much higher affinity, and takes much longer to disscociate after removal of Ca2+ by EGTA. By comparison, only 1:1 binding with high affinity was found between CaM and smooth muscle myosin light chain kinase. This emphasizes the importance of the unique amino acid sequence of the conserved N-terminal Orai segment for high CaM affinity and CDI. This work was supported by the state of Upper Austria (project DK NanoCell).

STIM1 and Orai represent the key components of Ca(2+) release-activated Ca(2+) channels. Activation of Orai channels requires coupling of the C terminus of STIM1 to the N and C termini of Orai. Although the latter appears to be central in the interaction with STIM1, the role of the N terminus and particularly of the conserved region close to the first transmembrane sequence is less well understood. Here, we investigated in detail the functional role of this conserved region in Orai3 by stepwise deletions. Molecular determinants were mapped for the two modes of Orai3 activation via STIM1 or 2-aminoethoxydiphenyl borate (2-APB) and for current gating characteristics. Increasing N-terminal truncations revealed a progressive decrease of the specific fast inactivation of Orai3 concomitant with diminished binding to calmodulin. STIM1-dependent activation of Orai3 was maintained as long as the second half of this conserved N-terminal domain was present. Further truncations abolished it, whereas Orai3 stimulation via 2-APB was partially retained. In aggregate, the N-terminal conserved region plays a multifaceted role in Orai3 current gating with distinct structural requirements for STIM1- and 2-APB-stimulated activation.

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca2+ release-activated Ca2+ (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca2+ stores are depleted. Here we show, utilizing a novel STIM1-derived Forster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.

Activation of immune cells is triggered by the Ca(2+) release-activated Ca(2+) current, which is mediated via channels of the Orai protein family. A key gating process of the three Orai channel isoforms to prevent Ca(2+) overload is fast inactivation, most pronounced in Orai3. A subsequent reactivation is a unique gating characteristic of Orai1 channels, whereas Orai2 and Orai3 currents display a second, slow inactivation phase. Employing a chimeric approach by sequential swapping of respective intra- and extracellular regions between Orai1 and Orai3, we show here that Orai1 specific proline/arginine-rich domains in the N terminus mediate reactivation, whereas the second, intracellular loop modulates fast and slow gating processes. Swapping C-terminal strands lacks a significant impact. However, simultaneous transfer of Orai3 N terminus and its second loop or C terminus in an Orai1 chimera substantially increases fast inactivation centered between wild-type channels. Concomitant swap of all three cytosolic strands from Orai3 onto Orai1 fully conveys Orai3-like gating characteristics, in a strongly cooperative manner. In conclusion, Orai subtype-specific gating requires a cooperative interplay of all three cytosolic domains.

Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca 2+ ) sensors that differentially regulate plasma membrane Ca 2+ release activated Ca 2+ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca 2+ -binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing “super-unstable” or “super-stable” STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca 2+ - and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca 2+ entry by STIM1 and STIM2.