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 Ca 2؉ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca 2؉ 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 Ca 2؉ 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.

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformationUpon depletion of ER calcium stores, STIM1 and ORAI1 associate and induce calcium release-activated calcium (CRAC) currents. This study reveals that STIM1 undergoes an intramolecular transition into an extended conformation that is involved in ORAI1 binding and activation.

In immune cells, generation of sustained Ca2؉ levels is mediated by the Ca 2؉ release-activated Ca 2؉ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca 2؉ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca 2؉ 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 Ca 2؉ 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.Store-operated Ca 2ϩ entry is key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca 2ϩ release-activated Ca 2ϩ (CRAC) 5 channel (2-6). However, its molecular components have remained elusive until 3 years ago; the stromal interacting molecule 1 (STIM1) (7, 8) and later on ORAI1 (9 -11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca 2ϩ sensor (7,8,12), and store depletion triggers its aggregation into puncta close to the plasma membrane, resulting in stimulation of CRAC currents (13,14). Its N terminus is located in the ER lumen and contains an EF-hand Ca 2ϩ binding motif that senses the ER Ca 2ϩ level and a sterile ␣ motif that is suggested to mediate homomeric STIM1 aggregation (15, 16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin (ERM)-like domain and a lysine-rich region have been proposed as essential for CRAC activation (15,17,18). ORAI1 has been assumed to act in concert with STIM1 (10,19,20), activating inward Ca...

The channel Orai1 requires Ca store depletion in the endoplasmic reticulum and an interaction with the Ca sensor STIM1 to mediate Ca signaling. Alterations in Orai1-mediated Ca 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 Ca 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 Ca permeation associated with gene transcription and provide a gating mechanism for Orai1.

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Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca 2؉ release-activated Ca 2؉ channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 M 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca 2؉ selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8 Å to more than 5.34 Å . In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.A major mechanism for many cell types to maintain longlasting elevation of intracellular Ca 2ϩ is the use of store-operated Ca 2ϩ influx (1). Initiated by the second messenger inositol 3-phosphate, Ca 2ϩ is released from the endoplasmic reticulum. Subsequently, Stim1 located in the endoplasmic reticulum senses via a luminal EF-hand the Ca 2ϩ content and redistributes after store-depletion to dynamically couple and activate the Orai1 channel, also named CRACM1 (2, 3). All three Orai members form highly Ca 2ϩ selective channels, and Orai1 has been suggested as the calcium release-activated calcium (CRAC) 6 channel in the plasma membrane (4, 5). Orai channels can be discriminated by distinct properties in feedback regulation to intracellular Ca 2ϩ and different pharmacological responses to 2-aminoethoxydiphenyl borate (2-APB; 50 M) (6, 7). Although Orai1 and Stim1 coexpressing cells were stimulated by low concentrations of 2-APB, high concentrations (50 M) completely inhibited Orai1 and partially suppressed Orai2 currents when coexpressed with Stim1 (6,8). This bimodal effect has also been observed for native CRAC channels in T-lymphocytes and mast cells (9). In contrast, coexpression of Orai1 and Stim2 resulted in robust stimulation by 2-APB (50 M), exhibiting a typical inward-rectifying current-voltage relationship of Orai1 (10, 11). It is proposed that 2-APB displaces the inhibitory calmodulin from the Stim2⅐Orai1 complex (10). Orai3, however, is exclusively stimula...

Background and purposePyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.Experimental approachPyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca2+ entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca2+ entry mediated by Orai channels. The consequences of inhibitory effects on Ca2+ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.Key ResultsPyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca2+ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca2+ entry as compared with TRPC3-mediated Ca2+ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.Conclusions and ImplicationsThe pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca2+ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca2+ channels.

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca2؉ release-activated Ca 2؉ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiledcoil domain C-terminally extended by ϳ50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca 2ϩ entry is a key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca 2ϩ release-activated Ca 2ϩ (CRAC) 5 channel (2-6). However, its molecular components have remained elusive until 4 years ago; the STIM1 (stromal interacting molecule 1) (7,8) and later on Orai1 (9 -11) have been identified as the two limiting components for CRAC activation. STIM1 is an ERlocated Ca 2ϩ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12,13). Its N terminus is located in the ER lumen and contains an EF-hand Ca 2ϩ -binding motif, which senses the ER Ca 2ϩ level, and a sterile ␣-motif, which is suggested to mediate homomeric STIM1 aggregation (14 -16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14,17,18). Three recent studies have independently identified the ezrinradixin-moesin ...