Defects in the STIM1 SOARα2 domain affect multiple steps in the CRAC channel activation cascade.

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. 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).

STIM1 phosphorylation at Y316 modulates its interaction with SARAF and the activation of SOCE and ICRAC.

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca2+ entry (SOCE). Hence, identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE is of interest. By performing a computational analysis, we identified that the Y316 residue is susceptible to phosphorylation. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and the Ca2+ release-activated Ca2+ current (ICRAC). STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to in cells with wild-type (WT) YFP-tagged STIM1. Additionally, the Y316F mutation altered the pattern of interaction between STIM1 and SARAF under resting conditions and upon Ca2+ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca2+-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by SARAF knockdown. Finally, in NG115-401L cells transfected with shRNA targeting SARAF, expression of STIM1 Y316F induced greater SOCE than STIM1 WT. Taken together, our results provide evidence supporting the idea that phosphorylation of STIM1 at Y316 plays a relevant functional role in the activation and modulation of SOCE.

A novel STIM1-Orai1 gating interface essential for CRAC channel activation.

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 Ca2+ release-activated Ca2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

A dual mechanism promotes switching of the Stormorken STIM1 R304W mutant into the activated state.

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.

Communication between N terminus and loop2 tunes Orai activation.

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.

Authentic CRAC channel activity requires STIM1 and the conserved portion of the Orai N terminus.

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.

Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription.

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.

Cholesterol modulates Orai1 channel function.

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation.

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.

A coiled-coil clamp controls both conformation and clustering of stromal interaction molecule 1 (STIM1).

Store-operated Ca(2+) entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1. Ca(2+) entry occurs through the plasma membrane resident Ca(2+) channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca(2+) store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.

STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry.

Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1-CC2 fragment in the apo and Orai1-bound states. Each CC1-CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2' helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM-Orai association pocket. STIM1 mutants disrupting CC1:CC1' interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices.

The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.

STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.

Ca(2+) release-activated Ca(2+) (CRAC) current, structure, and function.

Store-operated Ca(2+) entry describes the phenomenon that connects a depletion of internal Ca(2+) stores to an activation of plasma membrane-located Ca(2+) selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca(2+) sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca(2+)-selective pore-forming subunit of Ca(2+) release-activated Ca(2+) channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca(2+) currents.

Molecular determinants within N terminus of Orai3 protein that control channel activation and gating.

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.

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation.

Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (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 Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster 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.

A Ca2(+ )release-activated Ca2(+) (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2(+)-dependent inactivation of ORAI1 channels.

STIM1 and ORAI1, the two limiting components in the Ca(2+) release-activated Ca(2+) (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2-3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca(2+), a decrease in intracellular Ca(2+) chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba(2+) substitution for extracellular Ca(2+) completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca(2+) entry by triggering fast Ca(2+)-dependent inactivation of ORAI/CRAC channels.

Molecular determinants of the coupling between STIM1 and Orai channels: differential activation of Orai1-3 channels by a STIM1 coiled-coil mutant.

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca(2+) release-activated Ca(2+) current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by approximately 50 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.

A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Terminus Determine Coupling to ORAI1 Channels.

In immune cells, generation of sustained Ca(2+) 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.

2-aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size.

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 microm 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 3.8A to more than 5.34 A. 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.

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.

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.