Orai1 C-terminus Research Articles

The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry

The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis.

A pathogenic human Orai1 mutation unmasks STIM1-independent rapid inactivation of Orai1 channels.

Ca2+ release-activated Ca2+ (CRAC) channels are activated by direct physical interactions between Orai1, the channel protein, and STIM1, the endoplasmic reticulum Ca2+ sensor. A hallmark of CRAC channels is fast Ca2+-dependent inactivation (CDI) which provides negative feedback to limit Ca2+ entry through CRAC channels. Although STIM1 is thought to be essential for CDI, its molecular mechanism remains largely unknown. Here, we examined a poorly understood gain-of-function (GOF) human Orai1 disease mutation, L138F, that causes tubular aggregate myopathy. Through pairwise mutational analysis, we determine that large amino acid substitutions at either L138 or the neighboring T92 locus located on the pore helix evoke highly Ca2+-selective currents in the absence of STIM1. We find that the GOF phenotype of the L138 pathogenic mutation arises due to steric clash between L138 and T92. Surprisingly, strongly activating L138 and T92 mutations showed CDI in the absence of STIM1, contradicting prevailing views that STIM1 is required for CDI. CDI of constitutively open T92W and L138F mutants showed enhanced intracellular Ca2+ sensitivity, which was normalized by re-adding STIM1 to the cells. Truncation of the Orai1 C-terminus reduced T92W CDI indicating a key role for the Orai1 C-terminus for CDI. Overall, these results identify the molecular basis of a disease phenotype with broad implications for activation and inactivation of Orai1 channels.

Highlighting the Multifaceted Role of Orai1 N-Terminal- and Loop Regions for Proper CRAC Channel Functions.

Orai1, the Ca2+-selective pore in the plasma membrane, is one of the key components of the Ca2+release-activated Ca2+ (CRAC) channel complex. Activated by the Ca2+ sensor in the endoplasmic reticulum (ER) membrane, stromal interaction molecule 1 (STIM1), via direct interaction when ER luminal Ca2+ levels recede, Orai1 helps to maintain Ca2+ homeostasis within a cell. It has already been proven that the C-terminus of Orai1 is indispensable for channel activation. However, there is strong evidence that for CRAC channels to function properly and maintain all typical hallmarks, such as selectivity and reversal potential, additional parts of Orai1 are needed. In this review, we focus on these sites apart from the C-terminus; namely, the second loop and N-terminus of Orai1 and on their multifaceted role in the functioning of CRAC channels.

High affinity associations with α-SNAP enable calcium entry via Orai1 channels.

Molecular steps that activate store-operated calcium entry (SOCE) via Orai channel supramolecular complex remain incompletely defined. We have earlier shown that α-SNAP regulates the on-site functional assembly and calcium selectivity of Orai1 channels. Here we investigate the molecular basis of its association with Orai, Stim and find that the affinity of α-SNAP for Orai and Stim is substantially higher than previously reported affinities between Stim and Orai sub-domains. α-SNAP binds the coiled-coil 3 (CC3) sub-domain of Stim1. Mutations of Tryptophan 430 in Stim1-CC3 disrupted α-SNAP association and SOCE, demonstrating a novel α-SNAP dependent function for this crucial subdomain. Further, α-SNAP binds the hinge region near the C-terminus of Orai1 and an additional broad region near the N-terminus and Valine 262 and Leucine 74 were necessary for these respective interactions, but not Orai, Stim co-clustering. Thus, high affinity interactions with α-SNAP are necessary for imparting functionality to Stim, Orai clusters and induction of SOCE.

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

STIM1-Induced Conformational Transition of ORAI-1 Leads to Channel Activation

Calcium release-activated calcium (CRAC) channels in the plasma membrane are integral membrane proteins that play a central role in cellular signaling by generating the calcium influx. The calcium influx results in a decrease in the concentration of Ca2+ within the endoplasmic reticulum (ER) that triggers the immune system response. The stromal interaction molecule (STIM1) detects the decline of ER Ca2+ concentration and activates the channel. The molecular details of STIM1 interaction with Orai1 (a pore subunit of the channel) that causes the channel opening are not yet clear. In order to understand the molecular details of this signal transduction pathway, we developed all-atomic molecular models of the STIM1/Orai-1 complex and studied the binding of STIM1 to C-terminus of Orai-1 leading to conformational changes in Orai-1 protein complex followed by the activation of the CRAC channels. In this study, we examined the effect of different point mutations of the C-terminus of Orai-1 and ER tension on the STIM-induced conformational transition of Orai-1 that was further compared with previous experimental studies. Our results showed that the M1 helices of Orai1 are slightly bent in the region that spans the junction between the hydrophobic and basic portions of the pore. This raises the possibility that the conformation of the basic region could change, perhaps as part of the gating process, while the hydrophobic section and glutamate ring remain fairly fixed. Furthermore, we observed that the distance between M1 helices on the cytoplasmic site is increased resulting in the expected widening of the cytoplasmic region of the pore.

STIM Proteins Cluster Orai1 Channels and Modulate Receptor-Mediated Calcium Signals

Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels plays an indispensable role in receptor-evoked Ca2+ signaling. The two main CRAC channel facilitators are the PM Orai channels, and the ER Ca2+ sensors, STIM1 and STIM2. The molecular mechanism of STIM1/Orai1 coupling remains unsolved. Our recent work provided insight into the coupling mechanism between the Orai1 activating domain of STIM1 (SOAR) and the Orai1 C-terminus. Using rationally-designed mutational screens of the purported Orai1 interaction site within SOAR, we determined that mutating residue F394 to histidine completely prevents binding of full-length STIM1. We developed concatenated SOAR dimers containing one or two F394H mutations, and demonstrated that only a single functional subunit within a SOAR dimer is necessary for Orai1 binding. Current studies reveal that concatenated SOAR heterodimers (SH-S) are unable to form Orai1 clusters, whereas cells expressing wild-type dimers (S-S) retain this ability. The recent discovery of the splice variant of STIM2 (STIM2.1), which has an abrogated binding capacity similar to STIM1-F394H, provided us with another tool to investigate STIM/Orai1 coupling and its physiological role in receptor-evoked Ca2+ signals. Using concatenated heterodimers (SH-S or S2.1-S), we observed profound effects on dimer mobility and generation of ICRAC as compared to wild-type SOAR1 homodimers (S-S), in cells stably expressing Orai1. The physiological role of clustering Orai1 channels is suggested from co-expressing full length STIM1 and STIM2.1. We observe profoundly altered oscillatory responses in HEK cells induced by physiological levels of CCh. Our studies suggest that clustering may affect the ability for Orai1 channels to generate fully functional Ca2+ entry microdomains within ER-PM junctions. This provides strong evidence supporting the “unimolecular model” of STIM1-Orai1 coupling, which appears to be critical for channel clustering, ICRAC generation, and maintenance of Ca2+ oscillations.

Helix-Helix Contacts between the ORAI1 Pore Segment and the TM2/3 Ring Regulates STIM1-Mediated CRAC Channel Activation

Store-operated Orai1 channels mediate many critical cellular functions in animal cells ranging from gene expression to exocytosis. Activation of Orai1 channels by store depletion is driven by direct interactions between the Orai1 subunits with the endoplasmic reticulum Ca2+ sensor, STIM1, but how STIM1 binding transduces into opening of the Orai1 channel pore remains unclear. At the structural level, each Orai1 channel consists of six subunits with the transmembrane helices (TMs 1-4) of each subunit arranged in three concentric rings around a central aqueous pore formed by TM1. A prevailing model of channel gating postulates that STIM1 binding to the peripherally located Orai1 C-terminus is followed by a second, weaker STIM1 binding step to the centrally located N-terminus to drive pore opening. However, recent reports have cast doubt on the existence of a N-terminal STIM1 binding site, raising questions about how the gating signal is transmitted to the central pore. Here, we investigated the role of the transmembrane domains in this process and identified sixteen mutations in the non-pore lining TMs 2-4 that activate Orai1 channels in the absence of STIM1. Cysteine accessibility analysis and MD simulations indicate that the most robust of these activating mutations, H134S, induces rotation of the pore helix to move the side-chains of the gate formed by F99 aside, analogous to gating by STIM1. Further, atomic packing and mutational analysis revealed critical contact sites between TM1 and the TM2/3 helices, whose disruption by mutagenesis abrogated channel activation by STIM1 binding. We conclude that specific interactions between the transmembrane domains of Orai1 are critical for relaying the STIM1 activating signal from the C-terminus to the pore.

The STIM-Orai Pathway: Conformational Coupling Between STIM and Orai in the Activation of Store-Operated Ca2+ Entry.

Store-operated Ca2+ entry fulfills a crucial role in controlling Ca2+ signals in almost all cells. The Ca2+-sensing stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER) undergo complex conformational changes in response to depleted ER luminal Ca2+, allowing them to unfold and become trapped in ER-plasma membrane (PM) junctions. Dimers of STIM proteins trap and gate the plasma membrane Orai Ca2+ channels within these junctions to generate discrete zones of high Ca2+ and regulate sensitive Ca2+-dependent intracellular signaling pathways. The STIM-Orai activating region (SOAR) of STIM1 becomes exposed upon store depletion and promotes trapping of Orai1 at the PM. Residue Phe-394 within SOAR forms an integral part of the high-affinity Orai1-interacting site. Our results demonstrate that only a single active site within the dimeric SOAR domain of STIM1 is required for the activation of Orai1 channel activity. This unimolecular model is strongly supported by evidence of variable STIM1:Orai1 stoichiometry reported in many studies. We hypothesize that unimolecular coupling promotes cross-linking of channels, localizing Ca2+ signals, and regulating channel activity. We have also identified a key "nexus" region in Orai1 near the C-terminal STIM1-binding site that can be mutated to constitutively activate Ca2+ entry, mimicking STIM1 activated channels. This suggests that STIM1 mediates gating of Orai1 in an allosteric manner via interaction with the Orai1 C-terminus alone. This model suggests the dual role of STIM1 in regulating both localization and gating of Orai1 channels and has important implications for the regulation of SOCE-mediated downstream signaling and the kinetics of channel activation.

Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry.

Ca2+ influx by store-operated Ca2+ channels is a major mechanism for intracellular Ca2+ homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1.

Molecular Mechanisms of STIM1-Mediated Orai-1 Channel Activation

Immune response mechanisms at a cellular level are mostly triggered by a decrease in the concentration of Ca2+ within the endoplasmic reticulum (ER), followed by the opening of the calcium release-activated calcium (CRAC) channels that leads to increased intracellular Ca2+ concentrations. Recently, the stromal interaction molecule (STIM) was determined to be the ER Ca2+ sensor that activates the channel in response to the depletion of intracellular calcium content. However, the molecular details of STIM interaction with Orai that causes the channel opening are not yet known. Understanding these molecular processes would shed light on this signal transduction pathway and opens up new doors for tapping into such a mechanism with profound biological and clinical implications.CRAC channels in the plasma membrane are integral membrane proteins that play a central role in cellular signaling by generating the calcium influx. Orai protein is a pore subunit of the channel, which has 3 homologs (Orai-1, Orai-2, and Orai-3).We develop all-atomic molecular dynamics models of the STIM1/Orai-1 complex with the aim to demonstrate how the binding of STIM1 to C-terminus of Orai-1 will result in a conformational change in Orai-1 protein complex leading to the activation of CRAC channels. Different mutations of Orai-1 C-terminus are considered and the influence of factors such as ER tension on the enhancement of this conformational change is explored in accordance with experimental evidences.

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding.

Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.

Roles of the Orai1 C-Terminus and N-Terminus in 2-APB-Induced STIM1 Coupling

The small molecule, 2-aminoethoxydiphenyl borane (2-APB) has complex effects on store-operated Ca2+ entry (SOCE). Higher levels (50 μM) strongly inhibit STIM1-miedated Orai1 channel activation. However, using the poorly active STIM1 C-terminal domain (S1CT) 235-685), 50 μM 2-APB greatly enhances binding to and activation of Orai1 channels. We assessed which parts of the Orai1 channel were required for this coupling interaction. Using the Orai1 C-terminal truncation of Orai1 (removal of residues 267-301), 50 μM 2-APB caused neither binding between S1CT and truncated Orai1 nor Ca2+ entry. We replaced the missing Orai1 C-terminus with the FKBP-rapamycin binding (FRB) domain of the mTOR protein, and added the 12-kDa FK506- and rapamycin-binding protein (FKBP12) to the carboxyl end of S1CT. There was no Ca2+ entry even when S1CT and truncated Orai1(1-266) were physically tethered through rapamycin-induced FRB/FKBP12 interactions. However, the addition of 2-APB now recovered full Ca2+ entry. Thus, the C-terminus of Orai1 (267-301) is not specifically required and can be substituted by a simple binding interaction between FRB/FKBP12. We also revealed using FRET and fluorescence imaging, that the 267-301 C-terminus of Orai1 attached to the CFP-tagged PM-localization construct, was alone sufficient to trap S1CT upon the addition of 50 μM 2-APB. Finally, we identified a single residue (Leu-79) in the N-terminus of Orai1 that is critical for the effects of 2-APB on SOCE. The L79N Orai1 mutant completely abolished the activation effect of 50 μM 2-APB on S1CT, but only partially prevented the action of store-induced STIM1 activation of Orai1. Our data provide new insights into our understanding of 2-APP-induced activation of STIM1/Orai1 coupling.

Expression of ORAII, a plasma membrane resident subunit of the CRAC channel, in rodent and non-rodent species.

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed.

Mechanism of Activation of Store-Operated Calcium Entry by 2-Aminoethoxydiphenyl Borate

Store-operated Ca2+ entry (SOCE) is mediate by STIM-induced activation of Orai channels. The small molecule, 2-aminoethoxydiphenyl borate (2-APB), is known to have a biphasic effect on SOCE; lower 2-APB (≤10 μM) enhances, while higher levels of 2-APB (50-100 μM) are strongly inhibitory following a transient activation. But the mechanism by which 2-APB activates SOCe is still elusive. The effects of 2-APB were examined on coupling between various Orai1 mutants and STIM1, C-terminal (ct) STIM1 fragments, or the STIM-Orai activating region (SOAR) of STIM1, using a combination of imaging and whole-cell patch clamp analysis. Orai1 coupling with STIM1ct, STIM1ct-4EA and SOAR can be transiently activated by 2-APB at low levels (10 µM). The activation of SOCE by low 2-APB is induced by its action on the SOAR-Orai1 complex. We examined which domains/residues in Orai1 or in STIM1 are essential for the activating effect of 2-APB. Adjustment of cytosolic pH upward using nigericin abolished the action of 2-APB. In contrast, lower pH potentiated the activating effect of 2-APB. Our results reveal that the Orai1 C-terminus and N-terminus function in a concerted manner to mediate the activating effect of 2-APB on SOCE. Mutational analysis reveals that the negatively charged residues within the Orai1 C-terminal region are not required for the 2-APB-induced SOCE activation. Our studies provide new insights into the mechanism of 2-APB-induced activation of coupling between STIM and Orai and the triggering of SOCE.

Structural Requirements of N- and C-Terminal Orai Strands to allow Maximal Store-Operated Ca2+ Current Activation by STIM1

The Ca2+ sensor protein STIM1 anchored in the ER membrane and the pore-forming Orai protein in the plasma membrane represent the key components of Ca2+ release-activated Ca2+ (CRAC) channels. Recent publications have demonstrated that CRAC current activation requires 8 STIM1 molecules together with an Orai complex formed by 4 subunits. Channel gating necessitates direct binding of the cytosolic portion of STIM1 with both N- and C-termini of the Orai channel. A single point mutation L273S in Orai1 C-terminus abolishes the interaction with STIM1 and followingly the activation of Orai1 currents. A truncation till to the fourth amino acid of the N-terminal conserved region (aa73-89) results in the loss of STIM1 binding to the N-terminus and STIM1-mediated activation. We employed Orai1 dimers to identify whether all N-/C-terminal domains within an Orai concatamer are required to be intact for maximal Ca2+ current activation. Orai dimers with one wild-type and one L273S-mutant subunit displayed a similar extent of maximal currents like and an unchanged inactivation compared to wild-type proteins. These results suggest that either less than 8 STIM1 are even sufficient to activate Orai1 channels maximally or the L273S mutation is not sufficient to impair the recruitment of eight STIM1 proteins to an Orai1 oligomer containing wild-type and mutant C-termini with a 1:1 stoichiometry. Orai dimers containing one inactive N-truncation mutant and one wild-type form showed preserved but about 40% significantly reduced Orai1 currents. Hence, the lack of half of functional N-termini within an Orai channel already reduces the current extent. Interestingly, N-termini seem to contribute in a more dominant manner to the generation of maximal Ca2+ currents compared to Orai1 C-termini. (supported by T466 to I.D. and P22565 to C.R.)

A Basic Sequence in STIM1 Promotes Ca2+ Influx by Interacting with the C-Terminal Acidic Coiled Coil of Orai1

Store-operated Ca(2+) entry (SOCE) is a ubiquitous signaling process in eukaryotic cells in which the endoplasmic reticulum (ER)-localized Ca(2+) sensor, STIM1, activates the plasma membrane-localized Ca(2+) release-activated Ca(2+) (CRAC) channel, Orai1, in response to emptying of ER Ca(2+) stores. In efforts to understand this activation mechanism, we recently identified an acidic coiled-coil region in the C-terminus of Orai1 that contributes to physical association between these two proteins, as measured by fluorescence resonance energy transfer, and is necessary for Ca(2+) influx, as measured by an intracellular Ca(2+) indicator. Here, we present evidence that a positively charged sequence of STIM1 in its CRAC channel activating domain, human residues 384-386, is necessary for activation of SOCE, most likely because this sequence interacts directly with the acidic coiled coil of Orai1 to gate Ca(2+) influx. We find that mutation to remove positive charges in these residues in STIM1 prevents its stimulated association with wild-type Orai1. However, association does occur between this mutant STIM1 and Orai1 that is mutated to remove negative charges in its C-terminal coiled coil, indicating that other structural features are sufficient for this interaction. Despite this physical association, we find that thapsigargin fails to activate SOCE following coexpression of mutant STIM1 with either wild type or mutant Orai1, implicating STIM1 residues 384-386 in transmission of the Ca(2+) gating signal to Orai1 following store depletion.

STIM1, Orai1 and hTRPC1 are important for thrombin- and ADP-induced aggregation in human platelets

Ca 2+ entry, particularly store-operated Ca 2+ entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca 2+ content of the stores, where the intraluminal Ca 2+ sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca 2+ stores to the store-operated Ca 2+-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25–139) antibody, directed towards the Ca 2+-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288–301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557–571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca 2+ entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

Interference In Coiled-coil Mediated Coupling Between Stim1 And Orai Channels

STIM1 and ORAI1, the two limiting components in the CRAC signalling cascade, have been reported to couple tightly upon store-depletion culminating in CRAC current activation. Based on the homology within the ORAI protein family, an analogous scenario might be assumed for ORAI2 as well as ORAI3 channels as both are activated in a similar store- and STIM1-dependent manner. A combined approach of electrophysiology and confocal Förster Resonance Energy Transfer (FRET) microscopy revealed a general mechanism in the communication of STIM1 with ORAI proteins that involved the predicted second coiled-coil motif in STIM1 C-terminus and the conserved putative coiled-coil domain in the respective ORAI C-terminus. Of the latter, a much higher coiled-coil probability is predicted for ORAI2 as well as ORAI3 than for ORAI1, compatible with our observation that a single point coiled-coil mutation in ORAI1 C-terminus abrogated communication with STIM1 C-terminus, while an analogous mutation in ORAI2 and ORAI3 still allowed for their moderate activation. Conversely, destabilizing the second coiled-domain of STIM1 C-terminus by a single point mutation still enabled partial stimulation of ORAI2 and ORAI3 channels but not of ORAI1. A double mutation within the second coiled-coil motif of 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 mutual destabilization of putative coiled-coil domains in either C-terminus would be compatible with their heteromeric interaction.Supported by FWF P18169.