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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).

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...

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

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.

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca

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

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.

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.

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.

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.

A general cellular response following depletion of intracellular calcium stores involves activation of store-operated channels (SOCs). While Orai1 forms the native Ca 2+ release-activated Ca 2+ (CRAC) channel in mast and T cells, the molecular architecture of less Ca 2+ selective SOCs is insufficiently defined. Here we present evidence that diminished Ca 2+ selectivity and robust Cs + permeation together with a reduced fast inactivation are characteristics of heteromeric Orai1 and Orai3 channels in contrast to their homomeric forms. The first extracellular loop of these Orai isoforms differs by two aspartates replacing glutamates that affect the selectivity. Co-expression of an Orai3 mutant that mimicked the first loop of Orai1 with either Orai1 or Orai3 recovered or decreased Ca 2+ selectivity, respectively. Heteromeric Orai1/3 protein assembly provides a concept for less Ca 2+ -selective SOCs.

Dermaseptin PD-3-7 (aDrs) from frog skin contains three aspartic acid residues resulting in a negative net charge at neutral pH, as opposed to numerous other dermaseptins which are cationic helical antimicrobial peptides. Still, this peptide can be fitted into an amphipathic alpha helix by an Edmundson wheel projection. However, folding to the proposed helix was induced to only a low extent by zwitterionic vesicles or even detergents. Furthermore, no evidence of antibacterial or cytotoxic activity from soluble aDrs could be obtained. The peptide has an inherent propensity to an extended conformation in aqueous solution and self-assembles into amyloid fibrils in a reversible pH-controlled fashion, which was studied in some detail; above pH 5, the amyloid fibrils disassemble in a cooperative manner. This is probably caused by deprotonation of both side chain and terminal carboxyl groups, which results in intermolecular electrostatic repulsion. At neutral pH, this process proceeds instantaneously to the soluble form. Within the transition interval (pH 5-6.5), however, 'backward' granular aggregates, 10-500 nm in size, are formed. Such metastable amorphous aggregates, which are quickly released from an amyloid depot by a shift in pH, can mediate a strong cytotoxic effect. This activity does not involve lysis or interference with the cellular redox status, but apparently acts via an as yet unidentified mechanism. In this study, we present a new member of an emerging class of self-assembling frog skin peptides with extraordinary self-aggregation properties, which may potentially be relevant for biological processes. Structured digital abstract: * MINT-7256467: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by circular dichroism (MI:0016) * MINT-7255686: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by biophysical (MI:0013) * MINT-7256439: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence microscopy (MI:0416) * MINT-7256449: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by electron microscopy (MI:0040) * MINT-7256430: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence technologies (MI:0051).

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.

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca2+ signaling. A loss of Ca2+ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca2+ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear. Confocal Förster resonance energy transfer microscopy revealed that dynamic store-operated coupling of STIM1 to ORAI1 R91W was largely sustained similar to wild-type ORAI1. Characterization of various point mutants at position 91 by whole cell patch clamp recordings displayed that neutral or even negatively charged amino acids did not abolish ORAI1 function. However, substitution by hydrophobic leucine, valine, or phenylalanine resulted in non-functional ORAI1 channels, despite preserved STIM1 coupling. Besides conformational constraints at the N terminus/membrane interface predicted for the hydrophobic mutants, additional key factor(s) were suggested to determine ORAI1 functionality. Calculation of the probability for the 1st transmembrane domain and its hydrophobicity revealed a substantial increase for all hydrophobic substitutions that lead to non-functional ORAI1 R91X mutants in contrast to those with hydrophilic residues. Hence, increased hydrophobicity might lead to disrupted permeation/gating, as an ORAI1 channel with increased pore size and R91W mutation failed to recover activity. In conclusion, the increase in hydrophobicity at the N terminus/membrane interface represents the major cause for yielding non-functional ORAI1 channels.

In immune cells, generation of sustained Ca2+ levels is mediated by the Ca2+ release-activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca2+ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233–450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50–74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca2+ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233–420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233–485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.

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.

The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.

The Ca2+-selective TRPV6 as well as the L-type Ca2+ channel are regulated by the Ca2+-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca2+, employing Ca2+-imaging and patch-clamp techniques. Additionally, confocal Forster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein–protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca2+-insensitive mutant (CaMMUT) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca2+/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6–CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca2+-insensitive CaMMUT, however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaMMUT with rTRPV6, suggesting a strict Ca2+ dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca2+ levels, as observed by dynamic FRET measurements. An rTRPV6Δ695–727 mutant with the very C-terminal end deleted, yielded Ca2+ currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca2+ channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695–727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current which plays an important role in a broad range of cellular/physiological processes including T cell activation as well as cell proliferation, growth and apoptosis. Activation of Orai1 - the CRAC channel forming subunit - occurs via a physical interaction with the ER resident Ca2+ sensor protein STIM1. Upon ER Ca2+ store depletion, STIM1 undergoes extensive structural rearrangements resulting in an activated extended conformation. Interhelical rearrangements between the three cytosolic STIM1 coiled-coil (CC) domains drive this process therefore exposing SOAR/CAD allowing puncta formation and coupling to Orai1 in the cell periphery.Two years ago, the genetically inherited Stormorken Syndrome disease has been linked to the constitutively active human STIM1 R304W mutant (first published by Nesin et al.). In the present study, we focused on intra- and inter-molecular interactions specifically between the three cytosolic STIM1 CC domains comparing the STIM1 R304W mutant to the wildtype system as well as the constitutively active STIM1 L251S mutant employing electrophysiology, the YFP-OASF-CFP-FRET sensor and the recently described FRET based FIRE system. Our results revealed new insights into the mechanism how the disease related mutant R304W yields constitutive activity pointing to a novel effect distinct to the previously described constitutively active STIM1 CC1/CC3 mutants. (Supported by Austrian Science Fund (FWF) and science department of Upper Austria: P281203 to M.F and FWF-P27263 to C.R.)

Background: STIM1 and Orai1, reconstituting a main cellular Ca2+ entry pathway, interact via their cytosolic strands. Results: The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1. Conclusion: Several “hot spot” residues in the ETON region mediate STIM1 interaction, enabling conformational reorientation of the gate. Significance: Identification of critical residues for protein-protein interaction are fundamental to therapeutic drug development., STIM1 and Orai1 represent the two molecular key components of the Ca2+ release-activated Ca2+ 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 Ca2+ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca2+ 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.

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca(2+) entry and Ca(2+) release-activated Ca(2+) currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca(2+) selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca(2+) (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca(2+) entry pathways.

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., When endoplasmic reticulum calcium levels are low, STIM1 binds to and opens Orai1 channels in the plasma membrane to replenish calcium stores. Stathopulos et al. present solution structures of the STIM1 coiled-coil domain in the presence and absence of Orai1, revealing the structural basis for this interaction.

Rationale: Through largely unknown mechanisms, Ca 2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca 2+ entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca 2+ entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown. Objective: The goal of this study was to determine the agonist-evoked Ca 2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. Methods and Results: Ca 2+ imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca 2+ release-activated Ca 2+ channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca 2+ -selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca 2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC 4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC 4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation. Conclusions: These results demonstrate that distinct native Ca 2+ -selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC 4 acts through hitherto unknown intracrine mode to elicit store-independent Ca 2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Background: Heteromerization of TRP family members can affect their physiological and biophysical properties. Results: TRPV6 and TRPC1 associate via their ankyrin-like repeat domains. Co-expression of TRPV6 and TRPC1 reduced TRPV6 expression in the plasma membrane, and hence reduces TRPV6 influx currents. Conclusion: TRPC1 acts as negative regulator on TRPV6 mediated Ca2+ signaling. Significance: We show here a novel regulation in TRPV6 activity by formation of heterocomplexes with TRPC1., TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca2+ and Na+ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells.

A primary Ca2+ entry pathway in non-excitable cells is determined 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. Store depletion induces STIM1 oligomerization into puncta like structures, thereby triggering coupling to as well as activation of Orai1 channels. A C-terminal STIM1 domain couples to both N- and C-terminal, cytosolic strands of Orai, accomplishing gating of the channel. Here we highlight the inter- and intramolecular choreography of STIM1 and Orai proteins that govern in Ca2+ permeation.

Dermaseptin PD-3-7 (aDrs) from frog skin contains three aspartic acid residues resulting in a negative net charge at neutral pH, as opposed to numerous other dermaseptins which are cationic helical antimicrobial peptides. Still, this peptide can be fitted into an amphipathic alpha helix by an Edmundson wheel projection. However, folding to the proposed helix was induced to only a low extent by zwitterionic vesicles or even detergents. Furthermore, no evidence of antibacterial or cytotoxic activity from soluble aDrs could be obtained. The peptide has an inherent propensity to an extended conformation in aqueous solution and self-assembles into amyloid fibrils in a reversible pH-controlled fashion, which was studied in some detail; above pH 5, the amyloid fibrils disassemble in a cooperative manner. This is probably caused by deprotonation of both side chain and terminal carboxyl groups, which results in intermolecular electrostatic repulsion. At neutral pH, this process proceeds instantaneously to the soluble form. Within the transition interval (pH 5-6.5), however, 'backward' granular aggregates, 10-500 nm in size, are formed. Such metastable amorphous aggregates, which are quickly released from an amyloid depot by a shift in pH, can mediate a strong cytotoxic effect. This activity does not involve lysis or interference with the cellular redox status, but apparently acts via an as yet unidentified mechanism. In this study, we present a new member of an emerging class of self-assembling frog skin peptides with extraordinary self-aggregation properties, which may potentially be relevant for biological processes. Structured digital abstract: * MINT-7256467: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by circular dichroism (MI:0016) * MINT-7255686: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by biophysical (MI:0013) * MINT-7256439: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence microscopy (MI:0416) * MINT-7256449: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by electron microscopy (MI:0040) * MINT-7256430: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence technologies (MI:0051).

Calcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels, which serve essential functions ranging from regulation of transcription to cell growth. The best-characterized store-operated current, initially discovered in T lymphocytes and mast cells, is the Ca2+ release-activated Ca2+ (CRAC) current. The search for the molecular components of the CRAC channel has recently identified stromal interaction molecule 1 (STIM1) as the Ca2+ sensor in the endoplasmic reticulum (ER) and Orai1 as the CRAC channel pore. ER store depletion results in formation of STIM1 puncta that trigger Ca2+ influx via Orai1 channels. This review covers the role of domains within STIM1 and Orai and enlightens their function in the STIM1/Orai coupling process. Moreover, a molecular interpretation focuses on interactions between cytosolic portions of STIM1 and Orai together with a mechanistic view on the loss of function of the SCID (severe combined immunodeficiency)-linked Orai1 R91W mutant channel. The architecture of the selectivity filter of Orai channels is finally elucidated based on permeation properties of Orai pore mutants.

Ca(2+) entry in non-excitable cells is mainly carried by store-operated channels among which the CRAC channel is best characterized. Its two limiting molecular components are represented by the Ca(2+) sensor protein STIM1 located in the endoplasmic reticulum and Orai1 in the plasma membrane. STIM1 senses a decrease of the Ca(2+) content in internal stores and triggers its accumulation into puncta like structures resulting in coupling to as well as activation of Orai1 channels. The STIM1-Orai coupling process is determined by an interaction via their C-termini. This review highlights recent developments on domains particularly within the cytosolic part of STIM1 that govern this interaction.

In immune cells generation of sustained Ca2+ levels is mediated by the Ca2+ release activated Ca2+ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule (STIM1) that functions as a Ca2+ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of ER Ca2+ store leads to STIM1 multimerization into discrete punctae that co-cluster with ORAI1 thereby triggering coupling to and activation of ORAI1 channels. The C-terminus of STIM1 is sufficient for the activation of ORAI1 currents independent of store depletion. Here we unmasked an ORAI activating minimal fragment (OAMF) within STIM1 C-terminus that exhibited enhanced interaction with ORAI1 and resulted in three-fold increased Ca2+ currents. STIM1-OAMF still showed the ability of a homomeric interaction similar to longer fragments as well as the full-length form of STIM1 C-terminus. In contrast, further deletion of a thirty amino acid region resulted in a substantial reduction of homomeric interaction concomitant to a loss of coupling to as well as activation of ORAI1. In aggregate, we have identified two key regions within STIM C-terminus that govern ORAI1 activation. (Supported by PhD-Program W1201 from the FWF)

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.

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.

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Forster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.

Among store-operated Ca2+ entry (SOCE) pathways the Ca2+ release-activated Ca2+ (CRAC) channel is best characterized. STIM1 and Orai1 have been identified as the molecular key components of the CRAC channel. STIM1 acts as a Ca2+ sensor in the ER. Upon store-depletion STIM1 proteins oligomerize and translocate close to the plasma membrane. Thereby Orai proteins, representing pore-forming subunits in the plasma membrane, are activated by coupling to STIM1 multimers.Here we focused especially on the role of Orai N-termini. It is noticeable that this cytosolic strand includes an among all Orai proteins conserved region close to the first TM domain, which contains positively charged as well as hydrophobic amino acids.Up to now the role of these residues has not been clarified. Moreover this N-terminal conserved region functions not only as a binding partner for STIM1, but also for CaM and CRACR2A. The series of positively charged amino acids further resembles a PIP2 binding domain. With respect to these overlapping regions, we examined the role of positively charged and hydrophobic amino acids by generating point mutants. Single point mutations of positively charged amino acids reduced fast inactivation, possibly due to impairment of CaM binding. Replacement of hydrophobic amino acids did not affect fast inactivation excluding the disturbance of CaM binding. However, store-operated currents of these hydrophobic amino acid mutants were significantly increased, suggesting a possible connection with lipid-mediated processes.Mag. Lackner Barbara is a Recipient of a DOC/DOC-fFORTE-fellowship of the Austrian Academy of Sciences at the Institute of Biophysics at the Johannes-Kepler University of Linz.

Ca2+ influx in non-excitable cells is mainly carried by store-operated channels (SOCs), where Orai1 (CRACM1) and STIM1 represent the two molecular key players in this process. STIM1 functions as an endoplasmic reticulum located Ca2+ sensor and transmits the signal of store depletion to the plasma membrane by coupling to Orai1, which in turn causes channel activation. STIM1 C-terminus itself can act as a surrogate of full length STIM1 and is sufficient for the activation of Orai1 currents. 2-aminoethoxydiphenylborate (2-APB) has been shown to induce enhanced association of STIM1 C-terminus with Orai1 suggesting a conformational change within the former that drives this association. Indeed we were able to monitor 2-APB induced STIM1 C-terminal conformational rearrangement by fluorescence microscopy. In the absence of 2-APB the change in conformation was only seen for STIM1 C-terminus coupled to Orai1 but not for that part remaining in the cytosol suggesting this conformational change crucial for Orai1 binding. Moreover, we were able to mimic this conformational rearrangement by introducing selective point mutations into STIM1 C-terminus, which in line substantially increased binding to Orai1. In aggregate, our data support the theory of flexible regions within STIM C-terminus that undergo conformational rearrangement upon coupling to Orai1. (Supported by FWF-P21118)

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 Forster 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.

The endoplasmic reticulum Ca2+ sensor STIM1 forms together with the Ca2+ channel Orai1 the molecular basis for Ca2+ release activated Ca2+ (CRAC) channels. The recent crystal structure of Orai from Drosophila melanogaster shows a unique Ca2+ channel composed of a hexameric subunit complex. The pore structure is formed by transmembrane (TM) 1 helices, surrounded by two ring-like structures, formed by TM2 and TM3 as well as TM4. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques, molecular modeling and structure guided mutagenesis; we discovered a novel key mutation in the second trans-membrane helix of Orai1 that results in a Ca2+ selective, STIM1 independent, constitutively active current. Substitution of this essential residue to a hydrophobic amino-acid retained store-operated activation, yet with largely reduced Orai1 currents. In addition, we took advantage of the constitutively active Orai1 mutant, to evaluate reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning mutagenesis within the TM1 helix enabled identification of gating residues, the dimerization of which was altered in the constitutively opened and closed Orai1 channel conformation. In molecular dynamic simulations of an all-atom model of human Orai1 we will show the interaction network of these identified residues and predict implication of mutations on conformational changes. Our experiments will be summarized in a unique gating model, and we will moreover discuss how STIM1 binding might trigger the open channel conformation. This work was supported by the Austrian Science Foundation (FWF): P26067 to R.S. and P25172 to C.R. Irene Frischauf is an Elise Richter Scholarship holder: V286.

STIM1 and Orai1 are key players of the Ca2+-release activated Ca2+ (CRAC) current that plays an important role in T cell activation as well as mast cell degranulation. Activation of the CRAC channel forming subunit Orai1 occurs via a physical interaction with the Ca2+ sensor protein STIM1 when ER Ca2+ stores are depleted. Here, we show that this coupling process correlates with a refolding of STIM1 into an extended conformation by utilizing a novel STIM1-derived FRET sensor. This effect on STIM1 does not require external Ca2+ entry through activated CRAC channels, as a non-conducting Orai1 pore mutant is also to interact with STIM1 thereby switching it into its extended conformation. Furthermore, such extended conformation could be engineered by mutations in the cytosolic portion of STIM1 within the first and third coiled-coil domains. Introduction of these mutants exhibited strengthened interaction with Orai1 and leaded to constitutive CRAC current activation, even in the absence of store depletion. Hence, this STIM1-derived FRET sensor represents a promising tool for manipulating STIM1 coupling efficiencies with Orai1 based on conformational predictions.(supported by Austria Science Foundation P21118 and P22565).

Coexpression of STIM1, targeted to the endoplasmic reticulum and each of the three Orai (also termed CRACM) channels located in the plasma-membrane leads to store-operated, highly Ca2+ selective currents. While Orai1 has been reported to form the native Ca2+ release activated Ca2+ (CRAC) channels in human T-cells, the molecular architecture of less Ca2+ selective store-operated currents remains unknown. Here we show employing confocal fluorescence resonance energy transfer (FRET) that all three Orai proteins are able to form homo- and hetero oligomers. Overexpressed homomeric Orai1 or Orai3 together with STIM1, resulted in store-operated inward rectifying, highly Ca2+ selective currents, as resolved by whole-cell patch-clamp recordings. Coexpression of Orai1 together with Orai3 and STIM1 yielded similar store-depletion activated Ca2+ currents, yet with a leftward shifted reversal potential, pointing to less selective currents. In line, a tandem construct where Orai1was linked to Orai3 exhibited a similarly reduced Ca2+ selectivity that allowed for robust Cs+ permeation. Moreover, Orai3 pore mutants coexpressed with wild-type Orai3 affected Ca2+ and Cs+ selectivity/permeability. These results suggest that the divergent selectivities observed for endogenous store-operated channels might involve a heteromeric Orai1-Orai3 channel complex. Supported by FWF P18169.

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4 or TRPC5. Ca2+ and Na+ currents of TRPV6 over-expressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells.Supported by the Austrian Science Foundation (FWF): project P22747 to RS, project P21925 to KG, and project P18169 as well as P22565 to CR. IJ was supported by MCI fellowship (Ref. BES-2008-002875). IF is a Hertha-Firnberg scholarshipholder (T442).

STIM1 and Orai1 are key components of the Ca2+-release activated Ca2+ (CRAC) current that plays an important role in T cell activation as well as mast cell degranulation. Activation of the CRAC channel forming subunit Orai1 occurs via a physical interaction with the ER transmembrane Ca2+ sensor protein STIM1 when ER Ca2+ stores are depleted. This CRAC channel activation process is accompanied by a conformational change of STIM1 into an extended conformation together with puncta formation. Consequently SOAR/CAD is exposed during this process and drives oligomerization, probably by interhelical rearrangements between the three cytosolic STIM1 coiled-coil (CC) domains. Here we focused on intra- and inter-molecular interactions specifically between CC2/CC3 and the three α-helices comprising CC1. In an attempt to differentially examine and map possible interactions between these three α-helices and the SOAR/CAD comprising CC2 and CC3, a system termed “FRET-based Interactions in Restricted Environments (FIRE)” was developed. Furthermore, single point mutations were introduced into these helical fragments to eliminate or strengthen their interactions. In extension of these results, we additionally inserted point mutations and deletions into full length STIM1 and the YFP-OASF-CFP-FRET sensor for further functional analysis by patch-clamp and FRET measurements. Our results revealed new insights into the mechanism linking STIM1 oligomerization to the differential interactions of specific α-helices of CC1 with CAD/SOAR and allowed us to delineate a model describing STIM1 activation following store depletion. (supported by Austrian Science Fund (FWF): P22747 to R.S., P22565 and P25172 to C.R.)