Irene Frischauf - Academia.edu (original) (raw)

Papers by Irene Frischauf

In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store... more In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still being actively investigated, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating mechanism among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the “ANSGA” nexus mutations in TM4 of human Orai1 which mimic the STIM1-activated state of the channel. Investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, which directly control ...

Journal of Cell Science, 2019

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store- op... more Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store- operated calcium entry (SOCE). Identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE might be of interest. Using computational analysis, we have identified that the Y316 residue is susceptible to be phosphorylated. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells has resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and ICRAC. STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to YFP-STIM1-WT cells. Additionally, 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 tran...

The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels... more The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of...

Cells

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular funct... more All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved i...

Cancers

The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been r... more The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role...

International Journal of Molecular Sciences, 2021

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the e... more The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing ev...

Current Opinion in Physiology, 2020

Maintaining a precise calcium (Ca 2+) balance is vital for cellular survival. The most prominent ... more Maintaining a precise calcium (Ca 2+) balance is vital for cellular survival. The most prominent pathway to shuttle Ca 2+ into cells is the Ca 2+ release activated Ca 2+ (CRAC) channel. Orai proteins are indispensable players in this central mechanism of Ca 2+ entry. This short review traces the latest articles published in the field of CRAC channel signalling with a focus on the structure of the pore-forming Orai proteins, the propagation of the binding signal from STIM1 through the channel to the central pore and their role in human health and disease.

Cell Reports, 2020

Highlights d Compared with STIM1, STIM2 has 10 additional cytosolic cysteines d STIM2 oxidation i... more Highlights d Compared with STIM1, STIM2 has 10 additional cytosolic cysteines d STIM2 oxidation inhibits store-operated Ca 2+ entry d Redox proteomics identifies C313 as the redox sensor of STIM2 d Oxidation of C313 prevents STIM2-STIM2 oligomerization

Journal of Biological Chemistry, 2021

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion chan... more The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Cell Calcium, 2019

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell... more 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 2+ release-activated Ca 2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca 2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca 2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

Cell Calcium, 2018

A primary calcium (Ca2+) entry pathway into non-excitable cells is through the store-operated Ca2... more A primary calcium (Ca2+) entry pathway into non-excitable cells is through the store-operated Ca2+ release activated Ca2+ (CRAC) channel. Ca2+ entry into cells is responsible for the initiation of diverse signalling cascades that affect essential cellular processes like gene regulation, cell growth and death, secretion and gene transcription. Upon depletion of intracellular Ca2+ stores within the endoplasmic reticulum (ER), the CRAC channel opens to refill depleted stores. The two key limiting molecular players of the CRAC channel are the stromal interaction molecule (STIM1) embedded in the ER-membrane and Orai1, residing in the plasma membrane (PM), respectively. Together, they form a highly Ca2+ selective ion channel complex. STIM1 senses the Ca2+ content of the ER and confers Ca2+ store-depletion into the opening of Orai1 channels in the PM for triggering Ca2+-dependent gene transcription, T-cell activation or mast cell degranulation. The interplay of Orai and STIM proteins in the CRAC channel signalling cascade has been the main focus of research for more than twelve years. This chapter focuses on current knowledge and main experimental advances in the understanding of Orai1 activation by STIM1, thereby portraying key mechanistic steps in the CRAC channel signalling cascade.

Journal of Biological Chemistry, 2017

Edited by Roger J. Colbran Calcium (Ca 2؉) is an essential second messenger required for diverse ... more Edited by Roger J. Colbran Calcium (Ca 2؉) is an essential second messenger required for diverse signaling processes in immune cells. Ca 2؉ release-activated Ca 2؉ (CRAC) channels represent one main Ca 2؉ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca 2؉ sensor in the endoplasmic reticulum, and the Ca 2؉ ion channel Orai in the plasma membrane. After Ca 2؉ store depletion, STIM1 and Orai couple to each other, allowing Ca 2؉ influx. CRAC-/ STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca 2؉ selectivity, an increase in current density when switching from a Ca 2؉-containing solution to a divalent-free Na ؉ one, and fast Ca 2؉-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. Calcium (Ca 2ϩ) represents an important second messenger that is indispensable for various signaling processes in immune and other types of cells (1-3). The Ca 2ϩ release-activated Ca 2ϩ (CRAC) 3 channel, which is activated following intracellular Ca 2ϩ store depletion, represents one main Ca 2ϩ entry pathway (4, 5). CRAC channels are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1) and Orai (5-12). STIM1 represents an endoplasmic reticulum (ER)-located Ca 2ϩ sensing protein (6, 7, 13), whereas Orai forms the Ca 2ϩ-selective ion channel in the plasma membrane (5-8, 14-17). Upon depletion of Ca 2ϩ from the ER, STIM1 proteins oligomerize, move into discrete puncta at the plasma membrane (PM)-ER junctions, and couple to and activate Orai channels (16, 18-21). Subsequently, Ca 2ϩ permeates the Orai channel to enter the cell (22, 23). The Orai protein family includes three members, Orai1-3. They are all composed of cytosolic N-and C-terminal strands and four transmembrane domains (TM) connected via intracellular (TM2-TM3) or extracellular (TM1-TM2 and TM3-TM4) loops (17, 24, 25). Both N and C termini are required for STIM1-dependent Orai channel activity (20, 22, 26-30). The Orai C terminus forms the main binding site for STIM1 (20), whereas direct STIM1 binding to the N terminus is currently controversial (31). Based on the crystal structure of Drosophila Orai, Orai Ca 2ϩ ion channels are assumed to form hexameric complexes (32). STIM1-induced Orai channel pore opening involves a rotation of the hydrophobic region in TM1. However, it has so far remained unclear how this conformational change takes place. Mutagenesis studies have revealed that certain amino acids, like Gly 98 , Phe 99 , Val 102 , and Val 107 in TM1 (33-36), but also other TM residues, such as Leu 138 (37), Trp 176 (38), Thr 184 (36), and Pro 245 (28), or residues between TM4 and the C terminus (i.e. Leu 261-Val 262-His 264-Lys 265) (31) contribute to the maintenance of the closed state, as their point mutation leads to constitutively open channels. For this reason, it has been hypothesized that the open state is established upon global rearrangement of TM helices after STIM1 binding (28, 39, 40). CRAC/Orai channel currents exhibit a strongly inwardly rectifying current/voltage relationship with a reversal potential higher than ϩ50 mV (41, 42), which indicates one typical CRAC channel hallmark. The permeability for Ca 2ϩ is 1000 times larger than for Na ϩ (43). Orai channels conduct small monovalent ions, such as Na ϩ , Li ϩ , or K ϩ , as long as the monovalent solution lacks divalent ions. Monovalent Orai currents are inhibited by Ca 2ϩ concentrations in the micromolar range (43-48). Upon the switch from a Ca 2ϩ-containing to a divalentfree (DVF) Na ϩ-containing solution, CRAC/Orai currents have

Cells, 2022

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity’s knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic ...

Cancers

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel ... more The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in ...

International Journal of Molecular Sciences

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induc... more Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors mic...

Science Signaling

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

The Journal of biological chemistry, Jan 26, 2018

Carelease-activated Ca(CRAC) channels constitute the major Caentry pathway into the cell. They ar... more Carelease-activated Ca(CRAC) channels constitute the major Caentry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca-selective Orai channel and the Ca-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...

High-Power Laser Ablation VI, 2006

ABSTRACT We describe here the modification of various polymers (polytetrafluoroethylene, polyethy... more ABSTRACT We describe here the modification of various polymers (polytetrafluoroethylene, polyethyleneterephthalate, and polyvinyl alcohol) by UV-irradiation with wavelengths below 200 nm in an inert or reactive atmosphere. The light sources employed are F2- or excimer lasers and excimer lamps. The reactive gases include ammonia (NH3), acetylene (C2H2), and oxygen (O2). Photo-dissociated fragments of these gases can react with the polymers or be deposited thereon, resulting in new chemical groups at the surface. Special emphasis is put to improved adhesion of biological cells at these modified surfaces. Potentials applications include cell coated medical implants and prostheses as well as cell micro-arrays for high throughput screening.

In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store... more In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still being actively investigated, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating mechanism among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the “ANSGA” nexus mutations in TM4 of human Orai1 which mimic the STIM1-activated state of the channel. Investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, which directly control ...

Journal of Cell Science, 2019

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store- op... more Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store- operated calcium entry (SOCE). Identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE might be of interest. Using computational analysis, we have identified that the Y316 residue is susceptible to be phosphorylated. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells has resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and ICRAC. STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to YFP-STIM1-WT cells. Additionally, 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 tran...

The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels... more The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of...

Cells

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular funct... more All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved i...

Cancers

The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been r... more The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role...

International Journal of Molecular Sciences, 2021

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the e... more The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing ev...

Current Opinion in Physiology, 2020

Maintaining a precise calcium (Ca 2+) balance is vital for cellular survival. The most prominent ... more Maintaining a precise calcium (Ca 2+) balance is vital for cellular survival. The most prominent pathway to shuttle Ca 2+ into cells is the Ca 2+ release activated Ca 2+ (CRAC) channel. Orai proteins are indispensable players in this central mechanism of Ca 2+ entry. This short review traces the latest articles published in the field of CRAC channel signalling with a focus on the structure of the pore-forming Orai proteins, the propagation of the binding signal from STIM1 through the channel to the central pore and their role in human health and disease.

Cell Reports, 2020

Highlights d Compared with STIM1, STIM2 has 10 additional cytosolic cysteines d STIM2 oxidation i... more Highlights d Compared with STIM1, STIM2 has 10 additional cytosolic cysteines d STIM2 oxidation inhibits store-operated Ca 2+ entry d Redox proteomics identifies C313 as the redox sensor of STIM2 d Oxidation of C313 prevents STIM2-STIM2 oligomerization

Journal of Biological Chemistry, 2021

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion chan... more The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

Cell Calcium, 2019

Calcium signalling through store-operated calcium (SOC) entry is of crucial importance for T-cell... more 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 2+ release-activated Ca 2+ (CRAC) channel. STIM1, a key molecular component of this process, is located in the membrane of the endoplasmic reticulum (ER) and is initially activated upon Ca 2+ store depletion. This activation signal is transmitted to the plasma membrane via a direct physical interaction that takes place between STIM1 and the highly Ca 2+-selective ion channel Orai1. The activation of STIM1 induces an extended cytosolic conformation. This, in turn, exposes the CAD/SOAR domain and leads to the formation of STIM1 oligomers. In this study, we focused on a small helical segment (STIM1 α3, aa 400-403), which is located within the CAD/SOAR domain. We determined this segment's specific functional role in terms of STIM1 activation and Orai1 gating. The STIM1 α3 domain appears not essential for STIM1 to interact with Orai1. Instead, it represents a key domain that conveys STIM1 interaction into Orai1 channel gating. The results of cysteine crosslinking experiments revealed the close proximity of STIM1 α3 to a region within Orai1, which was located at the cytosolic extension of transmembrane helix 3, forming a STIM1-Orai1 gating interface (SOGI). We suggest that the interplay between STIM1 α3 and Orai1 TM3 allows STIM1 coupling to be transmitted into physiological CRAC channel activation.

Cell Calcium, 2018

A primary calcium (Ca2+) entry pathway into non-excitable cells is through the store-operated Ca2... more A primary calcium (Ca2+) entry pathway into non-excitable cells is through the store-operated Ca2+ release activated Ca2+ (CRAC) channel. Ca2+ entry into cells is responsible for the initiation of diverse signalling cascades that affect essential cellular processes like gene regulation, cell growth and death, secretion and gene transcription. Upon depletion of intracellular Ca2+ stores within the endoplasmic reticulum (ER), the CRAC channel opens to refill depleted stores. The two key limiting molecular players of the CRAC channel are the stromal interaction molecule (STIM1) embedded in the ER-membrane and Orai1, residing in the plasma membrane (PM), respectively. Together, they form a highly Ca2+ selective ion channel complex. STIM1 senses the Ca2+ content of the ER and confers Ca2+ store-depletion into the opening of Orai1 channels in the PM for triggering Ca2+-dependent gene transcription, T-cell activation or mast cell degranulation. The interplay of Orai and STIM proteins in the CRAC channel signalling cascade has been the main focus of research for more than twelve years. This chapter focuses on current knowledge and main experimental advances in the understanding of Orai1 activation by STIM1, thereby portraying key mechanistic steps in the CRAC channel signalling cascade.

Journal of Biological Chemistry, 2017

Edited by Roger J. Colbran Calcium (Ca 2؉) is an essential second messenger required for diverse ... more Edited by Roger J. Colbran Calcium (Ca 2؉) is an essential second messenger required for diverse signaling processes in immune cells. Ca 2؉ release-activated Ca 2؉ (CRAC) channels represent one main Ca 2؉ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca 2؉ sensor in the endoplasmic reticulum, and the Ca 2؉ ion channel Orai in the plasma membrane. After Ca 2؉ store depletion, STIM1 and Orai couple to each other, allowing Ca 2؉ influx. CRAC-/ STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca 2؉ selectivity, an increase in current density when switching from a Ca 2؉-containing solution to a divalent-free Na ؉ one, and fast Ca 2؉-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. Calcium (Ca 2ϩ) represents an important second messenger that is indispensable for various signaling processes in immune and other types of cells (1-3). The Ca 2ϩ release-activated Ca 2ϩ (CRAC) 3 channel, which is activated following intracellular Ca 2ϩ store depletion, represents one main Ca 2ϩ entry pathway (4, 5). CRAC channels are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1) and Orai (5-12). STIM1 represents an endoplasmic reticulum (ER)-located Ca 2ϩ sensing protein (6, 7, 13), whereas Orai forms the Ca 2ϩ-selective ion channel in the plasma membrane (5-8, 14-17). Upon depletion of Ca 2ϩ from the ER, STIM1 proteins oligomerize, move into discrete puncta at the plasma membrane (PM)-ER junctions, and couple to and activate Orai channels (16, 18-21). Subsequently, Ca 2ϩ permeates the Orai channel to enter the cell (22, 23). The Orai protein family includes three members, Orai1-3. They are all composed of cytosolic N-and C-terminal strands and four transmembrane domains (TM) connected via intracellular (TM2-TM3) or extracellular (TM1-TM2 and TM3-TM4) loops (17, 24, 25). Both N and C termini are required for STIM1-dependent Orai channel activity (20, 22, 26-30). The Orai C terminus forms the main binding site for STIM1 (20), whereas direct STIM1 binding to the N terminus is currently controversial (31). Based on the crystal structure of Drosophila Orai, Orai Ca 2ϩ ion channels are assumed to form hexameric complexes (32). STIM1-induced Orai channel pore opening involves a rotation of the hydrophobic region in TM1. However, it has so far remained unclear how this conformational change takes place. Mutagenesis studies have revealed that certain amino acids, like Gly 98 , Phe 99 , Val 102 , and Val 107 in TM1 (33-36), but also other TM residues, such as Leu 138 (37), Trp 176 (38), Thr 184 (36), and Pro 245 (28), or residues between TM4 and the C terminus (i.e. Leu 261-Val 262-His 264-Lys 265) (31) contribute to the maintenance of the closed state, as their point mutation leads to constitutively open channels. For this reason, it has been hypothesized that the open state is established upon global rearrangement of TM helices after STIM1 binding (28, 39, 40). CRAC/Orai channel currents exhibit a strongly inwardly rectifying current/voltage relationship with a reversal potential higher than ϩ50 mV (41, 42), which indicates one typical CRAC channel hallmark. The permeability for Ca 2ϩ is 1000 times larger than for Na ϩ (43). Orai channels conduct small monovalent ions, such as Na ϩ , Li ϩ , or K ϩ , as long as the monovalent solution lacks divalent ions. Monovalent Orai currents are inhibited by Ca 2ϩ concentrations in the micromolar range (43-48). Upon the switch from a Ca 2ϩ-containing to a divalentfree (DVF) Na ϩ-containing solution, CRAC/Orai currents have

Cells, 2022

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity’s knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic ...

Cancers

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel ... more The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in ...

International Journal of Molecular Sciences

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induc... more Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors mic...

Science Signaling

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

The Journal of biological chemistry, Jan 26, 2018

Carelease-activated Ca(CRAC) channels constitute the major Caentry pathway into the cell. They ar... more Carelease-activated Ca(CRAC) channels constitute the major Caentry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca-selective Orai channel and the Ca-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...

High-Power Laser Ablation VI, 2006

ABSTRACT We describe here the modification of various polymers (polytetrafluoroethylene, polyethy... more ABSTRACT We describe here the modification of various polymers (polytetrafluoroethylene, polyethyleneterephthalate, and polyvinyl alcohol) by UV-irradiation with wavelengths below 200 nm in an inert or reactive atmosphere. The light sources employed are F2- or excimer lasers and excimer lamps. The reactive gases include ammonia (NH3), acetylene (C2H2), and oxygen (O2). Photo-dissociated fragments of these gases can react with the polymers or be deposited thereon, resulting in new chemical groups at the surface. Special emphasis is put to improved adhesion of biological cells at these modified surfaces. Potentials applications include cell coated medical implants and prostheses as well as cell micro-arrays for high throughput screening.