Submitted by Sascha Berlansky BSc
Submitted at JKU Institute of Biophysics Department: Ion Channels
Supervisor Assoc. Univ.-Prof. Dr. Christoph Romanin
Characterization of Co-Supervisor Dr. Irene Frischauf, MLBT cancer related Orai1 May 2020 mutants A122T, N147S and P164H
Master thesis To obtain the academic degree of Master of Science In the Master’s program Molecular Biology
JOHANNES KEPLER UNIVERSITÄT LINZ Altenberger Straße 69 4040 Linz, Österreich jku.at DVR 0093696
ACKNOWLEDGEMENTS
First of all, I would like to thank A. Univ.-Prof. Dr. Christoph Romanin for giving me the opportunity to work in his team.
I am very grateful that my co-supervisor Dr. Irene Frischauf supported me throughout my time in this group with her knowledge. Thank you for taking the time to answer all my questions! This thesis would have never been written without your support.
I would also like to thank the entire Ion-Channel group for their friendly and patient support and all the practical advice I got from them. Special thanks to Dr. Marc Fahrner, Dr. Victoria Lunz, Ing. Sabine Buchegger, Adéla Tiffner MSc. and Sonja Lindinger MSc. for helping me with all those minor questions, which accumulate during a normal working day in the lab.
Finally, I would like to acknowledge that I am very fortunate to have Matthias Sallinger BSc. and Herwig Grabmayr MSc. at my side, not just coworkers, but also friends. Thank you for all the years of support, motivation and the fun we had together!
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EIDESSTATTLICHE ERKLÄRUNG
Ich erkläre an Eides statt, dass ich die vorliegende Masterarbeit selbstständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt bzw. die wörtlich oder sinngemäß entnommenen Stellen als solche kenntlich gemacht habe.
Die vorliegende Masterarbeit ist mit dem elektronisch übermittelten Textdokument identisch.
Linz, 21.05.2020 Ort, Datum
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Abstract
The role of Ca2+ as a signalling molecule is essential to fundamental cellular functions such as proliferation, cell growth, gene expression or the secretion of bioactive molecules. Intracellular Ca2+ homeostasis is maintained via regulatory systems such as store operated Ca2+ channels (SOCC), which are the key players of store-operated Ca2+ entry (SOCE).
Orai1 proteins build hexameric Ca2+ selective channels in the plasma membrane. Upon activation by STIM1, which acts as a Ca2+ sensor protein in the ER, the Ca2+release activated Ca2+ (CRAC) channels allow the influx of Ca2+ ions into the cell, replenishing the intracellular Ca2+ store within the ER.
Throughout years of research several disease-related STIM1 and Orai1 mutations have been identified. This thesis focuses on three cancer associated Orai1 mutants, taken from the cBioPortal database. The mutants A122T (malignant mixed Mullerian tumours of the uterus), N147S (germinal center B-cell type cancer) and P164H (lung adenocarcinoma) were investigated for their impact on pore structure at pore lining residues R91, F99 and E106 by utilizing a cysteine cross-linking approach. Furthermore, the electrophysiological channel characteristics were measured via whole cell patch clamp recordings, protein localization was verified by fluorescence microscopy and a NFAT screen was carried out to measure possible modulations of gene transcription downstream the Ca2+ signalling cascade.
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Kurzfassung
Die Rolle von Kalzium in der zellulären Signalübertragung ist fundamental für grundlegende Prozesse von Zellen, beispielsweise in der Proliferation, dem Zellwachstum, der Genexpression oder der Sekretion von bioaktiven Molekülen. Intrazelluläre Kalzium -Homöostase wird durch Systeme wie den speichergesteuerten Kalzium Kanal (store operated Ca2+ channels - SOCC) reguliert, welche Hauptakteure im speichergesteuerten Kalzium-Einlass (store-operated Ca2+ entry - SOCE) von Zellen sind.
Orai1 Proteine sind hexamere Kalzium-selektive Ionenkanäle in der Plasmamembran. Nach ihrer Aktivierung, welche durch STIM-Proteine, die als Kalziumsensoren in der Membran des endoplasmatischen Retikulum dienen, wird der Einstrom von Kalzium-Ionen aus dem extrazellulären Raum und somit das Auffüllen der intrazellulären Kalzium-Speicher im ER ermöglicht.
Durch jahrelange Forschung sind mehrere krankheitsassoziierte Mutationen von STIM1 und Orai1 identifiziert worden. In der folgenden Arbeit werden drei krebsassoziierte Orai1 Mutanten aus der Datenbank von cBioPortal untersucht. Die Mutanten A122T (Müllerscher Mischtumor), N147S (diffuses großzelliges B-Zelllymphom) und P164H (Adenokarzinom der Lunge) wurden auf strukturelle Änderungen der Kanalpore an den Positionen R91, F99 and E106, welche dem Inneren der Pore zugewandt sind, durch Cystein-Crosslinking untersucht. Zusätzlich wurden elektrophysiologische Kanalcharakteristiken mittels whole cell patch clamp gemessen, die Lokalisation der Proteine mit Fluoreszenzmikroskopie überprüft, sowie NFAT-Screening zur Überprüfung von möglichen Auswirkungen auf die Transkription von Genen durch Änderungen in der Signalkaskade durchgeführt.
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Contents
1. Introduction ...... 6 1.1. Calcium in cellular life ...... 6 1.1.1. Calcium functions ...... 6 1.1.1. Ca2+ binding motifs ...... 7 1.1.2. Ca2+ homeostasis ...... 8 1.1.3. Role of Ca2+ in cell cycle regulation ...... 10 1.2. Store operated calcium entry ...... 11 1.2.1. SOCE ...... 11 1.1.1. STIM ...... 12 1.1.2. Orai ...... 13 1.1.3. STIM/Orai interaction ...... 15 1.3. Calcium SOCE and cancer ...... 17 1.3.1. STIM, Orai and cancer ...... 17 1.3.1. A122T linked to malignant mixed Mullerian tumour of the uterus ...... 17 1.3.2. N147S linked to germinal center B-cell type cancer ...... 18 1.3.1. P164H linked to lung adenocarcinoma ...... 18 1.4. Task ...... 19 1.5. Research techniques ...... 21 1.5.1. SDS-Page ...... 21 1.5.2. Western blot and immunodetection ...... 22 1.5.3. Patch-clamp electrophysiology ...... 23 1.5.4. Confocal fluorescence microscopy ...... 24 2. Methods ...... 25 2.1. General information ...... 25 2.2. Plasmid construction ...... 26 2.2.1. PCR ...... 26 2.2.2. DPN I digestion & PCR validation ...... 28 2.2.3. Transformation into competent E. coli ...... 28 2.2.4. Miniprep ...... 29 2.2.5. Sequencing ...... 29 2.2.6. Midiprep ...... 30 2.2.7. Transfection ...... 30 2.3. Western blot ...... 31 2.3.1. Lysis and cysteine-crosslinking ...... 31
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2.3.2. SDS-Page ...... 32 2.3.3. Western blot ...... 33 2.3.4. Immunodetection ...... 34 2.4. Patch clamp measurements ...... 35 2.5. Confocal fluorescence microscopy ...... 36 3. Results ...... 38 3.1. Cysteine-crosslinking results ...... 38 3.2. Patch clamp results ...... 40 3.3. Confocal fluorescence microscopy ...... 41 3.3.1. Localisation Studies ...... 41 3.3.2. Nuclear Factor of Activated T-Cells (NFAT) screen ...... 42 4. Discussion ...... 44 4.1. General discussion ...... 44 4.2. A122T discussion ...... 44 4.3. N147S discussion ...... 45 4.4. P164H discussion ...... 45 5. References ...... 46 6. Addendum: Orai channels : key players in Ca2+ homeostasis ...... 49
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1. Introduction
1.1. Calcium in cellular life
1.1.1. Calcium functions Calcium (Ca2+) is a divalent cation with the atomic number 20. It is found in the second group of the periodic table, making it an alkaline earth metal fourth period of the periodic table of the elements alongside the other alkaline earth metals Beryllium (Be), Magnesium (Mg), Strontium (Sr), Barium (Ba), and Radium (Ra). [1]
Ca2+ also plays an important role in cellular life. It is necessary for essential cellular processes such as cell-differentiation, proliferation as well as apoptosis and several transcription factors (e.g. NFAT) are sensitive to changes in Ca2+ concentrations via the phosphatase Calcineurin. Ca2+ also plays a critical role for the release of neurotransmitters into the synaptic cleft by triggering vesicle fusion of neurotransmitter containing vesicles with the synaptic membrane via SNARE-protein complexes. [2]
Besides the already mentioned functions, Ca2+ is also responsible for the regulation of muscle contractions in smooth muscle tissue as well as skeletal muscle cells. [3] It has essential functions in the immune system and is required for the activation of T-cells by inducing the interaction between them and antigen presenting cells (APCs). [4] [5]
Ca2+ is found in very low concentrations in the intracellular space at approximately 100 nM and in an around 20,000-fold higher concentration in the mM range extracellularly. [6]
Figure 1. Biological functions of Ca2+
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1.1.1. Ca2+ binding motifs
In order to fulfill its biological functions, Ca2+ often binds to proteins that contain calcium binding motifs. Two of the best known motifs are the EF hand helix-loop-helix motif and the annexin Ca2+-binding motif. The annexin protein family binds Ca2+ via four repeat domains, each containing a calcium binding site (CBS) consisting of negatively charged glutamate and aspartate regions. [7] [8] Kretsinger et al labelled the binding motif „EF hand“ due to the structural similarity of the alpha helices to a human hand (see Figure 2). The EF hand motif consists of a pair of alpha helical domains that are connected by a loop of usually 12 amino acids. Five of these twelve amino acids contain negatively charged oxygen atoms, which help calcium binding. The remaining residues typically form a hydrophobic core that binds and stabilizes the two helices in their conformation. [9]
Figure 2. Left: Structure of the EF hand. The colored helices correspond to the fingers of the symbolic hand. Upon binding of the Ca2+ ion, the thumb helix moves to the open conformation. [9] Right: 3D-representation of the molecular Ca2+-binding geometry of an EF hand. [9]
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1.1.2. Ca2+ homeostasis
Mg2+ and K+ are the main cationic ions, which are found in a relative high concentration in the intracellular space, whereas Ca2+ and Na+ act as their extracellular counterparts. The concentration of Ca2+ in the extracellular space of animals ranges between values from 1 to 10 mM. In the cytosol, the concentration is much lower and only 100 nM of Ca2+ usually is measured. [8] This enormous 10,000-fold concentration gradient for Ca2+ across the cell membrane causes a strong driving force for Ca2+ influx. Measurements show that cells under resting conditions have a low membrane permeability to Ca2+ but a small increase in permeability leads to large Ca2+ influx. This is achieved via Ca2+ permeable ion channels in the plasma membrane. Various Ca2+ permeable channels exist: voltage-operated channels (VOC), second messenger-operated channels (SMOC), store-operated channels (SOC), receptor-operated channels (ROC) and the antiporter system Na+-Ca2+ exchanger (NCX). [10] An overview of some of the major regulators of Ca2+ is shown in Figure 3.
Figure 3. Depiction of some of the major regulators of calcium homeostasis. Ca2+-Pumps and Ca2+-channels (PMCA, NCX, and SOCE) regulate in- and out-flux the cytosol. G-protein– coupled receptors initiate signals that modify calcium stores downstream via signalling molecules such as IP3. Calcium ATPases SERCA or SPCA1 localized on organelles such as the golgi apparatus monitor and replenish intracellular storage sites. Sensing and replenishment of the ER/SR via Stim1 and ORAI1 are discussed in further chapters. [11]
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In order to keep Ca2+ homeostasis at physiological range, cells do not rely on Ca2+ channels alone, in which the influx of Ca2+ ions follows their electrochemical gradient. Cells need to build up and maintain the concentration of Ca2+ ions on both sides of the plasma membrane in order to function. This is achieved by ATP-driven Ca2+-pumps called „Ca2+ ATPases“. There are three known types of Ca2+ ATPases in vertebrates: plasma membrane Ca2+ ATPases (PMCAs), sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), and secretory pathway Ca2+ ATPases (SPCAs). [8]
Figure 4. Reaction mechanism of Ca2+-ATPases. In a simplified model, two functional states exist: E1 and E2. In the E1 state, in which the ATP is hydrolyzed, the pumps have high Ca2+ affinity while in the E2 state the affinity becomes much lower which leads to the release of Ca2+ at the extracellular side of the membrane. [12]
There are several inhibitors for different classes of ATPases, e.g. the Lanthanum ion La3+ or orthovanadate, which tend to inhibit all ATPases. La3+ inhibitions the ATPases by mimicking the divalent cationic charge of calcium, but it blocks the channel due to a higher atomic radius of rLanthanum = 195 pm (compared to rCalcium= 180 pm). Orthovanadate acts as a competitive inhibitor of ATPases, alkaline and acid phosphatases, and protein-phosphotyrosine phosphatases. [12] The specific inhibition of SERCAs triggered by thapsigargin (TG), which is ineffective against the other ATPases, is implemented in the NFAT-measurement experiments used for this thesis. Another way to artificially deplete Ca2+ stores of cells is via chelating the ions with suitable compounds such as glycol diamine tetraacetic acid (EGTA), which was used in patch clamp measurements.
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1.1.3. Role of Ca2+ in cell cycle regulation
The cell cycle consists of four phases: first gap phase (G1), the DNA-synthesis phase (S), the second gap phase (G2) and the mitosis phase (M). In order to ensure the proper division of the cells, there are control mechanisms termed “cell cycle checkpoints”. During these checkpoints, cells either continue with the next phase, initiate DNA repair mechanisms or apoptosis. If errors occur during these cell cycle checkpoints, cells that should initiate apoptosis due to irreparable damaged DNA or failed DNA repair mechanisms could continue with their mitotic division, leading to pathogenic conditions such as carcinogenesis. Essential regulators of the cell cycle are cyclins and cyclin-dependent kinases (CDKs). The expression as well as the activity of these regulators is dependent on Ca2+. [13]
Figure 5. Depiction of the cell cycle and Ca2+ regulated apoptosis [13]
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1.2. Store operated calcium entry
1.2.1. SOCE The concept of store-operated calcium entry, a process in which the release of stored calcium leads to the opening of plasma membrane calcium channels, has its roots in the late 1970’s, and was formalized in 1986 by Putney. [14]
It was discovered that upon the depletion off intracellular Ca2+ stores of the ER, Ca2+ influx occurred via the plasma membrane, resulting in replenishment of intracellular Ca2+ stores. This pathway was termed „store-operated Ca2+ entry“ (SOCE) in which a reduction of intracellular store concentration of Ca2+ leads to Ca2+ influx over the plasma membrane via store-operated Ca2+ channels (SOCCs). [10] The investigation of these Ca2+ entry pathways was done via electrophysiological techniques in mast cells, followed by the discovery, that SOCE takes place in nearly all cell types. [15] [16] [17] In 1992 Hoth & Penner termed these channels „Ca2+ release-activated Ca2+ (CRAC) channels“. [18]
The exact mechanisms and the unusual process that involves proteins from two different cellular compartments remained unclear in the early 1990s. CRAC channels were known to exhibit very specific properties that distinguished them from other known Ca2+ channels. These properties include a high Ca2+ selectivity with a permeability ratio of 1:1000 of Ca2+ to Na, a low single channel conductance with approximately 20fs as well as many known Ca2+-dependent feedback mechanisms. [10]
In 2005 Liou et al. identified the stromal interaction molecule 1 (STIM1) as a Ca2+ sensing protein in the membrane of the ER. [19] One year later Orai proteins, first termed “CRACM1”, were discovered by Feske et al. as the cause for severe combined immune deficiency via RNAi- approaches and genetic linkage analysis [20]
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1.1.1. STIM
The stromal interaction molecule (STIM) is a single-pass transmembrane protein located in the ER membrane that regulates store operated calcium entry (SOCE) via its function as a Ca2+ sensor. It senses a drop in ER Ca2+ concentration through its EF hand domain that binds to Ca2+ ions in resting state. [21] [22] [23] The N-terminus that contains a Ca2+ binding EF-hand motif as well as a sterile alpha motif (SAM) is located in the ER lumen (Figure 6). The interaction of the EF-hand originates from an aspartate and glutamate rich helix-loop-helix motif. [24] [25] [26] The C-terminus of STIM1 remains in the cytosol. It contains three coiled-coil regions (CC1, CC2, CC3), a serine-proline- as well as a lysine-rich region and the CRAC modulatory domain (CAD). While the N-terminus of STIM1 is crucial for of Ca2+ sensing, the C-terminus is essential for STIM1 oligomerization and its interaction with Orai1. Essential domains for Orai1 activation are OASF (Orai activating STIM fragment), CAD (CRAC activating domain), SOAR (STIM-Orai activating region), Ccb9 (Figure 7). [21, 27]
Figure 6. Depiction of hypothetical resting state of STIM1. ER lumen displayed in dark green (bottom), cytosol displayed in lighter green (top). Ca2+ is bound to the EF-Hand. [21]
Figure 7. Depiction of linearized STIM1, displaying essential regions. ER lumen displayed in dark green (left), cytosol displayed in lighter green (right). EF-Hand and SAM as well as CC3 and CC2 are shown as 3D models. [21]
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1.1.2. Orai
Orai proteins are tetra-spanning membrane proteins that form highly selective Ca2+-channels in the plasma membrane (Figure 8 and Figure 10). Their gating mechanism depends on the coupling to STIM1 proteins, which leads to an open state that allows the influx of Ca2+ ions into the cell. [28] [21] In mammals three types of highly conserved Orai proteins exist (Orai 1-3) which show tissue-specific expression levels. All three Orai proteins share an identical TM1 helix that forms the inner pore, surrounded by the other transmembrane helices. (Figure 10) The position E106 in TM1 was shown to be important for the selectivity for Ca2+ ions. Mutating a wild type glutamate to aspartate or glutamine leads to a vast alteration in ion selectivity of the channel. [29] A mutation to aspartate (E106D) seems to increase the diameter of pore, leading to a higher permeability for larger cations like Na+, Ba2+, and Sr2+. Exchanging the glutamate with an uncharged amino acid leads to a non-functional channel. [30] Position R91 in TM1 seems to function as an electrostatic gate. Mutations to hydrophilic residues allowed normal functioning of the channel whereas hydrophobic mutations such as the SCID mutant (R91W) lead to non- functional channels. [31, 32] Orai proteins have a mass of approximately 30 kDa and their C- and N-termini are both located in the cytoplasm. Two extracellular and one intracellular loop connect the four transmembrane helices of Orai. [28] A CRAC channel is formed by six Orai1 subunits, enabling the possibility of potential homo- and heteromeric channels. [33] All members of the Orai family contain a coiled-coil domain in their C-terminus that is essential for the interaction with STIM1. Likewise, the N-terminus, which is located close to the C-terminus in the cytoplasm, is important for the interaction with STIM1 and both termini are necessary for channel activation. [34] [35]
It is assumed that the assembly of CRAC channels by multimerisation of Orai subunits is realized by interaction of the transmembrane regions, since the deletion of the C-termini doesn’t affect channel assembly. [34] [36] Biochemical and fluorescence studies provided evidence for a tetrameric assembly model of the CRAC channel. However, the crystal structure of the Drosophila Orai protein revealed a hexameric assembly. [37] Although the crystal structure with its 3.35 Å resolution provides the best representation of an Orai structure, it should be stated that the mutant used in this experiment lacks parts of the C- and N-termini as well as the TM1- TM2 and the TM2-TM3 loops and contains mutations at positions C224S, P276R, P277R and C283T. In dOrai six TM1 helices form the pore that allows the Ca2+ ions to pass. The other transmembrane helices form the outer layers of the pore and offer structural support of the channel. [24] TM3 and TM4 are shown to be the most divergent domains between Orai isoforms. [21] [38] [39] Residues within TM3 also have been shown to modulate the gating and permeation even though they are not directly inside the pore. The current knowledge allows for a division of CRAC channel units into for parts: 1) a negatively charged selectivity filter composed of glutamates, 2) a hydrophobic region with three α-helical turns, 3) a basic domain that is suggested to act as an anion-coordinator (R91, K87, R83) and 4) a cytosolic domain containing two α-helical turns with a length of approximately 20 Å called “extended TM1 Orai1 N-terminal (ETON) region” that functions as a binding partner for STIM1. [21] [40]
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Figure 8. Left: Cartoon side view of an Orai1 monomer displaying N- and C-termini as well as all 4 transmembrane domains in different colours. Right: top view of an Orai hexamer, displaying a channel pore width of approximately 6 Å [21]
Figure 9. Cartoon representing a cross-section through the Orai1 pore, displaying pore-lining TM1 helices and the corresponding amino acids. Negatively charged amino acids are coloured in cyan, hydophobic amino acids in green and positive amino acids are shown in red. [21]
Figure 10. Approximate positions of A122 (red), N147 (green) and P164 (blue) are shown in a cartoon of a linearized Orai1 (top) as well as a membrane-embedded Orai1 cartoon model (left). Adapted from [21]
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1.1.3. STIM/Orai interaction
The coupling of the cytosolic strand of STIM1 to Orai1 domains induces a conformational change in the CRAC channel, opening the gate and thereby leading to Ca2+ influx into the cell. The interaction of Orai1 with STIM1 includes the cytosolic region of STIM1 as well as the C- terminus and the N-terminus of Orai1, also located in the cytosol. [21] As long as Ca2+ in the ER is abundant, it is bound to the EF-hand, keeping STIM1 in a resting state, and the STIM1 proteins remain uniformly distributed in the ER membrane. Upon store depletion of the ER the EF-hand senses a decrease in Ca2+ concentration and STIM1 alternates to its extended state. [28] The activated STIM1 proteins redistribute and oligomerize at ER-PM junctions, leading to the formation of STIM1 puncta. Orai1 proteins accumulate on the opposite side of the ER-PM junctions in the plasma membrane. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the plasma membrane, and STIM1 then traps Orai1 through binding of its calcium release- activated calcium activation domain, leading to an activated Orai1 (Figure 11). [41]
Figure 11. CRAC activation model. STIM1 is shown at resting state (left), transitioning to an active state, and finally activating Orai1 (right), leading to an influx of Ca+2 into the cytoplasm. [42]
During STIM1-Orai1 coupling of the Orai1 C-terminus and the coiled-coil 2 domain (CC2) of STIM1 involve mainly hydrophobic and ionic interactions. All members of the Orai family include a highly conserved N-terminal region (aa72-90) termed “extended transmembrane Orai1 N- terminal region” (ETON) that corresponds to the cytosolic TM1-extension. Mutants that lack the ETON (Δ1-76) or the whole N-terminus lose their function. [21] [40]
Both, the N- and C-termini of Orai1, are necessary for proper coupling to STIM1 and it has been suggested that STIM1 first binds to the Orai1 C-terminus before interacting with the N-terminus. [21] A Stim1 CC2 dimer binds to the C-termini of Orai1 and forms the hydrophobic and basic STIM-Orai association pocket (SOAP).
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Upon Stim1-Orai1-coupling, the channel opens and allows Ca2+ influx. Prakriya et al. had proposed a pore rotation model for channel opening, in which the torsion of the TM1 helix is linked with conformational changes in the Orai1 C-terminus after coupling to STIM1. In this model the hydrophobic side chains of pore-lining residues V102 and F99, which create a barrier for Ca2+ permeation in the closed state rotate approximately 20° outwards to open the channel. [43] [44]
In contrast, Dong et al. propose a „twist-to-open“ gating mechanism. By MD-simulations based on the dOrai structure they show that a coupling of TM1 to TM3 (R83-E149 and K85-E173) seems to be crucial for the activation of Orai channels and that a series of motions leads to channel opening. [45]
Frischauf et al have shown a small local widening of the pore (~1-2 Å) occurs during the opening of the channel, suggesting two gates in both the hydrophobic and basic region of the pore. Gain- of-function H134A mutants were shown to create hydrogen bonds between amino acid side chains that are facing the channels pore, such as S90 and R91. Additionally, this mutation decreases the hydrophobic gating barriers by creating a chain of water molecules through the channels pore. [46]
Long et al. crystallized the open dOrai channel by taking advantage of the H134A Orai1 gain-of- function mutation. [47] The open crystal suggests conformational changes and straightening of the TM4 and the extended TM4 region (M4ext) upon channel opening. [24]
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1.3. Calcium SOCE and cancer
1.3.1. STIM, Orai and cancer
Accumulating evidence suggests that SOCE plays a critical role in cancer cell proliferation, metastasis as well as tumor vascularization and antitumor immunity. [48] [49] [49] [48]. Ca2+ signalling via SOCE leads to different effects depending on the tumor stage. At tumor initiation stage, it induces genetic changes in premalignant cells. This can cause malignant transformations in cell, e.g. SOCE is responsible for the secretion of vascular endothelial growth factor (VEGF), which promotes the proliferation of endothelial cells. Different tumor tissues show overexpression of Orai1 and STIM1 as well as different mutations of Orai1 and STIM1 proteins. Targeting SOCE has significant potential as anticancer treatment. Pharmacological inhibition of Orai1 and STIM1 can diminish the growth of colorectal, breast, liver, melanoma and clear cell renal cancer cells. A knockdown of STIM1 was shown to support chemotherapy-induced apoptosis in pancreatic as well as lung cancer cells. It has been shown that Orai3 is involved in the tumorigenesis of estrogen receptor-positive breast cancer. [50] During angiogenesis, one of the hallmarks of cancer, the vascular endothelial growth factor (VEGF) and the corresponding VEGF-receptors induce Ca2+ store depletion, thereby inducing SOCE in human endothelial cells during angiogenesis. A knockdown of STIM or Orai1 was able to minimize VEGF-induced Ca2+ influx in human umbilical vein endothelial cells (HUVECs), leading to inhibition of cell proliferation and migration. [51] [50]
In this master thesis, three cancer related mutants (A122T, N147S and P164H) from the cBioPortal database are investigated (see task). A short summary of the related cancer types is provided in the following paragraphs.
1.3.1. A122T linked to malignant mixed Mullerian tumour of the uterus
A malignant mixed Mullerian tumour (MMMT), also termed “uterine carcinosarcoma”, is a rare type of tumor in the uterus, the ovaries, the fallopian tubes and other tissues that contains both carcinomatous (epithelial tissue) and sarcomatous (connective tissue) components. [52] These tumours are a dedifferentiated or metaplastic form of endometrial carcinoma, displaying histological features of both endometrial carcinoma and sarcoma. [53] Cervical MMMT usually occur in post-menopausal women with mean age at diagnosis of 61 to 69 years. The commonest clinical features are vaginal bleeding, abnormal vaginal cytology and polypoidal cervical mass. [54] [55] [56]. MMMT account for between 2-5% of all tumors derived from the uterus. [57] The survival of MMMT patients is determined primarily by depth of invasion and stage (four stages are classified). MMMTs are highly malignant whereas a stage I tumor has an expected five-year survival rate of 50%, while the overall five-year survival rate is less than 20%. [58]
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1.3.2. N147S linked to germinal center B-cell type cancer
Germinal centers (GCs) are sites within the lymphatic system and the spleen, in which mature B cells proliferate, differentiate and mutate their antibody genes during an immune response to an infection. They develop after the activation of follicular B cells by T-dependent antigens. [59] Germinal Center B-Cell type cancer is one of the three subtypes of diffuse large B-cell lymphoma that is known besides activated B-cell-like lymphoma and primary mediastinal B-cell lymphoma. [60] Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). The Germinal Center B-Cell Type seem to arise from normal germinal center B cells, whereas the activated B-cell-like type may arise from postgerminal center B cells that are arrested during plasmacytic differentiation, and PMBLs may arise from thymic B cells. [60] Usually germinal center B cells manifest many different hallmarks of cancer. Most of the B‐cell neoplasms originate from the GC reaction, and display many different point mutations, structural genomic lesions, and genetic and epigenetic clonal diversity. The dominant biological theme of germ cell derived lymphomas is mutation of genes, which are involved in epigenetic regulation mechanisms and immune receptor signalling that comes into play at critical transitional stages of the GC reaction. [61]
1.3.1. P164H linked to lung adenocarcinoma
Lung adenocarcinoma is the most common primary lung cancer which belongs to the non-small cell lung cancers (NSCLC) and has a strong association with previous smoking. However, it is also the most common subtype of lung cancer that is diagnosed amongst non- smokers. Adenocarcinoma of the lung usually evolves from the mucosal glands and represents about 40% of all lung cancers and remains the leading cause of cancer deaths. Lung adenocarcinoma usually occurs in the lung periphery, and in many cases, may be found in scars or areas of chronic inflammation. [62] Adenocarcinoma is mostly diagnosed at an advanced or metastatic stage, when patients show symptoms outside the respiratory tract, these extrapulmonary manifestations are termed „paraneoplastic syndromes”. One of the most common paraneoplastic syndromes associated with adenocarcinoma is hypercalcemia of malignancy. It is more common in squamous cell carcinoma but it can occur as well in adenocarcinomas. Cancer cells cause increased bone resorption by upregulating the activity of osteoclasts, leading to a breakdown of bone tissue followed by a release of Ca2+ into the bloodstream. This effect is caused by tumor cells that release the parathyroid hormone-related peptide (PTHrP), which has an upregulating effect on osteoclasts. Symptoms of elevated Ca2+ levels in the bloodstream include fatigue, increased thirst, constipation, polyuria and nausea. [63]. Studies of adenocarcinoma tumour tissues showed that Orai3 is overexpressed in lung adenocarcinomas when compared to non-tumour tissues by 66.5% compared to 32% overexpression for Orai2 and 31% for Orai1. [64]
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1.4. Task
Three cancer-related Orai1 mutations (A122T, N147S and P164H) from the cBioPortal database which are linked to different cancer types (see chapter Orai1 and cancer) were examined with a combination of three different analytical techniques:
Western Blotting for the analysis of structural changes at important channel residues R91 and F99 (both positions are supposed to act as a gate of the channel) as well as E106 (selectivity filter) via disulfide-crosslinking studies. Whole-Cell Patch Clamp measurements upon store depletion caused by EGTA to analyse changes in channel conductance caused by the mutations. Fluorescence Microscopy to A) investigate the localisation behaviour of the mutants via YFP- tags as well as B) analyse the effect of the mutations onto the Ca2+ dependent nuclear transcription factor (NFAT)
In order to examine if the introduced mutations have an impact onto the Orai1 pore, they were investigated via cysteine-crosslinking. The cysteine-mutations allow the formation of disulfide- bonds via crosslinking of residues in close proximity, leading to the formation of hOrai1-dimers. Comparing the rate of dimerization between metated and wild type proteins hints towards a structural change of the given positions, namely if the mutation leads to altered proximity of the hOrai1 monomeres at the given cysteine positions. To prevent unwanted crosslinking with natural occurring cysteines in hOrai1, only cysteine-free hOrai1 constructs (Δ64, C126,143,195V, N223A) were used for this experiment. The mutation N223A leads to a non- glycosylated mutant.
The patch clamp data provides insights into alterations in the ion conductance of the channel such as information if the channel is still functional, ion influx is reduced or increased or if the channel is constitutively open.
Finally, the NFAT screen delivers information of a change in transcriptional activation due to altered Ca2+ influx downstream in the Ca2+ signal cascade that might be caused by muations. Figure 15 depicts the workflow of this thesis.
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Figure 16: Depiction of the experimental workflow. Molecular biological preparation steps are shown in blue
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1.5. Research techniques
1.5.1. SDS-Page
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are used to separate proteins according to their molecular mass. Due to the strength of the bands also a semi-quantification of proteins is possible through this technique. Proteins are denatured by SDS molecules which act as detergents. The SDS molecules are charged negatively and shield the natural charges of different amino acids, thereby allowing a constant mass-to-charge-ratio of the amino acids, which enables an electrophoretic separation according to size in an electrical field in a polyacrylamide gel. The polyacrylamide gel consists of acrylamide molecules which are polimerized via the addition of ammonium persulfate (APS) and N,N,N,N- tetramethylenediamine (TEMED) that act as oxidizing agents. Different acrylamide concentrations determine the strength of the polymer-network, thereby modulating the pore size and separation strength of the PAGE-gel. The gel typically consists of two gels known as stacking and separation gel. The stacking gel is constituted of a lower acrylamide concentration and a more acidic pH. When a current is applied Cl- migrates faster towards the stacking gel due to their small size and their negative charge, whereas the SDS- protein complexes migrate slower due to their size. Glycine ions, which are uncharged in the stacking gel due to the lower pH migrate slow. However, when they reach the stacking gel, which has a higher pH, glycine gets deprotonated. This leads to an acceleration of the ions inside the gel that leads to sharper protein bands (termed “collection effect”). [65]
Figure 12. Schematic representation of a SDS-PAGE setup. Stacking gel is shown in green, running gel is displayed in grey. The anode, which applies an attractive force to the negative charge SDS-protein complexes is placed at the bottom of the setup, the cathode is positioned on top. [66]
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1.5.2. Western blot and immunodetection
Western blotting is usually used after SDS-PAGE to transfer the separated proteins onto a membrane (nitrocellulose or PVDF membrane) for detection purposes. The PVDF membrane is placed between a SDS-gel and a cathode. The negatively charged proteins migrate from the SDS-gel to the membrane via electrophoretic transfer. For detection of the proteins of interest, specific antibodies are used after free binding sides on the membrane are blocked (usually with BSA or non-fat dried milk powder). Detection takes place through an enzymatic reaction by antibody-labelled enzymes, such as horseradish peroxidase, which produce a luminescence signal that can be made visible on an X-ray film. [67]
Figure 13. Schematic representation of a western blot setup. On the left side the assembly of the sandwich is shown, the right side displays the electrophoretic transfer. The membrane needs to be placed towards the anode and the gel towards the cathode for correct protein transport. [67]
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1.5.3. Patch-clamp electrophysiology
The patch clamp measurements enable identification and classification of ion channels and their corresponding currents. In patch clamp the ion flow through one (single channels) or multiple channels (whole cell mode) is recorded after applying voltages. For this, a glass pipette tip with a diameter of approximately 1-5µm is attached to the membrane by approaching the tip to the cell membrane with a micromanipulator and forming the seal via a suction device. Changes in conductance (ions flowing through the channel) and kinetic properties of the cannel (opening/closing speed) of ion channels have been identified with this method. To record the data, two electrodes (usually Ag/AgCl electrodes) are necessary. One inside the glass tip and the second electrode is placed into the cell bath. The setup configuration for patch clamp measurements typically consists of a Faraday cage (isolation of the equipment from electrical noises), a vibration isolation table, one or several microscopes for cell imaging, micromanipulators to move and position the electrodes, low voltage amplifiers, a perfusion system (distribution of buffers and solutions) as well as a computer for data acquisition and stimulus generation. [68]
Figure 14. Schematic representation of different patch clamp recording setups. a) The cell attached mode is realised by suction without breaking of the membrane, in a whole cell as configuration the membrane is broken to access the cytosol of the cell with the electrode inside the pipette. b) Measurement recordings of cell-attached and whole cell mode show. Outwardly rectifying single channel currents as well as currents resulting from two channels are shown at the cell attached mode of the left side. In the whole cell mode outwardly rectifying total currents are shown below the whole cell configuration. Refer to main text. [69]
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1.5.4. Confocal fluorescence microscopy
Fluorescence microscopy is a technique that combines fluorescence modifications of proteins with microscopically detection techniques to increase the contrast between a signal (cells, tissues) and the background of a sample. Proteins of interest are labelled with different fluorophores, which allows the imaging of specific locations or interactions between proteins. Fluorescence dyes have specific absorption and excitation wavelengths. Fluorescence molecules occur in their so-called ground state (S0), in which electrons are excited upon exposure of specific wavelengths. This leads to a shift (termed “Stoaks shift”) to a higher energy state of electrons consisting of vibrational and rotational states (excited states S1,S2). The energy needed to shift an electron form S0 to the excited state is inversely related to the photon’s wavelength (E=h*c/λ with h as Plank’s constant c speed of light and λ wavelength of light in vacuum). Depending on the excitation wavelength, electrons undergo shifts either in the S1 or higher orbital numbers. The shift back from the excited state to the ground state is achieved by vibrational relaxation and photons of specific wavelengths are emitted. Alexander Jablonski described this process in a diagram named termed “Jablonski diagram”. [70] Confocal fluorescence microscopy describes the combination of fluorescence microscopy with a confocal microscopy system. Via a confocal laser-scanning microscope (CLSM) the out-of-focus haze can be improved, leading to a higher contrast for thicker samples. A laser beam is focused to a small spot of sample, leading to excitation of fluorescent molecules. The resulting emission is further imaged through a pinhole that blocks light out of the focus. [71]
Figure 15. Schematic representation of a confocal and spinning disk microscope setup. a) The excitation land emission pathways are focused at a specific point at the sample. Out of focus light is blocked via a pinhole. [72] b) Representative configuration of a spinning disk microscope. The collector disk and a pinhole disk scan spots on the sample with a high framerate. Emitted light from the sample is guided via a pinhole disk towards the camera system. [71]
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2. Methods
2.1. General information
Two different plasmid vectors were used: peYFP-C1 containing the hOrai1 sequence for the fluorescence studies and pcDNA 3.1/V5-His-TOPO containing Δ64 Cys-free (C126,143,195V-no glycosylation site N223A) hOrai1 mutants for crosslinking-studies.
PCR primers were designed using QuikChange® Primer Design Program by Agilent Technologies and manufactured by Eurofins Genomics oligonucleotide synthesis.
The DNA constructs are verified by sequencing by Eurofins Genomics. Sequencing results were translated into amino-acid sequences via ExPASy Translate Tool (SIB Swiss Institute of Bioinformatics), followed by alignments with the hOrai1 wildtype sequence for the validation of the constructs utilizing Clustal Omega Multiple Sequence Alignment Program (EMBL-EBI). Finished constructs were stored at -20°C. Plasmid concentrations for lipofection (BioRad) were determined with a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific).
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2.2. Plasmid construction
2.2.1. PCR The following mutations were inserted into the vectors (Figure 17) via site directed mutagenesis by PCR in a FlexCycler (Analytik Jena AG):
Table 1: List of constructs
Figure 17: Illustration of the used vector systems. pcDNA3.1/V5-His-TOPO vector from Invitrogen (left) and pEYFP-C1 vector from BD Bioscience Clontech (right).
Temperature Time Cycles 95°C 2 min 1 95°C 40 sec 60°C 40 sec 18 68°C 8 min 68°C 10 min 1 4°C -- Table 2. PCR-program for site directed mutagenesis
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Substance Volume [µL]
Nuclease free H2O (Promega) 37.75 Pfu polymerase 10x buffer (Promega 10x) 5 Dimethyl sulfoxide 1.5 dNTP mix 10mM (Promega) 1.25 Forward primer 125ng (Eurofins Genomics) 1.25 Reverse primer 125ng (Eurofins Genomics) 1.25 10-100µg Template DNA 1 Pfu polymerase (Promega) 1 Total volume 50 Table 3. PCR-program components
Point mutation Primer sequence
Fw. 5'-caggcactgaaggtgatgagcagcccc-3´ A122T Rev. 5'-ggggctgctcatcaccttcagtgcctg-3'
Fw. 5'-caccgcctcgatgctgggcaggatgca-3' N147S Rev. 5'-tgcatcctgcccagcatcgaggcggtg-3'
Fw. 5'-ggtcaaggagtcccaccatgagcgcatgc-3' P164H Rev. 5'-gcatgcgctcatggtgggactccttgacc-3'
Table 4. Mutants and corresponding primers (fw = forward primer, rev = reverse primer)
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2.2.2. DPN I digestion & PCR validation
To validate a successful PCR-reaction 15µl of the PCR samples were mixed with 3µl of loading dye (Orange 6x Loading dye Promega) and subjected to 1% agarose gel containing Midori Green Advance DNA/RNA stain (NIPPON Genetics Europe) for 20 minutes at 110V. The rest of the sample (35µl) and 1µl of the enzyme DPN I were incubated to ensure the degradation of the template plasmids that were methylated by E. coli and lack the desired mutations. A Lambda HindIII Marker (Promega) was used for size comparison. Figure 18 shows a representative gel electrophoreses with peYFP-C1 vectors containing hOrai1 mutants.
Figure 18. Depiction of the Lambda Hind III marker (left) [73], and gel electrophoresis (right). The distance of the fragments corresponds to the 6557bp band of the marker. This indicates successful PCR. Δ64 Orai1 contains 237 amino acids, which corresponds to 711bp for the insert. The vector has 5,5kb, therefore vector + insert have 6211bp.
2.2.3. Transformation into competent E. coli
To amplify the PCR product, the plasmids were transferred into competent E. coli. The E. coli, which were stored at -80°C were thawn on ice for 15 minutes. Next, 3µl of the DNA samples were added to 50µl of the E.coli, followed by incubation for 20 minutes on ice, followed by a 2 minute heat shock at 42°C. After the heat shock 250µl of pre-warmed LB medium were added to the samples, followed by incubation at 37°C whilst shaking for 1 hour for regeneration. After incubation the samples were pipetted into petri dishes containing kanamycin-agar for the peYFP-C1 vectors and ampicillin-agar plates for the pcDNA3.1/V5-His-TOPO vectors and incubated for growth overnight at 37°C.
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2.2.4. Miniprep
After overnight incubation of the transformed E. coli single colonies were picked and added to eprouvettes containing 3mL of LB-Medium and the corresponding antibiotic (Kanamycin / Ampicillin). The eprouvettes were incubated whilst shaking at 37°c overnight.
To purify the plasmids the PureYieldTM Miniprep protocol [74] by Promega was with the following modifications:
Elution (Step 10) was carried out with 60µl instead of 30µl and nuclease free H2O instead of the Promega Elution Buffer was used.
2.2.5. Sequencing
The Miniprep-DNA was sent off to Eurofins Genomics for sequencing. After receiving the sequence, it was verified if the desired mutations were successfully integrated into the plasmids by comparing the results to the wildtype. The wildtype Orai sequence was searched on PubMed (Accession number NM_032790.3), translated to amino acid code with Expasy Translate [75] and aligned by the clustal omega multiple sequence alignment tool. [76]
Figure 19. Representative sequence alignment of Δ64 hOrai1 (the first 63 amino acids are deleted in the wildtype) with E106C (yellow), A122T (red), cysteine-free (green) and the non- glycosylation mutation N223A (purple)
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2.2.6. Midiprep
After validation of the mutated plasmids the PureYieldTM Midiprep protocol [77] was carried out to further amplify the plasmids for transfection. For this, sequence-validated E. coli form the eprouvettes were added into 50mL of LB-medium with the corresponding antibiotic in an 250mL Erlenmeyer flask and incubated overnight at 37°c whilst shaking.
The flasks containing the transformed E.coli were centrifuged on the next day for 15 minutes at 5000 rpm and the supernatant was discarded. Next, the PureYieldTM Midiprep Quick protocol was carried out with a modification at Step 6: centrifugation for 20 minutes at 8000 rpm.
A NanoDrop measurement was done to determine the plasmid-concentration of the samples and the appropriate volume for the transfection was calculated (10µg of DNA per petri dish of HEK293 cells).
2.2.7. Transfection
To transfer the plasmids that were obtained via Midiprep into HEK293 cells, lipofection with TransFectinTM (Biorad) was carried out. For this, 800µl of transfection-DMEM (without FCS & antibiotics), a volume containing 10µg of plasmid-DNA and 10µL of TransFectinTM were mixed and incubated for 20 minutes to form DNA-liposome-complexes. Next, the solution was carefully added to HEK293 cells in petri dishes without washing away the adherent HEK293 cells and incubated for 20-22 hours at 37°C. The same treatment was carried out for the mock-samples that were used as blank values for the hOrai1 expression of the HEK293 cells, however these samples were prepared without the addition of the plasmid-DNA.
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2.3. Western blot
2.3.1. Lysis and cysteine-crosslinking
To purify the proteins the following lysis protocol was carried out after cells were transfected and incubated overnight:
Protocol Protein Purification out of HEK293 cells:
The medium is discarded and the cells are rinsed off the bottom of the Petri dish with 1mL HBSS (Biowest ) +1mM EDTA buffer. The solution is centrifuged for 1min at 4600rpm. The supernatant is discarded and the pellet resuspended in 1mL HBSS+1mM EDTA buffer, followed by centrifugation for 1min at 4600rpm. This washing step is then repeated. After the centrifugation the pellet is resuspended in 500µL Lysis Buffer, mixed with 15µL Protease Inhibitor and incubated on ice for 15min. Then the solution is pulled up and down a syringe (diameter 0,4x20mm, Braun) for 5 times. Next the solution is centrifuged for 10min at 4600rpm. The supernatant is now stored at -20°C. An aliquot of 21µL is treated with 5µL CuP-solution for 5min, then the reaction is stopped by addition of 5µL Quenching solution. The sample was incubated with 10µL Laemmli buffer for 10min at 55°C before loading 21µL onto the SDS-gel. The SDS-page is run at 150V for 70 min in a 12% gel (see 3.3.2).
To separate proteins from cell fragments, another centrifugation step for 10 minutes at 4600 rpm is performed. The supernatant that contains the hOrai1 proteins is transferred into fresh Eppendorf tubes and stored at -20°C.
Lysis buffer 20mM Tris-HCL 0.788g 2mM EDTA 0.146g 100mM NaCl 1.461g Glycerol 10% 25mL Table 5. Lysis buffer components
Cu2+-phenanthroline solution Quenching solution (pH 7.4)
CuSO4 1mM TrisHCl 50mM 1,10-phenantroline 1.3mM N-Ethylmaleimide 20mM NaCl 150mL EDTA 20mL DTT 0.3mM Table 6. Cu2+-phenanthroline and quenching solution components
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2.3.2. SDS-Page After 10 minutes incubation on 55°c to break secondary protein structures, 20µL of the samples are loaded on a 12% SDS-polyacrylamide in a chamber filled with 1x SDS-running buffer. For size determination 7µL of Precision Plus ProteinTM Dual Colour Standards (BioRad) was used. The power supply was set to 150V for 70 minutes.
12% running gel Stacking gel
Acrylamide 9,6mL Acrylamide 1,32mL
Tris [1,5M pH 8.8] 6,0mL Tris [0,5M pH 6.8] 2,5mL
SDS (10%) 240µL SDS (10%) 100µL
APS [500mg/ml, 35%] 180µL APS [500mg/ml, 35%] 60µL
ddH2O 7,72mL ddH2O 5,98mL
TEMED 1,2µL TEMED 10µL
Table 7. SDS-PAGE gel components
10x SDS running buffer [1L] 1x SDS-running buffer [3L]
Tris 30g 10x SDS-running buffer 300mL
Glycine 154g ddH2O 2700mL
SDS 10g
Table 8. Running buffer components
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2.3.3. Western blot
After electrophoretic separation the proteins were transferred to an Immuno-Blot PVDF membrane (BioRad). The membrane was activation by immersion it in ethanol and a sandwich- Western blot assembly is constructed in the order (also see Figure 20):
Support-grid (towards cathode) Fiber-pad Filter paper PVDF membrane Gel Filter paper Fiber-pad Support-grid (towards anode)
The Western-Blot assembly was placed into a chamber, surrounded with ice and filled with 1x blotting buffer. The power supply was set to 90V for 80 minutes.
Figure 20. Western-blot set-up: sandwich assembly is immersed in the blotting-buffer filled chamber. [78]
10x Blotting buffer [1L; pH 8.3] 1x Blotting buffer [1L]
10x blotting Tris 30,3g 100mL buffer
Glycine 171g ddH2O 700mL
Isopropanol 200mL [98,9% p.a.]
Table 9. Blotting buffer components
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2.3.4. Immunodetection
To block the unspecific binding sides of the PVDF-membrane a 5% milk powder PBS-solution is used. A rabbit anti-Orai1-antibody (Sigma; 1,0mg/mL) diluted 1:2000 in 5% milk-PBS solution was used as primary antibody, as secondary antibody an anti-rabbit antibody bound to a horse radish peroxidase (Sigma; 1,0mg/mL) diluted 1:5000 in PBS-T was used. For this, the membrane was put on a shaker for 1 hour. After blocking, the following steps were carried out on a shaker:
1. Incubation with primary antibody (anti Orai1 produced in rabbit) for 1hour 2. Washing 3x 5 min with PBST-buffer 3. Incubation with secondary antibody (anti rabbit) for 1 hour. 4. Washing 3x 5 min with PBST-buffer
After washing, the blotting-membrane was incubated in 1ml of luminol (BioRad) and 1ml of peroxide (BioRad) for 2 minutes to catalyze the light reaction necessary for the development of the X-ray film (GE Healthcare). The X-ray film was fixated on the membrane, kept in a hypercassette and developed in the dark room, using a developer- (Ilford) and a fixating-solution (Ilford).
10x PBS [1L; pH 7.2] 1x PBS [1l] 1x PBS-T [1L]
NaCl 80g 10x PBS 100mL 10x PBS 100mL
KCl 2g ddH2O 900mL ddH2O 900mL
Na2HPO4 20,1g Tween-20 1mL
NaH2PO4 1,7g
KH2PO4 2g
Table 10. PBS buffer components
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2.4. Patch clamp measurements
Patch clamp electrophysiology experiments were carried out 24 hours after transfection of the HEK293 cells. Experiments were carried out with the support of Herwig Grabmayr.
Whole cell currents were measured with an Axiovert S100 TV inverted microscope (Carl Zeiss), an Axopatch 200B microelectrode amplifier (Axon Instruments), a WR-89 three dimensional aqua purificate micromanipulator (Narishige International), and a pE Fluorescence LED illumination system (CoolLED Limited).
To evaluate the data and to control of the microelectrode amplifier, WinWCP software package (v4.2.2, University of Strathclyde) was used.
The cells located on glass dishes were washed with extracellular solution without Ca2+ to clean the cells from any remaining DMEM and a 10mM Ca2+ solution was used to fill the cell bath chamber. Triethylene glycol diamine tetra acetic acid (EGTA) triggers passive Ca2+ store depletion during the measurement. Two Ag/AgCl electrodes serving as recording and reference electrode and a voltage ramp in the range of -90mV to 90mV for one second was applied every five seconds with a holding potential set to 0mV. The current amplitudes were recorded at - 74mV, which is specific for Orai1 channels. For the correction of “normal” store-dependent currents, the current amplitude recorded shortly after the formation of whole-cell configuration has to be subtracted from all subsequent amplitudes. For constitutive currents, which show permanent Ca2+ influx, 10mM Ca2+ solution with additional 10µM Lanthanum (La3+) were added at the end of the experiment to inhibit the current.
For comparability of the experiments individual measurements where normalised by dividing all current amplitudes by the whole-cell capacitance.
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2.5. Confocal fluorescence microscopy
Confocal fluorescence microscopy experiments were carried out after 24-30 hours of HEK293 transfection at room temperature. All microscopy experiments were carried out with the support of Matthias Sallinger.
The cells were washed with 2mM Ca2+ extracellular solution to remove excessive DMEM.
The chamber was filled with extracellular solution. Depending on the type of experiment, various extracellular solutions containing 0mM Ca2+ or 1µM Thapsigargin (TG), to induce store-depletion were used.
All buffers used for patch clamp as well as for confocal fluorescence microscopy experiments are listed in Table 11. The experiments were performed using two different setups: For localisation studies a CSU-X1 Real-Time Confocal System (Yokogawa Electric Corporation) fitted with two CoolSNAP HQ2 CCD cameras (Photometrics), a dual port adapter (dichroic: 505lp, cyan emission filter: 470/24, yellow emission filter: 535/30, Chroma Technology Corporation). An Axio Observer.Z1 inverted microscope (Carl Zeiss) with two diode lasers (445 and 515 nm, Visitron Systems) which was placed on a Vision IsoStation anti-vibration table (Newport Corporation). The VisiView software package (v2.1.4, Visitron Systems) was used for image generation. Due to cross-excitation and crosstalk during the measurements appropriate cross-talk calibration factors were determined for each construct. Threshold determination and background subtraction for correct imaging was done. Images and the corresponding data were calculated on a pixel to pixel basis with a custom-made software integrated into MATLAB (v7.11.0, The MathWorks, Inc.) which implemented the method published by Zal and Gascoigne with a microscope-specific constant G parameter of 2.75.
For NFAT studies (described in the Results part) a QLC100 Real-Time Confocal System (VisiTech Int.) was connected to two Photometrics CoolSNAPHQ monochrome cameras (Roper Scientific) and a dual-port adapter (dichroic, 505lp; cyan emission filter, 485/30; yellow emission filter, 535/50; Chroma Technology Corp.) was used to record fluorescence images. The system was connected to an Axiovert 200 M microscope (ZEISS, Germany) in conjunction with two diode lasers (445 nm, 515 nm) (Visitron Systems).75 For controlling and image acquisition a VisiView 2.1.1 software (Visitron Systems) was used. Cross-excitation and cross-talk events were corrected as described in the paragraph above for the localisation studies.
The images and corresponding data values were too calculated on a pixel to pixel basis with a custom-made software integrated into MATLAB (v7.11.0, The MathWorks, Inc.) which implementing the method published by Zal and Gascoigne utilising a different microscope- specific constant G parameter of 2.0 compared to the localisation data evaluation.
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Extracellular Extracellular Extracellular Pipette solution 0mM Ca2+ (ph 2mM Ca2+ (ph 10mM Ca2+ (pH 7.2) 7.4) 7.4) (ph 7.4)
NaCl 8mM 140mM 140mM 145mM
KCl - 5mM 5mM -
CsCL - - - 5mM
CsMeSulf. 145mM - - -
MgCl2 5mM 1mM 1mM 1mM
HEPES 10mM 10mM 10mM 10mM
EGTA 20mM - - -
Glucose - 10mM 10mM 10mM
CaCl2 - - 2mM 10mM
Table 11. Components of the used solutions. Different experiments require various extracellular solutions. For store-depletion experiments 1µM TG can be added to the 0mM Ca2+ solution
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3. Results
Three cancer-related Orai1 mutations (A122T, N147S and P164H) from the cBioPortal database were structurally examined with western blotting (via a cysteine crosslinking assay), electrophysiological properties were measured with whole-cell patch clamp and effects on the transcription factor NFAT were observed with fluorescence microscopy studies. After verification of the desired point-mutations, followed by transfection of the plasmids into HEK239 cells, the influence of the given mutations on the hOrai1 pore architecture as well as functional features were analysed.
Results from cysteine crosslinking experiments, patch-clamp electrophysiology and different confocal fluorescence microscopy measurements are shown in that order in the following chapters.
3.1. Cysteine-crosslinking results
Figure 21. Depiction of used marker (75 and 25 kDa in red) (left) and example western blot, showing re-drawn marker (for visibility on the X-ray film. The Δ64 hOrai1 monomers have a mass of 23,7kDa and appear right below the 25kDa Marker
The blots were analysed with the software ImageJ (National Institute of Health). Rate of dimerization was calculated according to:
Dimers RateDimers = ∗100% Dimers + Monomers
Equation 1: Calculation of dimerization-rate
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n=3
Figure 22. Results of crosslinking experiments. The data was collected from three independent experiments. Degree of dimerization was calculated using Equation 1. Significant differences between cancer related mutants and their corresponding single point mutants are indicated by an asterisk.
Statistical evaluation was carried out using OriginLab Data Analysis Student’s t-test with a p- value of p< 0.05. Significant deviations could be observed for R91C + A122T, R91C + N147S, E106 + A122T and E106E + P164H. No significant deviations were found for all F99C mutants. All experiments were performed with HEK293 cells as model organism.
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3.2. Patch clamp results
To investigate the effects of the mutants related to functional electrophysiological characteristics on Orai1, whole cell patch clamp measurements were conducted. N-terminally labelled CFP- STIM1 proteins were coexpressed with N-terminally labelled YFP-Orai1 constructs. Current activation was measured over time with a gradual Ca2+ store depletion via EDTA. Inward currents were normalized to cell capacitance (pA/pF) for comparability between different cells.
CFP-STIM1 + YFP-Orai1 mutants wt A122T N147S P164H n = 10 n = 7 n = 6 n = 6 0 *
I (pA/pF) -5
0 50 100 150 200
Time (s)
Figure 23. Patch-clamp data of A122T (red), N147S (green) and P164H (blue) mutants as well as wild type (black) data. The data was normalized to cell capacitance. Error-bars signify standard error of the mean (SEM), significant difference in the activation of the channel is indicated by grey circles, regarding N147S and P164H.
No constitutive activation of the channels was identified with whole cell patch clamp. However, significant differences in the activation could be observed between the wildtype and N147S as well as wild type and P164H at 160s. The activation of the channel was slower, leading to decreased currents through the channel.
Statistical evaluation was carried out using OriginLab Data Analysis Student’s t-test with a p- value of p< 0.05. All experiments were performed with HEK293 cells as model organism.
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3.3. Confocal fluorescence microscopy
3.3.1. Localisation Studies HEK293 cells were transfected with CFP-STIM1-OASF fragment and the YFP-tagged hOrai1 mutations of interest to identify correct localisation of the proteins. The CFP-excitation was measured to detect membrane localisation as well as correct STIM1-Orai1 interaction. When OASF-Orai1 binding is unaffected, OASF translocates to the plasma membrane, indicating interaction with Orai1. Images were created using MATLAB (v7.11.0, The MathWorks, Inc.). Proper localisation for all Orai1 constructs could be observed.
Figure 24. Plasma membrane localization diagrams (left) and confocal fluorescence microscopy images (right) for WT-Orai1 and the corresponding mutations of interest recorded in the cellular centres (nuclei visible) of depicted cells.
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3.3.2. Nuclear Factor of Activated T-Cells (NFAT) screen Spinning disk confocal fluorescence microscopy was used to identify the nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). CFP-tagged NFAT constructs were cotransfected with Orai1 WT as well as the corresponding mutant constructs to identify the translocation of NFAT. The translocation was measured 24 hours after transfection. This approach identifies constitutively active Orai1 channels and constitutive Ca2+ entry. The NFAT- signalling cascade is activated via STIM1-Orai1 mediated Ca2+ entry. Increased intracellular Ca2+ concentration activates the Calmodulin/Calcineurin signalling cascade that dephosphorylates NFAT and leads to translocation of NFAT into the nucleus. This induces gene transcription. The percentage of cells with nuclear NFAT localization was determined using MATLAB (v7.11.0, The MathWorks, Inc.). [79]
30
25
20
15
10
5 cells with nuclear NFAT localization [%] localization NFAT nuclear cells with 0 WT A122T N147S P164H N=300 N=201 N=211 N=196
Figure 25. Percentage of cells with nuclear NFAT localization. Measurements were conducted 24 hours after co-transfection with YFP-Orai1 constructs in 2mM Ca2+ solution. No significant deviations were identified (t-test, p< 0.05).
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Figure 26. Representative NFAT fluorescence images. Images of cells coexpressing Orai1 mutant constructs (YFP labelled; left), CFP-NFAT (central) and overlay of the images (right). Nuclear NFAT localization is shown on the CFP pictures in green and Orai1 wildtype as well as mutated constructs are displayed in red (YFP pictures). Image colours are artificial.
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4. Discussion
Ca2+ release activated Ca2+ (CRAC) currents are induced via two proteins, a sensor molecule STIM1, which is located in the ER membrane and a channel protein Orai1, which is located in the plasma membrane. Gain- and loss of function mutations of these proteins are associated with several diseases.
The aim of this thesis was to identify if cancer related mutants A122T, N147S and P164H taken from the cBioPortal database show structural as well as functional anomalies compared to a wildtype hOrai1 protein.
4.1. General discussion
No significant deviations could be observed in the crosslinking studies for all of the F99C mutants, leading to the conclusion, that neither of the cancer related Orai1 mutants A122T, N147S and P164H are causing a structural change at position F99.
No significant deviations could be observed between wild type and A122T, N147S as well as P164H in the NFAT screens. This is further discussed for each mutant in the following paragraphs.
4.2. A122T discussion
Both positions, R91C as well as E106C, showed significant difference in the rate of dimerization for the A122T mutants. The rate of dimerization is lower in R91C + A122T compared to R91C, indicating a widening of the pore at position R91C. In contrast, E106C + A122T showed the opposite when compared to E106C: The mutation A122T seems to narrow the selectivity filter, based on the assumption of the underlying crosslinking-approach. These findings indicate a structural change, which affects the pore at its entrance that could lead to functional differences in the mutant. Position A122 is located in TM2, which is forming the second layer of the channel after the pore-forming TM1. Threonine, being a polar amino acid, whereas alanine, being unpolar due to its methyl-rest, have different electrochemical properties. A change from alanine to threonine incorporates an additional hydroxyl group into the TM2 structure. This hydroxyl group could form a hydrogen bond with surrounding binding partners, such as carboxyl groups, leading to structural differences of the channel. Also, due to the additional atoms in threonine, the steric character at position A122 changes, which, in turn, could also change the three- dimensional positioning of surrounding amino acids.
In patch clamp measurements no significant difference could be observed for A122T. This finding goes in line with the NFAT measurements, which also show no significant difference. Even if structural changes are indicated at certain positions by the crosslinking studies, the results of the electrophysiological studies as well as the NFAT screen indicate a similar functional behaviour of A122T mutants compared to wildtype channels.
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4.3. N147S discussion
Significant difference in dimerization rates was observed for the R91C mutants, which were lower compared to the wild type. This indicates a widening of the pore at position R91C. A change form asparagine to serine results in an exchange of a carboxyl-group to a hydroxyl- group in the amino acid rest. A carboxyl-group is able to form two hydrogen bonds with suitable neighbouring functional groups, such as amino groups (NH2), whereas a hydroxyl-group is only able to form a single hydrogen bond, leading to the conclusion, that the N147S mutant could form one hydrogen bond less than the naturally occurring asparagine. Asparagine is negatively charged and, due to two additional atoms, namely an additional carbon as well as a second oxygen atom, has different steric properties. The position N147 is located at the beginning of loop2, shortly after TM2. The detailed structural effect of an N147S on position R91C, which is located in the ETON region of the protein, that is located shortly before TM1 and critical to STIM coupling, is unclear, however it seems like a mutation in loop2 is able to modulate the positioning of R91C in the hexameric channel structure according to the crosslinking-assay.
In patch clamp studies N147S shows a significant deviation compared to the wild type. The activation seems to be slower in N147S mutants. As indicated in the crosslinking assay, the N147S mutation seems to widen the pore at position R91, which is next to the ETON region that spans from position A73-S90, a region that is essential for STIM-Orai interaction [40]. If position R91 is structurally modulated due to N147S, the mutation could also affect the neighbouring ETON region. A structural change in the ETON region could lead to a decreased coupling efficiency between STIM and Orai, leading to the observed slower activation in the whole cell patch clamp measurements. This reasoning is in conflict with the results of the localisation studies, which show a proper binding of OASF to the channel. However, it should be stated that OASF is a rather compact fragment of STIM, which could differ in the interaction with Orai1 compared to a full wild type STIM. [27] [80]
4.4. P164H discussion
E106C + P164H was the only mutant that showed significant deviations in the crosslinking studies. The rate of dimerization was almost doubled compared to E106C, leading to the conclusion, that a change from proline to histidine changes the pore structure and narrows it at the selectivity filter. Proline, a non-polar amino acid, known as “helix breaker” has a unique ring- forming rest group and zero possibilities for hydrogen bonds. Serine is a polar, positively charged amino acid with the ability to form three hydrogen bonds with suitable surrounding binding partners, such as hydroxyl groups (one hydrogen bond possible) and amino groups (two hydrogen bonds possible). P164 is located in loop2, close to TM3. [38] It has still to be examined, how exactly a mutation close to TM3 could lead to a narrowing of the selectivity filter, located in TM1. In patch clamp studies P164H shows a significant reduction of channel activation compared to the wild type. It is unclear, how the functional properties of the channel are changed by a mutation close to TM3, however loop2 seems to be responsible for the different, isoform-specific behaviour of Orai1 and Orai3 proteins in N-truncation mutants. The patch clamp results are in line with the findings of the crosslinking assay, a narrowing of the pore could lead to less influx of ions and therefore a decreased current.
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5. References
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28. Derler, I., I. Jardin, and C. Romanin, Molecular mechanisms of STIM/Orai communication. Am J Physiol Cell Physiol, 2016. 310(8): p. C643-62. 29. Yeromin, A.V., et al., Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature, 2006. 443(7108): p. 226-9. 30. Vig, M., et al., CRACM1 multimers form the ion-selective pore of the CRAC channel. Curr Biol, 2006. 16(20): p. 2073-9. 31. Derler, I., et al., A Ca2(+ )release-activated Ca2(+) (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2(+)-dependent inactivation of ORAI1 channels. J Biol Chem, 2009. 284(37): p. 24933-8. 32. Prakriya, M., Store-operated Orai channels: structure and function. Curr Top Membr, 2013. 71: p. 1-32. 33. Lis, A., et al., CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties. Curr Biol, 2007. 17(9): p. 794-800. 34. Muik, M., et al., Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem, 2008. 283(12): p. 8014-22. 35. Niu, L., et al., STIM1 interacts with termini of Orai channels in a sequential manner. J Cell Sci, 2020. 36. Li, Z., et al., Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release- activated Ca2+ channel activation. J Biol Chem, 2007. 282(40): p. 29448-56. 37. Cai, X., et al., The Orai1 Store-operated Calcium Channel Functions as a Hexamer. J Biol Chem, 2016. 291(50): p. 25764-25775. 38. Fahrner, M., et al., Communication between N terminus and loop2 tunes Orai activation. J Biol Chem, 2018. 293(4): p. 1271-1285. 39. Hoth, M. and B.A. Niemeyer, The neglected CRAC proteins: Orai2, Orai3, and STIM2. Curr Top Membr, 2013. 71: p. 237-71. 40. Derler, I., et al., The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1. J Biol Chem, 2013. 288(40): p. 29025-34. 41. Wu, M.M., E.D. Covington, and R.S. Lewis, Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions. Mol Biol Cell, 2014. 25(22): p. 3672-85. 42. Lab, Z., Stim & Orai Interaction Figure. 43. Yamashita, M., et al., STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate. Nat Commun, 2017. 8: p. 14512. 44. Yeung, P.S., M. Yamashita, and M. Prakriya, Pore opening mechanism of CRAC channels. Cell Calcium, 2017. 63: p. 14-19. 45. Dong, H., et al., Toward a Model for Activation of Orai Channel. iScience, 2019. 16: p. 356-367. 46. Frischauf, I., et al., Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription. Sci Signal, 2017. 10(507). 47. Hou, X., S.R. Burstein, and S.B. Long, Structures reveal opening of the store-operated calcium channel Orai. Elife, 2018. 7. 48. Chalmers, S.B. and G.R. Monteith, ORAI channels and cancer. Cell Calcium, 2018. 74: p. 160-167. 49. Faouzi, M., et al., Down-regulation of Orai3 arrests cell-cycle progression and induces apoptosis in breast cancer cells but not in normal breast epithelial cells. J Cell Physiol, 2011. 226(2): p. 542-51. 50. Xie, J., et al., SOCE and cancer: Recent progress and new perspectives. Int J Cancer, 2016. 138(9): p. 2067-77. 51. Abdullaev, I.F., et al., Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res, 2008. 103(11): p. 1289-99. 52. El-Nashar, S.A. and A. Mariani, Uterine carcinosarcoma. Clin Obstet Gynecol, 2011. 54(2): p. 292-304. 53. McCluggage, W.G., Malignant biphasic uterine tumours: carcinosarcomas or metaplastic carcinomas? J Clin Pathol, 2002. 55(5): p. 321-5. 54. Grayson, W., L.F. Taylor, and K. Cooper, Carcinosarcoma of the uterine cervix: a report of eight cases with immunohistochemical analysis and evaluation of human papillomavirus status. Am J Surg Pathol, 2001. 25(3): p. 338-47.
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55. Abell, M.R. and J.A. Ramirez, Sarcomas and carcinosarcomas of the uterine cervix. Cancer, 1973. 31(5): p. 1176-92. 56. Rajshekar, S.K., et al., Malignant mixed Mullerian tumour of the uterus. Ecancermedicalscience, 2013. 7: p. 302. 57. Chhieng, D.C. and G.P. Siegal, Updates in diagnostic pathology. Advances in experimental medicine and biology,. 2005, New York: Springer. xv, 197 p. 58. Aggarwal, M., Robbins & Cotran Pathologic Basis of Disease, 8th Ed. Anil Aggrawal's Internet Journal of Book Reviews, 2014. 13(1): p. 1-1. 59. Natkunam, Y., The biology of the germinal center. Hematology Am Soc Hematol Educ Program, 2007: p. 210-5. 60. Lenz, G., et al., Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad Sci U S A, 2008. 105(36): p. 13520-5. 61. Mlynarczyk, C., L. Fontan, and A. Melnick, Germinal center-derived lymphomas: The darkest side of humoral immunity. Immunol Rev, 2019. 288(1): p. 214-239. 62. Li, C. and H. Lu, Adenosquamous carcinoma of the lung. Onco Targets Ther, 2018. 11: p. 4829-4835. 63. Silvestri, N.J.P.N.T.T.G.A., Fishman's Pulmonary Diseases and Disorders, 5e. 2008: p. Chapter 118. 64. Benzerdjeb, N., et al., Orai3 is a predictive marker of metastasis and survival in resectable lung adenocarcinoma. Oncotarget, 2016. 7(49): p. 81588-81597. 65. Al-Tubuly, A.A., SDS-PAGE and Western Blotting. Methods Mol Med, 2000. 40: p. 391- 405. 66. Williams, L., SDS-Page Illustration. 67. Liu, Z.Q., T. Mahmood, and P.C. Yang, Western blot: technique, theory and trouble shooting. N Am J Med Sci, 2014. 6(3): p. 160. 68. Zhang, Y., et al., Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells. Methods Mol Biol, 2018. 1843: p. 1-16. 69. L., C., Patch-Clamp Techniques. Cell Physiology Source Book, (2012): p. 369- 381. 70. Lichtman, J.W. and J.A. Conchello, Fluorescence microscopy. Nat Methods, 2005. 2(12): p. 910-9. 71. Jonkman, J. and C.M. Brown, Any Way You Slice It-A Comparison of Confocal Microscopy Techniques. J Biomol Tech, 2015. 26(2): p. 54-65. 72. Andor, Confocal Dual Spinning Disk Illustration. 73. λ DNA-HindIII Digest. 74. PureYieldTM Miniprep Quick protocol. 75. Portal, S.B.R., ExPASy. 76. Clustal Omega Multiple Sequence Alignment Tool. 77. PureYieldTM Midiprep Quick protocol. 78. Technologies, L., General Western Blot Protocol. 79. Schober, R., et al., Sequential activation of STIM1 links Ca(2+) with luminal domain unfolding. Sci Signal, 2019. 12(608). 80. Muik, M., et al., STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation. EMBO J, 2011. 30(9): p. 1678-89.
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6. Addendum: Orai channels : key players in Ca2+ homeostasis
During my master thesis I had the opportunity to work as a co-author of a review about Orai proteins that was submitted to the jounal Current Opinion in Physiology.
The submitted version of the review is added as an addendum to this master thesis.
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COPHYS : Mechanisms of store-operated calcium entry – Orai channels Orai channels : key players in Ca2+ homeostasis
Matthias Sallinger, Sascha Berlansky and Irene Frischauf Life Science Center, Institute of Biophysics, Johannes Kepler University Linz, Austria correspondence to: [email protected] This work was supported by the Austrian Science Fund FWF P32075-B
Abstract: Maintaining a precise calcium (Ca2+) balance is vital for cellular survival. The most prominent pathway to shuttle Ca2+ into cells is the Ca2+ release activated Ca2+ (CRAC) channel. Orai proteins are indispensable players in this central mechanism of Ca2+ 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.
History| In 2006, RNA interference (RNAi) approaches together with human genetic linkage analysis identified a 33kDa (301 amino acids) cell surface protein genetically encoded on chromosome 121-3. First described as CRACM1 and later named after the goddesses of the seasons in Greek mythology – the Horae - this protein was then referred to as Orai1 and proven as the pore forming unit of one of the most prominent Ca2+ entry pathway – the Ca2+ release activated Ca2+ (CRAC) channel. The activation process of CRAC channels is highly unusual as it involves proteins residing within different cellular compartments: STIM1 acting as the Ca2+ sensor in the ER (for a detailed review on STIM proteins see Fahrner et al within this issue) and Orai1 as the pore-forming subunit in the plasma membrane.
Importance| Ca2+ signalling through CRAC channels is a complex network of intertwining pathways and tight regulation of Ca2+ is vital for cellular survival. Ca2+-dysregulation has been shown to be involved in several pathophysiological cellular malfunctions. Serious clinical human phenotypes result from loss- or gain-of-function (LOF and GOF, respectively) mutations4. Initially discovered in patients with severe combined immunodeficiency (SCID; R91W mutation leading to lack of CRAC channel function in T-cells1), Orai proteins have now been associated with various diseases5. Several mutations within transmembrane (TM) domains of Orai1 are the cause for tubular aggregate myopathy (TAM) (TM1: S97C, G98S, V107M TM2: L138F TM3: T184M TM4: P245L)6-9. A single nucleotide polymorphism in Orai1 (S218G10) is associated with atopic dermatitis and alterations in SOCE are related to end- stage human failing myocardium11. It has also been shown that Orai1 – 3 are upregulated in pregnancy and type 1 diabetes12. Accumulating evidence suggests that altered Ca2+ influx due to oncogenic remodelling of Orai proteins plays a critical role in cancer hallmarks like unrestricted proliferation, resistance to cell death, metastasis as well as tumour vascularization and antitumor immunity (reviewed in13,14). Orai1 and Orai3 proteins have been related to tumorigenesis of breast cancer as well as lung adenocarcinoma and play a role in cell migration and metastatic invasiveness14-19. In the context of breast cancer as well as prostate cancer cells, altered expression levels of Orai channels remodels the Ca2+ signalling pathways to avoid cell death14. Overexpression of Orai channels in prostate cancer has been linked with a decreased risk of recurrence after prostatectomy20. Angiogenesis, crucial for tumour development, has been shown to be dependent on calcium signalling21. SOCE is also responsible for the secretion of vascular endothelial growth factor (VEGF)15,22. Pharmacological inhibition of Orai can diminish the growth of colorectal, breast, liver, melanoma and clear cell renal cancer cells15. Understanding the molecular architecture and choreography of CRAC channels, therefore, has significant potential for therapeutic applications.
Orai proteins| There are three highly conserved homologous proteins of Orai1 known (Orai 1-3). They are located and distributed uniformly in the plasma membrane and consist of four, highly conserved transmembrane domains (Orai1: TM1-4; aa92-106, aa118-140, aa174-197 and aa236-258) sharing ~81-87% sequence similarity within TM2-4 and complete identity in the functionally critical TM1 helix. Both N- and C-terminal strands reside in the cytoplasm (Figure 1). The cytoplasmic strands exhibit considerable sequence homology within the segments concerned with direct STIM-Orai interaction. TM-domains are connected via two extra- (loop1, loop3) as well as one intracellular loop (loop 2), showing isoform specific characteristics and sequence deviations especially within the extracellular loop 3. Much less is known on the function of Orai2 and Orai3 compared to Orai1, therefore this review mainly focuses on the most prominent isoform, Orai1 (for a detailed review on Orai2 and Orai3 see23). In humans the Orai1 protein is expressed in a long (Orai1α) and a short (Orai1β) form 24,25 originating from alternative translation initiation at Met64 (for detailed reviews see ).
Orai1 structure| In 2012, against all expectations and countering the prevailing evidence for a tetramer, the crystal structure of the Drosophila melanogaster Orai (dOrai) showed that the channel is hexameric26. The closed dOrai channel structure has revealed that six monomers are arranged around a central axis, in which TM1 of each subunit forms the inner surface of the pore, thereby confirming earlier studies to identify pore-lining residues27. TM2-4 are positioned in concentric rings around this ion conducting pore26. dOrai proteins exhibit close similarity (~73% in TM regions) to human Orai1. This suggests and electrophysiological data strongly implies that also human Orai1 proteins assemble as hexamers28,29. Nonetheless, one should keep in mind that the dOrai crystal has been achieved by using various deletions and mutations, facilitating protein purification (reviewed in24). Store-dependent activation of CRAC channels involves direct binding of STIM1 to Orai1 as has been demonstrated by several groups24,27. Although a binding of STIM1 to the Orai1 N-terminus is still under debate, it has been shown via progressive N-terminal deletions that this region is essential for STIM1 coupling and that isolated fragments are able to bind to the STIM1 CAD domain27,30,31. Pre- binding of STIM1 to the Orai1 C-terminus followed by coupling to the Orai1 N-terminus in a stepwise manner has been suggested by Niu et al32. A 17 residues long conserved sequence, termed “extended transmembrane Orai1 N-terminal” (ETON, aa73-90) region is suggested to be involved during STIM1/Orai1 activation31. Amino acid residues located within the ETON region and at the beginning of TM1 (K85, S89, S90, R91) were suggested to play important roles during STIM-Orai coupling1,30,31. Yet, a full picture of the binding/activation mechanism between Orai1 N-terminus and STIM1 is still missing. The about 6Å narrow, ion-conducting pore of the Orai channel is formed by TM1 helices according to the dOrai crystal structure26 (Figure 1B). The channels entrance is surrounded by 6 highly conserved glutamate residues (E106 - selectivity filter) resulting in a hydrophobic surrounding and concentrated negative charges attracting Ca2+ ions25. Residues within TM1 that are discussed to have specific functions in channel gating are V102 (hydrophobic gate), G98 (gating hinge), L95 as well as F99 (hydrophobic gate)24,25. Additionally, R91, together with the formation of a water layer, may act as gate of the channel33. V102 and F99 have been proposed to work in concert forming a hydrophobic gate, as mutation to more polar amino acids leads to leaky gates34. Within the extracellular loop1 Frischauf et al. described a Ca2+ accumulating region (CAR) that enhances permeation at physiologically low Ca2+ levels via three negatively charged aspartates (D110, D112, D114)27,35. TM2 - 4 seem to stabilize the channel by forming a second layer around the central pore-forming TM1 helix26. Frischauf et al.33 identified several mutations (e.g. H134A, A137V) in TM2 affecting Orai channel gating. Especially the powerful H134A gain-of-function mutation developed constitutively active currents, completely independent of STIM1 activation. Using combined approaches including molecular dynamics simulations (MD-simulations), electrophysiology and cysteine-crosslinking, H134A proteins were shown to create hydrogen bonds between amino acid side chains facing the channels pore (S90 and R91). Additionally, mutating H134 to alanine decreases hydrophobic gating barriers by creating a chain of water molecules through the channels pore.
The role of the intracellular loop2 in humans is not fully clarified yet. It has been shown that STIM1 binds to loop2 in Caenorhabditis elegans, pointing to a different activation mechanism36. In addition, loop2 seems to be responsible for the different, isoform-specific behaviour of human Orai1 and Orai3 proteins shown with various N-truncated mutants that interact between residues Y80 in the N-terminus and N156 in loop237. Mutation of W176 to cysteine (W176C) and G183 to alanine (G183A) within TM3 has dramatic effects on gating and selectivity of the channel31. MD-simulations have predicted that also residue E190 contributes to selectivity and gating by reducing the number of water molecules in this region when mutated to glutamine38. The E190 residue was also shown to be responsible for the external pH sensitivity if Ca2+ is not present39. Niemeyer and colleagues discovered redox- dependent regulation of Orai1 proteins which they attributed to the cysteine 195 residue (C195) close to the extracellular loop3. There is no corresponding C195 residue within Orai3, 40 which makes Orai3 isoforms insensitive to H2O2-induced inhibition . The extracellular loop3 exhibits the lowest sequence-conservation among Orai isoforms. Molecular modelling together with MD-simulations identified loop3 as highly flexible region, electrostatically interacting with amino acids in the loop1 CAR, thereby modulating Ca2+ permeation10,35. In loop3, only Orai1 proteins contain an N-glycosylation site at position 223 (N223). Different glycosylation states of Orai1 might manipulate SOCE-mediated Ca2+ signalling, thereby playing a crucial role in pathophysiological processes involved in diseases and cancer related aberrations41. The outer shell of a hexameric Orai1 protein is formed by TM426. Quite recently the Long lab crystallized the open dOrai channel by taking advantage of the H134A Orai1 gain-of-function mutation33 (H206A in dOrai)42. The open crystal suggests conformational changes and straightening of the TM4 and the extended TM4 region (M4ext) upon channel opening. Additionally, it has been shown that the open pore is dramatically dilated, being ~10Å apart on the cytosolic end. A release of cytosolic latches between P245 (P288 in dOrai) and the SHK motif (aa263,264 and 265 in hOrai1) leading to a straightened TM4 helix, seems to expose cytosolic docking sites for STIM1. The TM4 helix bends at position P245 which enables cytosolic M4ext to point in opposite directions and interact through coiled-coils. The M4ext segment is widely accepted to be the major binding site for STIM1 and seems to be crucial in stabilizing the closed state of the channel30. The C-terminus is attached to TM4 via a flexible linker region that is required for coupling between Orai1 C-terminus and STIM143. Within this linker region one can find a five amino acid long sequence (aa 261–265, LVSHK) termed “nexus”. Mutation of these amino acids from LVSHK to ANSGA led to a constitutively active channel. Hydrophobic attachment of TM4 residues (L261, V262) to TM3 residue L174 is proposed by Zhou et al44. The cytosolic C-terminal strand of Orai is predicted to arrange in paired structures by forming coiled-coil interactions with other Orai1 subunits bending in opposite directions. Two hydrophobic residues (L273, L276) were shown to play significant roles during coiled-coil formation, STIM-Orai binding as well as channel activation24,45.
STIM1/Orai1 binding| Two models have been proposed to describe STIM1 binding to the cytosolic Orai1 C-terminus. The dimeric model postulates binding of a STIM1 dimer to a pair of M4ext, based on an NMR solution structure where the CC2 domains of CAD/SOAR fold as a binding pocket46. This binding model is not easily reconciled with the open dOrai structure. In the monomeric binding model, a STIM1 dimer engages only one C-terminus of Orai1, based on findings were a binding-deficient F394H STIM1 mutant within a STIM1 dimer is still able to activate Orai1 to its full extent47. With this model, one could possibly explain how the free CAD/SOAR of the dimer can crosslink Orai1 channels into clusters thereby slowing their diffusion48. Still, both models can represent different stages in the CRAC activation process. In earlier studies it has been hypothesized that the Orai1 C-termini straighten, which breaks their coiled-coil interaction allowing for STIM1 binding26. Which conformational rearrangements take place within Orai upon STIM binding, are only partially resolved. It is highly likely that STIM1 binding to one or both Orai1 termini induces signal propagation by exerting a force on the four TM regions. By now, there are several mutations identified that constitutively activate Orai1 – most of them located within TM2 to TM4 (H134A, P245L, W176C, G183A,T184M, ANSGA; Figure 1 A,B)7,33,44. It is the prevailing view that the closed channel is stabilized by multiple TM-interactions which are released upon the signal from STIM1 to open the gate allowing for Ca2+ influx.
Mechanisms of Ca2+ permeation| One essential characteristic of the CRAC channel is its very high selectivity for Ca2+ over Na+ and its small unitary current : features that point to an energetic barrier for ion passage through the pore – either by a purely physical constraint or the narrow pore diameter (~0,39nm)25,27,49. Therefore, conformational changes need to take place within the pore to allow for Ca2+ influx. The group of Prakriya has proposed a pore rotation model where torsion of the TM1 helix is linked with conformational changes in the Orai1 C-terminus upon STIM1 binding and channel opening50. Hydrophobic side chains of pore-lining residues V102 and F99 create a barrier for ion permeation in the closed state, which are rotated outwards (~20˚) upon STIM1 binding34. A recent study by Dong et al51 supports this ‚twist-to-open‘ gating mechanism. By the use of MD-simulations based on the dOrai structure they show that coupling of TM1 to TM3 (R83-E149 and K85-E173) is crucial for graded activation of Orai channels and that a series of motions lead to channel opening without relaxing structural integrity. In contrast, Frischauf et al have shown a small local widening of the pore (~1-2 Å) occurring during channel opening, proposing two gates in both the hydrophobic and basic region of the pore33. In line, the open dOrai crystal reveals a widening of the basic region in the channels pore although the limited resolution may have prevented the identification of a slight rotation of the pore helix42. A recent study combining crystallization and cryo-electron microscopy compared the closed and open state of the dOrai channel by use of the constitutively open dOrai P288L (P245L in hOrai1) mutant that mimics the action of STIM1 binding52. They propose a model for the conformational transduction pathway from the peripheral TM4 to the pore lining TM1 helix: Latched TM4 helices close the pore from the cytosolic side in closed channel configuration and Ca2+ flux is blocked by positive charge repulsion and anion plugs. Upon channel opening, the basic section of TM4 helices moves outwards, facilitating Ca2+ permeation (anion recruitment model). Lui and colleagues were not able to observe pore helix rotation comparing closed and open states of Orai similar to the results of Hou et al42, although both groups used two different open Orai mutants (P245L and H134A, respectively). Nowadays, MD-simulations are widely used to gain mechanistic insights into Orai channels, an approach that is nicely reviewed in53. Merging the two different gating models is therefore tricky as the basis used for MD-simulations differ (dOrai in34,51 and a modelled hOrai structure in33,35). Ultimately, an atomic-resolution structure is needed for resolving the native behaviour of human Orai1.
Undoubtedly, Orai1 TM domains are critically involved in transmitting the STIM1 signal to the ion-conducting pore54. Discovering the exact, physiological mechanism of the allosteric conformational switch necessary to convey the signal from STIM1 binding to Orai1 channel opening is an exciting focus for further research.
Figure 1. Schematic and structural representation of important domains in Orai1 proteins
A| Schematic representation of one hOrai1 monomer with important amino acids and regions highlighted. N – and C – termini are located inside the cytosol and range from aa1 – 91 and aa259-301, respectively. The ETON (aa73-90) region is highlighted in orange. The four TM- domains (TM1: aa92-106; TM2: aa118-140; TM3: aa174-197 and TM4: aa236-258) are connected via two extracellular loops (loop1 and loop3) and one intracellular loop (loop2). Important hOrai1 residues and domains highlighted in yellow represent unpolar, green basic, blue acidic and red neutral/polar side chains.
B| Cross section through a 3D model of a hexameric hOrai1 channel, displaying two hOrai1 monomers facing each other with TM2-TM4 stabilizing the pore-forming TM1-helices. Important amino acid residues are highlighted as in A. Additionally, side chains of amino acid residues facing the ion-conducting pore (left) and side chains of residues within TM2 and TM3 (right) crucial for stability of TM domains are depicted. (PDB number: 4HKR with adapted hOrai1 sequence described in Frischauf et al, Sci Signal 2015).
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** Hou X, Burstein SR, Long SB: Structures reveal opening of the store‐operated calcium channel Orai. eLife 2018, 7. The authors present the first structure of an open Orai pore using the constitutively open dOrai H206A mutant (corresponding to human Orai1 H134A). The open channel structure exhibits a large dilation of the inner pore and a straightening of TM4 and TM4ext helices. The authors propose a novel mechanism for channel activation in which the TM4ext helices act as latches keeping the channel closed in its resting state and uncross for STIM1 binding and opening of the pore.
* Yeh, Y. C., Lin, Y. P., Kramer, H. & Parekh, A. B: Single nucleotide polymorphisms in Orai1 associated with atopic dermatitis inhibit protein turnover, decrease calcium entry and disrupt calcium‐dependent gene expression. Hum Mol Genet 2019. This paper investigates single nucleotide polymorphisms in Orai1 which are associated with atopic dermatitis. Within this comprehensive study the authors show that disease‐related mutations in Orai1 are more abundantly expressed in the plasma membrane by escaping the cells’ degradation pathway and turnover more slowly than wildtype proteins. A mismatch in STIM/Orai1 stoichiometry in turn leads to the inhibition of Ca2+ entry.
* Dong, H. et al: Toward a model for activation of Orai channel. iScience 2019, 16. The authors propose a working model for the putative open state of the Drosophila Orai channel. Energetic, structural and experimental data suggests a ‘twist‐to‐open’ mechanism which disrupts the hydrophobic barrier in a way that it introduces small conformational changes in the pore allowing for ion permeation. A series of motions within the channel is needed to open the pore without affecting overall structural integrity.
** Liu, X. et al: Molecular understanding of calcium permeation through the open Orai channel. PLoS Biol 2019, 17. This paper gives insight into crystal and cryo‐electron structures of the constitutively open dOrai P288L mutant (corresponding to P245L in human Orai1 proteins). The authors propose a model, where the STIM1 signal is transduced from TM4 to TM1, culminating in an outward twist of the basic section of TM1. The so emerged wider pore aggregates anions that enhance the gradient across the plasma membrane facilitating Ca2+ permeation.
* Bohenry, D. et al: Mechanistic insights into the Orai channel by molecular dynamics simulations. Semin Cell Dev Biol 2019, 94. This review provides a first and comprehensive summary of how molecular dynamics simulations can be used to gain insight into Orai channel gating, selectivity and stability. It is explained how MD‐simulations can be applied in order to provide guidance for live‐cell experiments and how they can help to determine the molecular steps in the STIM1/Orai signalling cascade. References
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