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Thesis

Reference

Characterization of the trafficking and functional properties of the long STIM1 isoform

SAUC, Sophie

Abstract

Store-Operated Ca2+ entry (SOCE) is a ubiquitous mechanism of Ca2+ entry involved in many cellular processes. SOCE is triggered by the Ca2+ depletion of the Endoplasmic Reticulum (ER) which initiates the translocation of the ER Ca2+ sensor STIM1 to the plasma membrane (PM) where it aggregates into punctate structures corresponding to cortical ER subdomains (cER). At the PM, STIM1 gates its channel counterpart Orai1 allowing Ca2+ entry and ER refiling. This thesis work aimed at characterized a longer STIM1 splice variant, STIM1L, mainly expressed in skeletal muscle tissue. We demonstrated STIM1L inability to promote cER expansion contrary to STIM1 while mediating efficient SOCE. We confirmed the requirement of the lysine-rich tail of STIM1 to enlarge these PM contact sites. Finally, we studied STIM1L in the skeletal muscle tissue and localized it in the junctional part of the sarcoplasmic reticulum questioning its involvement as PM Orai1 partner in this context.

SAUC, Sophie. Characterization of the trafficking and functional properties of the long STIM1 isoform. Thèse de doctorat : Univ. Genève, 2017, no. Sc. 5164

DOI : 10.13097/archive-ouverte/unige:102871 URN : urn:nbn:ch:unige-1028712

Available at:

http://archive-ouverte.unige.ch/unige:102871

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

Département de biologie cellulaire FACULTE DES SCIENCES Professeur J.C. Martinou

Département de physiologie cellulaire FACULTE DE MEDECINE

et métabolisme Professeur N. Demaurex

Docteur M. Frieden

___________________________________________________________________

Characterization of the trafficking and functional properties of the long STIM1 isoform

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Sophie SAÜC de France

Thèse n° 5164

Genève 2017

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Table of contents

I. Résumé ... 4

II. Abstract ... 6

III. Introduction ... 8

1. Calcium signaling ... 8

1.1. Ca2+ dependent proteins ... 9

1.2. Ca2+ pumps and exchangers ... 10

1.3. Ca2+ channels ... 11

2. History of SOCE ... 13

2.1. From the founding studies to the concept of SOCE ... 13

2.2. SOCE associated currents ... 15

3. SOCE players ... 16

3.1. STIM/Orai discovery, and their structure ... 16

3.2. Sequential events from store depletion to Ca2+ entry (Figure III.7) ... 20

3.3. TRPC channels and other members of the Orai and STIM families ... 22

3.4. Physiological functions of SOCE ... 26

4. SOCE and skeletal muscle ... 31

4.1. Muscle architecture and physiology ... 31

4.2. Overview of skeletal muscle regeneration ... 35

4.3. SOCE in skeletal muscle ... 39

IV. Aim of the thesis ... 48

V. Experimental tools ... 49

1. Cells ... 49

2. Microscopy ... 51

3. Ca2+ imaging ... 52

4. STIM1L antibody production ... 55

VI. Publications ... 56

Publication 1 ... 56

Publication 2 ... 74

Publication 3 ... 86

VII. Unpublished data ... 97

1. Materials and methods ... 97

2. Results ... 101

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VIII. Discussion and perspectives ... 115 1. STIM1L traps and gates Orai1 channels without remodeling cortical ER ... 115 2. STIM1L localization in adult skeletal muscle ... 119 3. Development of a model to study STIM1L function with in vitro differentiated human muscle fibers ... 122 IX. Remerciements ... 125 X. References ... 127

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I. Résumé

La signalisation calcique régule de nombreux processus biologiques tels que la division cellulaire, la différenciation cellulaire, la contraction musculaire, etc. Classiquement, suite à un signal donné tel que : modification du potentiel de membrane, fixation d’un ligand, on assiste à une élévation de la concentration cytoplasmique de Ca2+ à l’origine d’une cascade de signalisation faisant intervenir des protéines du type calmoduline et permettant la mise en place du processus cellulaire dédié. Le Ca2+ à l’origine du signal pourra venir soit de l’extérieur de la cellule soit de l’un des compartiments cellulaires tels que le réticulum endoplasmique (RE) qui constitue un stock de Ca2+ important. Dans le cas du RE, la vidange des stocks est suivie par une entrée de Ca2+ cytosolique qui sera ensuite pompé par les SERCA pour sarco/endoplasmic reticulum Ca2+-ATPase afin de reconstituer les réserves. Cette entrée « capacitive » de Ca2+ ou SOCE pour Store Operated Ca2+ Entry est connue depuis longtemps mais les acteurs moléculaires supportant ce phénomène n’ont été que récemment identifiés.

STIM1 et son homologue STIM2 ont été mis en évidence en tant que protéines impliquées dans la régulation du SOCE en 2005 au cours d’un criblage à l’aide d’ARN interférant. Ces 2 protéines sont présentes dans la membrane du RE et sont capables de détecter des variations calciques à l’intérieur de cette organelle grâce à leur domaine de liaison au Ca2+. Lorsque la concentration de Ca2+ dans RE diminue, la molécule de Ca2+ liée à STIM est libérée provoquant une modification de conformation de la protéine. S’ensuit une oligomérisation de STIM qui migre au niveau de la membrane plasmique où elle recrute ses canaux partenaires, appelés Orai. L’interaction entre STIM et Orai permet l’ouverture de ce canal calcique et donc une entrée massive de Ca2+ dans le cytoplasme qui sera ensuite pompé par les SERCA pour permettre un remplissage des stocks. Ce phénomène est associé à un remodelage du RE qui forme alors de fines extensions sous membranaire mais toujours en lien avec le reste du RE. Ce RE, appelé RE cortical, est enrichi en molécules STIM1 et situé à moins de 20 nanomètres de la membrane plasmique.

En 2011, le laboratoire du Pr. Bernheim a identifié une nouvelle isoforme de STIM1 résultant d’un épissage alternatif de l’ARNm de STIM1 et apparaissant au cours de la différenciation musculaire de myoblastes en myotubes. Cette isoforme appelée STIM1L possède 106 aa supplémentaires situés entre les exons 11 et 12 de l’isoforme classique de STIM1. STIM1L induit un influx SOCE plus rapide que l’influx généré par STIM1 grâce à un pré- recrutement membranaire de la protéine au niveau de clusters qui colocalisent parfaitement avec les canaux Orai dans des myotubes.

Le but de ma thèse était l’étude de cette nouvelle isoforme afin d’en déterminer dans un premier temps les caractéristiques intrinsèques comparées à l’isoforme classique, STIM1, déjà très bien décrite et dans un deuxième temps, de déterminer sa fonction dans la physiologie du muscle squelettique adulte, tissu dans lequel elle est le plus abondamment exprimée.

Afin de pouvoir comparer STIM1 et STIM1L dans un modèle dépourvu de toutes isoformes endogènes pour simplifier l’analyse des résultats, nous avons décidé d’utiliser des

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fibroblastes de souris dont les gènes codant pour STIM1 et STIM2 (MEFDKO) avaient préalablement été invalidés et ré-exprimant soit STIM1 soit STIM1L. Nous avons ainsi pu montrer que STIM1L était capable de générer un influx SOCE d’amplitude comparable à celui généré par STIM1 sans toutefois induire de remodelage du RE visible en microscopie électronique. Une analyse détaillée des clusters formés par STIM1L en microscopie optique a permis de préciser ces données. En effet, suite à l’activation du SOCE, STIM1L est recruté à la membrane plasmique pour former de nouveaux clusters mais ceux-ci ne s’agrandissent pas contrairement à ceux induits par STIM1. Ce phénotype serait lié au masquage du domaine C terminal spécifiquement chez STIM1L due à un repliement différent de celui de STIM1 en raison de la région supplémentaire de 106 aa dans la partie cytosolique de la molécule. En effet, ce domaine riche en lysine et impliqué dans le recrutement de STIM1 à la membrane, permet aussi l’agrandissement des clusters formés par STIM1 comme nous avons pu le confirmer. Par ailleurs, la mesure de la vitesse d’activation du SOCE dans le modèle cellulaire utilisé n’a pas permis de récapituler les données obtenues dans les myotubes démontrant que la capacité de STIM1L à induire un influx rapide n’était pas une capacité intrinsèque de la molécule. Ainsi STIM1L, bien qu’étant capable de recruter des canaux Orai1 suite à la vidange des stocks calciques du RE, ne forme pas de clusters avec Orai1 avant l’activation du SOCE dans des MEFDKO, comme c’est le cas dans les cellules musculaires. Ces résultats suggèrent la présence spécifique dans le muscle d’un ou plusieurs partenaires responsables du pré- recrutement de STIM1L à la membrane et responsable de l’influx SOCE rapide décrit dans ce modèle.

La génération d’un anticorps dirigé contre STIM1L par la plateforme d’anticorps recombinants de la faculté de médecine de Genève, a permis dans la deuxième partie de ma thèse de préciser la localisation de la protéine endogène chez l’Homme dans le muscle adulte.

STIM1L est ainsi présent à la fois dans les fibres musculaires lentes et rapides, majoritairement au niveau de la partie longitudinale du réticulum sarcoplasmique (SR) et non au niveau de la partie jonctionelle apposée à la membrane où est localisé Orai1. Cette localisation, qui est la même pour l’isoforme STIM1, est surprenante et soulève la question du rôle de STIM1 et STIM1L dans le muscle squelettique adulte. Par ailleurs, le développement d’un modèle in vitro de différenciation de fibres musculaires humaines a donné des résultats prometteurs.

Pour la première fois à notre connaissance, nous avons pu obtenir des fibres présentant un stade avancé de maturation avec des triades montrant une orientation transversale caractéristique du muscle adulte. Ce modèle, après optimisation, permettra d’étudier la fonction de STIM1 et STIM1L dans des fibres musculaires humaines après invalidation spécifique de chacune des isoformes.

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II. Abstract

Ca2+ is a universal second messenger involved in the regulation of many cellular processes such as cell division, apoptosis or muscle contraction. At resting state, cytosolic Ca2+

concentration is maintained very low due to permanent activity of PMCA (plasma membrane Ca2+ ATPase) and SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) pumps located at the plasma membrane (PM) and at the membrane of the endoplasmic reticulum (ER), respectively. Following cell stimulation that frequently leads to ER Ca2+ depletion, a particular type of Ca2+ channels is getting activated, called Store Operated Ca2+ (SOC) channels. Once open, these SOC channels mediate Ca2+ influx within the cytosol that is pumped back by SERCA to refill the stores and generate Ca2+ signals that in turn activate various Ca2+ dependent pathways. The main molecules supporting this process have only been recently identified:

STIM1, the ER-located Ca2+ sensor and Orai, the PM Ca2+ channel.

Thanks to an EF hand domain present in the luminal part of the protein, STIM1 and STIM2 are able to detect Ca2+ variations within the ER. Upon ER Ca2+ depletion, Ca2+ bound to STIM is released which triggers a conformational change in the 3D structure of the protein. This mediates STIM oligomerization and subsequent translocation at the PM where it gates Orai Ca2+ channels allowing Ca2+ entry into the cytosol. SOCE is associated with ER remodeling characterized by formation of thin sheets of cortical ER located at less than 20 nm from the PM, enriched in STIM molecules and connected with the bulk ER.

In 2011, Pr. Bernheim’s lab identified a splicing isoform of STIM1 that is expressed during muscle differentiation. This isoform, named STIM1L, possesses a supplementary domain of 106 aa located between exon 11 and 12 of the classical STIM1. STIM1L has been shown to mediate faster SOCE than STIM1 in myotubes. This property is explained by STIM1L pre- recruitment at the PM where it co-localized with Orai1 channels before store depletion.

The goal of my thesis was first to identify the intrinsic properties of STIM1L compared to the already well-described STIM1 isoform and secondly to investigate about its function in adult skeletal muscle where this isoform is abundantly expressed. To study independently STIM1 and STIM1L, we chose a MEF (mouse embryonic fibroblasts) cell line derived from stim1-/- stim2-/- knock-out mice in which we re-expressed either YFP-STIM1 or YFP-STIM1L. We showed that STIM1L mediated SOCE with the same efficiency than STIM1 but without ER remodeling as visualized on electron microscopy images. Additionally, careful analysis of STIM1L clusters imaged by total internal reflection microscopy demonstrated that STIM1L formed new clusters but failed to enlarge them contrary to STIM1 upon ER depletion. We hypothesized that the C terminal part of STIM1L was masked due to the supplementary domain that would change the 3D structure of the molecule. Indeed, when the lysine rich domain of STIM1 is deleted, STIM1 clusters do not expand any more, mimicking STIM1L behavior. Moreover, STIM1L ability to induce fast SOCE was shown to be muscle specific as we failed to recapitulate it in other cell types. STIM1L does not form complex with Orai1 channels prior to SOCE activation contrary to what has been shown in muscle cells. This

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suggests the presence of STIM1L muscle specific partner(s) that are needed to maintain STIM1L at the PM for Orai1 pre-recruitment and fast SOCE that are muscle dependent.

During the second part of my thesis, we developed a specific STIM1L antibody which allowed us to detect the endogenous protein in adult muscle tissue in fast and slow muscle fibers.

Surprisingly, we found STIM1L and STIM1 mainly localized in the longitudinal part of the sarcoplasmic reticulum (SR) contrary to Orai1 described to be at the triads raising the question of the role of both isoforms in adult skeletal muscle. Finally, optimization of a cell culture method to differentiate human satellite cells into mature muscle fibers gave promising results.

We succeeded to obtain human muscle fibers with the beginning of transversely oriented triads a hallmark of adult skeletal muscle, which was to our knowledge never achieved. This model will be a precious tool to study STIM1 and STIM1L function in adult skeletal muscle physiology for future experiments.

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III. Introduction

1. Calcium signaling

Since the appearance of multicellular organisms, cells have developed a wide variety of signals to communicate between them such as hormones, neurotransmitters and paracrine signals.

However few molecules used as intercellular messengers are membrane permeant to allow direct transmission of the signal into the cell. Consequently, intracellular signaling pathways based either on proteins modifications (for example phosphorylation, acetylation, and methylation) or second messengers have been set up by cells to relay extracellular signals.

Broadly used by living organisms, from bacteria to mammalians, Ca2+ is one of these universal second messengers. The discovery of Ca2+ as second messenger is

often attributed to Sydney Ringer (figure III.1) in 1883 while he was studying heart contraction. In this seminal experiment, Ringer inadvertently exchanged distilled water that he usually used to prepare isolated rat heart infusing solution by London tap water.

Surprisingly, while beating normally when infused with tap water based solution, rat heart infused with distilled water based solution, that was deprived of any ions, failed to contract.

Subsequent experiments led him to identify Ca2+ as the responsible pipe water component for continuous isolated heart

beats (Ringer 1883). Half a century later, in 1947, Heilbrunn and Wiercinski’s study proved definitively the key role of Ca2+ in muscle physiology by injecting Ca2+ ions in muscle fibers to induce contractions (while Na+, K+ and Mg2+ failed) (Heilbrunn and Wiercinski 1947).

Comparable experiments of Ca2+ injections into other cell types followed with neurons (Miledi 1973) and mastocytes (Kanno, Cochrane et al. 1973) where such injections induced exocytosis of neurotransmitter vesicles and secretory granules respectively. In the field of embryogenesis, Timourian and co-workers demonstrated that application of a Ca2+ chelator at different sites on the surface of sea urchin eggs inhibited or not their division, highlighting the crucial role of Ca2+ in the first step of embryogenesis (Timourian, Clothier et al. 1972). In parallel, other experiments during the mid-1950 and 1960’s revealed the capacity of Ca2+ to cross mitochondrial membranes as well as the sarcoplasmic one (Carafoli 2003). The concept

Figure III.1: Sydney Ringer.

From Carafoli E., et al. (2003).

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of Ca2+ signaling was born and extensive studies were performed ever since to decipher the multiple Ca2+ signaling pathways used by cells to fulfill a wide diversity of functions. Spatially and temporarily encoded Ca2+ signals, specific of a defined cell type, allow a precise control of a huge variety of cellular processes (Berridge, Bootman et al. 2003). Whereas Ca2+ micro- domains restrict the regulation of Ca2+ dependent pathways within a certain region of the cell (spatial encoding), various duration and frequencies of Ca2+ signals determine temporal control of Ca2+ dependent cellular responses. For example, exocytosis and skeletal muscle contractions are fast processes (µs to ms range) compared to gene transcription which are controlled by Ca2+ transients that last longer and spread throughout the cell as Ca2+ waves.

Encoding of Ca2+ signals is based on the Ca2+ signaling machinery that is specific of a cell type (Berridge, Bootman et al. 2003). However one can classify the Ca2+ cellular toolkit in three main types of actors that are Ca2+ dependent proteins, Ca2+ pumps and exchangers, and Ca2+

channels.

1.1. Ca2+ dependent proteins

Each protein has a specific 3D shape and Ca2+, because of its charge and size, has the ability to fit within the multiple different conformations of protein binding sites. Ca2+ binding proteins possess a typical EF hand domain which has an α-helix-loop-α-helix 3D structure that allows Ca2+ ion to be sequestered within, by interaction with 6 residues located in the loop (figure III.2) (Zhou, Frey et al. 2009).

Proteins often have several EF hand domains conferring them a Ca2+ buffering capacity.

Proteins are dedicated to Ca2+ buffering in the cytoplasm such as parvalbumin, calbindin D-28 and calretinin while others like calnexin, calreticulin or calsequestrin binds Ca2+ in the endoplasmic and sarcoplasmic reticulum to increase the Ca2+ containing capacity of these organelles. Of note, some Ca2+ dependent proteins play a more active role such as Ca2+

dependent enzymes that are calpain proteases, nitric oxide synthases (endothelial and neuronal NOS), adenylyl cyclase (AC-I, AC-III, AC-V, AC-VI and AC-VIII) and phosphorylase kinase for instance. Transduction of Ca2+ signals by proteins is based on the modification of their 3D structure upon Ca2+ binding which changes their ability to interact with some potential partners. The most studied Ca2+ binding protein is calmodulin (CaM1-4) that can bind

2+

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functions from smooth muscle contraction (Tansey, Luby-Phelps et al. 1994) to long term memory mediated by GABAergic neurons (Lledo, Hjelmstad et al. 1995). Calmodulin domain also constitutes the core domain of cameleon Ca2+ probes based on FRET (Fluorescence resonance energy transfer), see materials and methods for further explanations, technology and designed to study Ca2+ variations in multiple organelles (Miyawaki, Llopis et al. 1997).

Figure III.2: The helix-loop-helix EF-hand Ca2+-binding motif.

Adapted from Zhou Y., et al. (2009)

(A) Cartoon illustration of the canonical EF-hand Ca2+-binding motif. The EF-hand motif contains a 29- residue helix-loop-helix topology, much like the spread thumb and forefinger of the human hand. Ca2+

is coordinated by ligands within the 12-residue loop (loop sequence positions 1, 3, 5, 7, 9 and 12).

Residue at position 12 serves as a bidentate ligand. The Ca2+-binding pocket adopts a pentagonal bipyramidal geometry. (B) 3D structure of a typical canonical EF-hand motif from calmodulin. Ca2+is chelated by ligands from a 12-residue loop.

1.2. Ca2+ pumps and exchangers

If Ca2+ buffering proteins participate in the maintenance of Ca2+ gradient between cytosol and external medium or internal stores, they are not sufficient. To this end, cells possess Ca2+

pumps and exchangers for efficient Ca2+ cytosolic removal. Ca2+ exchangers which have a low affinity for Ca2+ but a high velocity, are mainly dedicated to Ca2+ extrusion but in some particular condition can also mediate Ca2+ influx. Exchangers expressed in mammalian cells can be divided in 3 subtypes according to the ions exchanged: the NCX (Na+/Ca2+ exchanger) that is broadly expressed, the NCKX (Na+/ Ca2+-K+ exchanger) present only in photoreceptors, neurons, and skin and finally the NCLX (Na+/Ca2+-Li+ exchanger) that is the Ca2+ extrusion system of the mitochondria (Lytton 2007). Conversely, Ca2+ pumps have a high Ca2+ binding capacity and mediate only efflux outside the cell or pump it back to the ER/SR. They extrude

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2 or 1 Ca2+ molecule per 1 molecule of ATP hydrolyzed for the sarco-endoplasmic reticulum pumps called SERCA (smooth endoplasmic reticular Ca2+ ATPase) and for the plasma membrane pumps named PMCA (plasma membrane Ca2+ ATPase) respectively (Clapham 2007). A wide diversity of exchangers and pumps subtypes exist that display specific features (kinetics, capacity) participating in the fine tuning of Ca2+ signals.

1.3. Ca2+ channels

They can be classified according to the stimulus that triggers their gating. The most studied type of Ca2+ channels are voltage-gated Ca2+ channels expressed in excitable cells and activated upon membrane depolarization. Depending on the amplitude of the depolarization needed for their activation, the time they are opened and their localization, they are classified in 5 main families: T(transient and tiny current)-type, B(brain)-type, L(long-lasting)-type, N(neither nor B nor L)-type and P(Purkinje cell)-type voltage gated Ca2+ channels. They are all composed of different combinations of subunits but for each of them the subunit α1

constitutes the pore forming channel (Brini and Carafoli 2000). The L-type Ca2+ channels is the one expressed in skeletal muscle and will be discussed in details in the section III.4.1. of this introduction. Another type of Ca2+ channels is activated after fixation of a ligand and then called ligand gated Ca2+ channels (LGCC). IP3 (inositol 1,4,5-trisphosphate) receptor is an example of LGCC that is located within the ER membrane and activated upon IP3 fixation.

Cytosolic IP3 binding to its receptor, triggers its opening and Ca2+ release outside the ER. IP3 is produced together with diacylglycerol (DAG) after PIP2 (phosphatidylinositol 4,5- bisphosphate) hydrolysis by phospholipase C (PLC). PLC that is coupled to a PM receptor, becomes activated upon ligand fixation on its receptor. This Ca2+ signaling pathway regulates numerous cellular processes from fertilization to cell death. In muscle cells, Ca2+ release from SR, needed for the contraction, is mediated by the direct interaction between a PM voltage sensor and the ER resident Ca2+ channel, named Ryanodine receptor (RyR) (see section III.4.1.2. for further details). Emptying of the ER Ca2+ store by activation of IP3 pathway or RyR opening activates a fourth type of Ca2+ channels named store operated Ca2+ entry (SOCE) channels. SOCE channels are highly Ca2+ selective and will be discussed in details in the following sections. Finally, others channels belonging mainly to the TRP(Transient receptor potential) superfamily of cation channels can be activated by temperature, stretches,

2+

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the mitochondrial Ca2+ uniporter (MCU) participates in Ca2+ buffering as it allows Ca2+ entry within the mitochondrial matrix especially during cytosolic Ca2+ elevations. Ca2+ regulates several Krebs cycle enzymes then participating in mitochondrial metabolism regulation but also triggers apoptosis via cytochrome c release at high concentration (Chaudhuri and Clapham 2014). Then, mitochondria are involved in cytosolic Ca2+ buffering but also participate actively in some Ca2+ dependent pathways such as apoptosis.

In addition to their mode of activation, other features distinguish Ca2+ channels such as Ca2+

selectivity and kinetics of opening. For instance, voltage dependent Ca2+ channels have a fast kinetics of activation whereas IP3R are relatively slow to be activated. This heterogeneity of Ca2+ channels contributes substantially in the coding of Ca2+ signals. Cellular Ca2+ signals are generated upon the arrival of an external stimulus that leads to opening of Ca2+ channels located at the PM or in the ER/SR membrane. As mentioned above, Ca2+ raising within the cell is possible due to the huge Ca2+ gradient between the cytosol where Ca2+ concentration is around 100-200 nM and either external medium (10000 fold higher) or intracellular Ca2+

stores where Ca2+ level is around 400 µM range. Once in the cytosol, Ca2+ molecules bind to their target proteins modifying their conformation and activity. The Ca2+ dependent cellular functions turn on as long as high cytosolic Ca2+ level (or oscillations) is maintained. Conversely, as soon as Ca2+ channels close, Ca2+ concentration returns to its basal level due to the permanent activity of Ca2+ exchangers and pumps leading to the termination of Ca2+

dependent processes (figure III.3).

Among the different Ca2+ entry pathways described previously, SOCE have gained importance in the past few years, particularly since the discovery of its molecular players and their implication in several disorders and diseases. Description of SOCE properties and functions in diverse tissues, especially in skeletal muscle, will be the topic of the introduction of this thesis.

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Figure III.3: Ca2+ signaling machinery.

Adapted from Carafoli E., et al. (2003) and Berridge M., et al. (2003)

When cells are activated, Ca2+enters in the cell from the external medium or is released from internal Ca2+stores, the endoplasmic/sarcoplasmic reticulum (ER/SR). According to the stimulus activating the cell (stretch, ER Ca2+depletion, depolarization, ligand fixation, etc.), different types of Ca2+channel open: stretch activated Ca2+channel (SACC), store operated Ca2+ channel (SOCC), voltage gated Ca2+

channel (VGCC) or ligand gated Ca2+ channel (LGCC). Most of Ca2+(shown as grey circles) bound to Ca2+

binding proteins which are Ca2+ buffers or effectors. To return to cytosolic basal Ca2+ level, Ca2+is extruded from the cell by various exchangers and pumps. Na+ exchanger (NCX) and plasma-membrane Ca2+(PMCA) extrude Ca2+to the outside, whereas SERCA (smooth endoplasmic reticular Ca2+ATPase) pumps refill the ER. Mitochondria also have an active function during the recovery process as they sequester Ca2+rapidly through the mitochondrial Ca2+uniporter (MCU), and release it more slowly back into the cytosol to be dealt with by SERCA and PMCA.

2. History of SOCE

2.1. From the founding studies to the concept of SOCE

The concept of capacitative or store-operated Ca2+ entry was first described by James W.

Putney in 1986 (Putney 1986) in line with an original idea by G. Droogmans and R. Casteels (Casteels and Droogmans 1981). In this seminal paper, Putney proposed a model to explain the bi-phasic cytosolic Ca2+ elevation upon agonist stimulation that he described in 2 previous studies. As methods to visualize Ca2+ fluxes were limited at that time, he monitored cytosolic

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Ca2+ variations indirectly by measuring Ca2+ dependent K+ release in salivary and lacrimal gland upon cholinergic or adrenergic stimulation. He described that activation of PM cholinergic or adrenergic receptors induced a transient Ca2+ elevation due to liberation from internal pools that was followed by a plateau as Ca2+ from external medium entered in the cytosol (Putney 1977). In the following paper, he noted that a second agonist stimulation in absence of Ca2+

did not produce the first transient response unless Ca2+ was added temporary in the external medium between the 2 stimulations (Parod and Putney 1978). This experiment proved that Ca2+ entry was necessary to refill internal stores. The identification in 1983 of IP3 as second messenger mediating Ca2+ release from internal stores upon receptor activation (Streb, Irvine et al. 1983) led Putney to propose the visionary model for capacitative Ca2+ entry. He defined capacitative Ca2+ entry or SOCE as a process whereby Ca2+ store depletion due to IP3

stimulation induced Ca2+ influx to allow store refilling (see figure III.4).

Figure III.4: Original scheme from J.Putney in the seminal 1986 paper depicting the model of capacitative Ca2+ entry.

Agonist (Ag) binding to its receptor (RA) leads to breakdown of PIP2 into diacyglycerol (DG) and IP3. IP3

binds to a receptor (RI) on the endoplasmic reticulum (ER) which induces cytoplasmic Ca2+ release. The decrease in Ca2+ content of the ER relieves an inhibitory constraint on a direct pathway for Ca2+to enter the Ca2+store from the extracellular space. In the continued presence of IP3, Ca2+ will continue down its concentration gradient to the cytosol resulting in a sustained, Ca2+ entry phase of the response.

However, subsequent studies obliged Putney to revisit the model as they demonstrated that (i) Ca2+ did not enter stores directly from outside but transit via the cytosol and (ii) SOCE was elicited regardless of the mechanism of store emptying (Putney 1990). Indeed, even passive mechanisms like blockade of SERCA pumps by thapsigargin (Tg) induced SOCE (Takemura, Hughes et al. 1989). Then, the new and nowadays accepted definition of SOCE is a mechanism of Ca2+ entry triggered by Ca2+ store depletion that is associated with Ca2+ currents referred as ICRAC or ISOC depending on the characteristics of the current.

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2.2. SOCE associated currents

In 1989, Lewis and Cahalan by using patch clamp in whole cell and perforated configurations recorded for the first time a highly selective Ca2+ current in the activated human T lymphocyte upon antigen stimulation (Lewis and Cahalan 1989). Three years later, Hoth and Penner further described this current elicited by store depletion regardless of the pathway implicated (agonist stimulation or passive depletion by Tg) and named it: Ca2+ release-activated Ca2+

current (ICRAC) (Hoth and Penner 1992). In addition to be highly Ca2+ selective, the current recorded in mast cells was inward rectifying, inhibited by trivalent ions and also intracellular Ca2+ elevation (Hoth and Penner 1993). The latter property, now referred to as fast Ca2+- dependent inactivation (or CDI) together with others ICRAC features were confirmed by Lewis and colleagues in T lymphocytes (Zweifach and Lewis 1995). They also measured the unitary conductance of ICRAC channel around 24 fS by noise analysis (Zweifach and Lewis 1993). These findings allowed precise characterization of ICRAC current thatcontributed a decade latter to confirm Orai1-3 as prototypical ICRAC channels.

However, ER Ca2+ store depletion activates other types of Ca2+ currents in various cell types.

These currents, namely ISOC, have a higher conductance associated with a decreased Ca2+

selectivity (table III.1). ISOC currents are nowadays considered to be supported by homo or hetero channel complexes of subunits belonging to the Transient Receptor Potential family (TRP family) of cation channels (Parekh and Putney 2005) together with Orai channels.

The discovery of the pathway linking store emptying and PM Ca2+ channel activation came relatively recently compared to the identification of SOCE mechanism and its associated Icrac

current. It took almost 20 years between the publication of Putney’s article in 1986 and the identification of the ER Ca2+ sensor, STIM1, and its PM Ca2+ channel counterpart, Orai1, as the main molecular players supporting SOCE in 2005 and 2006 respectively.

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Table III.1: Biophysical properties of store-operated Ca2+ channels.

Adapted from Parekh A. B., et al. (2005)

This table summarizes differences between ICRAC and ISOC identified in various cell types.

3. SOCE players

3.1. STIM/Orai discovery, and their structure

Two independent studies using either Drosophila S2 cells or Hela cells for RNAi based screening, revealed STIM1 and STIM2 as the ER Ca2+ sensors involved in the SOCE pathway (Liou, Kim et al. 2005, Roos, DiGregorio et al. 2005). One year later, 3 other studies based also on genome wide RNAi screen in Drosophila S2 cells identified the channel responsible for the Icrac current associated with SOCE (Feske, Gwack et al. 2006, Vig, Peinelt et al. 2006, Zhang, Yeromin et al. 2006). Feske and co-workers named the 3 mammalian homologous to the Drosophila channel for the keepers of the gates of heaven: Orai1, Orai2 and Orai3.

Additionally, a genome-wide single-nucleotide polymorphism analysis associated a missense mutation (R91W) in Orai1 gene with the hereditary severe combined immune deficiency (SCID) syndrome, in which T lymphocytes were defective for SOCE, confirming Orai1 as key player in this pathway (Feske, Gwack et al. 2006). Finally, overexpression of STIM1 together with Orai1 recapitulating ICRAC current in heterologous system validated definitively that these 2 molecules were necessary and sufficient to mediate SOCE (Peinelt, Vig et al. 2006, Soboloff,

Current Conductance Selectivity Permeability ratio Cell type

ICRAC 0.02 pS; 110 Ca2+

Ba2+> Ca2+≥Sr2+ Ca2+:Na+; 1000:1

Mast cell RBL-1/-2H3 Jurkat T cells Hepatocytes Dendritic cells Megakaryocytes MDCK cells

ISOC

11 pS; 10 Ca2+ Ca2+>Na+ Ca2+:Na+; >10:1 Endothelia 1-2 pS; 100-160 Ca2+ Ba2+≥Ca2+>>K+ Ca2+:K+; 1000:1 A431 epidermal cells

2.7 pS; 90 Ca2+ Ca2+=Ba2+= Na+ Ca2+:Na+ :K+; 1 :1:1 Aortic myocyte 2.3 pS; 1.5 Ca2+ Ca2+>Na+ Ca2+:Na+; 50:1 Portal vein myocyte

5.4 pS; 20 Ca2+ ? ? Pulmonary artery myocytes

0.7 pS; 90 Ca2+ ? ? Mesangial cells

43 pS; 1.3 Ca2+ K+, Na+>Ca2+ Ca2+:Na+; 13:1 Pancreatic acinar cells

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Spassova et al. 2006). Subsequent publications allowed a deep characterization of STIM and Orai structure and function as well as of the mechanism of interaction between the 2 molecules.

3.1.1. Structure of STIM1 (figure III.5)

STIM1 is a transmembrane (TM) protein with two EF-hand binding site in the luminal part of the protein: a canonical one that binds Ca2+ and a hidden EF-hand that stabilizes the canonical binding site. These 2 domains form a stable 3D conformation with a sterile alpha motif (or SAM) that maintains the molecule in a quiescent state (Stathopulos, Zheng et al. 2008). In the cytosolic part of STIM1, 3 coiled-coil domains are juxtaposed. This region is involved in STIM1 higher order oligomers formation upon activation as well as Orai1 gating (Covington, Wu et al. 2010). Several studies have mapped more precisely the STIM1-Orai1 interacting domain and named it OASF (Orai-activating small fragment)(Muik, Frischauf et al. 2008), CAD (CRAC activating domain)(Park, Hoover et al. 2009), SOAR (STIM Orai-activating region)(Yuan, Zeng et al. 2009), and Ccb9 (coiled-coil domain region containing region b9)(Kawasaki, Lange et al.

2009). More precisely, the minimal domain for STIM1 dependent Orai1 gating is the CC2-CC3 region that is folded and maintained in this position through CC1 interaction when STIM1 is in inactive conformation (Muik, Fahrner et al. 2011, Fahrner, Muik et al. 2014). STIM1 possesses also a CRAC modulatory domain (CMD) also called inactivation domain of STIM (IDSTIM) involved in Ca2+-dependent inactivation (Derler, Fahrner et al. 2009, Lee, Yuan et al. 2009, Mullins, Park et al. 2009, Muik, Fahrner et al. 2011), a serine/proline domain and a poly-lysine region at the C terminal that participates in STIM1 targeting at the PM (Liou, Kim et al. 2005, Baba, Hayashi et al. 2006, Huang, Zeng et al. 2006, Smyth, Dehaven et al. 2006).

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Figure III.5: Functional domains within human STIM1.

Adapted from Muik M., et al. (2012).

From left to right: cEF, canonical EF-hand motif; nEF, hidden EF-hand motif; SAM, sterile alpha motif;

TM, transmembrane domain; CC1/CC2/CC3, coiled-coil domains 1-3; CMD, CRAC modulatory domain;

IDSTIM,inactivation domain of STIM1; S/P, serine/proline-rich region; K or PDB, polybasic cluster. The minimal functional regions within STIM1 are highlighted on the top: CAD, CRAC activating domain;

SOAR, stim Orai-activating region; Ccb9 coiled-coil domain region containing region b9; OASF Orai- activating small fragment. Note STIM1L supplementary domain at position 515 aa.

3.1.2. Structure of Orai1 (figure III.6)

Orai1 is composed of 4 transmembrane domains (TM) with the N and C terminus of the protein localized within the cell and implicated in Orai1-STIM1 interaction. The N terminus segment together with TM1 constitute the pore of the channel with the E106 residue being the selectivity filter (Vig, Peinelt et al. 2006, Gwack, Srikanth et al. 2007, McNally, Yamashita et al. 2009) and G98/R91 the gate of the pore (Zhang, Yeromin et al. 2011) (see figure III.6).

The channel itself is comprised of several Orai1 subunits of which TM1 faces the pore while TM2-4 constitute the outer part of the channel. The number of Orai1 molecules necessary to form a functional channel is still a matter of debate as some studies favors tetrameric configuration while crystallography of the Drosophila Orai1 channel revealed a hexameric structure in active state (Hou, Pedi et al. 2012). By recording ICRAC current in cells over- expressing preassembled tandem Orai1 multimers of 2, 3 or 4 subunits together with a dominant negative, Mignen’s study suggests tetrameric conformation as the functional channel (Mignen, Thompson et al. 2008). Others studies based on single-molecule imaging of 2 or 4 Orai1 subunits assembled in concatemers followed by counting of photo-bleaching step necessary to extinguish concatemers fluorescence leads to comparable conclusion (Ji, Xu et al. 2008, Penna, Demuro et al. 2008). However, regarding the high amino acid sequence

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homology between mammalian and Drosophila channel, it is likely that mammalian Orai1 channel would be also formed by 6 Orai1 subunits. Tetrameric configuration found in concatemer-based studies would result of rearrangement of tetrameric concatemeres into hexamers in the PM or artefacts as photo-bleaching imaging is not always precise due to fluorophore instability that can easily leads to under-estimation.

Figure III.6: Functional organization of Orai1.

Adapted from Prakriya M., et al. (2015).

The topology of Orai1 is illustrated, with selected functional domains noted by colored bars or circles.

The thick yellow bars in the NH2 and COOH termini depict putative STIM1 binding sites on Orai1. Yellow and red lines show the locations of mutations causing gain-of-function (i.e., STIM1-independent Orai1 activation) or loss-of-function phenotypes, respectively. Black lines indicate mutations affecting ion selectivity. Intracellular residues important for CDI are marked in purple (NH2 terminus and II–III loop) and selected residues important for STIM binding and gating are marked in red (NH2 and COOH termini).

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3.2. Sequential events from store depletion to Ca2+ entry (Figure III.7)

Figure III.7: Functional model of SOCE depicting events from ER Ca2+ depletion to Ca2+ entry via Orai1 channel.

From Prakriya M., et al. (2015).

At far left, STIM1 dimer is in its resting state bound to Ca2+and freely diffusing in the ER membrane.

Store depletion and Ca2+ unbinding from the EF hand initiates a conformational change of the cytosolic domains that allows the PBD to bind to PIP2 in the PM, trapping STIM1 at the ER-PM junction. In addition, CC2 helices in STIM1 move into an antiparallel configuration, and the CC3 helix disengages from CC2 to create a binding interface for the Orai1 COOH termini. STIM1 binding traps and activates the Orai1 channel. Only a single dimer is shown for clarity; higher-order STIM1 oligomers are thought to form after store depletion, and binding of multiple STIM1 dimers to Orai1 tetramers or hexamers is required for full activity.

When stores are filled, STIM1 molecules are localized throughout the ER and maintained inactive by Ca2+ binding to the EF-hand domain. The two EF-hand domains interact with the SAM in a folded and compact monomeric 3D structure while CAD domains of 2 STIM1 interact together forming dimers that further stabilizes the inactive state (Stathopulos, Li et al. 2006, Muik, Fahrner et al. 2009, Covington, Wu et al. 2010, Zhou, Srinivasan et al. 2013). Upon ER Ca2+ depletion, Ca2+ ion bound to the EF-hand is released which causes a destabilization of the molecule and its oligomerization via the EF hand domains (Stathopulos, Li et al. 2006, Stathopulos, Zheng et al. 2008). The STIM1 KD for Ca2+ is around 200 µM (Stathopulos, Li et al.

2006) which is in accordance with the ER Ca2+ concentration needed for STIM1 activation which are comprised between 187 µM and 210 µM (Luik, Wang et al. 2008). Multimerization

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is necessary and sufficient to induce STIM1 translocation to the PM (Zhang, Yu et al. 2005, Baba, Hayashi et al. 2006, Wu, Buchanan et al. 2006, Xu, Lu et al. 2006, Liou, Fivaz et al. 2007, Varnai, Toth et al. 2007, Luik, Wang et al. 2008). STIM1 translocation is accompanied by ER remodeling with the formation/extension of ER-PM contact sites where ER and PM are distant of 10 to 17 nm depending studies (Wu, Buchanan et al. 2006, Orci, Ravazzola et al. 2009). The poly-lysine domain of STIM1 while not necessary for sustained SOCE, stabilizes STIM1 at the PM through PIP2 and phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) interaction (Huang, Zeng et al. 2006, Liou, Fivaz et al. 2007, Park, Hoover et al. 2009). Once at the PM, STIM1 traps Orai1 channel that diffuses in the PM (Wu, Covington et al. 2014) and mediates its clustering (Xu, Lu et al. 2006). STIM1 interacts with Orai1 via its CAD domain (Luik, Wu et al. 2006, Barr, Bernot et al. 2008, Muik, Frischauf et al. 2008, Navarro-Borelly, Somasundaram et al. 2008) at a ratio of either 1:1 or 1:2 Orai1 channels forming hexameric structures (figure III.8) (Stathopulos, Schindl et al. 2013, Zhou, Wang et al. 2015).

Figure III.8: STIM1:Orai1 stoichiometric coupling models.

From Stathopulos P.B., et al. (2016)

(A) STIM1 dimers coupling to Orai1 dimers in a hexameric channel assembly derived from the solution NMR structure of STIM1 CC1-CC2 in a symmetric complex with two Orai1 C-terminal domains. (B) STIM1 dimers coupling to Orai1 monomers in a hexameric channel assembly derived from STIM1 CC2- CC3 dimer concatemers with an inactivating mutation in one subunit fully activating SOCE.

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A first interaction between the COOH terminus of Orai1 and STIM1 CAD domain occurs and allows a second interaction between STIM1 and Orai1 N terminal tail (Zheng, Zhou et al. 2013).

The precise mechanism of Orai1 gating is still ill-defined but specific amino acids in TM1 would play a crucial role (see section Orai1 structure) as mutations of either V102 or G98 lead to constitutive open channel. Orai1 opening allows Ca2+ entry and refilling of the stores as cytosolic Ca2+ is pumped back into the ER by SERCA (Liou, Fivaz et al. 2007). SOCE process ends when STIM1 and Orai1 dissociate. This is due to STIM1 conformational changes as Ca2+ ion binds to the STIM1 EF-hand region (Muik, Frischauf et al. 2008) but also because of cytosolic Ca2+ elevation that is necessary for STIM1 de-oligomerization (Shen, Frieden et al. 2011).

Finally, after de-oligomerization, Orai1 and STIM1 return to wide spread PM and ER localization respectively.

A mentioned above, STIM1 have been identified together with STIM2 as ER Ca2+ sensor supporting SOCE while Orai1 has been discovered concomitantly to other family members, Orai2 and Orai3 channels. Additionally, more recent studies have described splice variants to the canonical isoforms of which differences and specific functions begin to be unraveled. The next section will describe some features of the other members of the STIM1 and Orai1 family.

3.3. TRPC channels and other members of the Orai and STIM families 3.3.1. TRPC channels

TRPC (Transient Receptor Potential-Canonical) family of cation channels belong to the TRP (Transient Receptor Potential) superfamily that comprises six subfamilies: TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystic) and TRPML (mucolipin) in addition to the TRPC channels (figure III.9). The founding member of the family, the Drosophila trp channel, was identified in 1989 by Montell and colleagues while studied the pathway of phototactism in Drosophila melanogaster (Montell and Rubin 1989). Ever since 29 or 30 members (depending on the species) have been discovered in mammals, many of which participate in sensory signal transduction. Beyond their wide variety of ionic selectivity, mode of activation, and expression pattern, they share a structural homology. All members possess six transmembrane domains with the last two segments containing the selectivity filter.

Concerning the TRPC family, the seven members (TRPC1-7) are further characterized by a consensus motif present in the C terminal part of the molecule: Glu-TRP-Lys-Phe-Ala-Arg

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(EWKFAR) and can be activated downstream to the phospholipase C pathways. Their discovery date from the 1990’s while intensive researches aiming at identifying the Ca2+ channel supporting SOCE were pursued (Birnbaumer 2009). Whereas overexpression of several TRPCs (TRPC1-5, 7) increased SOCE, silencing of endogenous TRPC1, 2, 3, 4 and 7 decreased it in diverse cell lines (Zitt, Zobel et al. 1996, Vannier, Peyton et al. 1999, Liu, Wang et al. 2000, Freichel, Suh et al. 2001, Jungnickel, Marrero et al. 2001, Riccio, Mattei et al. 2002, Vazquez, Wedel et al. 2003, Lievremont, Bird et al. 2004, Yildirim, Kawasaki et al. 2005, Liu, Cheng et al.

2007). Additionally, heteromeric complexes between different TRPCs appear also store dependent in some cases (Zagranichnaya, Wu et al. 2005). However, electrophysiological experiments revealed that none of the TRPCs channels mediated currents displaying the features of ICRAC (Boulay, Zhu et al. 1997, Hurst, Zhu et al. 1998, Okada, Inoue et al. 1999) with high calcium selectivity and inward rectification with reversal potential > + 40mV. On the contrary, ISOC currents mediated by TRPC channels upon store depletion, together with Orai1 the prototypical ICRAC channels, are diverse showing a wide range of ionic selectivities associated with a higher conductance. Indeed, studies have shown Orai1 requirement for TRPC dependent SOCE as i) Orai down regulation abolishes SOCE despite TRPC and STIM1 expression and ii) Orai1-TRPC-STIM1 ternary complex was found in several cell types. Indeed, studies from Ambudkar’s lab revealed that ICRAC was necessary to mediate exocytosis of TRPC containing vesicles (Cheng, Liu et al. 2008, Cheng, Liu et al. 2011) although Orai1 and TRPC dependent functions are distinct. While ISOC generated by TRPC, STIM1 and Orai1 controls NFκB dependent pathways ICRAC supported by Orai1 and STIM1 regulates NFAT activation and subsequent downstream targets (Ong, Jang et al. 2012). Then, TRPC1 is nowadays considered as a store dependent channel but also TRPC4 and TRPC3 in some cell type while it is still a matter of debate for the others (Ong, de Souza et al. 2016). Indeed, if Muallem and co-workers demonstrated STIM1 ability to gate all TRPCs via electrostatic interactions between the negatively charged aspartate residues in the C terminus region of TRPCs and the positively charged lysine situated in the N terminus of STIM1, they do not interact directly in vivo (Lee, Yuan et al. 2010). Only evidences for TRPC1 and TRPC4 interaction together with STIM1 are supported by functional, FRET, TIRF and co-immunoprecipitation experiments (Huang, Zeng et al. 2006, Lopez, Salido et al. 2006, Ong, Cheng et al. 2007, Pani, Ong et al. 2008, Zeng, Yuan et al. 2008, Sundivakkam, Freichel et al. 2012). Thus, depending on the context (cell types,

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expression levels, etc.) the mode of activation of TRPC channels differs from store dependent, via STIM1 interaction, to store-independent giving them the ability to fine tune a wide range of physiological functions but complex to investigate.

Figure III.9: Scheme of the six TRP families.

Adapted from Montell, C. (2005).

Several domains are indicated: the six TM with the pore between the 5th and 6th TM, ankyrin repeats (A), coiled coil domain (cc), protein kinase domain, and the TRP domain.

3.3.2. STIM isoforms

 STIM1L

In the Bernheim’s laboratory a longer isoform of STIM1 has been identified as an alternative splicing product of the same gene occurring during myogenesis (Darbellay, Arnaudeau et al.

2011). This isoform called STIM1L has 106 amino acids more than STIM1 which are situated between exon 11 and 12 (see figure III.5). In human myotubes, STIM1L induces faster SOCE influx than the classical isoform due to its clustering near the PM and its colocalization with Orai1 before SR depletion. STIM1L is pre-localized close to the PM thanks to actin interaction throughout an Actin Binding Domain (ABD) located in the extended exon 11. Other studies have shown a higher binding capacity of STIM1L to TRPC channels either in heterologous system overexpressing TRPC3 or TRPC6 together with either STIM1 or STIM1L and in human primary myotubes with endogenous TRPC1 and TRPC4 (Horinouchi, Higashi et al. 2012, Antigny, Sabourin et al. 2017). While mainly expressed in skeletal muscle where STIM1L level are equivalent to STIM1 (Cully, Edwards et al. 2012), this isoforms is also less abundantly present in heart, brain, lung, liver and spleen at least in mice (Darbellay, Arnaudeau et al.

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2011). Another study found no STIM1L mRNA in several human tissues (heart, placentae, brain and leucocyte) as well as cell lines including pulmonary arterial smooth muscle cells (PASMC), human umbilical vein endothelial cells (HUVEC), Hela cells, and human embryonic kidney 293 (HEK293) cells, but confirms its presence in human skeletal tissue (Horinouchi, Higashi et al.

2012). The discrepancy between the 2 studies could be explained by differences between species or because of STIM1L expression variations during development. While STIM1L protein expression increases during in vitro human myoblasts differentiation and is present in human adult skeletal muscle (Darbellay, Arnaudeau et al. 2011), STIM1L is detected in neonatal rat ventricular myocytes (NRVMs) but not in adult (Luo, Hojayev et al. 2012). In the latter study, the authors also demonstrated that STIM1L was upregulated in adult heart when submitted to thoracic aortic constriction, a mouse model for blood overload induced hypertrophy and subsequent heart failure. This pathological process is mediated by NFAT signaling pathway activated via sustained elevated cytosolic Ca2+ concentration then indicating a role for STIM1L in Ca2+ signaling in heart in addition to SR refilling as suggested firstly in skeletal muscle (Darbellay, Arnaudeau et al. 2011).

 STIM2 and STIM2.1/ STIM2β

STIM2 was identified together with STIM1 through RNAi based screening for SOCE players (Liou, Kim et al. 2005, Roos, DiGregorio et al. 2005). The two molecules share 61% homology, mainly within the luminal part of both proteins. STIM2 has a lower affinity for Ca2+ (STIM2 KD

of isolated EF-hand-SAM domains 500 µM), rendering the molecule more sensible to slight ER Ca2+ variations compared to STIM1 Ca2+ (STIM1 KD of isolated EF-hand-SAM domains 200 µM)(Brandman, Liou et al. 2007, Zheng, Stathopulos et al. 2011). Consequently, STIM2 is believed to play an important role in basal Ca2+ homeostasis as it becomes active upon smaller Ca2+ depletion compared to STIM1. Recently, 2 groups identified an inhibitory STIM2 isoform namely STIM2.1 or STIM2β (Miederer, Alansary et al. 2015, Rana, Yen et al. 2015). This isoform is a STIM2 splice variant in which 8 supplemental amino acids are located into the CAD domain.

STIM2.1 or STIM2β inhibitory role is mediated through Orai1 interaction while this isoform forms multimers with either STIM1 or STIM2.

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3.3.3. Orai isoforms

All Orai proteins mediate ICRAC currents and are broadly expressed in mammals. They share high degree of identity (62%) especially in the TM domains (92%) and identical selectivity filter, conferring them similar properties in terms of store dependency for activation, high Ca2+

selectivity, I/V curve relationship, and drug sensitivity except for 2-ABP. Whereas Orai1 and Orai2 are inhibited by high dose of 2-APB (50mM), Orai3 is activated by the same amount of 2-ABP. Other slight distinctions are made between the 3 channels regarding reactive oxygen species sensitivity as well as fast and slow CDI with Orai3 being the more sensitive for fast CDI and Orai2 being insensitive to slow CDI. Additionally to their ability to mediate ICRAC current, Orai3 is believed to form heterologous Arachidonate-Regulated Ca2+ (ARC) channels with Orai1. ARC channels are pentameric complex of Orai1 and Orai3 subunits that are SOCE independent but activated by arachidonic acid (Hoth and Niemeyer 2013). Finally, an Orai1 isoform, Orai1β, has been identified as alternative translation initiation variant of the well- known Orai1 protein, its function is not yet defined (Fukushima, Tomita et al. 2012).

3.4. Physiological functions of SOCE 3.4.1. At the cellular level

SOCE has first been described as a process implicated in store refilling upon agonist induced store depletion (Putney 1986). Indeed Ca2+ release from internal stores participates in a wide variety of cell function such as activation of K+ channel in lacrimal gland (Parod and Putney 1978) via activation of IP3R as well as contraction upon repetitive stimulations in muscle cells via opening of ryanodine receptor (Kurebayashi and Ogawa 2001). Additionally, maintenance of ER Ca2+ concentration is crucial for protein synthesis and folding as several ER residing chaperone proteins are Ca2+-dependent including GRP94 (glucose-regulated protein 94), BiP (Binding immunoglobulin protein), calreticulin, calnexin, ERp57, and PDIA (Protein disulfide- isomerase) (Corbett and Michalak 2000, Gidalevitz, Stevens et al. 2013). Numerous studies have also highlighted an important role for SOCE in regulating directly Ca2+ dependent processes via local (Di Capite, Ng et al. 2009) or global cytosolic Ca2+ elevations. SOCE is an important regulator of gene transcription via activation of the calcineurin/NFAT (Nuclear factor of activated T-cells) pathway in several cell types among them T lymphocytes (Feske, Giltnane et al. 2001) and muscle cells (Stiber, Hawkins et al. 2008). Upon SOCE, Ca2+ binds to

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calmodulin allowing conformational change of the protein which can in turn interact and activate the serine/threonine phosphatase calcineurin. Activated calcineurin dephosphorylates NFAT which translocates into the nucleus and binds DNA on its target genes. In addition to gene transcription, SOCE controls exocytosis and arachidonic acid release necessary for inflammatory mediators production in RBL cells (Artalejo, Ellory et al. 1998, Ng, Nelson et al. 2009) as well as adenylyl cyclase and PM Ca2+ATPase activity (Cooper, Yoshimura et al. 1994), (Bautista and Lewis 2004) among other cellular functions explaining CRAC channelopathies.

3.4.2. At the whole body level

Genetic disorders associated with mutations in Orai1 or STIM1 gene have highlighted major roles of SOCE in human physiology including immunity, dental enamel formation, skeletal muscle and platelets development and function (figure III.10). Complementary to analysis of human patient’s phenotype, global and conditional KO mice for STIM1 and Orai1 permitted to gain insights in pathways impaired by SOCE deficiency in CRAC channelopathies.

Figure III.10: Disease phenotypes associated with mutations in ORAI1 and STIM1.

From Lacruz R.S., et al. (2015)

Null and LoF mutations in ORAI1 and STIM1 cause CRAC channelopathy,which is defined by (1) SCID- like immunodeficiency with recurrent and chronic infections, (2) autoimmunity due to autoantibody- mediated hemolytic anemia and thrombocytopenia, (3) muscular hypotonia, and (4) ectodermal dysplasia characterized by anhydrosis and defects in dental enamel development (left). GoF mutations in STIM1 and ORAI1 cause a spectrum of disease entities with partially overlapping symptoms: TAM, York platelet syndrome, and Stormorken syndrome. The arrows and white rectangles indicate organs and cell types affected by both LoF and GoF mutations in ORAI1 and STIM1 that result in similar disease manifestations, although their pathophysiology differs. AIHA, autoimmune hemolytic anemia; SCID, severe combined immunodeficiency.

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