Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

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Thesis

Reference

Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

DIDIER BULLA, Monica

Abstract

La régulation de la concentration calcique intracellulaire est essentielle à la survie et fonction de nombreux types cellulaires chez l'Homme. Un processus majeur régulant les signaux et l'homéostasie calciques associe la vidange du réticulum endoplasmique/sarcoplasmique, à l'activation de canaux Ca2+ sélectifs à la membrane plasmique. Ce processus est appelé store–operated Ca2+ entry (SOCE) et est médié par les protéines réticulaires STIM (stromal interaction molecule) et les canaux calciques membranaires ORAI. Le gain de fonction des isoformes STIM1 et ORAI1 est associé à la myopathie à agrégats tubulaires (MAT), une maladie génétique rare affectant la musculature striée squelettique et provoquant myalgie, fatigue et crampes musculaires. L'absence de thérapie pour les patients souffrant de la MAT souligne le besoin de renforcer notre compréhension du processus SOCE. Ce travail de thèse a donc pour but d'investiguer l'impact de trois mutations dans le gène ORAI1 récemment associées à la MAT.

DIDIER BULLA, Monica. Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy. Thèse de doctorat : Univ. Genève, 2018, no. Sc. Méd. 29

URN : urn:nbn:ch:unige-1138639

DOI : 10.13097/archive-ouverte/unige:113863

Available at:

http://archive-ouverte.unige.ch/unige:113863

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

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- 1 - UNIVERSITÉ DE GENÈVE

Département de physiologie cellulaire FACULTÉ DE MÉDECINE

et métabolisme Professeur N. Demaurex

___________________________________________________________________________

Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

THESE

présentée à la Faculté de Médecine de l’Université de Genève pour obtenir le titre de Docteur ès sciences médicales, MD–PhD

par

Monica DIDIER BULLA de

Genève, Suisse

Thèse n° 29

Genève 2018

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List of abbreviations

2–APB 2–aminoethoxydiphenyl borate ABD Actin–binding domain

AM Acetoxymethyl ester

ARC arachidonate–regulated calcium–selective BHQ 2,5–di(tert–butyl)–1,4–benzohydroquinone BTP2 3,5–bis(trifluoromethyl)pyrazole

C2 Conserved calcium binding domain CAD CRAC activation domain

CaM Calmodulin CASQ Calsequestrin CC Coiled–coil domain

CDI Calcium–dependent inactivation, FCDI (fast), SCDI (slow) cER Cortical ER

CK Creatine kinase

CN Calcineurin

CPA Cyclopiazonic acid CRACR CRAC channel regulator DAG Diacylglycerol

DHPR Dihydropyridine receptor DVF Divalent–free

ECC Excitation–contraction coupling EM Electron microscopy

ER Endoplasmic reticulum Erev Reversal potential

FRET Fluorescence resonance energy transfer H2O2 Hydrogen peroxide

HCX Proton-calcium exchanger HEK Human embryonic kidney cells I/V Current–voltage relationship

ICRAC Calcium release–activated calcium current ID Inhibitory domain

IP3 Inositol trisphosphate Kd Dissociation constant

KO/DKO Knock–out, double knock-out LGCC Ligand–gated calcium channel

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- 4 - MCS Membrane contact sites

MDS Molecular dynamic simulations MEF Mouse embryonic fibroblasts

NADH–TR Nicotinamide adenine dinucleotide–tetrazolium reductase NCX/NCLX Sodium-calcium exchanger

NFAT Nuclear factor of activated T cells NK Natural killer

NMDG N–methyl–d–glucamine

PIP2 Phosphatidylinositol 4,5–bisphosphate PKC Protein kinase C

PLC Phospholipase C

PM Plasma membrane

PMCA Plasma membrane calcium ATPase POST Partner of STIM

ROC Receptor–operated channel ROS Reactive oxygen species RyR Ryanodine receptor SAC Stretch–activated channel SAM Sterile alpha motif

SCDI Severe combined immunodeficiency SDH Succinic dehydrogenase

SERCA Sarco/endoplasmic reticulum calcium ATPase SOC Store–operated channel

SOCE Store–operated calcium entry SPCA Secretory–pathway calcium ATPase SR Sarcoplasmic reticulum

SS Signal sequence

STIM Stromal interaction molecule STIMATE STIM–activating enhancer

Syt Synaptotagmin

TAM Tubular aggregate myopathy

Tg Thapsigargin

TIRF Total internal reflection fluorescence

TM Transmembrane domain

TRP Transient receptor potential channel VOC Voltage–operated channel

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I. Résumé

La régulation de la concentration calcique intracellulaire est essentielle à la survie et à la fonction de nombreux types cellulaires chez l’Homme. Dans une cellule au repos, la concentration cytosolique de calcium (Ca²⁺) est faible. Avec l’arrivée d’un potentiel d’action ou l’activation d’un récepteur de surface, la mise en place de tout un système, composé de récepteurs, seconds messagers, canaux calciques, protéines liant le Ca²⁺ ou pompes et échangeurs membranaires permet d’orchestrer et réguler le signal calcique, et de finalement restaurer l’homéostasie cellulaire. La combinaison de tous ces acteurs diffère d’un tissu à l’autre pour répondre aux besoins spécifiques de chaque cellule.

Un processus majeur régulant les signaux et l’homéostasie calciques associe la vidange du réticulum endoplasmique/sarcoplasmique (RE/RS), lieu principal du stockage de Ca²⁺, à l’activation de canaux Ca²⁺ sélectifs à la membrane plasmique (MP). Ce processus est appelé store–operated Ca²⁺ entry (SOCE) et est médié par les protéines STIM (stromal interaction molecule) et ORAI. STIM est une protéine transmembranaire du RE comportant un domaine de liaison au Ca²⁺ (main EF) dans sa partie luminale. Lors de la déplétion des stocks calciques, la fuite des ions Ca²⁺ libèrent les protéines STIM est permettent leur oligomérisation et leur mouvement vers la MP où ils établissent un contact avec les canaux ORAI. STIM et ORAI se regroupent en « clusters » dans des zones restreintes où la membrane du RE s’appose à la MP appelées sites de contacts membranaires (SCM). Lors du remplissage des stocks, les protéines STIM quittent ces SCM et les canaux ORAI sont redistribués à la surface cellulaire. L’étude de la structure de l’isoforme Orai1 dans la Drosophile a permis de montrer que les sous–unités d’Orai1, composées de quatre domaines transmembranaires (TM1–TM4), s’assemblent symétriquement autour d’un axe central pour former un hexamère avec trois cercles concentriques : le premier formé de domaines TM1 est le pore du canal, le deuxième (fait de TM2 et TM3) et le troisième (TM4) maintiennent la structure. De nombreuses études ont permis d’identifier les acides aminés essentiels au maintien de la fonction d’ORAI1 tels que le filtre de sélectivité calcique (E106), ceux contrôlant l’ouverture du pore (R91 et V102), les résidus responsables de la sensibilité aux oxydants ou aux changements de pH, et ceux permettant la liaison de STIM1 à l’extrémité C-terminale d’ORAI1.

Une multitude de modulateurs permettent de potentialiser ou d’inhiber le processus SOCE, parmi lesquels des protéines cytosoliques, du RE ou de la MP, des facteurs physiques tels que le pH ou la température, des composés chimiques, et les ions Ca²⁺ eux–mêmes. Ainsi, la mise en place de mécanismes compensatoires permet le maintien de l’homéostasie calcique en cas de dysfonction des protéines STIM ou ORAI. Cependant, lorsque cette compensation devient insuffisante, l’altération des fonctions tissulaires mène au développement de maladies immunes ou musculaires. Le gain de fonction des isoformes STIM1 et ORAI1 est associé à la myopathie à agrégats tubulaires (MAT), une maladie génétique rare affectant la musculature striée squelettique et provoquant myalgie, fatigue et crampes musculaires. L’accumulation de tubules sarcoplasmiques dans les fibres musculaires ainsi que l’élévation des taux sériques de créatine kinase (CK) sont deux caractéristiques utilisées dans le diagnostic de la MAT. L’absence de thérapie pour les patients souffrant d’une telle maladie souligne le besoin de renforcer

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notre compréhension du processus SOCE et de ses dysfonctions, ainsi que l’urgence de développer de nouvelles stratégies thérapeutiques pour rétablir la balance calcique dans les tissus affectés.

Ce projet de thèse à pour but d’investiguer l’impact de trois mutations dans le gène ORAI1 récemment associées à la MAT : la substitution G98S dans le domaine TM1, la mutation V107M proche du filtre de sélectivité, et la substitution T184M dans le domaine TM3. La collaboration avec le groupe de Jocelyn Laporte à Strasbourg a permis la publication d’un premier article visant à déterminer l’impact clinique, histologique et fonctionnel de ces mutations, et d’établir une corrélation entre génétique et phénotype. La mutation G98S est associée à des symptômes musculaires et extra–musculaires sévères, la mutation V107M à des symptômes plus modérés, et la mutation T184M à une élévation asymptomatique des taux sanguins de CK.

Dans les trois cas, les biopsies musculaires montrent une accumulation comparable de tubules sarcoplasmiques et l’agrégation des protéines sarcoplasmiques STIM1, RyR1 et SERCA1.

L’étude microscopique des canaux ORAI1 mutés montre une distribution à la MP homogène au repos, et une cinétique de formation de « clusters » conservée après activation. L’utilisation de la sonde calcique intracellulaire Fura–2 a permis l’étude du processus SOCE et a montré une entrée excessive de Ca²⁺ dans les cellules HEK–293T sur–exprimant les différentes formes de canaux mutés. Lorsqu’ils sont exprimés dans une lignée de fibroblastes déficients en STIM, les canaux ORAI1–G98S et –V107M sont actifs de manière constitutive, le canal ORAI1–T184M n’étant activé qu’en présence de STIM. Aussi, la sévérité des phénotypes calciques de ces mutants corrèle étroitement avec l’intensité des signes cliniques observés dans les patients, la mutation G98S étant associée aux symptômes les plus handicapants. Dans la seconde partie du projet, nous avons cherché à décrire plus précisément comment les mutations V107M et T184M affectent les propriétés biophysiques du canal. L’étude des courants ioniques médiés par ces mutants montre que la mutation V107M perturbe la sélectivité au Ca²⁺ du canal ainsi que sa sensibilité aux modulations par le pH. La sensibilité au pH et aux oxydants est cependant conservée chez le mutant T184M. Les expériences de microscopie calciques révèlent une sensibilité à l’activation STIM1–dépendante accrue pour ces deux mutants, et établissent l’efficacité de la molécule GSK–7975A dans l’inhibition des deux canaux dérégulés. Par ailleurs, une collaboration a permis d’identifier un nouveau gène CASQ1 (calséquestrine 1) comme étant impliqué dans le développement de la MAT, où les mutations N56Y et G103D préviennent la polymérisation de la protéine.

Ce projet propose donc que les mutations G98S et V107M dans le domaine TM1 d’ORAI1 déstabilisent le pore du canal ; la mutation V107M déplace le filtre de sélectivité E106, augmentant la perméabilité aux cations monovalent et aux ions Ca²⁺, et diminuant sa sensibilité à la modulation par le pH ; les mutations V107M et T184M promeuvent la transmission du signal d’ouverture du canal médiée par STIM1. Pour finir, l’efficacité inhibitrice du GSK–7975A sur deux de ces canaux supporte la considération de cette molécule dans les futures stratégies thérapeutiques pour les maladies telles que la MAT.

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II. Abstract

The tight regulation of intracellular calcium (Ca²⁺) levels is essential for human cells survival and for the control of a broad range of cellular functions. In a resting cell, the cytosolic Ca²⁺

concentration is low. Upon voltage or agonist–induced activation, the fine orchestration of receptors, second messengers, Ca²⁺ channels, Ca²⁺–binding proteins and Ca²⁺ pumps or exchangers allows Ca²⁺ influx, regulates Ca²⁺ signals and restores Ca²⁺ homeostasis. The combination of these different Ca²⁺ actors varies among human tissues and tunes the Ca²⁺

signal respectively to each tissue specific function.

A major process controlling Ca²⁺ balance and signaling is store–operated Ca²⁺ entry (SOCE), a mechanism that combines Ca²⁺ release from endoplasmic/sarcoplasmic (ER/SR) Ca²⁺ stores and the opening of Ca²⁺ channels at the plasma membrane (PM). Store depletion is sensed by the ER transmembrane protein STIM (stromal interaction molecule) that carries a Ca²⁺ binding EF–hand motif in its luminal portion. Ca²⁺ unbinding from STIM allows the protein to oligomerize and move towards ER–PM membrane contact sites (MCS), where it binds, clusters and gates the ORAI Ca²⁺ selective channel. Once ER/SR stores are refilled, STIM proteins leave MCS, and ORAI channels close and redistribute along the PM. The recently published crystal structure of the Drosophila melanogaster Orai1 isoform showed that the four transmembrane domains (TM1–TM4) of the Orai1 subunit form a symmetric and hexametric structure with a central TM1 ring followed by two outers rings (made of TM2–TM3 and TM4 respectively).

Several other studies identified ORAI1 residues controlling essential features such as the Ca²⁺

selectivity filter E106, the R91 and V102 channel gates, three oxidant reactive cysteines, several internal and external pH sensing residues, and the C–terminal leucines essential for STIM1 binding.

An extensive list of modulators, among which ER, PM or cytosolic proteins, physical factors, drugs and Ca²⁺ itself, potentiate or inhibit SOCE. When the function of STIM or ORAI is affected, a number of compensation mechanisms take place to maintain Ca²⁺ homeostasis. But when these mechanisms are overloaded, tissue function is impaired, leading to immune or muscular diseases. Gain–of–function mutations in STIM1 and ORAI1 isoforms lead to tubular aggregate myopathy (TAM). TAM is a rare genetic skeletal muscle disorder leading to muscular pain, weakness and cramps. The accumulation of sarcoplasmic tubules in muscle fibers and the elevation of the muscle–damage marker creatine kinase (CK) are two characteristics of TAM used for diagnosis. The lack of therapeutics for this disorder strengthen the urge to understand how SOCE dysregulation induces TAM and to develop new tools to reestablish Ca²⁺ balances in affected tissues.

This project focuses on three newly described ORAI1 gain–of–function mutations associated with TAM: G98S standing in TM1, V107M neighboring the selectivity filter, and T184M in TM3. The collaboration with the group of Jocelyn Laporte in Strasbourg lead to a first publication aiming to determine the clinical, histological and functional impact of these mutations, and to establish a phenotype–genotype correlation. Mutation G98S lead to very severe muscular symptoms, mutation V107M was associated with moderated clinical signs, and the patient with mutation T184M showed asymptomatic CK elevations. All muscle biopsies

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showed similar accumulation of sarcoplasmic tubules and aggregation of the sarcoplasmic STIM1, RyR1 and SERCA1. Microscopy experiments showed a normal homogeneous distribution of mutated channels at rest and normal clustering kinetics upon activation. SOCE mediated by the mutants was studied thanks to the Ca²⁺ sensitive dye Fura–2 and was increased when each of the channels were expressed in HEK–293T cells. Additionally, ORAI1–

G98S and –V107M showed constitutive activity, as demonstrated by the manganese quench assay in STIM deficient mouse embryonic fibroblasts, whereas –T184M activity was only revealed in the presence of STIM. Notably, the severity of G98S, V107M and T184M phenotypes strongly correlated with the clinical observations in patients, with G98S being the most disabling. In the second part of the project, we proposed to further investigate how mutations V107M and T184M alter the biophysical properties of the channel. ORAI1 activity was assessed by patch clamp and revealed the loss of selectivity and pH sensitivity of the mutated V107M channel, while T184M pH and H2O2–mediated regulation were conserved.

Additionally, Ca²⁺ imaging experiments showed an enhanced sensitivity for STIM1–mediated gating and an efficient inhibition of both mutant activities with the ORAI specific blocker GSK–

7975A. Finally, participation in a third study identified CASQ1 (calsequestrin 1) as a new gene affected in TAM cases, where mutations N56Y and G103D impaired protein polymerization.

From this work, we propose that ORAI1 mutations G98S and V107M in the TM1 helix destabilize the channel pore; that additionally, mutation V107M displaces the E106 selectivity filter, increases monovalent cation and Ca²⁺ ions permeation, and decreases the channel sensitivity to acidic block; and that V107M and T184M mutations promote the transmission of the STIM1 gating signal to facilitate ORAI1 permeation. Finally, the consideration of GSK–7975A as a potential drug for TAM patients is supported by its efficient inhibition of the overactive V107M and T184M channels.

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III. Introduction

Calcium (Ca²⁺) stands among the most common intracellular second messengers and governs various cellular functions such as proliferation, differentiation, contraction, motility, secretion and apoptosis (Berridge et al., 2000; Clapham, 2007). Its essential role was first described by an experiment of Sydney Ringer, where the addition of CaCl2 to a heart perfusion restored and sustained ventricular contractions (Fig. 1) (Ringer, 1883). Over the past century, thorough studies allowed to portray a rather exhaustive picture of the Ca²⁺ signaling process.

However, how this universal second messenger could specifically regulate so many different cellular functions is still under intense investigation. The answer resides in the remarkable flexibility of the system, where elements of a wide–ranging Ca²⁺ signaling “toolkit” can be diversely arranged depending on tissue specificity (Berridge et al., 2003).

Figure 1. Effect of blood mixture and CaCl2 on heart ventricular contractions. From Ringer (1883).

The top panel shows the effect of substituting ventricular saline perfusion with a blood mixture (indicated by an arrow) on ventricular contractility. Solution substitution elicited contractions that weakened with time. The lower panel shows that CaCl2 perfusion (arrow) restored and sustained ventricular contractility.

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1. Tuning calcium signals

The resting cytosolic Ca²⁺ concentration averages approximatively 100 nM and drastically increases during a signaling event. The sources of Ca²⁺ ions are multiple: the extracellular environment contains ~ 1.2 mM Ca²⁺ in physiological conditions, and organelles such as the sarco/endoplasmic reticulum (SR/ER) – known as the main Ca²⁺ intracellular stores – the Golgi apparatus, mitochondria and vesicles hold Ca²⁺ ions in the μM to mM range (Ashby and Tepikin, 2001). The cell possesses an extensive Ca²⁺ signaling “toolkit” made of receptors, transducers, channels, Ca²⁺ binding proteins, pumps and exchangers that, combined, shape the spatio–

temporal pattern of the Ca²⁺ signal; the versatility of this signal determines the downstream cellular effect (Berridge et al., 2000; Berridge et al., 2003). In Purkinje neurons for example, a fast (microseconds) and confined submembrane presynaptic Ca²⁺ increase, or Ca²⁺

microdomain, controls vesicles fusion and neurotransmitters release in the synaptic cleft (Denk et al., 1995). In T lymphocytes however, global and repeated (minutes to hours) Ca²⁺ transients, or oscillations, are needed to promote gene expression and cell proliferation (Lewis, 2003).

These examples illustrate how tightly Ca²⁺ signals are regulated and how they affect cellular functions.

1.1. Receptors and transducers

To mobilize Ca²⁺ into the cytosol, a broad range of receptors at the cell surface translate various stimuli such as membrane depolarization, mechanic stress or presence of agonists, into cytosolic Ca²⁺ rises. The muscarinic, bradykinin or histamine G–protein coupled receptors associate the binding of an agonist to the activation of downstream cellular kinases (Gilman, 1987). In the case of tyrosine–kinase–linked receptors, the engagement of the appropriate signaling molecule prompts receptor multimerization and the generation of phosphorylation cascades (Ullrich and Schlessinger, 1990). In non–excitable cells, the stimulus is relayed by transducers (G proteins, Ras, etc.) to phospholipase C (PLC), that catalyzes the breakdown of phosphatidylinositol 4,5–bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3) (Kadamur and Ross, 2013). The latter binds to its respective ligand–gated Ca²⁺ channel IP3R at the ER membrane and allows the release of Ca²⁺ ions from intracellular stores (Fig. 2A) (Berridge, 1997). In muscles cells, membrane depolarization prompts Ca²⁺ entry by inducing a conformational change of the membrane voltage–sensitive Ca²⁺ channels DHPR (dihydropyridine receptor). This signal is transmitted to ryanodine receptors (RyR) – the SR

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equivalent of IP3R – that directly interact with DHPR and promotes Ca²⁺ release, through a process called excitation–contraction coupling (ECC) (Fig. 2B) (Wang et al., 2001; Protasi, 2002).

Figure 2. Ca²⁺ release from the ER/SR stores.

(A) Scheme of PLC-mediated IP3 production in a non-excitable cell. After activation of G-coupled receptors at the plasma membrane (PM), the G protein alpha subunit activates PLC. PLC catalyzes the breakdown of PIP2 from the PM inner leaflet into DAG and IP3. IP3 binds and gates its respective receptor IP3R on the ER membrane, allowing the release of Ca²⁺ ions from the stores.

(B) Excitation-contraction coupling (ECC) scheme in an excitable cell (muscle).

1) The propagation of an action potential (AP) from the neuromuscular junction (NMJ) depolarizes the muscle membrane (sarcolemma), inducing the opening of voltage-sensitive Ca²⁺ channels DHPR and leading to Ca²⁺ influx into the muscle fiber.

2) DHPR directly interact with RyR receptor-channels at the SR membrane. DHPR gating induces a conformational change of RyR that then opens and allows Ca²⁺ release from the SR stores.

3) Cytosolic Ca²⁺ elevations triggers the contraction of myofilaments.

4) SR stores are further refilled thanks to the SR Ca²⁺ ATPase SERCA pump.

B

A

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Each receptor, with its specific activation/regulation mechanisms, kinetics, transducers and PLC isoform therefore leads to different levels of IP3 generation and Ca²⁺ signaling patterns;

large and rapid Ca²⁺ transients control exocytosis while small, slow and long lasting Ca²⁺ waves are required for cell proliferation (Fig. 3). Besides, the phosphorylation state of IP3R receptors as well as Ca²⁺ ions themselves modulate the activity of IP3R. Elevated luminal Ca²⁺

concentrations or low cytosolic Ca²⁺ levels increase IP3R sensitivity to its ligand (Taufiq Ur et al., 2009; Vanderheyden et al., 2009). These numerous layers of regulation add complexity to Ca²⁺ signaling.

Figure 3. Spatio-temporal pattern of Ca²⁺ signaling. Modified from Berridge et al. (2003)

“On” reactions: Following a stimulus-mediated activation, second messengers trigger the opening of RyR or IP3R and subsequent Ca²⁺ release form the ER/SR stores. Ca²⁺ elevations are sustained by the opening of plasma membrane (PM) Ca²⁺ channels. Most Ca²⁺ ions are buffered by Ca²⁺-binding proteins. A small proportion though associates with Ca²⁺-dependent effectors to activate downstream cellular functions. The duration of the Ca²⁺ signal determines the outcome: brief Ca²⁺

elevations allow vesicular fusion and exocytosis while long-lasting Ca²⁺ waves promote gene transcription or cell proliferation.

“Off” reactions: The signal ends when Ca²⁺ ions are extruded by PM pumps or exchangers (PMCA and NCX), or are sequestered in ER/SR stores or mitochondria (by SERCA and mitochondrial uniporter, NCLX).

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1.2. Ca

²⁺

channels

To build complex Ca²⁺ signals, an IP3R single–channel event is transmitted to neighboring IP3R clusters through saltatory propagation and leads to their coordinated opening (Keizer et al., 1998). But in most cases, because of the limited Ca²⁺ capacity of the ER/SR stores, Ca²⁺

release alone is not sufficient to activate downstream pathways. Additional Ca²⁺ from the external compartment is then supplied by Ca²⁺ permissive channels at the plasma membrane (PM). These latter are divided in several classes: the voltage–operated channels (VOC), the stretch–activated channels (SAC), the receptor–operated channels (ROC), the store–operated channels (SOC) and the transient receptor potential (TRP) channels.

Among them, the most thoroughly studied and described are VOCs, which are further sub–

classified in L (long–lasting), T (transient), R (resistant or residual), N (neural), and P/Q (Purkinje, cerebellar) –types, depending on their tissue distribution, voltage sensitivity or gating kinetics. DHPR belongs to the L–type sub-family. Besides directly interacting with RyR, DHPR–mediated Ca²⁺ influx induces Ca²⁺ release from the SR, by a process called “Ca²⁺–induced Ca²⁺ release”. In muscle cells, Ca²⁺ entry through DHPR is not sufficient by itself, and summation of Ca²⁺ influx and release is necessary to sustain contraction (Endo, 2009).

ROCs opening relies on the action of agonists in a non voltage–dependent manner. They are mostly described in neurons, and are at the origin of several calcium–dependent signaling cascades controlling trafficking, synaptic plasticity, cell survival and death (Kennedy and Ehlers, 2006; Tada and Sheng, 2006). Glutamate binding on NMDA receptors, for instance, induces synaptic remodeling by evoking localized Ca²⁺ transients in dendritic spines (Hunt and Castillo, 2012).

The SOC channel group comprises ORAI and several TRP channels where gating is coupled to the depletion of ER/SR stores, a mechanism called store–operated Ca²⁺ entry (SOCE). SOCE and ORAI channels are further discussed in the Introduction points 2 and 3. TRP channels are mostly non–selective cation channels expressed in almost all mammalian cells and divided in seven subgroups: TRPC (“canonical”), TRPV (“vanilloid”), TRPM (“melastatin”), TRPN (“no mechanoreceptor potential C”), TRPA (“ankyrin”), TRPP (“polycystin”) and TRPML (“mucolipin”). Because of their small Ca²⁺ conductance, a long–lasting activity of TRP channels sustains the Ca²⁺ signal without risking Ca²⁺ overload and toxicity (Clapham et al., 2001; Minke and Cook, 2002). Therefore, TRPs are often involved in slow cellular processes such as cell proliferation or sustained muscle contraction (Birnbaumer, 2009). Their gating depends on various chemical and physical stimuli (ligand binding, second messenger, changes in

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temperature or mechanical force), and recent publications also linked TRPC1, TRPC4 and TRPC5 to store–depletion (Salido et al., 2009; Antigny et al., 2013; Antigny et al., 2017).

1.3. Ca

²⁺

–binding proteins

The availability of free cytosolic or ER/SR Ca²⁺ ions is actually very limited, as most of it is buffered by Ca²⁺ binding proteins: Ca²⁺ buffers and Ca²⁺ effectors, that mainly possess one or several putative helix–loop–helix EF–hand motifs. With their low Ca²⁺ affinity and high Ca²⁺

capacity, reticular Ca²⁺ buffers (e.g. calnexin, calreticulin, calsequestrin) are able to sequester large amounts of Ca²⁺ ions in the stores that are rapidly mobilized upon store depletion (Prins and Michalak, 2011). Cytosolic Ca²⁺ buffers (e.g. calbindin D–28, calretinin, parvalbumin) not only maintain Ca²⁺ gradients between the cytosol and Ca²⁺ rich compartments, but also shape the amplitude and the spatio–temporal magnitude of Ca²⁺ signals, depending on their Ca²⁺

binding ratio and diffusional range (Schwaller, 2010). Additionally, Ca²⁺ buffers, together with the Ca²⁺ extrusion system, prevent Ca²⁺ overload and toxicity (Farber, 1990; Carafoli et al., 2001; Celsi et al., 2009).

Mitochondria also play a major role in Ca²⁺ buffering and signaling. Even though mitochondria were the first organelles shown to be able to accumulate Ca²⁺ ions (Carafoli, 1979), their contribution to Ca²⁺ homeostasis was lengthily discussed. Extensive work over the past 30 years allowed the identification of the “MCU (mitochondrial Ca²⁺ uniporter) complex”

and confirmed its role in mitochondrial Ca²⁺ uptake and signaling (Baughman et al., 2011; De Stefani et al., 2011; Marchi and Pinton, 2014). It is now commonly accepted that mitochondria sense and relocate to Ca²⁺ microdomains to sequester Ca²⁺ ions (Quintana et al., 2006;

Quintana et al., 2007; Quintana and Hoth, 2012) and that they participate in Ca²⁺ redistribution by communicating with the ER/SR (Szymanski et al., 2017).

Besides buffering Ca²⁺ ions, some proteins that undergo conformational changes upon Ca²⁺

binding carry out biological functions. The most famous and universal Ca²⁺ effector is calmodulin (CaM), a protein possessing four EF–hands (Zhou et al., 2009) and being at the crossing point of many intracellular pathways. In its Ca²⁺ bound conformation, CaM activates specific kinases that in turn initiate metabolic cascades, gene expression, and many other processes (Vogel, 1994). The Ca²⁺–binding properties of CaM were exploited to develop FRET (fluorescence resonance energy transfer)–based Ca²⁺ sensors (Fig. 4) that, by introducing mutations modifying the protein affinity for Ca²⁺, could report Ca²⁺ changes in different cellular compartments (cytosol, ER, nucleus), in vitro and in vivo (Miyawaki et al., 1997; Fiala and Spall,

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2003). Among multifunctional Ca²⁺ binding proteins, S100 was shown to be engaged in the Ca²⁺–depended regulation of protein phosphorylation, enzymatic activity, cell structure, growth, differentiation and communication (Donato, 1999). Synaptotagmin 1 (Syt1) is a Ca²⁺

sensing transmembrane protein first described in neuronal synaptic vesicle docking and neurotransmitters release. Syn1 senses Ca²⁺ elevations thanks to its C2 (conserved Ca²⁺

binding) domains, and facilitates synaptic vesicle fusion (Fernandez-Chacon et al., 2001).

Similarly, the cytosolic protein kinase C (PKC) senses DAG and Ca²⁺ elevations and triggers vesicular fusion (Newton, 1995; Vaughan et al., 1998). The ER/SR resident stromal interaction molecules (STIMs) are major protagonists of the SOCE process that sense negative changes in the luminal Ca²⁺ content and couple store depletion to the gating of PM Ca²⁺ channels (discussed in section 2). Lastly, numerous Ca²⁺ sensitive enzymes (CaM kinase, PKC, adenylate cyclase, nitric oxide synthase, calcineurin, calpain, etc.) complete the very long list of Ca²⁺

effectors and illustrate again the central role of Ca²⁺ as a second messenger and the versatility of the Ca²⁺ signal (Berridge et al., 2003).

Figure 4. The FRET-based Ca²⁺ sensor “cameleon”. From Fiala and Spall (2003).

A cyan (ECFP) is connected to a yellow (EYFP) fluorescent protein by the Ca²⁺-binding region of CaM (gray) and the M13 peptide (orange). M13 is a synthetic peptide holding a CaM-binding domain. In the Ca²⁺ unbound form of the cameleon probe, ECFP emits a 485 nm light when excited at 440 nm.

Upon Ca²⁺ binding to CaM, the protein changes conformation and allows ECFP emission to excite EYFP. The reduction of 485 nm and the increase of 535 nm emissions indicate a Ca²⁺ rise.

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1.4. Ca

²⁺

pumps and exchangers

The term “Ca²⁺ homeostasis” implies a cellular equilibrium, meaning that whatever floods the cytosol needs to be extruded or taken up by organelles to restore the resting state. To do so, cells dispose of Ca²⁺ pumps and exchangers at plasma, ER/SR or mitochondrial membranes.

The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) uses the energy from ATP hydrolysis to actively import Ca²⁺ ions into the stores (Brini et al., 2000). Ca²⁺ buffers such as calreticulin for the ER or calsequestrin (CASQ1) for the SR sustain SERCA activity by decreasing the gradient of free Ca²⁺ ions in the reticulum lumen. Istaroxime, a drug undergoing phase III clinical trials for acute heart failure, displays lusitropic (improvement of diastole recovery) and inotropic (support of systolic contraction) characteristics by combining the inhibition of the PM Na⁺/K⁺– ATPase and the potentiation of the SERCA2a pump isoform, thereby regulating Ca²⁺ cycling (Khan et al., 2009). On the contrary, phospholamban and sarcolipin negatively regulate SERCA activity in skeletal and myocardial muscles (Espinoza-Fonseca et al., 2015). Thapsigargin (Tg), a plant–derived tumour inducing agent, commonly used in the Ca²⁺ experimental field, irreversibly blocks SERCA pumps to allow the passive depletion of the ER/SR stores (Thastrup et al., 1989). This leak is mediated by the translocon on the ER membrane (Flourakis et al., 2006). PMCA (plasma membrane Ca²⁺ ATPase) is the PM equivalent of SERCA. The expression of the four PMCA isoforms is tissue–specific. CaM and PM phospholipids were shown to respectively increase PMCA affinity for Ca²⁺ ions and keep the pump in a permanently activated state (Berrocal et al., 2017). Interestingly, Ca²⁺ itself is a powerful modulator of PMCA, where high Ca²⁺ concentrations upregulate PMCA expression levels and promote alternative splicing (Krebs, 2009). Lastly, the Golgi secretory–pathway Ca²⁺ ATPase (SPCA) is a non–selective divalent pump that takes up Ca²⁺ and Mn²⁺ not only to shape the Ca²⁺ signal, but also to regulate protein trafficking, maturation and secretion (Wuytack et al., 2002).

Because of their high–affinity/low–capacity for Ca²⁺ ions, Ca²⁺ ATPases are considered as fine tuners of cytosolic Ca²⁺ levels. Ca²⁺ exchangers though exhibit a low affinity but high capacity for Ca²⁺ ions and are preferentially expressed in excitable tissues (Blaustein and Lederer, 1999). NCX, the PM 3Na⁺/1Ca²⁺ antiporter, takes advantage of the Na⁺ gradient and electrochemical force to extrude cytosolic Ca²⁺ ions, at a very fast rate. The mitochondrial NCLX and HCX antiporters however, feed the cytosol with Ca²⁺ ions in exchange of Na⁺ or H⁺ respectively. As antiporters depend on the energy provided by the ions gradients, the Ca²⁺ flow direction can be reversed, and low extracellular Na⁺ contents favor Ca²⁺ uptake by NCX.

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The broad variety of Ca²⁺ channels, binding proteins, pumps and exchangers, as well as their respective kinetics and specific regulation illustrates the versatility of the Ca²⁺ signaling machinery. Each cell can therefore combine its own “Ca²⁺ toolkit” and organize it in localized independent or communicating subcellular complexes to pursue specialized tasks. Moreover, the faculty of Ca²⁺ ions themselves to directly or indirectly control the signal and remodel the

“Ca²⁺ toolkit” expression provides the cell with a fantastic adaptation capacity to compensate for gain or loss of functions. Nevertheless, when compensation is overcome, Ca²⁺ homeostasis is compromised, leading to impaired tissue functions and multisystemic diseases (Carafoli et al., 2001).

2. Store–operated Ca

²⁺

entry (SOCE)

In 1986, Putney proposed the model of “capacitative Ca²⁺ entry”, and placed IP3 at the central position of controlling and linking Ca²⁺ release to Ca²⁺ entry (Putney, 1986). He named the ER compartment the “Ca²⁺ capacitor”, based on the assumption that the ER was in continuity with the extracellular compartment, and that Ca²⁺ ions first transit through the ER before being delivered to the cytosol. Shortly after, it was uncovered that Ca²⁺ ions actually cross the PM before reaching the ER stores, and the original model was revised and renamed

“store–operated Ca²⁺ entry” (Putney, 1990).

At that time, a lot of effort was invested in the determination of the Ca²⁺ channel responsible for SOCE. The combination of Ca²⁺ imaging and whole–cell electrophysiology in rat mast cells and human T lymphocytes identified the current mediated by this SOCE channel (Hoth and Penner, 1992; Zweifach and Lewis, 1993). Recordings described a current with the following characteristics:

- slow development

- small amplitude (pA range)

- small unitary conductance (based on low–noise analysis) - inwardly rectifying current–voltage (I/V) relationship - very high Ca²⁺ selectivity: reversal potential (Erev) > 40 mV - Ca²⁺:Na⁺ permeability ratio > 1,000

- voltage–independent gating

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- activation by IP3 agonists, intracellular IP3 or Ca²⁺ chelators - potentiation by extracellular Ca²⁺

- inhibition by intracellular Ca²⁺, called “Ca²⁺–dependent inhibition” (CDI) - sensitivity to heavy metals (blocked by Gd³⁺, La³⁺, Ni²⁺ and Cd²⁺)

and consequently named it “Ca²⁺ release–activated Ca²⁺” (CRAC) current (ICRAC) (Fig. 5).

The introduction of SERCA inhibitors was decisive in the association of ICRAC to SOCE, and Tg, cyclopiazonic acid (CPA) or 2,5–di(tert–butyl)–1,4–benzohydroquinone (BHQ) are now intensively used to study the function of Ca²⁺ stores and unravel the complexity of Ca²⁺

signaling (Table 1) (Moore et al., 1987; Goeger et al., 1988; Thastrup et al., 1990; Lytton et al., 1991; Michelangeli and East, 2011). But, the major revolution was brought by wide RNA interference screenings and the identification of SOCE/CRAC key players: the Ca²⁺ channel ORAI, and its gating ligand STIM (Liou et al., 2005; Roos et al., 2005; Feske et al., 2006; Zhang et al., 2006; Soboloff et al., 2012).

Figure 5. The Ca²⁺ release-activated Ca²⁺ current (ICRAC).

ICRAC current-voltage (I/V) relationship recorded in a HEK-293T cell overexpressing STIM1 and ORAI1 and exposed to 10 mM CaCl2. The intracellular compartment is dialyzed with 10 mM EGTA and 2 µM thapsigargin to allow passive store depletion. The I/V curve shows an inwardly rectifying pattern with a very positive reversal potential (Erev), a sign of high Ca²⁺

selectivity.

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2.1. STIM and ORAI

STIM isoforms and splicing variants

The ubiquitous STIM family members are ER Ca²⁺ sensors highly conserved among species.

They are type I single–pass ER proteins with a luminal N–terminus and a cytosolic C–terminus.

In humans, the family includes two isoforms, STIM1 and STIM2 that share most of their sequence (Fig. 6). Their luminal portion holds an N–terminal ER targeting signal sequence (SS), two EF hand motifs, one canonical (cEF) and one non–canonical “hidden” (hEF), followed by a sterile alpha motif (SAM) and the transmembrane domain. The cytosolic portion possesses three coiled–coil domains (CC1, CC2 and CC3), an inhibitory domain (ID) involved in CDI, and a polybasic (lysine–rich) tail interacting preferentially with PIP2 to anchor the protein at the PM upon store depletion (Liou et al., 2007; Derler et al., 2009; Korzeniowski et al., 2009; Walsh et al., 2009; Soboloff et al., 2012; Bhardwaj et al., 2013; Bhardwaj et al., 2016). The main differences between STIM1 and STIM2 lie in the affinity of their respective EF hands for Ca²⁺

ions, and the affinity of their polybasic tail for PIP2. The high Ca²⁺ dissociation constant (Kd) of STIM2 (~ 0.4 mM, compared to ~ 0.2 mM for STIM1) predispose this isoform to sense very small changes in ER Ca²⁺ content (Zheng et al., 2008). Additionally, the high affinity of the STIM2 polybasic tail for lipids stabilizes the protein’s localization at the PM in resting conditions (Parvez et al., 2008), thereby allowing STIM2 to quickly respond to discrete ER Ca²⁺ variations and assigning STIM2 to more homeostatic roles.

Alternative splicing of STIM1 produces a longer isoform (STIM1L) with an additional cytosolic actin–binding domain (ABD) that influences the protein’s distribution (Darbellay et al., 2011; Sauc et al., 2015). STIM1L is mainly expressed in the heart, in the brain and in skeletal muscles. This isoform was shown to mediate rapid SOCE activation and, together with TRPC1 and TRPC4, seems to be involved in muscle differentiation and maturation (Antigny et al., 2013;

Antigny et al., 2017). Recent studies also identified a new STIM2 splicing variant with inhibitory properties: STIM2.1 or STIM2β (Miederer et al., 2015; Rana et al., 2015). STIM2.1 is ubiquitously but poorly expressed and its binding to ORAI channels is defective. This isoform travels to the PM by forming heterodimers with STIM1 or STIM2 proteins and exerts a direct allosteric inhibition on the channel. As STIM2.1 affinity for CaM is very high, an additional indirect modulation of channel gating by STIM2.1 via CaM in not excluded. Because STIM1 is

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the most studied isoform, and for simplicity reasons, this manuscript will further refer to STIM1 only.

STIM1 activation and SOCE

In resting conditions, Ca²⁺ ions are trapped in EF hand pockets and STIM1 dimers are kept in a folded “closed” conformation by intra– and intermolecular interactions (Fahrner et al., 2014). A recent study proposed that “sentinel” residues in CC1 prevent STIM1 extension and activation (Hirve et al., 2018). Upon store depletion, Ca²⁺ dissociation from the EF hands induces a luminal rearrangement of STIM1, the pairing of transmembrane domains (TMD) and CC1 regions, the protein elongation, and a subsequent high–order STIM1 oligomerization (Fig.

7) (Stathopulos et al., 2008; Stathopulos et al., 2013).This “open” STIM1 conformation exposes the coiled–coil CC2 and CC3 domains forming the CRAC activation domain (CAD; also referred

Figure 6. Stromal interaction molecules (STIM). Adapted from Bhardwaj et al. (2016).

Schematic outline of human STIM1, STIM1L (A) and STIM2 (B) isoforms, with 685, 791 and 746 amino acids respectively. N-termini are luminal, C-termini are cytosolic. SS: signal sequence, cEF and hEF: canonical and hidden EF-hand domains, SAM: sterile alpha motif, TMD: transmembrane domain, CC: coiled-coil domains 1 to 3, SOAR: STIM-ORAI activating region (referred as CAD in the text), ID: inactivation domain, ABD: actin-binding domain in STIM1L, S/P: serine/proline-rich regions, P/H: proline/histidine-rich region in STIM2, K: lysine-rich domains (with amino acid sequences). Cysteine residues are indicated with yellow circles.

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as STIM–ORAI activation region, SOAR), that will interact with and aggregate ORAI Ca²⁺

channels in clusters at the PM (Wu et al., 2006; Muik et al., 2009; Park et al., 2009; Yuan et al., 2009).

The interaction of ER resident proteins with PM channels requires the close apposition of the ER membrane to the PM (both separated by only 10–20 nm). Following stores depletion, ER remodeling and the organization of ER–PM membrane contact sites (MCS) were indeed described (Orci et al., 2009). Total internal reflection fluorescence (TIRF) microscopy, allowing Figure 7. Store-operated Ca²⁺ entry (SOCE). From Bhardwaj et al. (2016), redrawn from Soboloff et al. (2012).

1) At rest, inactive STIM1 and ORAI1 homodimers are homogenously distributed in ER and PM membranes respectively. Intra- and intermolecular interactions of STIM1 CC domains keep the dimer in a “closed” conformation.

2) The agonist-mediated activation of PLC induces PM PIP2 hydrolysis and results in IP3 production.

3) IP3 further binds to its respective receptor channel IP3R on the ER membrane, prompting Ca²⁺

release from the stores.

4) Ca²⁺ dissociation from STIM1 EF-hands activates STIM1 dimers: the interaction of their luminal EF-SAM domains and their CC domains, leading to the extension of the proteins and the exposure of CAD/SOAR domains and polybasic tails.

5) Activated STIM1 dimers oligomerize and translocate to ER-PM membrane contact sites (MCS), where they bind to PM PIP2.

6) STIM1 further binds and clusters ORAI1 channels.

7) The interaction of STIM1 CAD with ORAI1 induces channel gating and subsequent Ca²⁺ entry into the cytosol.

8) SERCA pumps recruited at ER-PM contact sites allow ER refilling and termination of the process.

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the study of events at close proximity to the PM, showed STIM1 diffusion and formation of puncta co–localizing with ORAI1 clusters (Fig. 8A and B) (Liou et al., 2005; Navarro-Borelly et al., 2008). These findings were further confirmed by electron microscopy and the observation of thin cortical ER (cER) structures facing the PM and enriched in STIM1 (Fig. 8C and D) (Orci et al., 2009). These MCS do not only occur at the PM, but were also described among ER and mitochondria, the Golgi apparatus, endo/lysosomes and phagosomes, and are thought to be major sites for Ca²⁺ signals and lipid transfer (Phillips and Voeltz, 2016).

ORAI isoforms and splicing variant

The Ca²⁺ selective ORAI channels (also known as CRACM – CRAC modulators) are largely present in all eukaryotic cells. Their name comes from Greek mythology, where Orai (or Horae) are the “gatekeepers of heaven”. The family comprises 3 isoforms, ORAI1, ORAI2 and ORAI3, Figure 8. ER-PM membrane contact sites (MCS). From Liou et al. (2005), Navarro-Borelly et al. (2008) and Orci et al. (2009).

(A) Redistribution of YFP-STIM1 into punctae at the PM following store depletion in HeLa cells, and assessed with TIRF. (B) Tg-induced ORAI1-CFP and STIM1-YFP molecular interactions assessed by FRET. (C) Electron micrograph showing the apposition of thin cortical ER (cER) sheets (asterisk) close to the PM (circle) in untransfected HeLa cells treated with Tg. (D) Cryo-immuno electron micrograph of YFP-STIM1 transfected HeLa cells. STIM1, detected with an anti-GFP antibody and gold particles, was enriched in cER (asterisk) facing the PM (circle).

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with high sequence homology but different tissue distribution, electrophysiological features and regulation mechanisms (Gwack et al., 2007; Hoth and Niemeyer, 2013).They are ~ 30 kDa proteins with four transmembrane domains (TM), cytosolic N– and C–termini, and assemble as homo– or heteromers to form a highly selective Ca²⁺ channel (Lis et al., 2007; Schindl et al., 2009). ORAI isoforms mainly differ in their sensitivity to the SOCE inhibitor 2–APB (2–

aminoethoxydiphenyl borate): while ORAI1 and ORAI2 are inhibited by high concentrations of the compound, 2–APB activates ORAI3 in a STIM–independent manner, alters the channel selectivity and abolishes CDI (Schindl et al., 2008; Zhang et al., 2008). ORAI3 and ORAI1 heteromerization forms the ARC (arachidonate–regulated Ca²⁺–selective) channel, related to SOCE and regulated by arachidonic acid (Mignen et al., 2008a). A recent study identified a shorter form of ORAI1 (ORAI1β) resulting from an alternative translation initiation and suggested that, by loosing a phospholipid binding domain, ORAI1β’s diffusion ability is increased (Fukushima et al., 2012). Overall, the ORAI1 isoform is the most studied and best described homolog, and is particularly investigated in the immune system as T lymphocytes, B lymphocytes and natural killer (NK) cells almost exclusively rely on ORAI1 activity (Feske et al., 2006). The thesis focuses on ORAI1, whose structure and properties are further developed in section 3.

Channel gating

ORAI1 gating is triggered by the interaction with the STIM1 CAD domain (Muik et al., 2009;

Park et al., 2009) and endogenous SOCE can be easily assessed with Ca²⁺ imaging experiments.

However, the extremely low unitary conductance of the channel (10–35 fS compared to > 20 pS for voltage–gated Ca²⁺ channels) makes single channel or endogenous Ca²⁺ current recording very difficult in most cell types (Prakriya and Lewis, 2006). In a divalent free solution, the Na⁺ unitary conductance though can reach ~ 1 pS and is sometimes used as an alternative for the evaluation of ORAI1 activity. ORAI1 and STIM1 overexpression increases the current amplitude up to 100 times and whole–cell configuration patch clamp is usually required to follow ORAI1–STIM1 mediated currents in isolated cells (Peinelt et al., 2006). STIM1–ORAI1 stoichiometry is also a strong determinant of channel trapping and gating. A minimum of two STIM1 molecules per ORAI1 subunit seems to be optimal to record maximal SOCE or ICRAC in Ca²⁺ imaging or patch clamp experiments. At high levels of ORAI1 expression, SOCE amplitude and ICRAC density drastically decrease (Hoover and Lewis, 2011). Overexpression of ORAI1 alone without STIM1 co–expression actually abolishes endogenous Ca²⁺ entry in several cell types

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(Soboloff et al., 2006; Cheng et al., 2008). Interestingly, CDI also depends on the STIM1:ORAI1 ratio, with stronger inhibition observed as the ratio increases (Scrimgeour et al., 2009).

2.2. SOCE modulation

Ca²⁺–dependent inactivation (CDI)

The first type of modulation is Ca²⁺ itself, through what is called Ca²⁺–dependent inhibition (CDI). Electrophysiological studies describe two different processes in which ICRAC density decreases over time, one fast (in the msec range, FCDI) and one slow (sec to min, SCDI) (Zweifach and Lewis, 1995a, b; Parekh, 1998). FCDI is revealed by imposing negative voltage ramps to store–depleted cells, and by blocking SERCA–mediated refilling with Tg. FCDI is observed even when the intracellular compartment is dialyzed with 10 mM EGTA. As EGTA only buffers Ca²⁺ ions at a minimal distance of 0.1 μm from the PM (Fierro and Parekh, 1999), it was proposed that FCDI is mediated by the accumulation of Ca²⁺ ions in the close vicinity of the channel intracellular pore that exert a direct negative feedback on the current. SCDI not only differs by its slower kinetics, but also by the fact that intracellular EGTA abolishes ICRAC slow inactivation. Although the underlying molecular mechanisms of SCDI are still unclear, this process is considered to finely tune CRAC activity by sensing more global intracellular Ca²⁺

elevations.

SOCE termination

While FCDI and SCDI occur in the absence of store refilling, SOCE termination requires the replenishment of Ca²⁺ stores. This step is essential but not sufficient to initiate STIM1 de–

oligomerization and dissociation from the PM, and only an additional local cytosolic Ca²⁺

elevation close to the STIM1–ORAI1 interface allows the deactivation of the SOCE process (Shen et al., 2011). Mitochondria play a central role in the regulation of Ca²⁺ levels in such microdomains. Their capacity to take up Ca²⁺ ions essentially depends on high Ca²⁺ gradients, and mitochondria were shown to relocate to T lymphocytes immune synapses where SOCE takes place (Quintana et al., 2007; Quintana and Hoth, 2012). Mitochondria are thought to decrease CDI, store refilling and impair SOCE termination, as blocking mitochondrial migration along microtubules towards the PM fails to sustain Ca²⁺ influx (Quintana et al., 2006).

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In addition to their ability to capture Ca²⁺ ions, mitochondria also regulate SOCE by producing reactive oxygen species (ROS). The oxidation of STIM1 cysteine residues enhances the protein multimerization and PM translocation, and is followed by Ca²⁺ entry, even in the absence of store depletion (Bhardwaj et al., 2016). In contrast, the addition of hydrogen peroxide (H2O2) to the external environment inhibits ORAI1 clustering and reduces SOCE (Bogeski et al., 2010; Alansary et al., 2016). Along with redox modulation, external and internal pH changes severely impact on ICRAC density, with current potentiation at basic pH and decline upon acidification (Beck et al., 2014; Tsujikawa et al., 2015). Besides, increasing temperature boosts ICRAC, probably by affecting lipid membrane composition. The current density – temperature relationship is non–linear and ICRAC drastically decays under 21°C (Somasundaram et al., 1996). One should carefully consider this property as most ICRAC whole–cell patch–clamp recordings are performed at room temperature (23°C) for simplicity.

Post–translational modifications

Post–translational modifications of STIM1 and ORAI1 alter their stability and function.

STIM1 glycosylation seems to be necessary for the protein translocation to the PM and the initiation of SOCE (Williams et al., 2002). On the contrary, the addition of a glycan to the unique ORAI1 glycosylation site reduces SOCE, probably by altering the channel interaction with other modulating proteins (Dorr et al., 2016). The high turnover of ORAI1 channels at the PM is due to their internalization in sub–plasmalemal endocytic vesicles, and their enrichment at ER–PM junctions by a STIM1–mediated trapping (Hodeify et al., 2015). And ORAI1 glycosylation might also promote this high turnover (Niemeyer, 2016). In addition, STIM1 phosphorylation prejudices STIM1–ORAI1 interaction and is thought to be a determinant of ER redistribution during the cell cycle (Preston et al., 1991). PKC–mediated phosphorylation of ORAI1 inhibits the channel activity in a Ca²⁺–dependent manner, consigning this post–transcriptional modification to the Ca²⁺ negative feedback loop (Kawasaki et al., 2010). Finally, ORAI1 and STIM1 ubiquitination by the ubiquitin ligases Nedd4-2 and POSH respectively, reduce their surface expression and limit SOCE (Keil et al., 2010; Lang et al., 2012).

Interacting proteins

Many STIM1–ORAI1 interacting molecules complete the set of positive and negative SOCE regulators (Srikanth et al., 2013). Among them is Junctate, an ER resident protein interacting

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with STIM1 and enhancing its recruitment to the PM, depending on the ER luminal Ca²⁺

content. Invalidating Junctate EF–hands induces puncta formation and SOCE activation in store–replete cells, and is an alternative mechanism to trigger STIM1 translocation (Srikanth et al., 2012). Another ER transmembrane protein named STIMATE (STIM–activating enhancer) directly interacts with STIM1 and supports its conformational change (Jing et al., 2015).

CRACR2A (CRAC channel regulator 2A) and its homolog CRACR2B are cytosolic Ca²⁺ sensors that associate with STIM1 and ORAI1 to form a stable ternary complex (Srikanth et al., 2010).

In its Ca²⁺–bound form, CRACR2A destabilizes the complex and ends SOCE. When Ca²⁺ binding is prevented by mutations in the respective motif, the excessive activity of the SOCE machinery leads to Ca²⁺ overload and cell death. The organization of STIM1–ORAI1 clusters at the PM appears therefore to be critical for SOCE regulation. The GTP–binding cytoskeleton filaments Septins (in particular Septin 4) were described as SOCE coordinators that assemble in ring structures to scaffold SOCE partners and prevent their diffusion along the plasma lipid bilayer.

Septins’ rearrangement organizes PIP2 rich membrane microdomains at ER–PM junctions, promotes STIM1 recruitment and stabilizes ORAI1 clusters (Sharma et al., 2013). The Ca²⁺

effector CaM is a prevalent negative modulator of SOCE/CRAC that, in the Ca²⁺–bound form, initiates STIM1–ORAI1, STIM1–PM and STIM1 multimers dissolution, hence resulting in SOCE termination (Li et al., 2017). CaM was proposed to associate with the CAD domain of STIM1 and to interact with the ORAI1 N–terminus (residues W76 and Y80, in the channel pore) to mediate CDI (Mullins et al., 2009; Liu et al., 2012; Bhardwaj et al., 2013). However the poor accessibility of the predicted CaM–binding motif to CaM questions this hypothesis. The ER Ca²⁺

binding protein SARAF (SOCE–associated regulatory factor) was suggested to elicit SCDI (Palty et al., 2012; Jha et al., 2013). SARAF travels to the PM together with STIM1 upon store depletion, senses global Ca²⁺ elevations and destabilizes STIM1–ORAI1 interactions to prevent stores overfilling. Golli is a member of the myelin basic protein family shown to be essential in T lymphocytes (Feng et al., 2004). By interacting with the STIM1 C–terminus, Golli negatively regulates SOCE and preserves Ca²⁺ homeostasis. However, this stranglehold can be reversed by the overexpression of STIM1 (Walsh et al., 2010). Upon store–depletion, POST (partner of STIM), mainly located at the ER membrane, associates with STIM1, ORAI1, SERCA and PMCA in large complexes, and sustains the accumulation of Ca²⁺ ions in the ER–PM cleft by inhibiting the PMCA pump (Krapivinsky et al., 2011). This short list of SOCE regulators is not exhaustive and new STIM1-ORAI1 partners are continuously identified. In the end, the combination of all of these molecular mechanisms and physical parameters are thought to support Ca²⁺ signaling

Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

Thesis

Reference

Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

Biophysical properties of the ORAI1 channel in the context of tubular aggregate myopathy

Table of contents

List of abbreviations

I. Résumé

II. Abstract

III. Introduction

1. Tuning calcium signals

1.1. Receptors and transducers

B

A

1.2. Ca

channels

1.3. Ca

–binding proteins

1.4. Ca

pumps and exchangers

2. Store–operated Ca

entry (SOCE)

2.1. STIM and ORAI

2.2. SOCE modulation