Characterization of the receptor activated and store operated Ca2+ entry pathways in endothelial cells

Thesis

Reference

Characterization of the receptor activated and store operated Ca2+

entry pathways in endothelial cells

GIRARDIN, Nathalie

Abstract

Endothelial cells form a monolayer lining all blood and lymph vessels and the heart cavity.

The strategic location of these cells at the interface between the flow of blood and surrounding tissues makes this "organ" multifunctional. Indeed, endothelial cells form a semipermeable barrier involved in local control of vascular tone, regulation of blood coagulation and adhesion of inflammatory cells, control the permeability of blood vessels or the process of angiogenesis.

Many, if not all of these regulatory mechanisms involve a calcium signal. Ca2+ is an intracellular second messenger involved in a wide range of physiological processes. But the process of Ca2+ entry in nonexcitable cells such as endothelial cells remains an enigma that is the subject of intense investigations. In these cells, in contrast to excitable cells, the channels involved in Ca2+ entry are not activated by membrane depolarization. The calcium signal consists of two phases: the addition of an agonist is followed by release of Ca2+ from the endoplasmic reticulum (ER) after the opening of the inositol 1,4,5-triphosphate (IP3). This initial elevation of [...]

GIRARDIN, Nathalie. Characterization of the receptor activated and store operated Ca2+ entry pathways in endothelial cells. Thèse de doctorat : Univ. Genève, 2010, no. Sc.

4184

URN : urn:nbn:ch:unige-57052

DOI : 10.13097/archive-ouverte/unige:5705

Available at:

http://archive-ouverte.unige.ch/unige:5705

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

UNIVERSITE DE GENEVE

Département de zoologie et biologie FACULTE DES SCIENCES

animale Professeur J.-L. Bény

Département de physiologie cellulaire FACULTE DE MEDECINE

et métabolisme Professeur N. Demaurex

Docteur M. Frieden

Characterization of the Receptor Activated and Store Operated Ca²⁺ Entry pathways in endothelial cells

THESE

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

par

Nathalie GIRARDIN de

Les Bois (Jura)

Thèse n°4184

Genève 2010

Table of contents

I. Résumé en français_____________________________________ 3 II. Abstract______________________________________________ 5 III. Introduction___________________________________________ 7

A. The endothelium

I. Structure______________________________________________ 7 II. Function______________________________________________ 7 B. Ca²⁺ signaling

I. Ca²⁺: a second messenger_________________________________ 9 II. Ca²⁺ homeostasis________________________________________ 10 III. Ca²⁺ influx_____________________________________________ 12

i. Store operated Ca²⁺ Entry (SOCE)____________________ 12 ii. Store independent Ca²⁺ entry or Receptor Activated

Calcium Entry (RACE)_____________________________ 17 C. Ca²⁺ signaling in Endothelial Cells____________________ 20 IV. Aims of the study_______________________________________ 22 V. Experimental tools_____________________________________ 23 VI. Publication I__________________________________________ 28 VII. Electrophysiological characterization of Ca²⁺entry in EC :

Single channel recording____________________ 44 VIII. Manuscript___________________________________________ 49 IX. Discussion and perspectives______________________________ 77 X. Remerciements________________________________________ 84 XI. References____________________________________________ 85

I. Résumé en français

Les cellules endothéliales forment une monocouche qui tapisse l'ensemble des vaisseaux sanguins et lymphatiques, ainsi que la cavité cardiaque. L'emplacement stratégique de ces cellules à l'interface entre le flux de sang et les tissus environnants rend cet « organe » multifonctionnel. En effet, les cellules endothéliales forment une barrière semi-perméable impliquée dans le contrôle local du tonus vasculaire, la régulation de la coagulation sanguine, l'adhérence des cellules inflammatoires, le contrôle de la perméabilité des vaisseaux ou le processus de l'angiogenèse. Beaucoup, sinon la totalité de ces mécanismes de régulation impliquent un signal calcique.

Le Ca²⁺ est un messager secondaire intracellulaire impliqué dans un large éventail de processus physiologiques. Depuis des années le processus d'entrée de Ca²⁺ dans les cellules dites non excitables, comme les cellules endothéliales, reste une énigme qui fait l'objet d'investigations importantes. Dans ces cellules, contrairement aux cellules excitables, les canaux impliqués dans l'entrée de Ca²⁺ ne sont pas activés par la dépolarisation membranaire.

Le signal calcique se compose de deux phases: l’’addition d’’un agoniste est suivie par la libération de Ca²⁺ du réticulum endoplasmique (RE) causée par l'ouverture du récepteur inositol 1,4,5-triphosphate (IP3). Cette première élévation du Ca²⁺ cytosolique est accompagnée par une entrée de Ca²⁺ extracellulaire, ce qui prolonge le signal calcique.

Dans la littérature, deux mécanismes d’’entrée calcique différents ont été décrits, chacun résultant probablement de l'activation de différents types de canaux. Un mécanisme reliant le niveau de remplissage du RE à l’’entrée calcique, et qui est appelé « Store-Operated Calcium Entry » (SOCE) et un autre mécanisme nécessitant la présence d'un agoniste pour être activé et qui est appelé « Receptor-activated Calcium Entry » (RACE). La voie SOCE est présente dans pratiquement tous les types cellulaires et peut être activée artificiellement par une réduction passive du Ca²⁺ dans le RE, avec par exemple de la thapsigargin (TG) qui bloque la pompe SERCA (Sarco/Endoplasmic Reticulum Ca²⁺-ATPase). Le RACE, lui est activé par la stimulation d’’un récepteur et la génération de seconds messagers. Mais il est difficile d'isoler expérimentalement le RACE du SOCE. En effet, la stimulation avec un agoniste conduit, au moins dans une certaine mesure, à une baisse du niveau calcique dans le RE due au relâchement de Ca²⁺ via les récepteurs IP3. Il est donc souvent supposé que le SOCE joue un rôle majeur pendant une réponse physiologique.

Le but de ma thèse est l’’étude de l’’entrée calcique dans les cellules endothéliales. J’’ai tout d’’abord participé au projet consistant à différencier pharmacologiquement le SOCE du RACE grâce à l’’imagerie calcique. Dans un deuxième temps, j’’ai utilisé l’’électrophysiologie, plus précisément le patch clamp en configuration « cellule attachée » et « cellule entière » afin de caractériser les canaux impliqués dans l’’entrée calcique après stimulation artificielle (SOCE) ou stimulation par un agoniste (RACE) et d’’évaluer la contribution de ces deux mécanismes durant une réponse physiologique.

Le premier projet est donc l’’utilisation de l’’imagerie calcique afin de différencier le RACE du SOCE. Pour réaliser ce projet, deux stimulations différentes ont été étudiées. Soit l’’addition d’’un agoniste (100PM histamine), soit l’’addition d’’un inhibiteur de la pompe SERCA (1PM TG), ce qui permet d’’activer préférentiellement l’’influx RACE ou l’’influx SOCE respectivement. Après avoir testé différents inhibiteurs connus de l’’influx calcique, il s’’est avéré dans un premier temps, que le La³⁺ à une concentration de 10PM était le seul inhibiteur permettant de différencier les deux types d’’influx, bloquant efficacement l’’influx SOCE mais n’’ayant que peu d’’effet sur l’’influx RACE. Hélas, après une étude plus approfondie de ce bloqueur, nous nous sommes rendus compte que ce résultat était en fait un artefact dû à la présence de phosphate dans l’’une des deux conditions expérimentales. En effet

le phosphate ajouté en présence de La³⁺ a le pouvoir de le chélater, réduisant fortement sa concentration libre. L’’utilisation d’’histamine chlore nous a démontré que le La³⁺ était tout aussi inefficace que les autres inhibiteurs dans la discrimination des deux influx. Par contre dans la même étude nous avons pu confirmer, en mesurant la variation de Ca²⁺ libre directement dans la lumière du RE, que la déplétion en Ca²⁺ du RE est très faible lors de l’’activation par un agoniste, ce qui signifie que le SOCE n’’est pas activé dans ces conditions, favorisant l’’influx de type RACE. La sensibilité aux inhibiteurs des mitochondries permet de différencier les deux influx. En effet, l’’inhibition des mitochondries diminue le SOCE sans affecter le RACE. Cette étude a donc permis de montrer que dans les cellules endothéliales, l’’entrée calcique après activation par l’’histamine arrive à maintenir un niveau calcique élevé dans le RE et que le SOCE n’’est pas ou peu activé. La réponse calcique soutenue en présence de l’’agoniste est donc essentiellement due à un influx RACE.

L’’absence d’’inhibiteur permettant de différencier l’’influx calcique via le SOCE ou le RACE en imagerie calcique, nous a fait changer de méthode et nous tourner vers le patch clamp. Notre première approche a été en configuration dite « cellule attachée », permettant directement la caractérisation des canaux impliqués dans l’’influx activé par déplétion passive ou par ajout d’’un agoniste. Cette configuration nous a permis de caractériser un canal activé par l’’histamine mais pas par la déplétion du RE. Ce canal a une conductance d’’environ 25pS, est entrant rectifiant, cationique non sélectif et est Ca²⁺ dépendant.

Cette approche expérimentale étant très délicate et totalement dépendante de la présence du canal sous la pipette, nous avons décidé de changer de configuration et avons opté pour l’’utilisation du patch perforé en configuration dite « cellule entière », afin de caractériser non pas directement les canaux, mais les courants activés lors d’’une entrée calcique via SOCE ou via RACE. En effet cette configuration permet l’’enregistrement des courants de la cellule entière, ce qui ne dépend donc plus de la présence ou non de canaux sous la pipette. Cette approche nous a permis d’’enregistrer les courants activés par l’’histamine et par la TG dans différentes conditions expérimentales. L’’adition d’’histamine ou de TG en présence de 10mM de Ba²⁺ et de 2mM de Ca²⁺ dans le milieu extra cellulaire, active un courant avec les caractéristiques du courant CRAC (Ca²⁺-release activated Ca²⁺), comme la rectification entrante, l’’inhibition par le La³⁺ et le comportement dans un milieu extra cellulaire sans cation divalent. Dans ces conditions il n’’est donc pas possible de les différencier. Par contre, en présence de 10mM de Ca²⁺ et en l’’absence de Ba²⁺, les courants activés par la TG et l'histamine sont clairement différents. En effet, alors que la TG active toujours un courant entrant rectifiant, mais plus petit qu’’en présence de Ba²⁺, l’’histamine active un courant sortant rectifiant, bloqué par le Ba²⁺. Nous avons également montré que, pendant les enregistrements à voltage clampé, le RE est plus appauvri en Ca²⁺ en présence de 10mM Ba²⁺ qu’’en présence de 10mM Ca²⁺ dans le milieu extracellulaire, lors d’’une activation par l’’histamine. Ceci explique l'activation d'un courant CRAC-like (en raison de la déplétion des stocks) lors de l’’addition d’’histamine en présence de Ba²⁺. En milieu 10mM Ca²⁺, le courant sortant activé par l'histamine augmente lorsque le Na⁺ extracellulaire est enlevé. De plus ce courant est inhibé par le Ni²⁺ et par le KB-R7943, deux inhibiteurs connus de l’’échangeur Na⁺/Ca²⁺ (NCX), ce qui nous fait penser que la NCX est impliquée dans l’’entrée calcique activé par l’’histamine. Le Ni²⁺ et le KB-R7943 inhibent aussi la réponse calcique après addition d'histamine, mais pas la réponse après addition de TG. Ces données nous ont conduit à conclure que dans des conditions physiologiques, une partie de l’’influx RACE est du à la NCX, travaillant dans le mode inverse. Cet influx est suffisant pour ne pas appauvrir le RE en Ca²⁺, empêchant donc l'activation d’’un influx SOCE.

II. Abstract

Endothelial cells form a monolayer lining all blood and lymph vessels and the heart cavity. The strategic location of these cells at the interface between the flow of blood and surrounding tissues makes this "organ" multifunctional. Indeed, endothelial cells form a semipermeable barrier involved in the local control of the vascular tone, the regulation of the blood coagulation and the adhesion of inflammatory cells, the control the permeability of blood vessels or the process of angiogenesis. Many, if not all of these regulatory mechanisms involve a calcium signal.

Ca²⁺ is an intracellular second messenger involved in a wide range of physiological processes. But the process of Ca²⁺ entry in nonexcitable cells such as endothelial cells remains an enigma that is the subject of intense investigations. In these cells, in contrast to excitable cells, the channels involved in Ca²⁺ entry are not activated by membrane depolarization. The calcium signal consists of two phases: the addition of an agonist is followed by the release of Ca²⁺ from the endoplasmic reticulum (ER) after the opening of the inositol 1,4,5-triphosphate (IP3). This initial elevation of cytosolic Ca²⁺ is accompanied by a Ca²⁺ entry from the extracellular medium, thereby prolonging the Ca²⁺ signal.

In the literature, two different mechanisms of Ca²⁺ entry have been described, each one probably resulting from the activation of different types of channels: a mechanism linking the filling level of the ER to the Ca²⁺ entry, which is called ““Store-Operated Calcium Entry””

(SOCE) and another mechanism requiring the presence of an agonist to be activated and which is called ““Receptor-Activated Calcium Entry”” (RACE). The SOCE pathway is present in virtually all cell types and can be artificially activated by a passive depletion of Ca²⁺ in the ER, for example with the thapsigargin (TG) which blocks the pump SERCA (Sarco/

Endoplasmic Reticulum Ca²⁺-ATPase). The RACE pathway is activated after stimulation of a receptor and the generation of second messengers. But it is difficult to isolate experimentally the RACE from the SOCE pathway. Indeed, the stimulation with an agonist leads potentially to a depletion of Ca²⁺ in the ER, due to release of Ca²⁺ via the IP3 receptors. It is often assumed that SOCE plays a major role during a physiological response.

The purpose of my thesis is the study of calcium entry in endothelial cells. I first participated in the project to pharmacologically differentiate the SOCE from the RACE using Ca²⁺-imaging. In a second time, I used electrophysiology, specifically the patch clamp in

““cell-attached”” and ““whole-cell”” configuration to characterize the channels involved in Ca²⁺

entry after an artificial stimulation (SOCE) or a stimulation by an agonist (RACE) and assess the contribution of these two mechanisms in a physiological response.

The first project is the use of Ca²⁺ imaging to differentiate the RACE from the SOCE pathway. For this project, two stimuli were investigated. Either the addition of an agonist (100µM histamine), or an inhibitor of the SERCA pump (1µM TG), which preferentially activates the RACE influx or SOCE influx, respectively. After testing various inhibitors of Ca²⁺ influx, it was found initially that 10µM of La³⁺ was the only inhibitor that differentiated the two influxes, effectively blocking the SOCE influx but had little effect on the RACE influx. Unfortunately, after further study of the blocker, we realized that this result was an artifact due to the use of histamine phosphate. Indeed, the added phosphate in the presence of La³⁺ has the power to bind it, greatly reducing its free concentration. The use of histamine chloride has shown that La³⁺ was also ineffective in the discrimination of the two influxes.

But in the same study we have confirmed by measuring the change in free Ca²⁺ directly into the ER lumen, that the ER depletion is very weak when cells are activated by an agonist. We have also shown that the depolarization of mitochondria by the use of oligomycin and rotenone can differentiate the two influxes. Indeed, the inhibition of mitochondria decreases the SOCE pathway without affecting the RACE pathway. This study has shown that in

endothelial cells, Ca²⁺ entry after activation by histamine is able to maintain a high Ca²⁺ level in the ER and thus that SOCE pathway is little or not activated. The sustained Ca²⁺ response in the presence of agonist is mainly due to an RACE influx.

The absence of inhibitor to differentiate Ca²⁺ influx via the SOCE or RACE pathways in Ca²⁺ imaging, made us change the method and we used the patch clamp. Our first approach was the ““single channel”” configuration to characterize the channel(s) involved in the influx activated by passive depletion or addition of an agonist. This configuration allowed us to characterize a channel activated by histamine but not by depletion of the ER. This nonselective cation channel has a conductance of about 25pS, is inwardly rectifyied, and is Ca²⁺ dependent.

This experimental approach being very delicate and totally dependent on the presence of the channel under the pipette, we decided to change configuration and have opted for the perforated patch configuration of the ““whole cell”” to characterize not directly the channels, but the currents activated during a Ca²⁺ entry via SOCE or via RACE pathways. This approach allowed us to record currents activated by histamine and TG in different experimental conditions. The addition of histamine or TG in presence of 10mM Ba²⁺ and 2mM Ca²⁺ in the extracellular environment, activate both a current with the characteristics of the CRAC current (Ca²⁺ release-activated Ca²⁺), such as the inward rectification , the inhibition by La³⁺ and the behaviour in an divalent cation free environment. In these conditions it is not possible to differentiate them. On the other side, in presence of 10mM Ca²⁺ and without Ba²⁺, the currents activated by TG and histamine are clearly different.

Indeed, while the TG still activates an inward current, but smaller than in the presence of Ba²⁺, histamine activates an outward rectifying current, blocked by Ba²⁺. We also showed that during the voltage-clamp recordings, the ER is more Ca²⁺ depleted in presence of 10mM Ba²⁺

than in presence of 10mM Ca²⁺ in the extracellular medium, during histamine activation. This explains the activation of a CRAC-like current (due to depletion of stores) during the addition of histamine in presence of Ba²⁺. In 10mM Ca²⁺, the outward current activated by histamine increases when the extracellular Na⁺ is removed, is inhibited by Ni²⁺ and KB-R7943, two known inhibitors of the Na⁺/Ca²⁺ exchanger (NCX), thus we suggests that the NCX is involved in Ca²⁺ entry activated by histamine. In Ca²⁺ imaging, the Ni²⁺ and the KB-R7943 inhibit also the Ca²⁺ response after histamine addition, but not the response after TG addition.

These data led us to conclude that in physiological conditions, a part of RACE influx is due to the NCX working in the reverse mode. This influx is sufficient to prevent an important store depletion, thus the activation of SOCE influx is minimal.

III. Introduction

A. The endothelium

I. Structure

The endothelium represents the inner cellular layer lining of the blood and lymphatic vessels as well as the heart cavity (Fig. 1). Due to the extraordinary length of the vascular system, in a person with a body weight of 70kg, the endothelium covers an area of approximately 700m² and weighs about 1kg (Sumpio et al. 2002). The strategic location at the interface between the blood flow and the surrounding tissues make this cellular layer a dynamic and multifunctional ““organ””. The endothelium is a type of epithelial tissue, which differs from other epithelia because it derives from the embryonic mesoderm, while the others derive from the ectoderm or endoderm.

In an adult the endothelium is composed of approximately 1.6x10¹³ endothelial cells (EC), which are aligned in the direction of blood flow. Although ECs are typically flat, the shape of cells varies across the vascular tree (Aird 2007). ECs are interconnected and connected with the basal lamina (a structure of collagen synthesized by the endothelium) by hemidesmosomes and desmosomes. It is therefore a tissue that can withstand high mechanical stress, but that is permeable to small solutes and water.

Figure 1: Human artery

Adapted from http://www.britannica.com

II. Function

The most obvious function of the endothelium is to keep the blood inside blood vessels, while allowing the exchange of nutrients. In this function, endothelial cells and basal lamina cooperate by acting as a molecular sieve. But the endothelium performs many other functions by exerting significant paracrine actions on the underlying smooth muscle cells or endocrine actions on circulating blood elements, such as platelets and white blood cells. By

these actions, this semi-permeable barrier is involved in the local control of the vascular tone, the regulation of blood coagulation, the adhesion of inflammatory cells, the control of the vessel permeability or the angiogenesis process (Michiels 2003). The production and release of different vasoactive compounds like nitric oxide (NO), prostacyclin or endothelin-1(ET-1), of pro and anticoagulant factors, like von Willebrand factor (vWF) and many other indispensable products, allow EC to accomplish these functions and to regulate all the processes relating to vessel physiology (Fig. 2). These mediators are released in response to multiple chemical stimuli, including shear forces exerted by blood flow, certain hormones (catecholamines, vasopressin), thrombin or autacoids (bradykinin, histamine, endothelin-1). At resting state, ECs have anticoagulative, antithrombotic and profibrinolytic properties. But when activated by hormones or by a lesion, EC switches towards a procoagulative, proproliferative and vasoconstricting state (Sumpio et al. 2002).

The disturbance in ECs functions that is induced by diverse intrinsic and extrinsic factors, is reflected by a decreased of NO bioavailability, an inappropriate regulation of the vascular smooth muscle tonicity, an impaired antithrombogenic properties, and a perturbed angiogenic competence. These dysfunctions are the source of various cardiovascular pathologies, including hypertension, atherosclerosis or heart failure (Feletou and Vanhoutte 2006); (Goligorsky et al. 2000); (Goligorsky 2005). Many, if not all of these regulatory mechanisms imply directly or indirectly a cytosolic Ca²⁺ signal, for instance for the production and release of nitric oxide (NO) or prostacyclin (Luckhoff and Busse 1990).

Figure 2: Known secretory/expression products of endothelial cells relating to vessel physiology (Sumpio et al. 2002).

C. Ca

signaling

I. Ca²⁺: a second messenger

Until an inadvertent error in 1883 from a researcher in the field of heart physiology, Sydney Ringer, Ca²⁺ in human body was only known to be an important component of bones and teeth. Ringer was surprised that isolated rat hearts contracted beautifully in tap water

solution used, whereas in subsequent experiments, the heart failed to contract in distilled water. In trying to understand the reason for the different behaviour of hearts in the two media, Ringer had found that the ion responsible was Ca²⁺. It was the beginning of the concept that Ca²⁺ is a fundamental carrier of messages (Ringer 1883); (Carafoli 2003).

Around 0.7% of Ca²⁺ is circulating. This circulating Ca²⁺ is a keystone of cellular biology signalling. The Ca²⁺ ion is a ubiquitous and universal second messenger. Each cell emits, collects and analyzes a vast amount of information. A multitude of chemical compounds as different as polypeptides or steroid travel throughout our body and carries information from one cell to another. These carriers of information have been called first messengers. They will stimulate the target cells, either directly, as do steroids or indirectly, by causing the increase of intracellular metabolites known as second messenger. If we assume a known first messenger and a target cell, we define a second messenger coupled to the first messenger for the following properties: the first messenger triggers an increase in intracellular metabolite and an artificial increase of the metabolite triggers totally or partially same cellular event as the first messenger. Ca²⁺ meets this definition (http://www.universalis.fr).

PMCA Ca²⁺entry NCX channels

mNCX 3Na⁺

Ca²⁺

MCU

3Na⁺

Ca²⁺

SERCA Ca²⁺

RyR IP₃R ^IP3

ER [Ca²⁺]

§200-500µM Mito [Ca²⁺]

§0.5µM Cytosol [Ca²⁺]

§100nM

Extra-cellular space [Ca2+]

§2mM

PMCA Ca²⁺entry NCX channels

mNCX 3Na⁺

Ca²⁺

MCU

3Na⁺

Ca²⁺

SERCA Ca²⁺

RyR IP₃R ^IP3

ER [Ca²⁺]

§200-500µM Mito [Ca²⁺]

§0.5µM Mito [Ca²⁺]

§0.5µM Cytosol [Ca²⁺]

§100nM Cytosol [Ca²⁺]

§100nM

Extra-cellular space [Ca2+]

§2mM

Extra-cellular space [Ca2+]

§2mM Figure 3 : Ca²⁺ toolkit

Cells possess an important Ca²⁺

signaling toolkit composed of channels, pumps and exchangers embedded in diverse membranes.

Mitochondria have a role of cytosolic Ca²⁺ buffer by taking up Ca²⁺ through mitochondrial Ca²⁺ uniporter (MCU) and releasing it through mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). High Ca²⁺ concentration in the endoplasmic reticulum is maintained by the sarco- endoplasmic Ca²⁺ ATPase (SERCA) and the release of Ca²⁺ arises from either Inositol-1,4,5 triphosphate receptor (IP₃R) or Ryanodine receptor (RyR). The plasma membrane Ca²⁺

ATPase (PMCA) and the Na⁺/Ca²⁺

exchanger (NCX) extrude Ca²⁺ from the cells and different channels are involved in the Ca²⁺ entry.

Letm1 H⁺ Ca²⁺

II. Ca²⁺ homeostasis

Ca²⁺ as a ubiquitous second messenger is responsible for controlling many different cellular processes, such as the cell growth and the cell death, the muscle contraction, the neuronal excitability or the cell differentiation. At rest the intracellular Ca²⁺ concentration is very low (100nM). But when cells are activated this level rises to roughly 1000nM. It is this elevation of intracellular Ca²⁺, or more precisely the versatility of the Ca²⁺ signalling mechanism in terms of speed, amplitude and spatio-temporal patterning, that regulates so many functions. The level of intracellular Ca²⁺ is determined by the balance between the introduction of Ca²⁺ into the cytoplasm (the ON mechanism) and the Ca²⁺ removal to restore the resting state (the OFF mechanism), by the combined action of a complex machinery composed of Ca²⁺ channels, pumps and exchangers. Each cell type has its own toolkit to create Ca²⁺ pulses with different spatial and temporal properties (Fig. 3). It is a variation of the ON/OFF mechanisms reaction that allows these Ca²⁺ transients.

i. The ON mechanisms

The cells have an access to two different sources of Ca²⁺ to elevate the intracellular Ca²⁺ concentration. The first is Ca²⁺ from internal stores and the second is Ca²⁺ from extra- cellular space. The major internal Ca²⁺ store is the endoplasmic reticulum (ER), or the equivalent organelle, the sarcoplasmic reticulum (SR) of muscle cells, with a free Ca²⁺

concentration between 200-500µM. This high luminal concentration is maintained by sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) pumps located in the ER membrane. These pumps actively transport Ca²⁺ from the cytoplasm to the lumen of the ER, against the chemical gradient, using ATP. The Ca²⁺ is then sequestered in the organelle by the high concentration of specialized buffer molecules such as calsequestrin, creating equilibrium between bound and free Ca²⁺ (Clapham 1995). Ca²⁺-release from these internal stores is mediated by different channels among which the inositol-1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) families that are the most abundant. The principal activator of IP3R is, as suggested by its name, the second messenger IP3 (inositol-1,4,5-trisphosphate).

Indeed, stimulation of cells with an agonist activates phospholipase C (PLC), leading to the generation of IP3 and diacylglycerol (DAG). Different mechanisms can activate the PLC, such as G-protein-coupled receptors, tyrosine––kinase-coupled receptors or activation trough Ras.

The IP3 generated is able to circulate in the cytosol and binds IP3R (Berridge 1993); (Meyer et al. 1988). The cytosolic Ca²⁺ itself is the second essential regulator of IP3R and its action can be both stimulatory and inhibitory. Indeed low concentrations of Ca²⁺ (100––300 nM) are

stimulatory, while above 300 nM, Ca²⁺ becomes inhibitory (Bootman and Lipp 1999). IP3Rs are also regulated by many additional signals such as ATP or cAMP, but IP3 and Ca²⁺ are the obligate physiological agonists, without which IP3R cannot be activated (Marchant and Taylor 1997); (Foskett et al. 2007).

The direct regulation by the Ca²⁺ is known as Ca²⁺-induced Ca²⁺-release (CICR) mechanism. The CICR is the main activation mechanism of the other ligand-activated receptor/channel present in the ER membrane: the RyR. Indeed, without Ca²⁺, the receptor can not have its maximum effect or can not be activated. The Ca²⁺ activation shows a bell- shaped dependence (it is inactive at low nM concentrations of Ca²⁺, active at low ȝM concentrations of Ca²⁺ and inactivated by high concentrations of Ca²⁺ that are in the mM range) (Bezprozvanny et al. 1991). The binding site for Ca²⁺ is on the cytosolic face, leading to a positive feedback that will cause a greater influx of Ca²⁺ in the cytosol (Endo et al. 1970).

The RyR are particularly important in neurons and skeletal muscles. Other second messengers are involved in the activation of this receptor, such as the cADP ribose or the cAMP (Rakovic et al. 1999); (Noguchi et al. 1997). But like the IP3R, without Ca²⁺, RyR could not be opened.

The second source is the extracellular Ca²⁺. To use this infinite pool, the cells have families of Ca²⁺ entry channels located at the plasma membrane. The description of these channels and the mechanism of Ca²⁺ influx will be discussed later, in other chapters.

ii. The OFF mechanisms

Once the Ca²⁺ has performed its signalling functions, it has to be removed from the cytoplasm so that the cell returns to its resting state. To do this, the cell is equipped with pumps, and exchangers. The plasma membrane Ca²⁺ ATPase (PMCA) actively extrudes Ca²⁺

outside the cell and the Na⁺/Ca⁺ exchanger (NCX) located at the plasma membrane drives Ca⁺ ions out, allowing a return to resting state. These two proteins also maintain the low Ca²⁺

concentration vital for the cell. In addition to these plasma membrane proteins, the SERCA pumps return Ca⁺ to the internal stores, leading also to a Ca²⁺ decrease in the cytosol (Berridge et al. 2000); (Berridge et al. 2003).

The mitochondrion is another important component of the OFF mechanism. In addition to its role of ATP production, mitochondria are also involved in cellular Ca²⁺

homeostasis, by rapidly sequestering Ca²⁺ during the developpement of the Ca²⁺ signal and then releases it back slowly during the recovery phase (Arnaudeau et al. 2001). The mitochondria can accumulate Ca²⁺ and are thereby able to buffer local Ca²⁺. Several proteins are involved in the mitochondrial Ca²⁺ buffering. First a mitochondrial Ca²⁺ uniporter (MCU)

drives a rapid Ca²⁺ uptake, using the electrochemical gradient created by the extrusion of protons. This uniporter operates at micromolar concentration of cytosolic Ca²⁺ (Rizzuto and Pozzan 2006). The molecular identity of this uniporter is still unknown, but two members of the uncoupling protein (UCP) family, UCP2 and UCP3 where shown to be indispensable for mitochondrial Ca²⁺ uptake. But it remains unclear whether these two proteins represent the channel proteins responsible for mitochondrial Ca²⁺ sequestration or if they are regulators of its activity (Trenker et al. 2007). Recently another mitochondrial player has been discovered by genome-wide RNAi screening, which is involved in the slow uptake of Ca²⁺ across the inner mitochondrial membrane, at submicromolar concentration of cytosolic Ca²⁺. This protein named Letm1 is an exchanger: the uptake of Ca²⁺ is coupled with the extrusion of H⁺ (Jiang et al. 2009). The protein involved in the extrusion of Ca²⁺ is also an exchanger. Once the cytosolic Ca²⁺ has returned to its resting level, the mitochondrial Na⁺/Ca²⁺ exchanger (mNCX) pumps the large load of Ca²⁺ back into the cytoplasm, from which it is either return to the ER or removed from the cell (for review see (Bernardi 1999)).

III. Ca²⁺ influx

The signals produced by Ca²⁺ release from Ca²⁺ stores are limited by the capacity of the ER. Thus a lot of Ca²⁺ signals are supported by a Ca²⁺ influx that results from the opening of Ca²⁺ channels located at the plasma membrane. These channels are defined by the way they are activated, such as Voltage-operated Ca²⁺ channels (VOCCs), Receptor-activated Ca²⁺

channels (RACCs) or Store-operated Ca²⁺ channels (SOCCs).

Voltage-operated channels as their name suggested, are activated by membrane depolarization and mediate Ca²⁺ influx. Because my thesis has been performed on endothelial cells, which are non-excitable cells and thus do not express these types of channels, they will not be discussed further. So this introduction will be focused on SOCCs and RACCs.

i. Store-operated Ca²⁺ entry (SOCE)

The SOCE pathway is present in virtually every cell type and is highly conserved among species (Mohamed et al. 2003). The regulation of store-operated Ca²⁺ channels (SOCC) is directly linked to the Ca²⁺ filling state of the endoplasmic reticulum. When the cellular Ca²⁺ pools are depleted, SOCC are activated, leading to Ca²⁺ influx. A part of this cytosolic Ca²⁺ is then used by the SERCA pumps for Ca²⁺ store refilling.

The theory of channels directly regulated by the level of the ER filling was first proposed in 1986 by Putney (Putney 1986). This idea comes from a series of experiments in

parotid acinar cells investigating the relationship between Ca²⁺ release from ER, Ca²⁺ entry, and store refilling. Indeed, when stores were full, Ca²⁺ influx did not occur but, as the stores emptied, Ca²⁺ entry developed. This mechanism was originally called capacitative Ca²⁺ entry (CCE). The first studies on the SOCE were performed with physiological stimulations that trigger Ca²⁺ release from stores through IP3 receptor. But the discovery of a specific inhibitor of the SERCA, thapsigargin (TG), which enables the activation of SOCE artificially, without the production of any second messenger or the activation of an upstream intracellular pathway, has been a great advance in the study of SOCE. TG by blocking the SERCA prevents the filling of the ER. So, a slow and passive depletion of the stores happens due to the ER Ca²⁺ leak, which can not be compensated (Takemura et al. 1989).

To date, the best characterized SOCE current is the CRAC current (Ca²⁺ release activated Ca²⁺), which is mainly found in blood cells like T lymphocytes. First described by Hoth and Penner in mast cells, this current is very small, exhibits a strong inward rectification, and is highly Ca²⁺ selective (Hoth and Penner 1992);(Fasolato et al. 1993).

However, its tiny unitary conductance, estimated by noise analysis at 24fS prevents its recording using the single channel configuration (Parekh and Putney 2005); (Zweifach and Lewis 1993). The ICRAC has been largely studied and has some well known electrophysiological behaviours. Firstly, the current magnitude is amplified in divalent-free (DVF) conditions. In absence of extracellular divalent ions, ICRAC conducts Na⁺ in place of Ca²⁺, due to the removal of Ca²⁺ block, leading to a significantly larger current (Bakowski and Parekh 2002). This large Na⁺-current exhibits a rapid depotentiation resulting from the dissociation of Ca²⁺ from an external site. The binding of Ca²⁺ on this site is required for the full activation of the CRAC channels (CRACC) (Su et al. 2004); (Zweifach and Lewis 1996).

Another known characteristic of the CRAC current, is the rapid inactivation, due to an intracellular Ca²⁺-binding site on the CRACC. This constitutes a local fast negative feedback mechanism (Hoth and Penner 1993). ICRAC is not the only current supporting SOCE. SOCE currents are heterogeneous and depend on the cell types studied. Several compounds have been reported to inhibit SOCC, such as La³⁺, Gd³⁺, SK&F96365 or 2-APB. All these compounds are not selective for ISOC but block also some Cl^- channels or nonselective cations channels at the same concentrations (Franzius et al. 1994). This lack of specific inhibitors is a major obstacle to differentiate a real ICRAC from other ISOC sometimes known as CRAC-like current.

After the discovery that activation of a Ca²⁺ entry was directly linked to ER, the attention was focused on the nature of the signal linking intracellular Ca²⁺ stores to SOCC.

Over the years, many theories have emerged. Among these models, three different types of signals have been proposed to explain this link between ER and plasma membrane. Putney et al. have been first to put forward the idea that a diffusible messenger might link the store emptying to activation of SOCC (Putney 1990). But the first experimental evidence for such a messenger was provided by the Tsien’’s group, who described a factor in extracts of stimulated Jurkat T cells that increased Ca²⁺ entry in several cell types (Randriamampita and Tsien 1993). This factor was termed CIF for calcium influx factor. The original extract activating Ca²⁺ entry in non stimulated cells had an effect when added in the extracellular solution. It is the Hanley’’s group, which made an important step in CIF purification by proving the original extract was a mix of different factors. They have used different purification techniques to isolate a specific component that act exclusively when microinjected into the cells (Thomas and Hanley 1995); (Kim et al. 1995). Further evidence validating the CIF model came from the groups of Marchase and Bolotina. Indeed these groups have extracted CIF from other systems (yeast and platelets) and showed the product to have the same properties than CIF extracted from Jurkat T cells. Moreover they have contributed to go further in the purification of this factor (Csutora et al. 1999). So CIF is a diffusible factor that is produced when stores are depleted and activates SOCC (Trepakova et al. 2000). The mechanism of activation was also resolved. Indeed, when released from the stores, CIF induces the displacement of inhibitory calmodulin from the plasma membrane variant of Ca²⁺-independent phospholipase A2 (iPLA2), which transduces the signal to SOCC leading to their opening (Smani et al.

2004). But in spite of these studies showing the presence and the biological activity of CIF, detected by different groups in a variety of cell types (for review see (Bolotina and Csutora 2005)), the existence of this factor remains very controversial. Indeed, its molecular identity is still unknown and the isolated factor differs from one laboratory to another. Moreover, the current activated by CIF has not the same sensitivity to La³⁺ and CIF is relatively stable.

These two last points are not compatible with the endogenous ISOC, leading to some controversy about the involvement of CIF in SOCE activation (Putney et al. 2001).

The two other ideas advanced involve direct physical interactions between ER and plasma membrane. In the exocytosis model, depletion of stores causes the fusion of vesicles containing SOCC with the plasma membrane (Fasolato et al. 1993). But there are a few problems with this model. First, the exocytosis mechanism requires Ca²⁺, whereas the activation of SOCE occurs maximally when the cytosolic Ca²⁺ concentration is very low.

Moreover two studies have demonstrated activation of SOCC following the excision of membrane patches, suggesting that the channels are present in the plasma membrane before

the stores depletion (Zubov et al. 1999); (Braun et al. 2001). In the conformational coupling model, the communication between the ER and the plasma membrane involves a direct protein-protein interaction (Irvine 1990). A key feature of this model is that IP3 receptor

““senses”” when the store is empty and can then transmit this information to the SOCC, leading to SOCE activation via a direct protein-protein interaction (Berridge 1995). This model was proposed based on the example of the direct protein-protein interaction involved for the communication between the plasma membrane L-type voltage-dependent Ca²⁺ channels and the RyR, in skeletal muscle (Ebashi 1991).

A great step forward to understand the mechanism of SOCE activation was achieved by the molecular identification of two proteins directly involved in SOCE pathway: STIMs and ORAIs proteins. STIM was originally identified in a library screen by its ability to confer binding of pre-B lymphocytes to stromal cell and was first named SIM (Stromal interaction molecule) (Oritani and Kincade 1996), converted into STIM, due to its function in STIMulating calcium influx. The STIM gene family is ubiquitous. Whereas one gene is present in Drosophilia, there are two genes expressed in mammals (Williams et al. 2001). The key breakthroughs in identifying the proteins involved in SOCE came from RNAi screening in 2005 (Liou et al. 2005); (Roos et al. 2005), demonstrating that STIM1 is essential for SOCE. STIM1 is localised in the ER membrane and in the plasma membrane. It is a single membrane-spanning domain protein containing two protein-protein interaction domains, an EF-hand Ca²⁺-binding domain and a sterile alpha motif (SAM) protein interaction domain.

The amino-terminus is predicted to be either within the ER lumen or facing the extracellular space. Thanks to its EF-hand domain localised in the ER lumen, STIM1 is able to sense the Ca²⁺ filling state of the organelle. Following Ca²⁺ store emptying, STIM1 aggregates forming punctate structures anchored in the ER that re-localise near the plasma membrane. This formation of puncta is followed by the activation of SOCE (Liou et al. 2005); (Zhang et al.

2005). STIM1 triggers SOCE but does not form the SOCC itself. The RNAi screening was also the approach to discover the channel supporting Ca²⁺ influx. Three independent research groups performed a screening, leading to the identification of several additional genes required for SOCE, including ORAI, also named CRACM (Feske et al. 2006); (Vig et al.

2006); (Zhang et al. 2006). A parallel genetic screen performed by one of the three groups led to the discovery that a point mutation in Orai1 is the cause of a rare disease, the severe combined immunodeficiency (SCID). The SCID patients lack functional CRAC in T lymphocytes (Feske et al. 2006). The ORAI family has three members in mammals, which are each composed of four trans-membrane domains. There are two evidences showing that it is

ORAI1, which forms the CRACC. The first one is the amplification of the CRAC current when STIM1 and ORAI1 are overexpressed together in cells and secondly the ion selectivity is altered when a glutamate residue is mutated in the ORAI1 gene (Peinelt et al. 2006);

(Soboloff et al. 2006); (Yeromin et al. 2006); (Prakriya et al. 2006).

STIM1 functions as an ER Ca²⁺ sensor that regulates the ORAI1 channel opening.

When the store is full, Ca²⁺ is bound to the EF-end domain of STIM1 present in the ER lumen and in basal conditions STIM1 forms dimers (Penna et al. 2008). But when the ER is depleted, STIM1 releases Ca²⁺, leading to its oligomerization (Fig. 4a). This oligomerization precedes and triggers translocation of STIM1 to the plasma membrane and the activation of ORAI1 channel activity (Liou et al. 2007); (Muik et al. 2008); (Luik et al. 2008). ORAI1 is also predominantly a dimer in the plasma membrane under resting conditions. But the interaction with the C-terminus of STIM1 induces ORAI1 dimers to dimerize, forming tetramers that constitute the Ca²⁺-selective pore (Fig 4b) (Penna et al. 2008).

Evidences demonstrate that ORAI1 channels support ICRAC in particular in blood cells.

But some other channels are good candidates for SOCC, especially the members of transient receptor potential (TRP) channel family. The TRP proteins superfamily is a diverse group of cation permeable channels. TRP channels are present in virtually all mammalian cell types

Figure 4 : A. In the basal state, that means when ER is filled and Ca²⁺ bounds to the STIM EF-hand domain, STIM molecules are dimers. ER depletion leads to the Ca²⁺ unbinding from the EF-hand domain of STIM. This molecular switch is followed by the STIM oligomerization and translocation to ER-plasma membrane junctions.

B. STIM oligomerization and translocation induces Orai channels to cluster in the adjacent plasma membrane. Two STIM can activate a single CRAC channel consisting of an Orai tetramer (Cahalan 2009)

EF-Hand SAM

ERM domain

A B

ERM domain

EF-Hand SAM

and this superfamily includes 56 members, divided in seven subfamilies: TRPC (C for canonical), TRPM (M for melastatin), and TRPV (V for vanilloid), TRPN (N for NOMPC from no mechanoreceptor potential-c), TRPA (A for ankrin-like with transmembrane domain 1), TRPML (ML for mucolipin) and TRPP (P for Polycystin) (for reviews see: (Clapham 2003); (Montell 2005); (Nilius et al. 2005)). Studies on the involvement of TRPC members in SOCE have shown that STIM1 interact directly with endogenous TRPC1, TRPC4 and TRPC5 and indirectly with TRPC3 and TRPC6 (Yuan et al. 2007); (Worley et al. 2007).

Interestingly, they reported that STIM1 is not required for the channels function, but rather for the channel activity if it works as SOCE. In another report from Ambudkar’’s lab, the authors demonstrated an interaction between STIM1, ORAI1 and TRPC1. These three proteins seem to form a tertiary complex essential for SOCE in salivary glands (Ong et al. 2007).

Despite the evidence that the SOCE is a ubiquitous pathway and essential to maintain adequate Ca²⁺ levels in the ER, that is critical for a variety of cellular functions (such as correct folding and processing of synthesis proteins), it is clear that it is not the only pathway for Ca²⁺ entry in non-excitable cells. Examination of the literature indicates that in normal physiologically relevant responses, evidences of the essential role of the SOCE is largely limited to hematopoetic cells (Zweifach and Lewis 1993); (Lewis and Cahalan 1989); (Hoth and Penner 1993). Most of the other studies concerning Ca²⁺ entry in other cell types use conditions leading to depletion of the stores with pharmacological agents such as TG or high concentration of IP3 or by inducing passive depletion of stores with intracellular Ca²⁺-buffers.

All these methods force the system to reveal the presence of SOCE in the cells, but do not demonstrate the relevance of this pathway in physiological conditions. So it is not surprising that there are evidences demonstrating the existence of alternative store-independent pathways (Shuttleworth 1996); (Shuttleworth et al. 2004).

ii. Store-independent Ca²⁺ entry or receptor-activated Ca²⁺ entry (RACE) The RACE pathway requires the presence of an agonist to be activated and is independent of Ca²⁺ stores filling state. Its activation results from the production of second messengers, such as Ca²⁺ itself, IP4 (Luckhoff and Clapham 1992), arachidonic acid (Mignen et al. 2005) or diacylglycerol (Hofmann et al. 1999). But as for SOCE before the discovery of STIM1 and ORAI1, the field of RACE suffers from two major problems: the lack of specific channel inhibitor, and the unknown molecular identity of the channel(s) responsible for this type of Ca²⁺ entry. The previous studies on RACE have shown evidences that a great variety of ionic channels are potential players involved in this pathway and that might be possibly

activated by various mechanisms, which depends on the cellular system investigated. To date, the best characterized RACE current is the arachidonic-regulated Ca²⁺ (ARC) entry, which was described originally by the group of Trevor Shuttleworth (Mignen and Shuttleworth 2000).

a. Arachidonic-regulated Ca²⁺ entry (ARCE)

The ARC current requires arachidonic acid to get activated, but is independent from store depletion. The ARC channel (ARCC) has much in common with the CRACCs: it displays small conductance values, its current-relationships are inwardly rectifying with very positive reversal potentials due to a high selectivity to Ca²⁺ and it is also blocked by La³⁺ and Gd³⁺. However, unlike the CRACCs, ARCCs have no Ca²⁺-dependent fast inactivation and are insensitive to 2-APB. Moreover the sequential activation in the same cell of the two different conductances are additive, confirming the co-existence of these two distinct pathways (Shuttleworth 2009). Until recently, the molecular identity of proteins involved in ARC pathway was unknown. There were just evidences excluding members of the TRP channel family (Shuttleworth et al. 2004). However, the studies revealing the important role of ORAI1 and STIM1 in SOCE gave the idea to the Shuttleworth’’s lab to test the involvement of these proteins in the ARC pathway. They have demonstrated that STIM1 protein levels altered the activity of ARCC. But unlike the CRACCs, ARCC is exclusively dependent on the expression of STIM1 that resides in the plasma membrane (Mignen et al. 2007). The similarity between ICRAC and IARC suggests that the channels supporting these two currents are molecularly related. So they tested the involvement of ORAI family. Whereas the CRACC pore appears to be comprised of only ORAI1, ORAI1 and ORAI3 appear to contribute to the ARCC pore (Mignen et al. 2008). Then, albeit the similitude between IARC and ICRAC, the two currents are developing under distinct modes of activation and the channels involved seem to be formed by different multimers.

b. RACE and the TRP channels

On the contrary to ARCC and to CRACC, the other RACC described are generally non Ca²⁺ selective. This point and the fact that RACC are activated by different second messengers suggest that the most promising candidates to support RACE are members of the TRP channel family. The TRP channels are mostly non-selective cation channels and are gated by a great variety of mechanisms such as heat, osmotic cell swelling, store depletion, mechanical stretch or second messengers (DAG, Ca²⁺, PIP3, 5,6-epoxyeicosatrienoic acid,

ADP-ribose, arachidonic acid, ATP……) (for review see (Venkatachalam and Montell 2007);

(Clapham 2003); (Montell 2005)). The interest is focused particularly on members of the subfamily of TRPC, because the common feature of this subfamily is the activation through pathways that engage the PLC (Zhu et al. 1996); (Okada et al. 1998); (Boulay et al. 1997).

But the mechanism, through which the TRPC members are activated, differs. Both IP3 and DAG are produced upon activation of PLC, suggesting different activation modes: activation due to the release of Ca²⁺ from ER or a direct activation by DAG (Montell 2005). It has been proved that TRPC3/6 and 7 are gated by direct exposure to DAG (Plant and Schaefer 2003);

(Kamouchi et al. 1999); (Vazquez et al. 2006); (Vennekens et al. 2002), whereas TRPC1, 4 and 5 are activated through a store-operated mechanism (Zhu et al. 1996); (Philipp et al.

1996); (Zitt et al. 1996). Other TRP family members are good candidates to support RACE, as TRPM2, TRPM4 and TRPM5 that have been shown to be activated by intracellular Ca²⁺

(Kraft and Harteneck 2005 146).

c. RACE and the Na⁺/Ca²⁺ exchanger

Beside ionic channels, another plasma membrane ion shuttling protein gained very much attention recently, the Na⁺/Ca²⁺ exchanger (NCX). This exchanger normally in the so- called forward mode represents a Ca²⁺ extrusion system, which uses the Na⁺ gradient to transport Ca²⁺ out of the cell, with a stochiometry of 3Na⁺ for 1Ca²⁺ (Blaustein and Lederer 1999). But the NCX can work also in the reverse direction and moves Ca²⁺ into the cells, depending on the electrochemical driving force. The net Ca²⁺ movement mediated by the exchanger may change direction when the membrane potential varies or when the cytosolic Na⁺ or Ca²⁺ concentration is altered.

So, a very important part of the exchanger function may be to promote Ca²⁺ entry.

Experimental evidences that NCX participates in Ca²⁺ entry were obtained in different type of cells like smooth muscle cells (Zhang et al.

2005), platelets (Harper and Sage 2007) or endothelial cells (Paltauf-Doburzynska et al.

2000). An important condition to reverse the

Figure 5 : A non-selective cation channel (for example a member of the TRP family) is activated upon cell stimulation, leading to a Na⁺ loading. This Na⁺ influx near the Na⁺/Ca²⁺ exchanger (NCX) reverses its mode and extrudes the excess of Na⁺ leading to a Ca²⁺ influx. This mechanism implies that the channel and the NCX are close.

mode of the NCX implies a Na⁺ loading procedure. This Na⁺ entry is believed to happen through non selective cation channels (NSCC). If the Na⁺ loading reaches a sufficient high level, the NCX could reverse its mode to extrude the excess of Na⁺, leading to a Ca²⁺ entry in a localized region of the plasma membrane (Arnon et al. 2000). This process depends also on the cytosolic Ca²⁺ concentration as well as on the membrane potential. Studies in platelets, HEK293 cells and smooth muscle cells have demonstrated a functional link between NSCC and NCX. The NSCC allowing a Na⁺ loading in these cells are part of the TRPC subfamily (Harper and Sage 2007); (Rosker et al. 2004); (Lemos et al. 2007); (Poburko et al. 2007).

C. Ca

signaling in Endothelial Cells

The stimulation of endothelial cells (EC) with an inositol 1,4,5-triphosphate- (IP3) generating agonist (like histamine) leads to a biphasic increase of cytosolic Ca²⁺

concentration. Indeed, the agonist upon binding to its receptor activates G-protein which in turn stimulates the phospholipase C (PLC), resulting in the production of DAG and IP3. The initial cytosolic Ca²⁺ elevation is due to the Ca²⁺ release from the ER through IP3-sensitive channels (Berridge 1993). This Ca²⁺ release is associated with a sustained Ca²⁺ influx from the outside (Himmel et al. 1993). The EC are non excitable cells: they do not have voltage- dependent channels. So Ca²⁺ entry occurs through voltage insensitive channels. This cytosolic Ca²⁺ elevation is accompanied by a membrane hyperpolarization due to Ca²⁺-activated K⁺ channels opening (Nilius and Droogmans 2001). The hyperpolarization represents a feed forward mechanism for Ca²⁺ entry as it increases the electrical driving force for Ca²⁺ to enter the cell. While the mechanism leading to the release of Ca²⁺ from the intracellular stores is well described, the mechanism of Ca²⁺ entry in EC as well as the molecular identity of the proteins involved in this influx is very controversial.

SOCE is frequently referred to as the main pathway for Ca²⁺ entry and thus is essential to a proper function of EC (Freichel et al. 2001); (Abdullaev et al. 2008). This pathway was reported for several EC types such as Human Umbilical Vein Endothelial Cells (HUVECs) (Oike et al. 1994), bovine/rabbit aorta (Vaca and Kunze 1994); (Sasajima et al. 1997) or bovine pulmonary artery (Pasyk et al. 1995). But the electrophysiological profile of the SOCE conductance is very different from one study to another, reporting conductance from

<0.5pA/pF to 5pA/pF (Fasolato and Nilius 1998); (Brough et al. 2001). The molecular composition of the SOCC in EC is also a highly controversial topic. Several groups propose different members of the TRPC family to mediate SOCE (Brough et al. 2001); (Moore et al.