Calcium signaling

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

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

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

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

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

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

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

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

Figure III.1: Sydney Ringer.

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

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

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

channels.

1.1. Ca²⁺ dependent proteins

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

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

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

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

2+

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

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

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

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

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

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

1.2. Ca²⁺ pumps and exchangers

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

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

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

1.3. Ca²⁺ channels

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

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

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

2+

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

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

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

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

dependent processes (figure III.3).

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

Figure III.3: Ca²⁺ signaling machinery.

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

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

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

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