Physiological functions of SOCE

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

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

3.4.2. At the whole body level

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

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

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

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

 Immunity

The best characterized role for SOCE concerns immune cell development and physiology. Most endogenous patch-clamp recordings of ICRAC were realized in RBL (Rat Basophilic Leukemia) cells or Jurkat cells that are a human T lymphocyte cell line (Hoth and Penner 1992, Zweifach and Lewis 1993). Both cell lines display recordable ICRAC contrary to HEK 293T cell line that, despite a measurable SOCE in Ca²⁺ imaging, does not allow endogenous ICRAC recordings. These findings likely reflect highest STIM1 and Orai1 expression levels in immune cells to ensure efficient SOCE explaining SCID-like syndrome in patients harboring mutations in either Orai1 or STIM1 gene. Despite several reports on SOCE deficient B cells, macrophages, mast cells, dendritic cells and neutrophils (Braun, Gessner et al. 2009, Steinckwich, Schenten et al. 2011, Nunes, Cornut et al. 2012, Felix, Crottes et al. 2013, Zhang, Clemens et al. 2014), impaired immune response in these patients, seems mainly due to defective T cell (Feske, Muller et al.

1996, Feske, Giltnane et al. 2001, McCarl, Khalil et al. 2010) and Natural Killer (NK) cell (Maul-Pavicic, Chiang et al. 2011, Schaballie, Rodriguez et al. 2015) ability to mediate cytotoxicity and cytokine production (including TNF-α and IFN-γ production) (Feske, Draeger et al. 2000, Fuchs, Rensing-Ehl et al. 2012, Chou, Badran et al. 2015). This explains the increased sensibility to viral infections but also the patient propensity to develop certain type of malignancies such as sarcomas (Byun, Abhyankar et al. 2010). Concerning the other immune cells, findings in patients compared to mice in vivo and in vitro studies are not always concordant. Although stim1^-/-stim2^-/- mice show preserved antigen-specific antibody responses (Matsumoto, Fujii et al. 2011), the absence of certain specific antibodies are noted for some patients with CRAC channelopathy previously exposed to the corresponding pathogens (via vaccination for example) (Schlesier, Niemeyer et al. 1993, Le Deist, Hivroz et al. 1995, Feske, Muller et al.

1996, McCarl, Picard et al. 2009, Picard, McCarl et al. 2009). This apparent discrepancy could originate from defect in T helper cells mediated B cell activation rather than impaired B cell function by itself (Lacruz and Feske 2015). Regarding innate immune cells, while several mice models with either Orai1 or STIM1 KO gene demonstrate altered chemotaxis, Reactive Oxygen Species (ROS) production and phagocytosis in neutrophils (Steinckwich, Schenten et al. 2011, Nunes, Cornut et al. , Zhang, Clemens et al. 2014, Steinckwich, Myers et al. 2015), the same cells from patients with loss-of-function mutations in the same genes have only slightly decreased SOCE and preserved immune functions. Differences between mice models and

human patients might be explained by species specificity due to implication of other Orai and STIM isoforms in human (Lacruz and Feske 2015).

 Autoimmunity

Lymphoproliferation, autoimmune hemolytic anemia and thrombocytopenia which are respectively destruction of erythrocytes and platelets by its own immune cells, are all features of autoimmune disorder. This characteristic of the clinical phenotype of STIM1 deficient patients (Picard, McCarl et al. 2009, Byun, Abhyankar et al. 2010, Fuchs, Rensing-Ehl et al.

2012) (that is less common in Orai1 deficient patients (McCarl, Picard et al. 2009)) can be surprising in the light of previously described impaired T cell functions. A subset of T cell population, namely Tregs for regulatory T cells, which controls tolerance to self-antigens of the other T cells (effector T cells) is down-regulated in patients and would be the cause of autoimmunity (Picard, McCarl et al. 2009, Fuchs, Rensing-Ehl et al. 2012). The restricted autoimmune disorders to only blood cells would be due to residual Tregs ability to maintain effector T cell tolerance to self-antigens as well as reduced T cell efficacy to fulfill their functions (Lacruz and Feske 2015).

 Platelets function

The most compelling evidence for SOCE involvement in platelets function comes from patients with gain-of-function mutations in Stim1, who develop York platelet or Stormorken syndrome (Stormorken, Holmsen et al. 1995, Misceo, Holmgren et al. 2014, Morin, Bruechle et al. 2014, Nesin, Wiley et al. 2014). In particular, these hereditary diseases are characterized by thrombocytopenia and mild bleeding diathesis clearly linking SOCE to platelet function.

Indeed, over-activated SOCE is associated with activated platelets prior to any type of stimulation. These platelets show increased α-granule secretion and exposed phosphatidylserine at their PM among other marks of activation (Bergmeier, Oh-Hora et al.

2009, Gilio, van Kruchten et al. 2010, Ahmad, Boulaftali et al. 2011, Nakamura, Sandrock-Lang et al. 2013). These findings are in accordance with patients and KO mice harboring loss-of-function mutations in SOCE genes, for whom these 2 platelet loss-of-functions are impaired while not inducing as drastic bleeding diathesis phenotype as for patients with York platelet or Stormorken syndrome (Lacruz and Feske 2015).

 Dental enamel formation

Dental enamel from patients with CRAC channelopathy appears hypo-mineralized and thus more prone to abrasion. This leads to almost complete dental enamel elimination in the childhood rendering tooth more sensible to caries (Feske, Gwack et al. 2006, McCarl, Picard et al. 2009, Picard, McCarl et al. 2009, Fuchs, Rensing-Ehl et al. 2012). Ameloblasts that are cells responsible for enamel formation, ensure Ca²⁺ transport from blood vessel at their basal pole to their apical pole in contact with the forming enamel. Ca²⁺ excreted by ameloblasts participates in hydroxyapatite formation which is a crystalline Ca²⁺ phosphate, the main component of enamel (Smith 1998). SOCE pathway would be implicated in Ca²⁺ uptake from blood vessel explaining the poor enamel mineralization in Orai1 and STIM1 deficient patients (Hubbard 1996, Hubbard 2000, Lacruz and Feske 2015).

 Skeletal muscle development and function

A common feature of patients with loss-of-function or gain-of-function mutations in Orai1 and STIM1 gene is skeletal myopathy. While patients with gain-of-function mutation mainly suffer from tubular aggregate myopathy (TAM) that is marked by decreased fatigue resistance associated with muscle pain and cramping mainly in the lower limbs (Lee and Noguchi 2016), patients with loss-of-function mutations show generalized muscle weakness sometimes associated with muscle fiber type II atrophy (Lacruz and Feske 2015). Involvement of SOCE in muscle development and physiology will be discussed in greater details in the following sections.

In addition to hereditary diseases associated with mutations in Orai1 and STIM1, studies have revealed SOCE implication in an increasing number of other physiological and pathophysiological functions including neurological processes, tumorigenesis, bone and skin formation, exocrine activity of lacrimal and mammary glands and reproduction (Putney, Steinckwich-Besancon et al. 2016). Careful analysis of conditional Orai and STIM KO mice have greatly participated in discovery of SOCE involvement in these functions as global KO mice for either STIM1 or Orai1 die soon after birth (Gwack, Srikanth et al. 2008, Oh-Hora, Yamashita et al. 2008, Stiber, Hawkins et al. 2008, Varga-Szabo, Braun et al. 2008). Conditional mice models will be of importance in the future to better decipher pathways regulated by SOCE in already

identified SOCE dependent physiological functions as well as to complete our knowledge regarding the other processes that it controls.