TRPC channels and other members of the Orai and STIM families

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

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

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

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

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

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

Adapted from Montell, C. (2005).

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

3.3.2. STIM isoforms

 STIM1L

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

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

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

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

 STIM2 and STIM2.1/ STIM2β

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

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

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

3.3.3. Orai isoforms

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

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

3.4. Physiological functions of SOCE