IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake

Andras T. Deak 1 Sandra Blass 1 Muhammad J. Khan 1 Lukas N. Groschner 1 Markus Waldeck-Weiermair 1 Seth Hallstro m 0 Wolfgang F. Graier 1 Roland Malli () 1 0 The Institute of Physiological Chemistry, Center of Physiological Medicine, Medical University of Graz , 8010-Graz , Austria 1 The Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz , 8010-Graz , Austria Mitochondria contribute to cell signaling by controlling storeoperated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca2+ channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca2+ uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+ buffering or ER Ca2+ depletion by sarcoplasmic or endoplasmic reticulum Ca2+ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca2+ micro-domains. - INTRODUCTION Store-operated Ca2+ entry (SOCE) is a common form of Ca2+ influx that is linked to important physiological functions of different cell types (Parekh and Putney, 2005). One characteristic feature of SOCE is its activation upon depletion of the endoplasmic reticulum (ER) Ca2+ store (Putney, 1986). With the identification of the key molecular constituents of SOCE the stromal interacting molecule 1 (STIM1) (Zhang et al., 2005; Liou et al., 2005) and the plasma membrane Ca2+-pore-forming Orai1 (Vig et al., 2006; Zhang et al., 2006) the clarification of the elusive molecular mechanism of SOCE has become possible (Soboloff et al., 2012). When the concentration of Ca2+ in the ER ([Ca2+]ER) is reduced, Ca2+ dissociates from the luminal EFhand domain of the ER-membrane-spanning STIM1, initiating its oligomerization (Liou et al., 2007). Subsequently, STIM1 oligomers translocate to subplasmalemmal ER domains, where they form higher-order aggregates, which appear as the so-called STIM1 punctae (Park et al., 2009). In this form, STIM1 couples with and activates Orai1 (Park et al., 2009) and other storeoperated channels (Cheng et al., 2013), resulting in Ca2+ entry. Apart from this function, STIM1 has been shown to regulate the activity of ion pumps (Manjarres et al., 2010; Ritchie et al., 2012), enzymes (Lefkimmiatis et al., 2009) and cell adhesion proteins (Shinde et al., 2013), pointing to a fundamental role of Ca2+-dependent STIM1 oligomerization in cell signaling. Long before the identification of STIM and Orai proteins and their role in SOCE, mitochondria were shown to contribute to the regulation of SOCE in immune cells (Hoth et al., 1997). Although the exact mechanisms by which mitochondria facilitate SOCE are still unclear, it is assumed that the ability of mitochondria to buffer Ca2+ counteracts the Ca2+-dependent inactivation of this Ca2+-sensitive Ca2+ entry pathway (Demaurex et al., 2009; Parekh, 2008b). In addition, mitochondrial Ca2+ uptake upon cell stimulation has been suggested to cause a more pronounced depletion of Ca2+ from the ER that consequently facilitates SOCE (Demaurex et al., 2009). Because proteins that mediate mitochondrial Ca2+ uptake have been identified only recently, the contribution of mitochondrial Ca2+ uptake to SOCE has only been investigated indirectly until now. In many studies, mitochondrial Ca2+ uptake was diminished by chemical uncouplers, such as carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) or antimycin A, an inhibitor of complex III of the respiratory chain, which resulted in a significant reduction in SOCE (Naghdi et al., 2010; Hoth et al., 1997; Glitsch et al., 2002). In line with these findings, energized mitochondria were shown to result in increased SOCE (Gilabert and Parekh, 2000). Although these findings point to the importance of mitochondria in SOCE regulation, the actual role of mitochondrial Ca2+ uptake in the process of SOCE activation and maintenance under physiological conditions remains elusive. In this study, mitochondrial Ca2+ uptake in HeLa cells was strongly diminished by a stable knockdown of either MCU (Baughman et al., 2011; De Stefani et al., 2011) (MCUKD) or UCP2 (Trenker et al., 2007; Trenker et al., 2008) (UCP2KD), two proteins of the inner mitochondrial membrane, which have been shown to be involved in mitochondrial Ca2+ uptake (WaldeckWeiermair et al., 2013). MCU, the proposed core component of a ubiquitous mitochondrial Ca2+ channel, contributes to mitochondrial Ca2+ uptake regardless of the source and mode of Ca2+ mobilization (Baughman et al., 2011; De Stefani et al., 2011), whereas UCP2 has been shown to mediate principally the uptake of Ca2+ from areas of high Ca2+ concentration Ca2+ micro-domains, which are formed upon ER Ca2+ release (Waldeck-Weiermair et al., 2013). Accordingly, the individual knockdown of these proteins enabled us to distinguish whether mitochondrial uptake of intracellularly released Ca2+ or of Ca2+ entering the cell is determinant for SOCE. Our data demonstrate that UCP2- and MCU-dependent mitochondrial uptake of inositol1,4,5-trisphosphate (IP3)-dependent intracellularly released Ca2+ represents an essential step in the activation of STIM1 and, hence, SOCE. Correlations between the dynamics of ER Ca2+ depletion and STIM1 oligomerization upon different modes of Ca2+ mobilization suggest that mitochondrial Ca2+ sequestration predominately shapes IP3-mediated cytosolic Ca2+ microdomains, which facilitates STIM1 oligomerization under physiological conditions of cell stimulation. In addition, MCU-dependent mitochondrial buffering of entering Ca2+ is crucial for the maintenance of SERCA-inhibition-induced SOCE signals. In summary, we highlight a special and tight regulation of STIM1 activation and SOCE maintenance by mitochondrial Ca2+ uptake upon physiological and nonphysiological stimuli. RESULTS Stable knock-down of either MCU or UCP2 inhibits mitochondrial Ca2+ uptake and impairs STIM1 oligomerization upon IP3-mediated Ca2+ release In line with recent reports (Baughman et al., 2011; De Stefani et al., 2011; Waldeck-Weiermair et al., 2013) a stable knockdown (...truncated)