STIM2 protein regulates Orai1-mediated store-operated Ca2+ entry in cardiomyocytes

Abstract

Stromal interaction molecules, STIM1 and STIM2, are ER Ca 2+ sensors that initiate store-operated Ca 2+ entry (SOCE). It has been recently identified three STIM2 splice variants (STIM2.1, 2.2 and 2.3), STIM2.2 being SOCE activator whereas STIM2.1 will be SOCE inhibitor. The role of STIM1-mediated SOCE has been highlighted in cardiac pathobiology but the one of STIM 2 .1 and STIM2.2 is unknown. To assess whether and how STIM2.1 and STIM2.2 regulate SOCE in neonatal rat ventricular cardiomyocytes (NRVMs). To analyze the function of STIM2.1 and STIM2.2 variants, the NRVMs were transfected with STIM2.2 or STIM2.1 in combination or not with STIM1 or dn-Orai1 106A mutant plasmids or with a mixture of 4 STIM2 siRNAs. At rest, both native STIM2 and exogenous STIM2.2 proteins present a reticular organization. Live cell confocal imaging in STIM2.2 overexpressing cells showed that STIM2.2 is aggregated and translocated towards the sarcolemma following the SR Ca 2+ store depletion. With Ca 2+ imaging, we found that STIM2.2 overexpression significantly increases SOCE, similarly to STIM1 overexpression, but the STIM1 and STIM2.2 co-expression has no additional effect. Importantly, the STIM2.2-enhanced SOCE is suppressed by functional Orai1 inhibition, either by co-expressing STIM2.2 with the dn-Orai1 106A mutant or with the selective Orai1 inhibitor, JPIII. In addition, the JPIII-sensitive Isoc current is also higher in STIM2.2 overexpressing cells. Nonetheless, silencing of endogenous STIM2 also increases SOCE. By co-immunoprecipitation, we found that STIM2, STIM1 and Orai1 interact together suggesting that all these proteins form a complex and constitute functional SOCE machinery. We demonstrated that, in overexpressing system, STIM2.2 functions together with STIM1 as a positive regulator of Orai1-mediated SOCE and that, in endogenous system, the STIM2.1 variant may be the predominant variant that negatively regulates SOCE in NRVMs.