Excessive cytosolic calcium ion (Ca(2+)) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca(2+) entry (CCE) is a process whereby depletion of intracellular Ca(2+) stores causes the activation of plasma membrane Ca(2+) channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)-resident Ca(2+) sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca(2+) concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca(2+) accumulation in neurons. Neurons from Stim2(-/-) mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2(-/-) mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca 2؉ entry (SOCE) with Orai1 as principal Ca 2؉ entry channel. Both proteins contribute to collagendependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca 2؉ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1 ] i rises are required for the procoagulant response (1, 2). The latter is achieved by a Ca 2ϩ -activated scramblase mechanism disturbing the normal phospholipid asymmetry in the plasma membrane, with, as a result, the exposure of phosphatidylserine (PS) 5 at the outer membrane surface (3, 4). Exposed PS provides high affinity binding sites for key coagulation factors and, thereby, facilitates the assembly of tenase and prothrombinase complexes, which are responsible for the formation of factor Xa and thrombin, respectively (3). Because thrombin is one of the most potent platelet agonists, the procoagulant platelet response triggers a potent positive feedback loop of platelet and coagulation activation. Recent in vivo studies have indicated that PS exposure and ensuing thrombin generation are key regulatory events in murine arterial thrombus formation (5, 6).Whereas stored platelets may expose procoagulant PS in a Ca 2ϩ -independent way, PS exposure in activated platelets relies on a high and prolonged rise in cytosolic [Ca 2ϩ ] i (7). Platelet stimulation with single G protein-coupled agonists, like thrombin and ADP, results in limited PS exposure (8, 9), but stimulation of the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI), with ligands such as collagen-related peptide (CRP) or convulxin, results in appreciable procoagulant activity (10, 11). Combined stimulation of the collagen and thrombin receptors though results in high PS exposure, likely because these agonists use different signaling pathways for mobilizing cytosolic Ca 2ϩ (1). Although thrombin transiently activates G q ␣ and phospholipase C␤2/␤3 isoforms, activation of GPVI causes a more persistent activation of the phospholipase C␥2 isoform (2, 12). For PS exposure, entry of extracellular Ca 2ϩ is required, complementing the Ca 2ϩ -mobilizing effect of phospholipase C stimulation, to reach sufficiently high [Ca 2ϩ ] i (10,13,14).

Calcium (Ca2+) signaling in T lymphocytes is essential for a variety of functions, including the regulation of differentiation, gene transcription, and effector functions. A major Ca2+ entry pathway in nonexcitable cells, including T cells, is store-operated Ca2+ entry (SOCE), wherein depletion of intracellular Ca2+ stores upon receptor stimulation causes subsequent influx of extracellular Ca2+ across the plasma membrane. Stromal interaction molecule (STIM) 1 is the Ca2+ sensor in the endoplasmic reticulum, which controls this process, whereas the other STIM isoform, STIM2, coregulates SOCE. Although the contribution of STIM molecules and SOCE to T lymphocyte function is well studied in vitro, their significance for immune processes in vivo has remained largely elusive. In this study, we studied T cell function in mice lacking STIM1 or STIM2 in a model of myelin-oligodendrocyte glycoprotein (MOG35–55)-induced experimental autoimmune encephalomyelitis (EAE). We found that STIM1 deficiency significantly impaired the generation of neuroantigen-specific T cell responses in vivo with reduced Th1/Th17 responses, resulting in complete protection from EAE. Mice lacking STIM2 developed EAE, but the disease course was ameliorated. This was associated with a reduced clinical peak of disease. Deficiency of STIM2 was associated with an overall reduced proliferative capacity of lymphocytes and a reduction of IFN-γ/IL-17 production by neuroantigen-specific T cells. Neither STIM1 nor STIM2 deficiency altered the phenotype or function of APCs. These findings reveal a crucial role of STIM-dependent pathways for T cell function and activation under autoimmune inflammatory conditions, establishing them as attractive new molecular therapeutic targets for the treatment of inflammatory and autoimmune disorders.

Ca 2+ influx by store-operated Ca 2+ channels is a major mechanism for intracellular Ca 2+ homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca 2+ store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 interaction, followed by reinteraction with STIM1 and dissociation from Orai1. Co-expression of SARAF and either Orai1 or various N-terminal deletion Orai1 mutants did not alter SARAF-Orai1 interaction; however, expression of C-terminal deletion Orai1 mutants or blockade of the C-terminus of Orai1 impair the interaction with SARAF. These observations suggest that SARAF exerts an initial positive role in the activation of SOCE followed by the facilitation of SCDI of Orai1.