Faculty Bibliography

Store-operated Ca²⁺ entry (SOCE) through Ca²⁺ -release activated Ca²⁺ (CRAC) channels is arguably the most important mechanism to generate Ca²⁺ signals in lymphocytes and other cells of the immune system. ORAI1 functions as the pore subunit of the CRAC channel and is activated by stromal interaction molecules (STIM) 1 and 2 in response to depletion of ER Ca²⁺ stores. The pathway is essential for functional immune responses involving T cells, mast cells and potentially other cells in the immune system. This presentation will discuss mechanisms of CRAC channel activation, phenotypes of human patients and mice lacking functional ORAI1 and STIM1 and the potential role of SOCE, ORAI1 and STIM1 in inflammatory diseases

BACKGROUND: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca(2+) influx through Ca(2+)-release activated Ca(2+) (CRAC) channels encoded by the gene ORAI1. OBJECTIVE: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca(2+) influx and CRAC channel function. METHODS: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. RESULTS: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca(2+) influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. CONCLUSION: Ca(2+) influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients

Store-operated Ca(2+) entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels. In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP(3)) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca(2+) loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex, and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca(2+) release and Ca(2+) reloading) and that any potential interference between Ca(2+) release and Ca(2+) influx is minimized by the spatial separation of the two processes.

Store-operated Ca2+ entry (SOCE) is a mechanism used by many cells types including lymphocytes and other immune cells to increase intracellular Ca2+ concentrations to initiate signal transduction. Activation of immunoreceptors such as the T-cell receptor, B-cell receptor, or Fc receptors results in the release of Ca2+ ions from endoplasmic reticulum (ER) Ca2+ stores and subsequent activation of plasma membrane Ca2+ channels such as the well-characterized Ca2+ release-activated Ca2+ (CRAC) channel. Two genes have been identified that are essential for SOCE: ORAI1 as the pore-forming subunit of the CRAC channel in the plasma membrane and stromal interaction molecule-1 (STIM1) sensing the ER Ca2+ concentration and activating ORAI1-CRAC channels. Intense efforts in the past several years have focused on understanding the molecular mechanism of SOCE and the role it plays for cell functions in vitro and in vivo. A number of transgenic mouse models have been generated to investigate the role of ORAI1 and STIM1 in immunity. In addition, mutations in ORAI1 and STIM1 identified in immunodeficient patients provide valuable insight into the role of both genes and SOCE. This review focuses on the role of ORAI1 and STIM1 in vivo, discussing the phenotypes of ORAI1- and STIM1-deficient human patients and mice

Store-operated Ca(2+) entry (SOCE) is a universal mechanism to increase intracellular Ca(2+) concentrations in non-excitable cells. It is initiated by the depletion of ER Ca(2+) stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca(2+) release activated Ca(2+) (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca(2+) influx and CRAC currents (I(CRAC)) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I(CRAC) consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca(2+) stores

A mutation in ORAI1, the gene encoding the pore-forming subunit of the Ca(2+)-release-activated Ca(2+) (CRAC) channel, abrogates the store-operated entry of Ca(2+) into cells and impairs lymphocyte activation. Stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum activates ORAI1-CRAC channels. We report on three siblings from one kindred with a clinical syndrome of immunodeficiency, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and defective enamel dentition. Two of these patients have a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca(2+) influx.

The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses

ORAI1 is a pore subunit of the store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1(-/-) mice. Orai1(-/-) mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1(-/-) mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1(-/-) mice, but B cells showed a substantial decrease in Ca(2+) influx and cell proliferation in response to B-cell receptor stimulation. Naive and differentiated Orai1(-/-) T cells showed substantial reductions in store-operated Ca(2+) entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null