Analysis of Ca channel localized on secretory granules by lipid bilayer that mimics exocytotic sites

肥大细胞通过分泌炎症介质在过敏反应中发挥重要作用。这些介质的胞外分泌（脱颗粒）是由细胞内Ca2+浓度的增加引起的。细胞外Ca2+通过质膜进入的主要途径是通过Ca2+释放激活钙（CRAC）通道进行储存操作的Ca2+进入。对于CRAC通道之一的Orai，有三种同工异构体：Orai、Orai-1、-2和-3。Orai-1负责肥大细胞的脱颗粒。我们发现Orai-2主要定位在分泌颗粒上，而Orai-1和-3定位在肥大细胞的质膜上。当Orai-2基因被敲除时，Ca2+库中的Ca2+释放明显减弱，但不影响thapsigarin诱导的Ca2+释放。当细胞受到抗原刺激时，敲低细胞的脱肉芽明显被抑制。这些结果表明，Orai-2通过Ca2+储存释放Ca2+来调节脱粒。

Mast cells play an essential role in allergic responses by secreting inflammatory mediators. The exocytotic secretion of these mediators (degranulation) is triggered by the increase in intracellular Ca2+ concentration. The main pathway for the entry of extracellular Ca2+ across the plasma membrane is store-operated Ca2+ entry through Ca2+ release-activated calcium (CRAC) channels. For Orai, one of CRAC channels,there are three isoforms, Orai, Orai-1, -2 and -3. Orai-1 is responsible for degranulation in mast cells. We found that Orai-2 was mostly localized on secretory granules, while Orai-1 and -3 were localized on the plasma membrane in mast cells. When Orai-2 was knocked down, Ca2+ release from Ca2+ store was significantly attenuated but that induced by thapsigargin was not affected. When cells were stimulated with antigen, degranulation was significantly inhibited in knockdown cells. These results suggest that Orai-2 regulates degranulation through Ca2+ release from Ca2+ store.