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

肥大细胞通过分泌炎性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)是由细胞内钙离子浓度增加触发的。细胞外钙离子跨膜进入的主要途径是通过钙离子释放激活钙通道(CRAC)的储存操作的钙离子内流。对于CRAC通道之一的Orai，有三种亚型，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定位于肥大细胞的质膜上。当Orai-2被击倒时，钙库中的钙离子释放显著减少，而thapsigargin诱导的钙库中的钙释放不受影响。当细胞被抗原刺激时，击倒细胞的脱颗粒明显受到抑制。这些结果提示ORAI-2通过钙库释放钙来调节脱颗粒。

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.