Calcium Release-Activated Calcium current - Investigation of a calcium signaling pathway in non-excitable cells

细胞内钙离子浓度([Ca~(2+)]i)的变化通过调节细胞过程在细胞对细胞外环境的反应中起着关键作用1，2.细胞内钙离子从细胞内储存物释放和细胞外钙离子进入产生细胞内钙信号。在不可兴奋的哺乳动物细胞中，不同的钙离子储存的耗尽导致储存操作的钙内流(SOC)。研究最多的是钙释放激活钙电流(ICRAC)5。ICRAC在许多细胞过程中发挥重要作用，如钙信号、基因表达和细胞增殖6-8。然而，ICRAC蛋白的分子同一性仍然不清楚，尽管朝着这一目标的证据正在出现。生理上，细胞内的钙储存是通过与位于内质网(ER)10、11膜上的肌醇-1，4，5-三磷酸受体(InsP3R)结合来耗尽的。我建议对选择的siRNA进行膜片钳分析，这些siRNA导致荧光成像平板阅读器(FLIPR)预筛选中钙信号显著减少(特定目标1)。已知InsP3R的三种亚型(类型I、类型II和类型III)。根据一组特定的InsP3R的表达，可以产生不同的细胞钙存储。我建议评估每一种InsP3R亚型在其通过ICRAC结合到B淋巴细胞内钙内流的能力方面的贡献(特定目标2)。

Changes in intracellular calcium concentration ([Ca2+]i) play a critical role in cellular responses to the extracellular environment by regulating cellular processes1, 2. Release of Ca2+ from intracellular stores and entry of extracellular Ca2+ generate intracellular calcium signals3. Depletion of distinct Ca2+ stores results in store-operated Ca2+ influx (SOC) in non-excitable mammalian cells4. The best studied SOC current is the Calcium Release Activated Calcium current (ICRAC)5. ICRAC plays an important role in many cellular processes such as Ca2+ signaling, gene expression and cell proliferation6-8. However, the molecular identity of the ICRAC protein is still unclear, although evidence towards this goal is emerging9. Physiologically, intracellular Ca2+ stores are depleted by binding inositol-1,4,5-trisphosphate (InsP3) to the inositol-1,4,5-trisphosphate receptor (InsP3R) located in the membrane of the endoplasmic reticulum (ER)10, 11.A small interference RNA (siRNA) approach based on the genome of Drosophila melanogaster has been initiated to identify the ICRAC protein. I propose patch-clamp analyses of selected siRNAs that lead to a significantly reduced Ca2+ signal in a Fluorometric Imaging Plate Reader (FLIPR) pre-screen (Specific Aim 1).Three isoforms of the InsP3R are known (type I, II and III). Distinct cellular Ca2+ stores could be generated depending on the expression of a specific set of InsP3R. I propose to evaluate the contribution of each InsP3R isoform in its ability to couple to Ca2+ influx through ICRAC in B lymphocytes (Specific Aim 2).