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

细胞内钙浓度（[Ca2+]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关键作用1,2。细胞内Ca2+的释放和细胞外Ca2+的进入产生细胞内钙信号3。在不可兴奋的哺乳动物细胞中，不同Ca2+存储的消耗导致存储操作的Ca2+内流（SOC）。研究得最好的SOC电流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Ca2+信号，基因表达和细胞增殖6-8。然而，ICRAC蛋白的分子特性仍然不清楚，尽管朝着这一目标的证据正在出现。生理上，细胞内Ca2+储存通过将肌醇-1,4,5-三磷酸（InsP3）与内质网（ER）膜上的肌醇-1,4,5-三磷酸受体（InsP3R）结合10,11而消耗。一种基于黑腹果蝇基因组的小干扰RNA （siRNA）方法已经开始鉴定ICRAC蛋白。我提出膜片钳分析的选择sirna，导致显着减少Ca2+信号在荧光成像板阅读器（FLIPR）预筛选（具体目标1）。已知有三种InsP3R同工异构体（I型、II型和III型）。不同的细胞Ca2+储存可能取决于一组特定的InsP3R的表达。我建议评估每个InsP3R异构体在其通过B淋巴细胞ICRAC偶联Ca2+内流的能力中的贡献（Specific Aim 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).