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

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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).