Regulation of intracellular calcium signaling and phosphoinositide metabolism by IRBIT

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Project Summary Epilepsy and Alzheimer’s disease affect more than nine million people in the United States. Despite their severity and decades of research, we still lack an understanding of the molecular mechanisms governing these diseases and other related neuropathological conditions. We propose that a recently discovered protein, IRBIT (IP3R binding protein released with inositol 1,4,5-trisphosphate), functions as a regulator of two major signaling pathways, lipid-based phosphoinositide signaling and release of calcium ions from intracellular organelles into the cytoplasm, that have both been shown to be critically involved in these neurological diseases and other pathological conditions. Based on this hypothesis, we will characterize how IRBIT regulates these signaling pathways by (i) characterizing calcium signaling in wild-type and IRBIT-/- human embryonic kidney (HEK293) cells, (ii) utilizing these cell lines to analyze phosphoinositide composition by lipid mass spectrometry and determine the kinetics of phosphoinositide metabolism using fluorescent phosphoinositide-binding proteins in live- cell confocal imaging experiments, and (iii) using an unbiased whole-genome expression analysis via RNA-Seq in wild-type and IRBIT-/- human embryonic kidney cells to assess how IRBIT’s activity influences expression of genes involved in these two signaling networks. The data from the proposed experiments will allow us to gain insight into a novel mechanism by which one protein, IRBIT, regulates two signaling pathways that are of critical importance in practically all mammalian cells. This enhanced understanding of these signaling pathways and their regulator mechanisms has the potential to further our knowledge about the molecular nature of debilitating diseases such as epilepsy and Alzheimer’s and allow us to develop improved treatment strategies for patients affected by these conditions.