PROJECT SUMMARY The objective of this project is to characterize the molecular mechanisms of the multi-modal ligand- dependent gating of inositol 1,4,5-trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed endoplasmic reticulum (ER) Ca2+ channels that play a key role in the maintenance of Ca2+ homeostasis by conducting Ca2+ stored in the ER into the cytoplasm. In the cytosol, numerous Ca2+-regulated proteins sense changes in local and global Ca2+ concentrations to regulate diverse cellular process including fertilization, cell death and differentiation. Because of the diversity of processes regulated by cytoplasmic Ca2+, IP3R-mediated Ca2+ conductance is precisely regulated by ions, small molecules and protein co-factors. The two primary regulatory ligands of IP3Rs are inositol 1,4,5-trisphosphate (IP3) and cytoplasmic Ca2+, both of which bind the channel to modulate the gating state of the pore. Whereas IP3 binding activates IP3Rs, cytoplasmic Ca2+ both activates the channel at low concentrations and inhibits the channel at high concentrations. The biphasic relationship between Ca2+ and IP3R activity ensures that cytoplasmic Ca2+ concentrations are properly regulated. Besides IP3 and Ca2+, IP3R activity is further shaped in cell-specific manner by other small molecules such as ATP, by enzymes that post-translationallly modify IP3Rs and by numerous protein co- factors. How the effects of these various factors are synthesized to determine IP3R gating state and thus regulate cellular Ca2+ signalling remain poorly understood at a molecular level. As dysregulation of IP3R activity is linked to cardiac disease, cancer, neurological disorders and other pathologies, understanding the regulation of IP3Rs will have broad relevance to human health and disease. The proposal aims to employ structural, biochemical and biophysical approaches to develop a mechanistic understanding of IP3R regulation, focusing on Ca2+, IP3 and three protein co-factors of the Bcl-2 family: Bcl-2, Mcl-1 and Bcl-xL. With these approaches we aim to understand i) how IP3 and Ca2+ binding jointly stabilize IP3Rs in an active conformation, ii) how excess Ca2+ inhibits ion conduction and iii) how the protein co-factors Bcl-2, Mcl-1 and Bcl-xL modulate channel activity in the presence of IP3 and Ca2+. The proposed studies will reveal principles of how multiple stimuli are integrated to regulate ion channel function. Due to the broad physiological role of IP3Rs, the finding derived from these studies will be relevant to a number of fields including ion channels, Ca2+ signaling, cell death and neurobiology. !