Project Summary/Abstract Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels localized to the endoplasmic reticulum (ER) membranes in almost every cell type. The rapid flux of Ca2+ through IP3R channels from the ER to the cytosol is central to numerous and markedly different cellular actions, ranging from contraction to secretion, from proliferation to cell death. Despite established significance of IP3Rs in physiology and pathology, the molecular mechanisms underlying function of these channels, both in native and disease states, remain poorly understood. The long-term goals of our research are to understand the mechanisms of ion permeation and gating in the family of IP3R channels, and how intracellular binding partners regulate the channel function. This proposal builds on extensive advances we made recently in structural studies of neuronal type 1 IP3R (IP3R1), the predominant type of IP3-gated Ca2+ release channel in cerebellar Purkinje cells. We aim to uncover high-resolution architecture of the entire tetrameric IP3R1 and to delineate conformational changes in the channel that underlie its gating motion and regulation by an array of intracellular molecules ranging from ions and small chemical compounds to proteins. Our research efforts will include cryo- EM structure determination, biochemistry, biophysical, mutagenesis and electrophysiological studies to address channel structure-function. Built upon the complementary expertise of established investigators with compelling preliminary data support, the proposed studies will unveil the structural and mechanistic basis for IP3R function and will elucidate how defects in mechanisms regulating the channel’s gating can lead to abnormal cell Ca2+ levels underlying numerous diseases. Our research is innovative since little is known at the atomic level about the IP3R function. With these studies accomplished, we will establish a detailed structural framework for understanding how the IP3R selectively senses and decodes multiple ligand-binding signals into gating motions that enable the passage of Ca2+ through the channel. This knowledge is crucial for developing new ways to control channel function. Overall, the proposed studies are highly significant, as they will provide valuable mechanistic insights into Ca2+ transfer across biological membranes illuminating the pathological consequences of deregulated Ca2+ signaling, that will ultimately aid in search for novel therapies targeting the IP3R channel family.