PROJECT SUMMARY Inositol 1,4,5-triphosphate receptors (IP3Rs) integrate diverse signals generated by hormones, growth factors, neurotransmitters, and changes in metabolic state to modulate downstream signaling in all cell types. IP3Rs are ligand-gated ion channels that are further regulated by allosteric and covalent mechanisms, mediating Ca2+ release from the endoplasmic reticulum (ER). The resulting increases of cytoplasmic and mitochondrial Ca2+ concentrations regulate many physiological processes (e.g., learning, memory, membrane trafficking, synaptic transmission, secretion, motility, membrane excitability, gene expression, cell division, and apoptosis). Furthermore, pathological dysregulation of IP3Rs and calcium signaling is implicated in cancer, neurodegenerative, autoimmune, and metabolic diseases, making IP3Rs promising targets for treatment of these diseases. Despite recent advances in structural studies, fundamental questions regarding the mechanisms of ligand interactions and channel gating remain mostly unanswered, in part because of the large size and complexity of IP3Rs and the limited availability of specific pharmacological tools. In this proposal, we will (Aim 1) combine cryo-electron microscopy (cryo-EM) and X-ray crystallography in conjunction with functional IP3R assays based on fluorescence-based calcium imaging to elucidate the general themes of IP3R gating cycle and molecular basis for receptor inhibition by small molecules. Our recently published data revealed that the IP3 binding site is occupied by a loop that we have termed the self-binding peptide (SBP), which is located distantly in the primary sequence. We hypothesize that the SBP is a novel regulatory site in IP3Rs that can modulate the apparent affinity for IP3, and thereby Ca2+ channel activity, and that the divergence of SBP sequences between IP3R subtypes contributes to their distinct regulatory properties. We will perform (Aim 2) functional and structural studies on IP3R subtypes and SBP mutants to test this hypothesis and identify the structural determinants of this interaction. Completion of these aims will yield unparalleled mechanistic insight into IP3R gating and regulation, potentially leading to the development of novel and specific pharmacological modulators of IP3Rs. In addition to being used as a long-sought research tools to study IP3Rs, these compounds will serve as a starting point for development of novel therapeutic approaches to treat diseases associated with aberrant IP3R activity.

PROJECT NARRATIVE Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP3Rs) is essential for the regulation of numerous physiological processes. Deregulation of IP3Rs leads to pathological changes in calcium signaling and is implicated in the pathogenesis of diverse diseases including cancer, neurodegenerative, autoimmune, and metabolic diseases. The goal of the proposed study is to elucidate the molecular mechanism of IP3R activity and regulation paving the road for development of novel therapeutic agents to control IP3R activity in the diseased state.