Mechanisms of inositol trisphosphate receptor-mediated calcium signaling

Project Summary/Abstract The overall objective of this research program is to understand the molecular mechanisms of intracellular calcium signals that regulate multiple diverse cellular functions, both healthy and pathological. Many of these signals are due to opening of inositol 1,4,5-trisphosphate receptors (IP3Rs), the most widely expressed ligand-gated Ca2+ channels. IP3Rs reside primarily in the membranes of endoplasmic reticulum (ER) from which they release Ca2+ when they bind both IP3 and Ca2+.The proposed studies focus on uncovering the structural and mechanistic basis for ligand binding, ion permeation and gating in the family of IP3R channels. IP3Rs work as Ca2+ signaling hubs through which diverse extracellular stimuli and intracellular inputs are processed and then integrated to result in delivery of Ca2+ from the ER lumen to generate cytosolic Ca2+ signals with precise temporal and spatial properties. However, an in-depth understanding of the comprehensive allosteric mechanism governing control of intracellular Ca2+ signals via IP3R channels is still lacking. Our structural and functional studies accomplished over the last years, establish the molecular architecture of neuronal IP3R subtype 1 (IP3R1) channel providing critical insights into channel gating and ligand-binding. Recent advances in our work coupled to our knowledge and expertise over two decades of research on calcium channels, guide our current research strategy. In this proposal, we seek to address the fundamental questions of how ligands and modulatory proteins affect the conformational landscape of IP3R, and how conformational changes in channel structure control and shape its function. We are pursuing a multidisciplinary approach that includes biochemical, biophysical, mutagenesis, electrophysiological, cryo-EM and cryo-ET studies to dissect structure-function of IP3Rs. We will use a deep learning neural network-based method to extract conformational motions of the IP3R domains directly from cryo- EM images. Continuing with IP3R1, we will elucidate the molecular and structural mechanism for channel gating and regulation at atomic level detail and define its subcellular organization and dynamics. Another effort will be directed at defining the structural basis for isoform-specific properties of IP3Rs. To this end, we will determine high-resolution structures of other IP3R subtypes in multiple functional states. Our structural findings will be validated by extensive functional analysis. We aim to elucidate the mechanisms of how IP3Rs sense multiple cellular signals and decode them into cytosolic Ca2+ signals to ensure appropriate regulation of downstream signaling pathways. The proposed studies will provide a structural framework and the mechanistic knowledge required for the development of new ways to control IP3R function.