Analysis of the structural basis for the gating and regulation of the human type 3 Inositol Trisphosphate Receptor

Abstract

The inositol 1,4,5triphosphate (IP3) receptor (IP3R) mediates Ca2+ release from the endoplasmic reticulum (ER) to control cytoplasmic and organellar Ca2+ concentrations. IP3Rs are co-activated by IP3 and Ca2+, inhibited by Ca2+ at high concentrations, and potentiated by ATP. Calcium-mediated signaling through IP3Rs is essential for regulating numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP3Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we aim to elucidate the structural basis for the gating cycle and activation of type 3 IP3R (IP3R-3), in addition to describing novel regulatory strategies which modulate receptor activity. This goal is divided into three main projects described here. For project 1, we identify and describe a novel, regulatory self-binding peptide (SBP) in a ligand-free human IP3R-3 structure determined to 3.8 Å via cryo-EM. Biochemical experiments were used to determine the SBP’s ability to compete with the endogenous ligand IP3 for access to the binding site. Project 2 details the determination of an ensemble of cryo-EM IP3R-3 structures that includes the active conformation, an inhibited conformation, and three pre-active states, providing snapshot structures that show the propagation of conformational changes from the ligand-binding site to the pore opening. Project 3 reports on the current progress for generating an IP3R structure bound to the non-specific, commercially available IP3R-antagonist 2-APB (2-Aminoethoxydiphenyl borate), which would elucidate its mechanism of action. The data shown here provide insights into the molecular mechanisms underlying IP3R activation and regulation.