Structural characterization of inositol-1,4,5-triphosphate receptors

Mitochondria are dynamic and complex organelles that are essential for a wide range of metabolic and signaling processes. Mitochondrial dysfunction is strongly associated with a growing number of metabolic and neurodegenerative diseases and cancer. A crucial regulator of mitochondrial functions is mitochondrial Ca2+ influx, which relies on synapse-like contact sites with the endoplasmic reticulum (ER). Sustained Ca2+ transfer into mitochondria at these contact sites is necessary to maintain synthesis of ATP and mitochondrial substrates, whereas excessive or reduced Ca2+ transfer leads to initiation of apoptotic cell death or autophagy, respectively. Recent studies link aberrant Ca2+ signaling at ER-mitochondria contact sites to the onset and progression o many metabolic diseases including diabetes-2 and obesity related illnesses. Despite recent identification of components of the Ca2+ transfer machinery at ER-mitochondria contact sites and their emergence as potential therapeutic targets, the molecular mechanisms underlying regulation of mitochondrial Ca2+ influx is poorly understood, hampering development of tools to fine tune Ca2+ flux in diseased states. One of the long-term goals of my research program is to uncover the molecular mechanisms underlying the activity and regulation of inosito1-1,4,5-triphosphate receptors (IP3Rs), the key regulator of Ca2+ transfer into mitochondria, using cutting edge structural biological methods including X-ray crystallography and cryo-electron microscopy (cryo-EM) together with biophysical, biochemical and functional methods. The aim of this proposal is to i) perform identification and biochemical, pharmacological and structural characterization of IP3R domains that can be expressed as isolated entities, and ii) express and purify intact recombinant IP3R suitable for the subsequent structural and functional analysis. These studies will facilitate the necessary tools to address more specific questions regarding to IP3R activity and regulation and would pave the path for discovery of novel tools to control the receptor activity in diabetes and obesity as well as other diseases where aberrant IP3R activity is involved.