Molecular regulation of the CRAC channel signaling pathway

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Project Summary/Abstract The calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediate Ca2+ influx in response to Ca2+ store depletion in the endoplasmic reticulum (ER), generating Ca2+ signals critical for many cellular processes. Activation of CRAC channels requires the interaction of Orai and STIM proteins, which are the plasma membrane Ca2+ channel and the Ca2+ sensor in the ER membrane; however, the underlying molecular mechanism remains incompletely understood. Mammals, including humans, have three Orai channels (Orai1, Orai2, and Orai3) and two STIM proteins (STIM1 and STIM2), whereas Drosophila has only one set of Orai and STIM. Both loss- and gain-of-function mutations that underlie human diseases have been identified in human Orai1 and STIM1. The physiological roles of Orai2 and Orai3 are less well understood, but there is emerging evidence showing that they could form heteromeric channels with Orai1, which might play roles distinct from channels formed by Orai1 and STIM1. The long-term goal of my research program is to study the molecular regulation of the CRAC channel signaling pathway in order to understand the molecular mechanisms of the diverse functions the CRAC channels play in different tissues and cells under physiological and pathological conditions. Initially, we are prioritizing structure-function studies of the Drosophila Orai-STIM complex and human CRAC channels to launch my research program. In this proposed research, we aim to address key knowledge gaps in (1) the biochemistry and structural biology of the Orai-STIM interaction, (2) the structure and function of human CRAC channels, and (3) regulation of CRAC channels by cellular factors and channel modulators. We will bring innovative approaches and apply the Principal Investigator’s experience in structural biology and ion channel structure-function studies to address these questions. Specifically, we will determine the atomic structure of the Drosophila Orai-STIM complex as well as structures of human Orai channels and human Orai-STIM complexes. We will also develop novel functional approaches and tools for structure-guided functional analysis of CRAC channels. In addition, we propose to study the regulation of the channel structure and function by small molecule modulators including lipids and pharmacological drugs. Finally, we will use functional, biochemical, and structural biology methods (including both X-ray crystallography and cryo-electron microscopy) to characterize the protein-protein interaction of Orai or STIM with a multitude of endogenous protein regulators. In the next five years, we aim to gain a deeper mechanistic understanding of the regulation of the CRAC channel signaling pathway and construct the foundation of knowledge and research tools to understand and study the broader signaling network in which the CRAC channels reside.