Structural basis of gating in the Ca2+ release-activated Ca2+ channels

PROJECT SUMMARY/ABSTRACT Ca2+ release-activated Ca2+ (CRAC) channels are highly Ca2+-selective ion channels critical for regulating Ca2+ homeostasis and signal transduction in many cells. These channels are activated in response to the depletion of endoplasmic reticulum (ER) Ca2+ stores and are expressed in both excitatory and non-excitatory cells. Mutations in CRAC channel genes have been linked to severe immunodeficiencies, autoimmunity, and debilitating myopathies, underscoring their physiological significance. CRAC channels are composed of two proteins: Orai, the pore-forming protein in the plasma membrane, and STIM1, an ER-resident protein that activates Orai upon Ca2+ store depletion. Despite significant progress, the structural mechanisms underlying Orai activation remain poorly understood. A previous study revealed the structure and architecture of the wild-type, closed state structure of the Drosophila melanogaster Orai (dOrai), showing a hexameric assembly with concentric transmembrane domains that surround a central pore. However, the structural details of the open state, including the conformation of pore-lining residues and the intracellular domains, remain unresolved. A 2020 cryoEM structure of a constituently open dOrai mutant offered preliminary insights into the open channel structure but suffered from poor resolution and constraints imposed during the sample preparation. As a result, fundamental questions about the structure and conformation of the channel in the open configuration persist. In this proposal, I aim to address these gaps using structural techniques and functional approaches to study the mechanism of CRAC channel activation and inhibition by CRAC channel antagonists. In Aim 1, I will use cryoEM to analyze the structures of three well-studied dOrai open mutants which we hypothesize represent different intermediate open states within the dOrai gating cycle. In Aim 2, I will study the effects of specific inhibitors on the structure of dOrai and identify their binding sites. I will further extend the structural results by functionally validating the binding sites through molecular dynamic simulations and electrophysiology studies. Insights gained from these structures will advance our understanding of CRAC gating and aid future mechanism- based drug discovery efforts targeting CRAC channels.