Structural and molecular mechanisms of CRAC channel gating

About This Grant

Project Summary/Abstract Store-operated Ca2+ release-activated Ca2+ (CRAC) channels play a central role in cellular Ca2+ signaling across diverse cell types including T-cells, microglia, and astrocytes. Activated by the endoplasmic reticulum (ER) Ca2+ sensor, STIM1, CRAC channels regulate a wide variety of effector cellular functions including transcription, metabolism, and cell proliferation. Their unique biophysical properties, characterized by exceptional Ca2+-selectivity, store-operated activation, and distinct gating modes, make them fascinating models for understanding the mechanisms of Ca2+ permeation and gating. Moreover, because CRAC channels sit squarely at the center of cellular Ca2+ signaling networks in many cells, aberrant CRAC channel function is implicated in the etiology of diseases ranging from immunodeficiency to allergies. Previous work on the molecular choreography of the CRAC channel activation process has revealed that opening of CRAC channels is mediated by direct interactions between the pore-forming Orai proteins with STIM1. Despite progress in elucidating the activation process of CRAC channels, numerous key aspects of the molecular and structural mechanisms of STIM1-Orai1 binding, channel pore opening, and regulation of gating remain unknown. Here, we propose a multi-disciplinary approach that will integrate photocrosslinking of Orai1 with the catalytic domain of STIM1 using unnatural amino acids, structural analysis of open Orai channel variants using cryo-electron microscopy, and use of fluorinated phenylalanine analogs to elucidate the molecular and structural basis of STIM1-Orai1 binding and the gating of Orai1 channels. Our goals are to: (1) determine the molecular and structural basis of the Orai1-STIM1 binding interface, (2) determine the structures of three gain-of function Orai mutants (including a human disease mutation) with distinct biophysical and functional properties via Cryo-electron microscopy and compare these to the closed, wildtype channels, and, (3) examine the contributions of electrostatic interactions involving the channel gate that mediate the opening of the pore. These studies will significantly advance our understanding of the molecular and structural mechanisms of CRAC channel gating, reveal how disease mutations alter these processes, and ultimately provide new therapeutic strategies for targeting CRAC channels in disease.