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Proc Natl Acad Sci U S A. 2018 May 29;115(22):E5193-E5202. doi: 10.1073/pnas.1718373115. Epub 2018 May 14.

Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating.

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Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.
Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4.
Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611;


Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1-4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.


CRAC channels; Orai1; STIM1; calcium; store-operated calcium entry

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