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

Priscilla S.W. Yeung, Megumi Yamashita, Christopher E. Ing, Régis Pomès, Douglas M. Freymann, Murali Prakriya*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)E5193-E5202
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number22
StatePublished - May 29 2018


  • CRAC channels
  • Calcium
  • Orai1
  • STIM1
  • Store-operated calcium entry

ASJC Scopus subject areas

  • General


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