Mapping interactions between the CRAC activation domain and CC1 regulating the activity of the ER Ca2+ sensor STIM1

Nisha Shrestha, Ann Hye-Ryong Shim, Mohammad Mehdi Maneshi, Priscilla See-Wai Yeung, Megumi Yamashita, Murali Prakriya*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Stromal interaction molecule 1 (STIM1) is a widely expressed protein that functions as the endoplasmic reticulum (ER) Ca2+ sensor and activator of Orai1 channels. In resting cells with replete Ca2+ stores, an inhibitory clamp formed by the coiled-coil 1 (CC1) domain interacting with the CRAC-activation domain (CAD) of STIM1 helps keep STIM1 in a quiescent state. Following depletion of ER Ca2+ stores, the brake is released, allowing CAD to extend away from the ER membrane and enabling it to activate Orai1 channels. However, the molecular determinants of CC1–CAD interactions that enforce the inhibitory clamp are incompletely understood. Here, we performed Ala mutagenesis in conjunction with live-cell FRET analysis to examine residues in CC1 and CAD that regulate the inhibitory clamp. Our results indicate that in addition to previously identified hotspots in CC1⍺1 and CC3, several hydrophobic residues in CC2 and the apex region of CAD are critical for CC1–CAD interactions. Mutations in these residues loosen the CC1-CAD inhibitory clamp to release CAD from CC1 in cells with replete Ca2+ stores. By contrast, altering the hydrophobic residues L265 and L273 strengthens the clamp to prevent STIM1 activation. Inclusion of the inactivation domain of STIM1 helps stabilize CC1–CAD interaction in several mutants to prevent spontaneous STIM1 activation. In addition, R426C, a human disease–linked mutation in CC3, affects the clamp but also impairs Orai1 binding to inhibit CRAC channel activation. These results identify the CC2, apex, and inactivation domain regions of STIM1 as important determinants of STIM1 activation.

Original languageEnglish (US)
Article number102157
JournalJournal of Biological Chemistry
Issue number8
StatePublished - Aug 2022


  • CRAC channels
  • Orai1
  • STIM1
  • calcium signaling
  • store-operated calcium entry

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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