TY - JOUR
T1 - STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy
AU - Navarro-Borelly, Laura
AU - Somasundaram, Agila
AU - Yamashita, Megumi
AU - Ren, Dongjun
AU - Miller, Richard J.
AU - Prakriya, Murali
N1 - Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2008/11/15
Y1 - 2008/11/15
N2 - Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca2+ and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.
AB - Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca2+ and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.
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U2 - 10.1113/jphysiol.2008.162503
DO - 10.1113/jphysiol.2008.162503
M3 - Article
C2 - 18832420
AN - SCOPUS:56649094581
SN - 0022-3751
VL - 586
SP - 5383
EP - 5401
JO - Journal of physiology
JF - Journal of physiology
IS - 22
ER -