Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

Sisi Zheng, Lijuan Zhou, Guolin Ma, Tian Zhang, Jindou Liu, Jia Li, Nhung T. Nguyen, Xiaoyan Zhang, Wanjie Li, Robert Nwokonko, Yandong Zhou, Fukuan Zhao, Jingguo Liu, Yun Huang, Donald Gill, Youjun Wang

Research output: Contribution to journalArticle

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Abstract

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1–491 and STIM1 1–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

Original languageEnglish (US)
Pages (from-to)1555-1567
Number of pages13
JournalPflugers Archiv European Journal of Physiology
Volume470
Issue number10
DOIs
StatePublished - Oct 1 2018

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Oligomerization
Chemical activation
Cells
Calcium
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
Genes
Cell membranes
Cluster Analysis
Switches
Null Lymphocytes
Molecules
Proteins
Homeostasis
Cell Membrane

All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Physiology (medical)

Cite this

Zheng, Sisi ; Zhou, Lijuan ; Ma, Guolin ; Zhang, Tian ; Liu, Jindou ; Li, Jia ; Nguyen, Nhung T. ; Zhang, Xiaoyan ; Li, Wanjie ; Nwokonko, Robert ; Zhou, Yandong ; Zhao, Fukuan ; Liu, Jingguo ; Huang, Yun ; Gill, Donald ; Wang, Youjun. / Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines. In: Pflugers Archiv European Journal of Physiology. 2018 ; Vol. 470, No. 10. pp. 1555-1567.
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title = "Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines",
abstract = "Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70{\%} of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1–491 and STIM1 1–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.",
author = "Sisi Zheng and Lijuan Zhou and Guolin Ma and Tian Zhang and Jindou Liu and Jia Li and Nguyen, {Nhung T.} and Xiaoyan Zhang and Wanjie Li and Robert Nwokonko and Yandong Zhou and Fukuan Zhao and Jingguo Liu and Yun Huang and Donald Gill and Youjun Wang",
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Zheng, S, Zhou, L, Ma, G, Zhang, T, Liu, J, Li, J, Nguyen, NT, Zhang, X, Li, W, Nwokonko, R, Zhou, Y, Zhao, F, Liu, J, Huang, Y, Gill, D & Wang, Y 2018, 'Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines', Pflugers Archiv European Journal of Physiology, vol. 470, no. 10, pp. 1555-1567. https://doi.org/10.1007/s00424-018-2165-5

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines. / Zheng, Sisi; Zhou, Lijuan; Ma, Guolin; Zhang, Tian; Liu, Jindou; Li, Jia; Nguyen, Nhung T.; Zhang, Xiaoyan; Li, Wanjie; Nwokonko, Robert; Zhou, Yandong; Zhao, Fukuan; Liu, Jingguo; Huang, Yun; Gill, Donald; Wang, Youjun.

In: Pflugers Archiv European Journal of Physiology, Vol. 470, No. 10, 01.10.2018, p. 1555-1567.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

AU - Zheng, Sisi

AU - Zhou, Lijuan

AU - Ma, Guolin

AU - Zhang, Tian

AU - Liu, Jindou

AU - Li, Jia

AU - Nguyen, Nhung T.

AU - Zhang, Xiaoyan

AU - Li, Wanjie

AU - Nwokonko, Robert

AU - Zhou, Yandong

AU - Zhao, Fukuan

AU - Liu, Jingguo

AU - Huang, Yun

AU - Gill, Donald

AU - Wang, Youjun

PY - 2018/10/1

Y1 - 2018/10/1

N2 - Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1–491 and STIM1 1–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

AB - Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca 2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca 2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca 2+ and Ca 2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca 2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca 2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca 2+ but is dispensable for the maintenance of long-term ER Ca 2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM1 1–491 and STIM1 1–666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.

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M3 - Article

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