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Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines.
Pflugers Arch. 2018 10; 470(10):1555-1567.PA

Abstract

Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ 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 Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ 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 Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ 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, STIM11-491 and STIM11-666 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.

Authors+Show Affiliations

Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China. Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing, 102206, People's Republic of China.Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China.Center for Translational Cancer Research, Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX, 77030, USA.Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China.Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China.Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China.Center for Translational Cancer Research, Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, TX, 77030, USA.Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing, 100875, China.Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing, 100875, China.Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing, 102206, People's Republic of China.Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, College of Biological Science and Engineering, Beijing University of Agriculture, Beijing, 102206, People's Republic of China.Institute of Biosciences and Technology, Department of Molecular and Cellular Medicine, Texas A&M University College of Medicine, Houston, TX, 77030, USA. yun.huang@ibt.tamhsc.edu.Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA. dgill4@pennstatehealth.psu.edu.Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing, 100875, People's Republic of China. wyoujun@bnu.edu.cn.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29934936

Citation

Zheng, Sisi, et al. "Calcium Store Refilling and STIM Activation in STIM- and Orai-deficient Cell Lines." Pflugers Archiv : European Journal of Physiology, vol. 470, no. 10, 2018, pp. 1555-1567.
Zheng S, Zhou L, Ma G, et al. Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines. Pflugers Arch. 2018;470(10):1555-1567.
Zheng, S., Zhou, L., Ma, G., Zhang, T., Liu, J., Li, J., Nguyen, N. T., Zhang, X., Li, W., Nwokonko, R., Zhou, Y., Zhao, F., Liu, J., Huang, Y., Gill, D. L., & Wang, Y. (2018). Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines. Pflugers Archiv : European Journal of Physiology, 470(10), 1555-1567. https://doi.org/10.1007/s00424-018-2165-5
Zheng S, et al. Calcium Store Refilling and STIM Activation in STIM- and Orai-deficient Cell Lines. Pflugers Arch. 2018;470(10):1555-1567. PubMed PMID: 29934936.
* Article titles in AMA citation format should be in sentence-case
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 L, AU - Wang,Youjun, Y1 - 2018/06/22/ PY - 2018/03/08/received PY - 2018/06/07/accepted PY - 2018/05/24/revised PY - 2018/6/24/pubmed PY - 2019/9/5/medline PY - 2018/6/24/entrez KW - 2-APB KW - Calcium KW - Endoplasmic reticulum KW - Orai1 KW - SOCE KW - STIM1 SP - 1555 EP - 1567 JF - Pflugers Archiv : European journal of physiology JO - Pflugers Arch VL - 470 IS - 10 N2 - Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ 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 Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ 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 Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ 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, STIM11-491 and STIM11-666 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. SN - 1432-2013 UR - https://www.unboundmedicine.com/medline/citation/29934936/Calcium_store_refilling_and_STIM_activation_in_STIM__and_Orai_deficient_cell_lines_ L2 - https://dx.doi.org/10.1007/s00424-018-2165-5 DB - PRIME DP - Unbound Medicine ER -