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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 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, 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.

SUBMITTER: Zheng S

PROVIDER: S-EPMC6153602 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Zheng Sisi S Zhou Lijuan L Ma Guolin G Zhang Tian T Liu Jindou J Li Jia J Nguyen Nhung T NT Zhang Xiaoyan X Li Wanjie W Nwokonko Robert R Zhou Yandong Y Zhao Fukuan F Liu Jingguo J Huang Yun Y Gill Donald L DL Wang Youjun Y

Pflugers Archiv : European journal of physiology 20180622 10

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+ ...[more]

PMID: 29934936

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Project description:Tight spatiotemporal regulation of intracellular Ca2+ plays a critical role in regulating diverse cellular functions including cell survival, metabolism, and transcription. As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.

Dataset Information

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

Publications

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

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