Interplay between ER Ca2+ Binding Proteins, STIM1 and STIM2, Is Required for Store-Operated Ca2+ Entry.

Heather Nelson,Michael Roe,Colin Leech,Richard Kopp

doi:10.3390/ijms19051522

Heather Nelson, Michael Roe + Show 2 more

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https://doi.org/10.3390/ijms19051522

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Abstract

Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca2+ signaling pathway in many types of cells, is activated by the direct interaction of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum (ER) Ca2+-binding protein, with Ca2+-selective Orai1 channels localized in the plasma membrane. While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.

Highlights

Dynamic changes in the concentration of free calcium ions within the cytosol ([Ca2+]c) are universal intracellular signals that act over a wide temporal and spatial range to control many cell functions
N-arachidonoyl glycine (NAGly), an endocannabinoid that reversibly inhibits Store-operated Ca2+ entry (SOCE) by disrupting stromal interaction molecule 1 (STIM1)-Orai1 interactions [29], partially inhibited SOCE in NIH 3T3 cells; the peak increase in cytosolic [Ca2+] and AUC60 were reduced by 70 ± 2% and 71 ± 2%, respectively (Figure 1B,D)
Knockdown of stromal interaction molecule 2 (STIM2) in HeLa cells, B cells, MDA-MB-231 cells, and mouse embryonic fibroblasts (MEFs) resulted in a small reduction of SOCE, while a more significant reduction in SOCE was seen when STIM2 was silenced in murine T cells, cortical neurons, mast cells, and Xenopus oocytes [3,21,22,40,41,42,53,54]

Summary

Introduction

Dynamic changes in the concentration of free calcium ions within the cytosol ([Ca2+]c) are universal intracellular signals that act over a wide temporal and spatial range to control many cell functions. Ca2+ signals are generated by the mobilization of calcium into the cytosol via influx through Ca2+-permeable ion channels in the plasma membrane (PM) and following release of Ca2+ from sites of intracellular sequestration. Store-operated Ca2+ entry (SOCE), a specific type of Ca2+ influx mechanism, is an important Ca2+. In siRNA screens, independent laboratories identified stromal interaction molecule 1 (STIM1) as an ER Ca2+ sensor responsible for the activation of SOCE [2,3]. After this discovery, genome-wide RNAi screens revealed that Orai was the ion-conducting pore subunit of store-operated channels (SOCs) [4,5]

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