Abstract
Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.
Highlights
Calcium ion (Ca2+ ) is essential for life, regulating many crucial cellular functions like contraction, migration and proliferation [1]
human embryonic kidney (HEK) cell lines (IP3 Rs-TKO) with CRISPR/Cas9 genomic editing technology using procedures similar to previous reports [17]. These IP3 Rs-TKO cells were generated from two separate HEK cell lines stably expressing genetically encoded Ca2+ indicators (GECI): GCaMP6m [29], a cytosolic Ca2+
Further results showed that overexpression of IP3 R3 could significantly increase Orai1 protein levels in GIPK cells (Figure 3C). These findings demonstrated that diminished store operated Ca2+ entries (SOCE) in IP3 Rs-TKO cells are caused by reduced expression of Orai1 protein, and that overexpression IP3 R3 could largely restore both Orai1 levels and SOCE responses
Summary
Calcium ion (Ca2+ ) is essential for life, regulating many crucial cellular functions like contraction, migration and proliferation [1]. Cytosolic Ca2+ signals are mainly controlled through a combination of two closely linked processes–Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry across the plasma membrane (PM) [2] Much of the former event is mediated by inositol 1,4,5-trisphosphate receptor (IP3 Rs), Ca2+ release channels resident in ER membranes [3,4,5,6]. There is one report showing that mutation in IP3 Rs could inhibit SOCE in Drosophila cells, likely through impairments of coupling between STIM1-Orai1 [27]. It still remains elusive whether IP3 Rs could regulate SOCE through other means. Our findings suggest that IP3 R3 maybe one key player in coordinating ER-mediated Ca2+ signalling
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