| Summary: | Being the largest the Ca<sup>2+</sup> store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca<sup>2+</sup> signalling often involves both Ca<sup>2+</sup> release via inositol 1, 4, 5-trisphosphate receptors (IP<sub>3</sub>R) and store operated Ca<sup>2+</sup> entries (SOCE) through Ca<sup>2+</sup> release activated Ca<sup>2+</sup> (CRAC) channels on plasma membrane (PM). IP<sub>3</sub>Rs are functionally coupled with CRAC channels and other Ca<sup>2+</sup> handling proteins. However, it still remains less well defined as to whether IP<sub>3</sub>Rs could regulate ER-mediated Ca<sup>2+</sup> signals independent of their Ca<sup>2+</sup> releasing ability. To address this, we generated IP<sub>3</sub>Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP<sub>3</sub>Rs-TKO, IP<sub>3</sub>Rs-DKO), and systemically examined ER Ca<sup>2+</sup> dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca<sup>2+</sup> leakage and refilling, as well as SOCE were all significantly reduced in IP<sub>3</sub>Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP<sub>3</sub>R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP<sub>3</sub>R3 is one crucial player in coordinating ER-mediated Ca<sup>2+</sup> signalling.
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