Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells

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.

Keywords: IP3R; NEDD4L; Orai1; SERCA; calcium; store-operated Ca2+ entry.