The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: Possible physiological significance of specific cleavage by caspase 3

The type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) is an intracellular Ca²⁺ channel protein that plays crucial roles in generating complex Ca²⁺ signalling patterns. IP₃R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP₃R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP₃R1 or IP₃R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP₃-generating agonist ATP and Ca ²⁺ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP₃R1-casp) or the channel domain alone (GFP-IP₃R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca²⁺ concentration. Interestingly, store-operated (-like) Ca²⁺ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca²⁺ stores of cells expressing GFP-IP₃R1-casp or GFP-IP₃R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca²⁺. We therefore propose that the channel domain of IP₃R1 tends to remain open and that the large regulatory domain of IP₃R1 is necessary to keep the channel domain closed. Thus cleavage of IP₃R1 by caspase 3 may contribute to the increased cytosolic Ca²⁺ concentration often observed in cells undergoing apoptosis. Finally, GFP-IP₃R1-casp or GFP-IP₃R1-ES can be used as a novel tool to deplete intracellular Ca²⁺ stores.