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

Tomohiro Nakayama, Mitsuharu Hattori, Keiko Uchida, Takeshi Nakamura, Yoko Tateishi, Hiroko Bannai, Miwako Iwai, Takayuki Michikawa, Takafumi Inoue, Katsuhiko Mikoshiba

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)299-307
Number of pages9
JournalBiochemical Journal
Volume377
Issue number2
DOIs
Publication statusPublished - 2004 Jan 15
Externally publishedYes

Keywords

  • Calcium
  • Caspase
  • Green fluorescent protein (GFP)
  • Inositol 1,4,5-trisphosphate receptor (IPR)
  • Store-operated calcium entry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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