title: 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.
abstract: The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca(2+) channel protein that plays crucial roles in generating complex Ca(2+) signalling patterns. IP(3)R1 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 IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP(3)-generating agonist ATP and Ca(2+) leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca(2+) concentration. Interestingly, store-operated (-like) Ca(2+) entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca(2+) stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca(2+). We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca(2+) concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca(2+) stores.
authors.affiliation: Division of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Nakayama
authors.forename: Mitsuharu
authors.initials: M
authors.lastname: Hattori
authors.forename: Keiko
authors.initials: K
authors.lastname: Uchida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Yoko
authors.initials: Y
authors.lastname: Tateishi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Bannai
authors.forename: Miwako
authors.initials: M
authors.lastname: Iwai
authors.forename: Takayuki
authors.initials: T
authors.lastname: Michikawa
authors.forename: Takafumi
authors.initials: T
authors.lastname: Inoue
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Mikoshiba
mesh_terms: D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015640:Ion Channel Gating; D017136:Ion Transport; D008164:Luminescent Proteins / Q000235:genetics; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002118:Calcium