title: Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.
abstract: Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca(2+)-release channels with each subunit containing a binding site for IP3in the amino terminus. We provide evidence that four IP3molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca(2+)release suggested that IP3Rs are maximally occupied by IP3before substantial Ca(2+)release occurs. We showed that ligand binding-deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca(2+)release and electrophysiological properties of native IP3Rs. However, IP3failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca(2+)release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca(2+)release.
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Kamil J
authors.initials: KJ
authors.lastname: Alzayady
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Rahul
authors.initials: R
authors.lastname: Chandrasekhar
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Wagner
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Petegem
authors.affiliation: Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA. david_yule@urmc.rochester.edu.
authors.forename: David I
authors.initials: DI
authors.lastname: Yule
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D064113:CRISPR-Cas Systems; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000235:genetics; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry* / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D021122:Protein Subunits; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium