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Sci Signal. 2016 Apr 5;9(422):ra35. doi: 10.1126/scisignal.aad6281.

Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.

Author information

1
Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA.
2
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
3
Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642, USA. david_yule@urmc.rochester.edu.

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.

PMID:
27048566
PMCID:
PMC4850551
DOI:
10.1126/scisignal.aad6281
[Indexed for MEDLINE]
Free PMC Article

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