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- Title
Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca<sup>2+</sup> release.
- Authors
Alzayady, Kamil J.; Liwei Wang; Chandrasekhar, Rahul; Wagner II, Larry E.; Van Petegem, Filip; Yule, David I.
- Abstract
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the amino terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentrationresponse relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ 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 Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed 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 IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding-competent ligandbinding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.
- Subjects
STOICHIOMETRY; INOSITOL trisphosphate; CALCIUM channels; BINDING sites; MONOMERS
- Publication
Science Signaling, 2016, Vol 9, Issue 422, p1
- ISSN
1945-0877
- Publication type
Article
- DOI
10.1126/scisignal.aad6281