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

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Sci. Signal.  05 Apr 2016:
Vol. 9, Issue 422, pp. ra35
DOI: 10.1126/scisignal.aad6281

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Intracellular calcium regulates such specialized processes as muscle contraction, neurotransmitter release, and insulin secretion and such general cellular processes as gene expression, proliferation, and cell death. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) plays key roles in increasing intracellular calcium by functioning as a channel that releases Ca2+ from the endoplasmic reticulum. The IP3R is a tetramer with each subunit having a binding site for IP3. Alzayady et al. determined how many subunits have to bind IP3 for the channel to open by engineering versions of the receptor expressed as a single protein with different numbers of IP3-binding sites. Analysis of these concatenated receptor proteins revealed that channel activity required IP3 bound to each of the four binding sites, which ensures that cells do not discharge calcium unless the signal to do so is strong enough. A similar approach could be used to define how heterozygous mutations in IP3R subunits produce disease.

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 concentration-response 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 ligand-binding 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.

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