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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 …