Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca ²⁺ release

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 N-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.

Emergence of ion channel modal gating from independent subunit kinetics

“…This yields a channel with multiple open states, consistent with experimental observations (37). However, by itself this rule is inconsistent with the recent discovery that channel opening requires all subunits to be bound by IP 3 (48). Therefore, we impose the additional constraint that the channel must be fully occupied by activating ligands to open.…”

“…However, we differ by considering separately the role of binding occupancy as a permissive factor. Whereas the necessity of full occupancy by IP 3 that we have included in the model has been established experimentally (48), the location of Ca 2+ binding sites and stoichiometry of regulation are not yet known. In our model, each subunit must have an occupied activating Ca 2+ site and an unoccupied inhibitory site for the channel to open.…”

“…If the methods of ref. 48, which constructed channels with mutations in the IP 3 binding domain of a known number of subunits, can be extended to the mutations identified by ref. 55, then the model makes strong, parameter-free predictions of channel behavior.…”

Noise and sensitivity in cell signalling

“…This yields a channel with multiple open states, consistent with experimental observations [165]. However, by itself this rule is inconsistent with the recent discovery that channel opening requires all subunits to be bound by IP 3 [7].…”

“…If the methods of ref. [7], who constructed channels with mutations in the IP 3 binding domain of a known number of subunits, can be extended to the mutations identified by ref. [161], then the model makes strong, parameter free predictions of channel behavior.…”