Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. II. Modulation of the receptor in permeabilized cells by the cytosolic compartment

Engagement of the IP₃ receptor by its ligand releases Ca²⁺ from intracellular stores of the rat basophilic leukemia (RBL) cell. The IP₃ receptor in washed permeabilized cells has high affinity (K_d = 1.2±0.3 nM) for [³H]IP₃ and is not sensitive to physiological concentrations of Ca²⁺. Moreover, washed permeabilized cells only release small amounts of Ca²⁺ when stimulated with IP₃. When [³H]IP₃ binding to permeabilized cells is performed in the presence of cytosolic constituents (unwashed cells), the IP₃ receptor has a lower affinity for [³H]IP₃ (K_d from 20 to 100 nM) and has enhanced Ca²⁺ release. Cytosolic supernatant, prepared by centrifugation of permeabilized cells and added back to washed permeabilized cells, decreases [³H]IP₃ binding in a dose-dependent manner and increases the amount of Ca²⁺ released by IP₃. Depletion of either MgATP or IP₃ in the cytosolic supernatant does not affect the supernatant's ability to decrease [³H]IP₃ binding. Though MgATP competitively inhibits [³H]IP₃ binding, it cannot fully account for the shift in K_d or the modulation of IP₃-stimulated Ca²⁺ release in the presence of cytosol. These findings suggest that components present in the cytosolic supernatant modulate the function of the IP₃ receptor by maintaining it in a low affinity state capable of promoting Ca²⁺ release.