IP₃ mediated global Ca²⁺ signals arise through two temporally and spatially distinct modes of Ca²⁺ release

The 'building-block' model of inositol trisphosphate (IP₃)-mediated Ca²⁺ liberation posits that cell-wide cytosolic Ca²⁺ signals arise through coordinated activation of localized Ca²⁺ puffs generated by stationary clusters of IP₃ receptors (IP₃Rs). Here, we revise this hypothesis, applying fluctuation analysis to resolve Ca²⁺ signals otherwise obscured during large Ca²⁺ elevations. We find the rising phase of global Ca²⁺ signals is punctuated by a flurry of puffs, which terminate before the peak by a mechanism involving partial ER Ca²⁺ depletion. The continuing rise in Ca²⁺, and persistence of global signals even when puffs are absent, reveal a second mode of spatiotemporally diffuse Ca²⁺ signaling. Puffs make only small, transient contributions to global Ca²⁺ signals, which are sustained by diffuse release of Ca²⁺ through a functionally distinct process. These two modes of IP₃-mediated Ca²⁺ liberation have important implications for downstream signaling, imparting spatial and kinetic specificity to Ca²⁺-dependent effector functions and Ca²⁺ transport.