Termination of Ca²⁺ puffs during IP₃-evoked global Ca²⁺ signals

We previously described that cell-wide cytosolic Ca²⁺ transients evoked by inositol trisphosphate (IP₃) are generated by two modes of Ca²⁺ liberation from the ER; 'punctate' release via an initial flurry of transient Ca²⁺ puffs from local clusters of IP₃ receptors, succeeded by a spatially and temporally 'diffuse' Ca²⁺ liberation. Those findings were derived using statistical fluctuation analysis to monitor puff activity which is otherwise masked as global Ca²⁺ levels rise. Here, we devised imaging approaches to resolve individual puffs during global Ca²⁺ elevations to better investigate the mechanisms terminating the puff flurry. We find that puffs contribute about 40% (∼90 attomoles) of the total Ca²⁺ liberation, largely while the global Ca²⁺ signal rises halfway to its peak. The major factor terminating punctate Ca²⁺ release is an abrupt decline in puff frequency. Although the amplitudes of large puffs fall during the flurry, the amplitudes of more numerous small puffs remain steady, so overall puff amplitudes decline only modestly (∼30%). The Ca²⁺ flux through individual IP₃ receptor/channels does not measurably decline during the flurry, or when puff activity is depressed by pharmacological lowering of Ca²⁺ levels in the ER lumen, indicating that the termination of punctate release is not a simple consequence of reduced driving force for Ca²⁺ liberation. We propose instead that the gating of IP₃ receptors at puff sites is modulated such that their openings become suppressed as the bulk [Ca²⁺] in the ER lumen falls during global Ca²⁺ signals.