Termination of Ca puffs during IP-evoked global Ca signals.

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