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.