Plasmalemmal Phosphatidylinositol-4,5-Bisphosphate Level Regulates the Releasable Vesicle Pool Size in Chromaffin Cells

During exocytosis, certain phospholipids may act as regulators of secretion. Here, we used several independent approaches to perturb the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] level in bovine chromaffin cells to investigate how changes of plasmalemmal PI(4,5)P2affect secretion. Membrane levels of PI(4,5)P2were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2signal was enriched in submicrometer-sized clusters. In parallel patch-clamp experiments on intact cells, we measured the secretion of catecholamines. Overexpression of phosphatidylinositol-4-phosphate-5-kinase Iγ, or infusion of PI(4,5)P2through the patch pipette, increased the PI(4,5)P2level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2from the membrane and abolished secretion. An inhibitor of phosphatidylinositol-3 kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, led to a transient increase in the PI(4,5)P2level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2levels led to correlated changes in the size of two releasable vesicle pools, whereas their fusion kinetics remained unaffected. We conclude that during both short- and long-term manipulations of PI(4,5)P2level secretion scales with plasma membrane PI(4,5)P2content and that PI(4,5)P2has an early effect on secretion by regulating the number of vesicles ready for release.