title: Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells.
abstract: 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)P2 affect secretion. Membrane levels of PI(4,5)P2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5)P2. The specific PI(4,5)P2 signal 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 I, or infusion of PI(4,5)P2 through the patch pipette, increased the PI(4,5)P2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5)P2 from 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)P2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5)P2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5)P2 levels 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)P2 level secretion scales with plasma membrane PI(4,5)P2 content and that PI(4,5)P2 has an early effect on secretion by regulating the number of vesicles ready for release.
authors.affiliation: Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Ira
authors.initials: I
authors.lastname: Milosevic
authors.forename: Jakob B
authors.initials: JB
authors.lastname: Sørensen
authors.forename: Thorsten
authors.initials: T
authors.lastname: Lang
authors.forename: Michael
authors.initials: M
authors.lastname: Krauss
authors.forename: Gábor
authors.initials: G
authors.lastname: Nagy
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
authors.forename: Erwin
authors.initials: E
authors.lastname: Neher
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000096:biosynthesis*; D051381:Rats; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002867:Chromones; D009025:Morpholines; D019269:Phosphatidylinositol 4,5-Diphosphate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one