Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I␥, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.exocytosis ͉ fusion pore ͉ granule ͉ phosphatidylinositol-4,5-bisphosphate ͉ secretion S trong evidence has implicated phosphoinositides (PIs) in the regulation of membrane traffic, including exocytosis (1-4). A first clue suggesting a direct role of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] in secretion came from its requirement for Ca 2ϩ -dependent exocytosis in broken chromaffin cells, independent from its phospholipase C (PLC)-mediated cleavage (5). Subsequently, a PI(4)P 5-kinase activity was implicated as a critical factor in the Ca 2ϩ -activated secretion of large dense-core vesicles (LDCVs) from permeabilized PC12 cells, a chromaffin cell line (6). In addition, the molecular characterization of the exocytic machinery revealed that several proteins that directly or indirectly participate in the priming and fusion of neurosecretory vesicles contain PI(4,5)P 2 -binding domains (1, 4, 7). For example, synaptotagmin, a putative Ca 2ϩ sensor in the exocytosis of synaptic vesicles and LDCVs, binds PI(4,5)P 2 by means of C2 domains, and in vitro assays showed that this interaction is important in membrane fusion (8-10). Ca 2ϩ -dependent activator for secretion (CAPS), which is required for Ca 2ϩ -evoked LDCV secretion (11), binds PI(4,5)P 2 by means of a PH domain (12). Moreover, manipulations that artificially mask available PI(4,5)P 2 , such as overexpression of a PI(4,5)P 2 -binding module, the PH domain of PLC␦1, inhibits LDCV exocytosis (13,14). Disruption of the function of Arf6, a positive regulator of PI(4,5)P 2 synthesis by means of its action on type I phosphoinositide-4-phosphate (PIP) kinases (15, 16), impairs neuroendocrine secretion (14, 17); conversely, stimulation of Arf6 enhances secretion (18). Diacylglycerol, a metabolic product of PI(4,5)P 2 , is also implicated in synaptic vesicle and LDCV fusion with the plasma membrane (PM). Diacylglycerol, whose levels can be regulated not only by PLC-mediated cleavage of PI(4,5)P 2 but also by alternative metabolic pathways, binds to Munc13, and this interaction plays an essential role in the priming reaction of exocytosis (19)(20)(21)(22).PI(4,5)P 2 can be generated by type I and type II PIP kinases, which function as PI(4)P 5-kinases and PI(5)P 4-kinases, respectively ...

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