A pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P-2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P-2 as being important in exocytosis

Kinetically distinct steps can be distinguished in the secretory response f rom neuroendocrine cells with slow ATP-dependent priming steps preceding th e triggering of exocytosis by Ca2+, One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P-2) thro ugh lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5- P-2 is usually thought to reside on the plasma membrane. However, because p hosphatidylinositol 4-kinase is an integral chromaffin granule membrane pro tein, PtdIns-4,5-P-2 important in exocytosis may reside on the chromaffin g ranule membrane. In the present study we have investigated the localization of PtdIns-1,5-P-2 that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically bi nds PtdIns-4,5-P-2 and is fused to green fluorescent protein (GFP). The PH- GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodami ne-neomycin, which also binds to PtdIns-4,5-P-2, showed a similar subcellul ar localization. The transiently expressed PH-GFP inhibited exocytosis as m easured by both biochemical and electrophysiological techniques. The result s indicate that the inhibition was at a step after Ca2+ entry and suggest t hat plasma membrane PtdIns-4,5-P-2 is important for exocytosis, Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIn s-4,5-P-2 in some manner alters calcium channel function in chromaffin cell s.