| Autor: |
Holz, Ronald W., Hlubek, Michael D., Sorensen, Scott D., Fisher, Stephen K., Balla, Tamas, Ozaki, Shoichiro, Prestwich, Glenn D., Stuenkel, Edward L., Bittner, Mary A. |
| Zdroj: |
Journal of Biological Chemistry; June 2000, Vol. 275 Issue: 23 p17878-17885, 8p |
| Abstrakt: |
Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca2+. One of these priming steps involves the maintenance of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P2is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P2important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P2that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4,5-P2and 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. Rhodamine-neomycin, which also binds to PtdIns-4,5-P2, showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca2+entry and suggest that plasma membrane PtdIns-4,5-P2is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P2in some manner alters calcium channel function in chromaffin cells. |
| Databáze: |
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