Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells
Plasmalemmal phosphatidylinositol-4,5-bisphosphate level regulates the releasable vesicle pool size in chromaffin cells
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DOI：
10.1523/jneurosci.3761-04.2005
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发表时间：
2005-03-09
影响因子：
5.3
通讯作者：
Neher, E
中科院分区：
文献类型：
--
作者：
Milosevic, I;Sorensen, JB;Neher, E
在胞吐作用期间，某些磷脂可作为分泌的调节剂。在此，我们使用了几种独立的方法来干扰牛嗜铬细胞中磷脂酰肌醇-4，5-二磷酸[PI（4，5）P-2]的水平，以研究质膜PI（4，5）P-2的变化如何影响分泌。PI（4，5）P-2的膜水平通过分析用PI（4，5）P-2特异性荧光探针标记的质膜菌苔的图像来估计。特异性PI（4，5）P-2信号在亚微米大小的簇中富集。在完整细胞的平行膜片钳实验中，我们测量了儿茶酚胺的分泌。过表达磷脂酰肌醇-4-磷酸-5-激酶I γ，或通过贴片移液管输注PI（4，5）P-2，增加了质膜中的PI（4，5）P-2水平并增强了分泌。表达的膜靶向肌醇5-磷酸酶结构域的synaptojanin 1消除PI（4，5）P-2从膜和废除分泌。磷脂酰肌醇-3激酶抑制剂2-（4-吗啉基）-8-苯基-4H-1-苯并吡喃-4-酮导致PI（4，5）P-2水平短暂升高，这与分泌增强有关。长时间孵育后，PI（4，5）P-2水平下降，分泌受到抑制。动力学分析表明，PI（4，5）P-2水平的变化导致两个可释放囊泡池大小的相关变化，而它们的融合动力学不受影响。我们的结论是，在短期和长期操作的PI（4，5）P-2水平分泌规模与质膜PI（4，5）P-2的内容和PI（4，5）P-2有一个早期的影响，通过调节准备释放囊泡的数量分泌。
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) P-2] level in bovine chromaffin cells to investigatehowchanges of plasmalemmal PI(4,5) P-2 affect secretion. Membrane levels of PI(4,5) P-2 were estimated by analyzing images of lawns of plasma membranes labeled with fluorescent probes specific for PI(4,5) P-2. The specific PI(4,5) P-2 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 Igamma, or infusion of PI(4,5) P-2 through the patch pipette, increased the PI(4,5) P-2 level in the plasma membrane and potentiated secretion. Expression of a membrane-targeted inositol 5-phosphatase domain of synaptojanin 1 eliminated PI(4,5) P-2 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) P-2 level that was associated with a potentiation of secretion. After prolonged incubation, the level of PI(4,5) P-2 decreased and secretion was inhibited. Kinetic analysis showed that changes in PI(4,5) P-2 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) P-2 level secretion scales with plasma membrane PI(4,5) P-2 content and that PI(4,5) P-2 has an early effect on secretion by regulating the number of vesicles ready for release.