Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
PLoS One. 2013 Jul 2;8(7):e67561. doi: 10.1371/journal.pone.0067561. Print 2013.
The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca(2+) channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.
外泌体细胞复合物亚基 Sec5 是 RalA-GTP 酶的下游效应物，可促进 RalA-外泌体相互作用和外泌体组装，从而将分泌颗粒固定在质膜的停靠部位。我们最近报道，RalA 通过将胰岛素分泌颗粒固定在 Ca(2+)通道上，辅助兴奋小体组装，从而部分调节胰岛β细胞中的两相胰岛素分泌。在这里，我们通过膜电容测量和全内反射荧光显微镜评估β细胞胞吐作用，以研究 Sec5 在调节胰岛素分泌中的作用。Sec5 存在于人和啮齿动物胰岛β细胞中，定位于胰岛素颗粒上。在大鼠 INS-1 细胞中耗尽 Sec5 蛋白会抑制去极化诱导的从易释放池和从储备池动员的成熟胰岛素颗粒的释放。胰岛素胞吐作用的这种减少主要归因于新到的胰岛素颗粒的募集和胞吐作用的减少，这些颗粒在质膜上的停靠时间很短，但它们包含了更大比例的双相葡萄糖刺激胰岛素分泌。Sec5 蛋白敲低对预停靠颗粒的影响很小，除非受到 KCl 去极化的强烈刺激。总之，β细胞中的新到胰岛素颗粒比预停靠颗粒对外泌体调节更敏感。
