Gao Z, Reavey-Cantwell J, Young R A, Jegier P, Wolf B A
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2000 Nov 17;275(46):36079-85. doi: 10.1074/jbc.M004284200.
Synaptotagmins (Syt) play important roles in Ca(2+)-induced neuroexocytosis. Insulin secretion of the pancreatic beta-cell is dependent on an increase in intracellular Ca(2+); however, Syt involvement in insulin exocytosis is poorly understood. Reverse transcriptase-polymerase chain reaction studies showed the presence of Syt isoforms III, IV, V, and VII in rat pancreatic islets, whereas Syt isoforms I, II, III, IV, V, VII, and VIII were present in insulin-secreting betaTC3 cell. Syt III and VII proteins were identified in rat islets and betaTC3 and RINm5F beta-cells by immunoblotting. Confocal microscopy showed that Syt III and VII co-localized with insulin-containing secretory granules. Two-fold overexpression of Syt III in RINm5F beta-cell (Syt III cell) was achieved by stable transfection, which conferred greater Ca(2+) sensitivity for exocytosis, and resulted in increased insulin secretion. Glyceraldehyde + carbachol-induced insulin secretion in Syt III cells was 2.5-fold higher than control empty vector cells, whereas potassium-induced secretion was 6-fold higher. In permeabilized Syt III cells, Ca(2+)-induced and mastoparan-induced insulin secretion was also increased. In Syt VII-overexpressing RINm5F beta-cells, there was amplification of carbachol-induced insulin secretion in intact cells and of Ca(2+)-induced and mastoparan-induced insulin secretion in permeabilized cells. In conclusion, Syt III/VII are located in insulin-containing secretory granules, and we suggest that Syt III/VII may be the Ca(2+) sensor or one of the Ca(2+) sensors for insulin exocytosis of the beta-cell.
突触结合蛋白（Syt）在钙离子诱导的神经递质释放过程中发挥着重要作用。胰腺β细胞的胰岛素分泌依赖于细胞内钙离子浓度的升高；然而，Syt在胰岛素释放过程中的作用却鲜为人知。逆转录聚合酶链反应研究表明，大鼠胰岛中存在Syt同工型III、IV、V和VII，而胰岛素分泌细胞系betaTC3细胞中则存在Syt同工型I、II、III、IV、V、VII和VIII。通过免疫印迹法在大鼠胰岛、betaTC3细胞和RINm5Fβ细胞中鉴定出了Syt III和VII蛋白。共聚焦显微镜显示，Syt III和VII与含胰岛素的分泌颗粒共定位。通过稳定转染使RINm5Fβ细胞（Syt III细胞）中Syt III的表达量增加了两倍，这赋予了胞吐作用更高的钙离子敏感性，并导致胰岛素分泌增加。在Syt III细胞中，甘油醛+卡巴胆碱诱导的胰岛素分泌比对照空载体细胞高2.5倍，而钾离子诱导的分泌则高6倍。在透化的Syt III细胞中，钙离子诱导的和马斯托帕兰诱导的胰岛素分泌也增加了。在过表达Syt VII的RINm5Fβ细胞中，完整细胞中卡巴胆碱诱导的胰岛素分泌以及透化细胞中钙离子诱导的和马斯托帕兰诱导的胰岛素分泌均有所增强。总之，Syt III/VII定位于含胰岛素的分泌颗粒中，我们认为Syt III/VII可能是β细胞胰岛素释放的钙离子传感器或其中之一。
