The first C2 domain of synaptotagmin is required for exocytosis of insulin from pancreatic β‐cells: action of synaptotagmin at low micromolar calcium-猫眼课题宝

突触结合蛋白的第一个 C2 结构域是胰腺 β 细胞中胰岛素的胞吐所必需的：突触结合蛋白在低微摩尔钙条件下的作用

DOI：

发表时间：

1997

期刊：

EMBO Journal

影响因子：

11.4

通讯作者：

中科院分区：

生物学1区

文献类型：

作者：

J. Lang;M. Fukuda;Hui Zhang;K. Mikoshiba;C. Wollheim

Ca2+和磷脂结合蛋白synaptotagmin参与神经胞吐作用。其在体内的确切作用和Ca2+亲和力尚不清楚。我们研究了它在胰岛素分泌中的可能功能，胰岛素分泌在10 μM胞质游离Ca2+的刺激下最大。Northern和Western blots结果显示，在胰腺β细胞系RINm5F、INS - 1和HIT - T15中存在表征良好的synaptotagmin亚型I和II。亚细胞分离和共聚焦显微镜显示它们主要存在于含胰岛素的分泌颗粒中，而仅少量存在于突触囊泡样微泡中。针对synaptotagmin I或II的第一个C2结构域Ca2+依赖性磷脂结合位点的抗体或Fab片段抑制Ca2+刺激，但不抑制GTPγS诱导的链溶素- O -渗透性INS - 1和HIT - T15细胞的胞外分泌。野生型synaptotagmin II的瞬时表达并未改变HIT - T15细胞的胞吐。然而，Ca2+依赖性磷脂结合位点的第一个C2结构域（Δ180-183, D231S）的突变再次仅抑制Ca2+‐，而不抑制GTPγS‐引起的胞外分泌。相反，第二个C2结构域的IP4结合位点（Δ325-341; K327,328,332Q）的突变不会改变胞外分泌。两个C2结构域（Δ180-183 /K327,328,332Q）突变的Synaptotagmin II比突变体Δ180-183诱导了更大的抑制作用，这表明对第二个C2结构域有离散的需求。因此，synaptotagmin亚型调节低微摩尔Ca2+发生的胞外事件。

The Ca2+‐ and phospholipid‐binding protein synaptotagmin is involved in neuroexocytosis. Its precise role and Ca2+‐affinity in vivo are unclear. We investigated its putative function in insulin secretion which is maximally stimulated by 10 μM cytosolic free Ca2+. The well‐characterized synaptotagmin isoforms I and II are present in pancreatic β‐cell lines RINm5F, INS‐1 and HIT‐T15 as shown by Northern and Western blots. Subcellular fractionation and confocal microscopy revealed their presence mainly on insulin‐containing secretory granules whereas only minor amounts were found on synaptic vesicle‐like microvesicles. Antibodies or Fab‐fragments directed against the Ca2+‐dependent phospholipid binding site of the first C2 domain of synaptotagmin I or II inhibited Ca2+‐stimulated, but not GTPγS‐induced exocytosis from streptolysin‐O‐permeabilized INS‐1 and HIT‐T15 cells. Transient expression of wild‐type synaptotagmin II did not alter exocytosis in HIT‐T15 cells. However, mutations in the Ca2+‐dependent phospholipid binding site of the first C2 domain (Δ180–183, D231S) again inhibited only Ca2+‐, but not GTPγS‐evoked exocytosis. In contrast, mutations in the IP4‐binding sites of the second C2 domain (Δ325–341; K327,328,332Q) did not alter exocytosis. Synaptotagmin II mutated in both C2 domains (Δ180–183/K327,328,332Q) induced greater inhibition than mutant Δ180–183, suggesting a discrete requirement for the second C2 domain. Thus, synaptotagmin isoforms regulate exocytotic events occurring at low micromolar Ca2+.