Sørensen Jakob B, Fernández-Chacón Rafael, Südhof Thomas C, Neher Erwin
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Membranbiophysik, Am Fassberg 11, D-37077 Göttingen, Germany.
J Gen Physiol. 2003 Sep;122(3):265-76. doi: 10.1085/jgp.200308855.
We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.
我们通过分析携带突变型突触结合蛋白C2A结构域的小鼠品系中大型致密核心囊泡胞吐作用的细胞内Ca2+依赖性，验证了一个长期存在的假说，即突触结合蛋白1是快速神经分泌的Ca2+传感器。该突变（R233Q）导致Ca2+依赖性磷脂与突触结合蛋白的双C2A - C2B结构域结合的解离常数（KD）增加了两倍。利用笼锁钙的光解和电容测量，我们发现突变细胞的分泌具有较低的分泌速率、较长的分泌延迟以及较高的细胞内Ca2+分泌阈值，这是由于快速胞吐作用的Ca2+传感器的表观KD增加了两倍。单个安培融合事件未发生变化。我们得出结论，Ca2+依赖性磷脂与突触结合蛋白1的结合反映了胞吐作用的细胞内Ca2+依赖性。
