DOC2B acts as a calcium switch and enhances vesicle fusion.
DOC2B acts as a calcium switch and enhances vesicle fusion.
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DOI：
10.1523/jneurosci.0538-08.2008
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发表时间：
2008-07-02
期刊：
影响因子：
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通讯作者：
Ashery U
中科院分区：
文献类型：
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作者：
Friedrich R;Groffen AJ;Connell E;van Weering JR;Gutman O;Henis YI;Davletov B;Ashery U
钙依赖的胞吐作用受大量蛋白质的调节。DOC2B是一种突触蛋白，在细胞内钙浓度小幅升高后移位到质膜(PM)。本研究的目的是探讨DOC2B在钙触发胞吐中的作用。利用生化和生物物理测量，我们证明DOC2B的C2a结构域以钙依赖的方式直接与PM相互作用。使用电生理、形态和全内反射荧光测量相结合的方法，我们发现DOC2B起到了启动因子的作用，并增加了融合能力囊泡的数量。比较野生型DOC2B和突变的DOC2B在重复刺激过程中的分泌，发现DOC2B在重复刺激过程中也促进儿茶酚胺的分泌，DOC2B必须转移到PM才能发挥促进作用，这表明其活性依赖于钙。DOC2B在PM发挥作用的假说得到以下发现的支持：DOC2B影响单个小泡的融合动力学，并与PM SNARES(可溶性NSF附着受体)相互作用。我们得出结论，DOC2B是一种钙依赖的启动因子，其在PM的活性使融合孔有效扩张，导致儿茶酚胺释放增加。
Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.