Cai Haijiang, Reim Kerstin, Varoqueaux Frederique, Tapechum Sompol, Hill Kerstin, Sørensen Jakob B, Brose Nils, Chow Robert H
Department of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA 90089, USA.
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19538-43. doi: 10.1073/pnas.0810232105. Epub 2008 Nov 25.
SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping the complex and inhibiting membrane fusion. However, several other studies also suggest that CPXs are positive regulators of neurotransmitter release. Thus, whether CPXs are positive or negative regulators of exocytosis is not known, much less the stage in the vesicle life cycle at which they function. Here, we systematically dissect the vesicle stages leading up to exocytosis using a knockout-rescue strategy in a mammalian model system. We show that adrenal chromaffin cells from CPX II knockout mice exhibit markedly diminished releasable vesicle pools (comprising the readily and slowly releasable pools), while showing no change in the kinetics of fusion pore dilation or morphological vesicle docking. Overexpression of WT CPX II-but not of SNARE-binding-deficient mutants-restores the size of the the releasable pools in knockout cells, and in WT cells it markedly enlarges them. Our results show that CPXs regulate the size of the primed vesicle pools and have a positive role in Ca(2+)-triggered exocytosis.
SNARE 介导的胞吐作用是一个多阶段过程，是突触传递和激素释放的核心。复合体蛋白（CPXs）是一种小蛋白，能非常迅速且高亲和力地结合到 SNARE 核心复合体上，最近有人提出它们通过钳制该复合体并抑制膜融合来抑制胞吐作用。然而，其他一些研究也表明 CPXs 是神经递质释放的正向调节因子。因此，CPXs 是胞吐作用的正向还是负向调节因子尚不清楚，更不用说它们在囊泡生命周期中发挥作用的阶段了。在这里，我们在一个哺乳动物模型系统中使用基因敲除 - 拯救策略，系统地剖析了导致胞吐作用的囊泡阶段。我们发现，来自 CPX II 基因敲除小鼠的肾上腺嗜铬细胞表现出可释放囊泡池（包括快速和缓慢可释放池）明显减少，而融合孔扩张动力学或囊泡形态对接没有变化。野生型 CPX II 的过表达——而不是缺乏 SNARE 结合能力的突变体的过表达——可恢复敲除细胞中可释放池的大小，并且在野生型细胞中会使其明显增大。我们的结果表明，CPXs 调节引发的囊泡池大小，并在 Ca(2+) 触发的胞吐作用中起正向作用。
