Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo
Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo
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DOI：
10.1073/pnas.1409311111
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发表时间：
2014-07-15
影响因子：
11.1
通讯作者：
Littleton, J. Troy
中科院分区：
文献类型：
--
作者：
Cho, Richard W.;Kuemmel, Daniel;Littleton, J. Troy
复合蛋白(CPX)是一种陷阱结合蛋白，在无钙内流的情况下，通过阻断突触小泡的自发融合来调节神经传递，同时促进动作电位的诱发释放。先前的研究表明，CPX可能通过反式相互作用使多个SNAR复合体交联化，从而发挥融合钳的作用。在Ca~(2+)内流过程中，CPX预计会发生构象转换，以顺式结合的方式塌陷到单一的SNARE复合体上，从而激活囊泡释放。为了在体内测试这个模型，我们在果蝇身上进行了CPX蛋白的结构-功能研究。利用cpx突变体破坏SNAR交联的遗传拯救方法，我们发现，预测可以阻止反式SNAR阵列形成的操作破坏了cpx的钳制功能。出人意料的是，这些突变体挽救了动作电位触发的释放，表明CPX的反式SNARE交联不是触发诱发融合的先决条件。相反，破坏CPX介导的顺式-圈套相互作用的突变是从开放构象转换到闭合构象所必需的，尽管它们正常地钳制自发释放，但未能挽救cpx突变体中诱导的释放缺陷。我们在体内的遗传操作支持CPX交联模型做出的几个预测，但意想不到的结果表明，可能存在其他机制来调节CPX对圈套介导的融合的影响。我们的发现还表明，CPX的抑制和激活功能在基因上是可以分离的，并可以映射到不同的分子机制，这些机制对SNARE融合机制进行了不同的调节。
Complexin (Cpx) is a SNARE-binding protein that regulates neurotransmission by clamping spontaneous synaptic vesicle fusion in the absence of Ca2+ influx while promoting evoked release in response to an action potential. Previous studies indicated Cpx may cross-link multiple SNARE complexes via a trans interaction to function as a fusion clamp. During Ca2+ influx, Cpx is predicted to undergo a conformational switch and collapse onto a single SNARE complex in a cis-binding mode to activate vesicle release. To test this model in vivo, we performed structure-function studies of the Cpx protein in Drosophila. Using genetic rescue approaches with cpx mutants that disrupt SNARE cross-linking, we find that manipulations that are predicted to block formation of the trans SNARE array disrupt the clamping function of Cpx. Unexpectedly, these same mutants rescue action potential-triggered release, indicating trans-SNARE cross-linking by Cpx is not a prerequisite for triggering evoked fusion. In contrast, mutations that impair Cpx-mediated cis-SNARE interactions that are necessary for transition from an open to closed conformation fail to rescue evoked release defects in cpx mutants, although they clamp spontaneous release normally. Our in vivo genetic manipulations support several predictions made by the Cpx cross-linking model, but unexpected results suggest additional mechanisms are likely to exist that regulate Cpx's effects on SNARE-mediated fusion. Our findings also indicate that the inhibitory and activating functions of Cpx are genetically separable, and can be mapped to distinct molecular mechanisms that differentially regulate the SNARE fusion machinery.