Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.
Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.
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DOI：
10.1371/journal.pbio.1002267
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发表时间：
2015-10
期刊：
影响因子：
9.8
通讯作者：
Südhof TC
中科院分区：
文献类型：
--
作者：
Bacaj T;Wu D;Burré J;Malenka RC;Liu X;Südhof TC
在前脑神经元中，Ca2+通过结合synaptotagmin-1和-7触发易释放囊泡的胞出，从而分别诱导快速和缓慢的囊泡胞出。synaptotagmin-1或-7的功能丧失分别选择性地损害了快速和慢速释放期，但不会改变通过高渗蔗糖刺激释放测量的囊泡易释放池（RRP）的大小，也不会改变囊泡启动到RRP的速率。然而，我们发现突触蛋白-1和-7同时丧失功能会显著降低RRP的容量，而囊泡启动进入RRP的速度同样没有改变。在缺乏synaptotagmin-1和-7的神经元中，synaptotagmin-1或-7都足以恢复RRP的大小。尽管RRP大小的维持与Ca2+无关，但synaptotagmin-1或synaptotagmin-7的Ca2+结合序列（包含在其C2结构域的柔性顶环序列中）的突变阻止了这些synaptotagmin维持RRP大小的能力。两个突触塔蛋白都与SNARE复合物结合；顶部环突变破坏了RRP的维持，从而减少了SNARE复合物的结合。因此，synaptotagmin-1和-7除了在Ca2+释放的不同阶段触发非冗余功能外，还在维持RRP的能力方面执行冗余功能。Synaptotagmins不仅在触发突触囊泡融合中发挥钙传感器的作用，而且还有助于突触囊泡融合的准备，这是为随后触发胞外分泌准备的囊泡的充分启动所必需的。神经元通过称为突触的特殊接触点相互交流。突触前神经元在神经末梢的突触前囊泡中储存化学神经递质。在突触传递过程中，突触前囊泡与质膜融合，将其神经递质内容释放到突触间隙，激活突触后受体。神经递质释放是一个多阶段的过程，需要启动突触囊泡进入一个容易释放的囊泡池。当动作电位（一种沿神经元传递的瞬态电信号）侵入神经末梢时，它促进细胞外钙离子（Ca2+）的流入，进而触发启动囊泡的融合，从而导致神经递质释放。先前的研究证实，synaptotagmins作为Ca2+释放传感器，并且在没有动作电位的情况下抑制突触囊泡的自发融合。在大脑前部的大多数神经元中，突触塔敏-1和突触塔敏-7这两种突触塔敏分别介导神经递质的快慢释放。我们现在表明，除了它们作为Ca2+传感器和融合钳的非重叠作用外，synaptotagmin-1和-7在维持易释放的囊泡池中发挥重要的重叠功能。这一功能是由两个突触tagmins冗余执行的；因此，只有当两个synaptotagmins都被删除时，易释放池的损害才会显现出来。这些结果将突触tagmins的功能扩展到Ca2+触发释放的上游步骤，并表明突触tagmins尽管结构域简单，但在神经递质释放中执行多个顺序角色。因此，突触tagmins协调Ca2+触发的胞外分泌的多个阶段，确保快速突触传递突触神经元之间的快速信息传递。
In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of synaptotagmin-1 or synaptotagmin-7—which are contained in flexible top-loop sequences of their C2 domains—blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release. Synaptotagmins function not only as calcium sensors in triggering synaptic vesicle fusion but also contribute to the preparation of synaptic vesicles for fusion, being required for full priming of vesicles ready for subsequent triggering of exocytosis. Neurons communicate with each other at specialized contact points called synapses. Presynaptic neurons store chemical neurotransmitters within presynaptic vesicles at the nerve terminal. During synaptic transmission, the presynaptic vesicles fuse with the plasma membrane, releasing their neurotransmitter content into the synaptic cleft to activate postsynaptic receptors. Neurotransmitter release is a multistage process that requires the priming of synaptic vesicles into a readily-releasable pool of vesicles. When an action potential—a transient electrical signal that travels along the neuron—invades a nerve terminal, it promotes the influx of extracellular calcium ions (Ca2+) that, in turn, trigger fusion of primed vesicles, thereby causing neurotransmitter release. Previous studies established that synaptotagmins function as Ca2+ sensors for release and, additionally, inhibit spontaneous fusion of synaptic vesicles in the absence of an action potential. In most neurons of the anterior part of the brain, two synaptotagmins, synaptotagmin-1 and -7, mediate fast and slow neurotransmitter release, respectively. We now show that in addition to their nonoverlapping roles as Ca2+ sensors and fusion clamps, synaptotagmin-1 and -7 perform an essential overlapping function in maintaining the readily-releasable pool of vesicles. This function is redundantly performed by both synaptotagmins; therefore, an impairment of the readily-releasable pool manifests only when both synaptotagmins are deleted. These results extend the functions of synaptotagmins to steps upstream of Ca2+ triggering of release and suggest that synaptotagmins, despite their simple domain structure, perform multiple sequential roles in neurotransmitter release. Thus, synaptotagmins coordinate multiple stages of Ca2+-triggered exocytosis, ensuring fast synaptic transmission for rapid information transfer between neurons at synapses.