Mechanisms of Fast Neurotransmitter Secretion

调节神经递质的胞吐是神经元间通讯的基本过程。 神经递质的分泌被广泛认为是所有真核细胞所共有的组成性囊泡胞吐的严格调节形式。 在神经终末，去极化引起的Ca 2+通过离子通道进入突触囊泡，促使突触囊泡融合，将神经递质释放到突触后细胞，许多实验室结合生物化学和遗传学方法建立了胞吐和胞吞机制的分子模型。特别是，最近的进展揭示了SNARE或核心复合物驱动组成性膜融合。然而，介导囊泡对接，在易释放池中捕获融合感受态囊泡，完成从传感器到融合机器的Ca 2+信号传导，调节囊泡融合的概率或调节易释放池的大小的机制知之甚少。我们已经启动了遗传筛选以鉴定在神经末梢介导Ca 2+触发胞吐的进一步组分，并鉴定了几个新的突变，这可能会影响神经传递其中，突变B682是特别有趣的，因为它抑制了一个独特的活性依赖性的诱发释放的损失，这与自发神经递质释放的同时增加，提示受损的可释放囊泡池。一个统一的假设表明，B682蛋白介导突触囊泡成熟的后期步骤，在“容易释放的囊泡池”中积累有融合能力的囊泡。具体来说，B682可以“钳”这些融合囊泡，使他们等待融合触发Ca 2+信号。为了测试这一假设（并探索其他的，如果有必要），功能丧失和获得B682突变将检查其对NMJ突触功能的影响。这些作用将通过电生理学、Ca 2+成像、FM 1 -43成像、共聚焦和电子显微镜以及生物化学等多学科方法进行研究。前两个目标将为评估B682的突触作用奠定基础。具体来说，我们将验证B682确实是一种突触前蛋白，影响突触传递的生理步骤，但不影响神经肌肉突触发生。一旦这些点完全建立，第三个目标将测试B682蛋白质假说的本质。具体而言，它将测试B682突变不会损害Ca 2+进入和/或排出的预测。此外，该目的将测试B682突变体中的异常囊泡分布是否是由囊泡再循环缺陷、囊泡运输至活性区的步骤和/或重新填充容易释放的囊泡池引起的。总之，这些研究将有助于更好地了解介导神经末梢快速、同步神经递质胞吐调节的分子机制。

Regulated neurotransmitter exocytosis is a fundamental process for the intercellular communication among neurons. It is widely accepted that neurotransmitter secretion is a tightly regulated form of constitutive vesicular exocytosis shared by all eukaryotic cells. At the nerve terminal, a depolarization-induced Ca2+ influx through ion specific channels triggers the fusion of synaptic vesicles, which expel their neurotransmitter cargo onto the postsynaptic cell.A combination of biochemical and genetic approaches by many laboratories has lead to molecular models describing exocytotic and endocytotic mechanisms. In particular, recent advances uncovered the SNARE- or core complex driving constitutive membrane fusion. However, little is known about mechanism that mediate vesicle docking, arrest fusion-competent vesicles in the readily releasable pool, accomplish Ca2+ signaling from the sensor to the fusion machinery, adjust the probability of vesicle fusion, or regulate the size of readily releasable pool.We have initiated a genetic screen to identify further components mediating Ca2+-triggered exocytosis at nerve terminals and identified several novel mutations, which potentially affect neurotransmission. Of these, the mutation B682 is especially interesting because it inhibits a unique activity-dependent loss of evoked release, which coincides with a simultaneous increase in spontaneous neurotransmitter release, suggestive of an impaired releasable vesicle pool. A unifying hypothesis suggests that B682 protein mediates a late step in synaptic vesicle maturation that accumulates fusion-competent vesicles in the "readily releasable vesicle pool". Specifically, B682 may "clamp" these fusogenic vesicles such that they await the fusion-triggering Ca2+ signal.To test this hypothesis (and explore others if warranted), loss- and gain-of-function B682 mutations will be examined for their effects on synaptic function at NMJs. These effects will be examined by a multi-disciplinary approach including electrophysiology, Ca2+ imaging, FM1-43 imaging, confocal and electron microscopy, and biochemistry.The first two Objectives will lay basic and essential groundwork for evaluating B682's synaptic role. Specifically, we will verify that B682 is indeed a presynaptic protein that affects a physiological step of synaptic transmission but not neuromuscular synaptogenesis. Once these points are fully established the third Objective will test the essence of the B682 protein hypothesis. Specifically, it will test the prediction that B682 mutations do not impair Ca2+ entry and/or extrusion. In addition, this objective will test whether the abnormal vesicle distribution in B682 mutants is caused by a defect in vesicle recycling, steps of vesicle trafficking to active zones and/or refilling the readily releasable vesicle pool. Together, these studies will help to better understand the molecular mechanisms that mediate the regulation of fast, synchronous neurotransmitter exocytosis at nerve terminals.