Mechanisms of Fast Neurotransmitter Secretion

受调节的神经递质胞吐是神经元细胞间通讯的一个基本过程。人们普遍认为，神经递质分泌是所有真核细胞共有的一种受到严格调控的组成性囊泡胞吐形式。在神经末梢，去极化诱导的Ca2+内流通过离子特异性通道触发突触囊泡融合，将其神经递质货物排出到突触后细胞。许多实验室将生化和遗传方法结合起来，建立了描述胞外和内吞机制的分子模型。特别是，最近的进展揭示了驱动本构膜融合的SNARE或核心复合物。然而，介导囊泡对接，在易释放池中阻止融合能力囊泡，完成从传感器到融合机制的Ca2+信号传递，调节囊泡融合的概率，或调节易释放池大小的机制知之甚少。我们已经启动了一项基因筛选，以确定在神经末梢介导Ca2+触发的胞外分泌的进一步成分，并确定了几个可能影响神经传递的新突变。其中，突变B682特别有趣，因为它抑制了一种独特的活性依赖性的诱发释放损失，这与自发性神经递质释放同时增加相吻合，表明可释放囊泡池受损。一个统一的假设认为，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”中积累融合能力囊泡。具体来说，B682可能“夹住”这些融合囊泡，使它们等待触发融合的Ca2+信号。为了验证这一假设（并在必要时探索其他假设），B682功能丧失和功能获得突变将被检测其对NMJs突触功能的影响。这些影响将通过多学科方法进行检查，包括电生理学，Ca2+成像，FM1-43成像，共聚焦和电子显微镜，以及生物化学。前两个目标将为评价B682的突触作用奠定基础和必要的基础。具体来说，我们将验证B682确实是一种突触前蛋白，它影响突触传递的生理步骤，但不影响神经肌肉突触发生。一旦这些观点完全确立，第三个目标将测试B682蛋白质假说的本质。具体来说，它将测试B682突变不会损害Ca2+进入和/或挤压的预测。此外，本目的将测试B682突变体中异常的囊泡分布是否由囊泡循环、囊泡运输到活性区域的步骤和/或重新填充容易释放的囊泡池的缺陷引起。总之，这些研究将有助于更好地理解介导神经末梢快速、同步的神经递质胞外分泌调节的分子机制。

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.