Mechanisms of calcium-dependent neurotransmitter release in health and disease

项目摘要/总结 所有已知的认知、情感和相关行为过程都依赖于由神经元集合形成的回路。 神经元之间的高保真通信需要神经递质的调节释放， 通常包含在突触前末梢的膜封闭囊泡中。在大多数神经元中，来自 电压门控通道作用于突触前蛋白，触发这些囊泡与血浆的融合 膜的神经递质快速释放的主要Ca 2+感受器是突触结合蛋白的成员， (Syt)家族，主要是Syt-1。Syt-1中的从头错义突变已在患有以下疾病的人类患者中发现： 严重的全球发育迟缓，强调了这种蛋白质在大脑功能中的重要作用。一 一对密切相关的蛋白质Doc 2 α和Doc 2 β（统称“Doc 2”）具有相似的结构特征，但 与Syt-1相比，在更慢的时间尺度上触发释放。Syt-1和Doc 2都包含串联C2结构域 以钙离子依赖的方式与细胞膜相互作用。但尽管进行了深入的研究， Syt-1和Doc 2作用于突触前膜和其他蛋白质以触发融合。候选机制 包括（1）Syt-1/Doc 2对突触前膜的作用，以及（2）与可溶性N- 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白，其催化膜 核聚变 该提案旨在解决关于Syt-1/Doc 2-膜和Syt-1/Doc 2-膜的主要未回答的问题。 1/Doc 2-SNARE相互作用，使快速，Ca 2+触发的膜融合。使用一套生物物理学 方法，这些实验将定义SNARE和生理磷脂如何合作，以塑造 Syt-1/Doc 2-膜融合之前、期间和之后的膜界面。来自人类的Syt-1突变 患者，其中两个尚未在文献中描述，将使用以下组合进行研究： 生物物理方法和活神经元中谷氨酸释放的高速成像。通过定义关键 Syt-1的结构-功能关系，这些结果将建立一个生物物理和生理基础， Syt-1突变导致人类患者患病。总之，拟议的实验站在显着 加深我们对健康和疾病中神经传递机制的理解。

PROJECT ABSTRACT/SUMMARY All known cognitive, affective, and related behavioral processes rely on circuits formed by neuronal ensembles. High-fidelity communication between neurons requires the regulated release of neurotransmitters, which are usually contained in membrane-enclosed vesicles at presynaptic terminals. In most neurons, Ca2+ influx from voltage-gated channels acts upon presynaptic proteins to trigger fusion of these vesicles with the plasma membrane. The principal Ca2+ sensors for fast neurotransmitter release are members of the Synaptotagmin (Syt) families, principally Syt-1. De novo missense mutations in Syt-1 have been found in human patients with profound global developmental delays, underscoring the essential role this protein plays in brain function. A pair of closely-related proteins, Doc2α and Doc2β (collectively “Doc2”), have similar structural features but trigger release on a slower timescale as compared to Syt-1. Both Syt-1 and Doc2 contain tandem C2 domains that interact with membranes in a Ca2+-dependent fashion. But despite intensive study, it remains unclear how Syt-1 and Doc2 act upon presynaptic membranes and other proteins to trigger fusion. Candidate mechanisms include (1) the action of Syt-1/Doc2 on presynaptic membranes, and (2) direct interactions with soluble N- ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, which catalyze membrane fusion. This proposal seeks to address major unanswered questions about the Syt-1/Doc2—membrane and Syt- 1/Doc2—SNARE interactions that enable fast, Ca2+-triggered membrane fusion. Using a set of biophysical approaches, these experiments will define how SNAREs and physiologic phospholipids cooperate to shape the Syt-1/Doc2—membrane interface before, during, and after membrane fusion. Syt-1 mutations from human patients, two of which have not yet been described in the literature, will be studied using a combination of biophysical approaches and high-speed imaging of glutamate release in live neurons. By defining critical structure-function relationships in Syt-1, these results will establish a biophysical and physiologic basis for how Syt-1 mutations cause disease in human patients. Together, the proposed experiments stand to significantly deepen our mechanistic understanding of neurotransmission in health and disease.