Role of CSP/Hsc70 in Regulated Neurotransmitter Release

描述（申请人提供）：神经递质的释放在许多步骤中受到调节，这部分地赋予突触其可塑性、适应性和个体性。两个关键步骤，钙离子进入和囊泡融合，似乎是由突触囊泡相关的半胱氨酸串蛋白（CSP），但在“相反”的方式调节。以前的工作支持这一假设，即CSP通过抑制突触前Ca 2+进入来减少释放，并通过促进Ca 2+触发融合的下游步骤来增加释放。CSP可能与Hsc 70一起指导Ca 2 v通道、G蛋白、突触融合蛋白和突触结合蛋白之间的蛋白质相互作用。为了更好地理解CSP的作用，我们需要知道：（1）CSP是否确实促进了神经末梢Ca 2+通道的G β/γ抑制，（2）其他已知的CSP相互作用介导了CSP的哪些功能，以及（3）哪些功能由神经末梢的PKA磷酸化调节。为了解决这些问题，我建议测试上述假设，利用遗传模型系统果蝇研究系统有针对性的CSP突变对神经肌肉接头的神经传递的影响，完成一个完整的体内结构/功能分析。具体而言，目标1将（a）将J-、L-、C-和Ct-结构域的蛋白质相互作用与CSP的突触作用相关联，包括Ca 2+进入、Ca 2+触发的融合、释放的短期可塑性和Ca 2+稳态。目的1还将（B）解决神经末梢处PKA介导的CSP磷酸化的意义。目的2将确定（a）CSP是否对Ca 2+进入和/或（B）囊泡融合的G β/γ抑制至关重要。从这个系统的分析，一个实质性的框架将出现理解CSP的明显相反的行动。这项工作还将扩大我们对突触传递的重要调控机制及其与神经系统功能可塑性和人类健康关系的理解。

DESCRIPTION (provided by applicant): Neurotransmitter release is regulated at many steps, which in part confers upon synapses their plasticity, adaptability, and individuality. Two key steps, Ca2+ entry and vesicle fusion, appear to be regulated by the synaptic vesicle-associated cysteine-string protein (CSP) - but in "opposing" ways. Previous work supports the hypothesis that CSP reduces release by inhibiting presynaptic Ca2+ entry and increases release by promoting a downstream step of Ca2+-triggered fusion. Together with Hsc70, CSP might direct protein interactions among Ca 2v channels, G proteins, syntaxin, and synaptotagmin. To better understand CSP's action, we need to know: (1) whether CSP indeed promotes Gbeta/gamma inhibition of Ca2+ channels at nerve terminals, (2) which of the other known CSP interactions mediates which function of CSP, and (3) which functions are regulated by PKA-phosphorylation at nerve terminals. To resolve these issues, I propose to test the above hypotheses by exploiting the genetic model system Drosophila to examine the effects of systematically targeted CSP mutations on neurotransmission at neuromuscular junctions, accomplishing a complete in vivo structure/function analysis. Specifically, Aim 1 will (a) correlate protein interactions of the J-, L-, C-, and Ct-domain with CSP's synaptic roles, including Ca2+ entry, Ca2+-triggered fusion, short-term plasticity of release, and Ca2+ homeostasis. Aim 1 will also (b) resolve the significance of PKA-mediated phosphorylation of CSP at nerve terminals. Aim 2 will determine (a) whether CSP is critical for Gbeta/gamma inhibition of Ca2+ entry and/or (b) vesicular fusion. From this systematic analysis a substantial framework will emerge for understanding the apparently opposing actions of CSP. The proposed work will also expand our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health.