CSP/Hsc70 in Regulated Neurotransmitter Release

描述(由申请人提供)：神经递质的释放在许多步骤中被调节，这在一定程度上赋予突触可塑性、适应性和个性。两个关键步骤，钙离子进入和囊泡融合，似乎受到突触囊泡相关的半胱氨酸串蛋白(CSP)的调节--但方式相反。以往的工作支持CSP通过抑制突触前Ca~(2+)进入而减少释放，通过促进Ca~(2+)触发的融合下游步骤而增加释放的假说。CSP与Hsc70共同参与了钙通道、G蛋白、突触素和突触素之间的相互作用。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钙通道的Gbeta/Gamma抑制；(2)在其他已知的CSP相互作用中，哪些是调节CSP功能的；(3)哪些功能是由神经末梢的PKA磷酸化调节的。为了解决这些问题，我建议通过利用遗传模型系统果蝇来检验上述假设，以检测系统靶向CSP突变对神经肌肉接头处神经传递的影响，完成完整的体内结构/功能分析。具体地说，目标1将(A)将J-、L-、C-和Ct-结构域的蛋白质相互作用与CSP的突触作用联系起来，包括钙进入、钙触发融合、短期释放可塑性和钙稳态。Aim 1还将(B)解决PKA介导的神经末梢CSP磷酸化的意义。目的2将确定(A)CSP是否在抑制钙离子内流和/或(B)囊泡融合中起关键作用。从这一系统分析中，将出现一个实质性的框架，用于理解CSP明显相反的行动。这项拟议的工作还将扩大我们对突触传递的重要调控机制以及它们与神经系统功能可塑性和人类健康的关系的理解。

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.