HSC70 FOR NEUROTRANSMITTER RELEASE

两个条件似乎是神经递质快速同步释放的必要条件：第一，钙离子进入动力学的最佳调整；第二，钙离子进入部位与钙/2+进入部位非常接近的最佳组装，以确保钙/2+离子的短扩散通道。我们先前对果蝇半胱氨酸串蛋白(CSP)突变的遗传分析表明，CSP介导了神经递质的同步释放，而不是囊泡循环。脊椎动物CSP的体外研究表明，CSP与牛70kD热休克同源蛋白(HSC70)存在协同作用，在囊泡再循环过程中，HSC70可能参与了囊泡去涂层的过程。结合遗传学和生化证据，我们认为CSP可能与HSC70协同作用，通过引导HSC70进入同步释放机制并刺激其活性来调节其同步释放。该模型假定HSC70在神经递质释放中具有新的功能。这一建议的重点将是在体内严格检验这一假说，通过分析缺乏HSC70和/或CSP功能的果蝇突变株的同步神经递质释放，已经鉴定出五个不同的结构性表达的热休克同源基因(HSC1-5)，它们都与牛HSC70有显著的序列同源性。然而，这两种基因都没有苍蝇突变的报道。由于研究正确的同源蛋白对我们的成功至关重要，我们在目标1中建议通过HSC70与CSP的生化相互作用来鉴定真正的HSC70同源蛋白。为了测试HSC70的一些功能，我们在目标2中建议确定HSC70在体内对同步释放和囊泡回收的作用。因此，我们将研究HSC70功能缺失对突变果蝇的影响。具体地说，我们将使用电生理记录和幼虫神经肌肉连接处的FM1-43成像来证明神经传递的任何损害。在目标3中，我们建议测试CSP和Fly HSC70是否在体内协同作用。这将通过分析双突变果蝇的同步释放和囊泡回收来实现，这些双突变果蝇在体内同时缺乏CSP和HSC70的协同作用。这将通过对同时缺乏CSP和HSC70功能的双突变果蝇的同步释放和囊泡回收的分析来实现。或者，我们将确定HSC70的过度表达是否能够挽救由于CSP功能丧失而导致的神经递质释放缺陷。这也将有助于确定发射机版本中CSP和HSC70功能的层次顺序。这项工作可能有助于理解神经递质同步释放的分子机制，这是调节神经系统功能可塑性的基本条件之一。这一认识将有助于对抗突触功能障碍对人类生活的戏剧性影响。

Two conditions appear essential for fast synchronous neurotransmitter release: First, an optimal adjustment of CA/2+ entry kinetics and second, and optimal assembly of the CA2+ entry site in close proximity to the Ca/2+ entry site in close proximity to the Ca/2+ sensor site to ensure a short diffusion passage for Ca/2+ ions. Our previous genetic analysis of cysteine string protein (Csp) mutations in Drosophila indicates that CSP mediates synchronous neurotransmitter release but not vesicle recycling. In vitro studies of vertebrate CSP suggest a cooperative interaction of CSP with bovine 70kD heat shock cognate protein (HSC70), which is suggested to mediate the uncoating of clathrin-coated vesicles during vesicle recycling Combining the genetic and biochemical evidence, we propose that CSP may cooperatively interact with HSC70 to mediate synchronous release by directing HSC70 to the synchronous release machinery and stimulating its activity. This model postulates a novel function for HSC70 in neurotransmitter release. The focus of this proposal will be to stringently test this hypothesis in vivo by analyzing synchronous neurotransmitter release in mutant Drosophila strains which lack HSC70 and/or CSP function in Drosophila, five distinct constitutively expressed heat shock cognate genes (HSC1-5) have been identified which all share a significant sequence homology with bovine HSC70. However, no fly mutations have been reported for either gene. Since it will be critical for our success to study the correct homologous protein, we propose in Aim 1 to identify the true HSC70 homologue by its biochemical interaction with CSP. To test some of the suggested functions of HSC70., we propose in Aim 2 to determine the in vivo role of HSC70 for synchronous release and for vesicle recycling. Therefore, we will study the effects caused by the loss of HSC70 function in mutant Drosophila. Specifically, we will use electrophsiological recordings and FM1-43 imaging at the larval neuromuscular junction to demonstrate any impairment of neurotransmission. In Aim 3 we propose to test whether CSP and fly HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 function. Alternatively, we will determine whether the over expression of HSC70 is able to rescue to neurotransmitter release defect caused by the loss of CSP function. This will also facilitate to determine the hierarchical order of CSP and HSC70 functions in transmitter release. The proposed work may significantly help to understand the molecular mechanism of synchronous neurotransmitter release which is one of the basic conditions mediating the functional plasticity of our nervous system. This understanding will help to fight the dramatic effects of synaptic dysfunction for human life.