HSC70 FOR NEUROTRANSMITTER RELEASE

快速同步释放神经递质有两个必要条件：首先，CA/2+进入动力学的最佳调整；其次，CA2+进入位点的最佳组装靠近CA/2+传感器位点的CA/2+进入位点，以确保CA/2+离子的短扩散通道。我们之前对果蝇中半胱氨酸串蛋白（Csp）突变的遗传分析表明，Csp介导同步神经递质释放，但不参与囊泡循环。脊椎动物体外研究表明，CSP与牛70kD热激同源蛋白（HSC70）协同相互作用，可能介导囊泡循环过程中笼蛋白包被囊泡的脱包。结合遗传和生物化学证据，我们提出CSP可能与HSC70协同相互作用，通过引导HSC70进入同步释放机制并刺激其活性，从而介导同步释放。该模型假设HSC70在神经递质释放中的新功能。本研究的重点是通过分析果蝇中缺乏HSC70和/或CSP功能的突变菌株的同步神经递质释放来严格验证这一假设，已经鉴定出5个不同的组成性表达的热休克同源基因（HSC1-5），它们都与牛HSC70具有显著的序列同源性。然而，这两种基因在果蝇中均未发生突变。由于研究正确的同源蛋白对我们的成功至关重要，我们在Aim 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。测试HSC70的一些功能。我们建议在Aim 2中确定HSC70在体内同步释放和囊泡循环中的作用。因此，我们将研究HSC70功能缺失对突变果蝇的影响。具体来说，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伤。在Aim 3中，我们提议在体内测试CSP和飞行HSC70是否协同相互作用。这将通过分析同时缺乏CSP和HSC70的双突变蝇在体内的同步释放和囊泡循环来实现。这将通过分析同时缺乏CSP和HSC70功能的双突变蝇的同步释放和囊泡循环来实现。或者，我们将确定HSC70过表达是否能够挽救因CSP功能丧失而导致的神经递质释放缺陷。这也有助于确定CSP和HSC70功能在发射机释放中的等级顺序。研究结果有助于进一步了解神经递质同步释放的分子机制，而神经递质同步释放是调节神经系统功能可塑性的基本条件之一。这种理解将有助于对抗突触功能障碍对人类生活的巨大影响。

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.