Synaptotagmin C2B Domain as a Ca2+ Sensing Module

描述（由申请人提供）：本提案旨在了解神经元中膜融合的基本方面。膜融合由SNARE蛋白（囊泡膜上的v-SNARE，靶膜上的t-SNARE）介导，并受到调节因子的严格控制。我们主要集中在Ca 2+触发的突触囊泡的胞吐，这是由Ca 2+结合蛋白synaptotagmin（syt）1控制。Syt I通过与t-SNARE和膜的直接物理相互作用而起作用。这一提议继续了我们对syt I的研究，但也扩展了我们的工作，以解决该蛋白质的其他十五种异构体的功能。目的1a采用一个确定的重组系统来检验syt基因家族以有助于赋予细胞内膜融合反应特异性的方式分化的假设。我们假设特异性部分是通过在不同亚细胞区室中syt和t-SNARE的不同亚型之间的选择性配对来实现的。我们将确定syt-SNARE配对代码，并将其与这些蛋白质在神经元中的分布进行比较。在目标1 B中，我们继续追求我们的目标，即重建快速的膜融合，重现神经元中突触小泡胞吐的快速动力学。这是确定胞吐作用的精确分子机制的关键一步。在目标2中，我们将通过直接分析从敲除/敲入小鼠中分离的突触囊泡的融合活性，弥合目标1中使用的最小融合测定与突触传递研究（例如，下文的目标3）之间的差距。这种方法补充电生理分析，因为它报告了缺乏特定蛋白质的囊泡的内在融合特性。这些实验也将使我们有可能解决我们在目标1中无法以活性形式重建的突触囊泡蛋白的功能。在目标3中，我们将测试这一假设，即不同的异构体的syt已经分化，赋予突触不同的动力学成分的释放。我们预测，syt异构体与快速动力学介导同步传输，而syt异构体与较慢的动力学介导异步传输。这些研究将为神经元回路中的信息流提供新的见解。总之，这里提出的实验将为我们理解突触处的膜融合提供关键信息。这将有助于在突触传递受损的疾病状态下改变神经元之间的通信。

DESCRIPTION (provided by applicant): This proposal is aimed at understanding fundamental aspects of membrane fusion in neurons. Membrane fusion is mediated by SNARE proteins (v-SNAREs on the vesicle membrane, t-SNAREs on the target membrane) and is tightly controlled by regulatory factors. We have largely focused on the Ca2+triggered exocytosis of synaptic vesicles, which is controlled by the Ca2+binding protein synaptotagmin (syt) 1. Syt I operates through direct physical interactions with t-SNAREs and membranes. This proposal continues our studies of syt I but also extends our work to address the functions of the other fifteen isoforms of this protein. Aim 1a employs a defined reconstituted system to test the hypothesis that the syt gene family has diverged in ways that help confer specificity to intracellular membrane fusion reactions. We hypothesize that specificity is achieved, in part, through selective pairing between different isoforms of syt and t-SNAREs in various sub-cellular compartments. We will determine the syt-SNARE pairing-code and compare this with the distribution of these proteins in neurons. In Aim 1 b we continue to pursue our goal of reconstituting rapid membrane fusion that recapitulates the rapid kinetics of synaptic vesicle exocytosis in neurons. This is a key step toward defining the precise molecular mechanism that underlies exocytosis. In Aim 2 we will bridge the gap between the minimal fusion assay used in Aim 1 and studies of synaptic transmission (e.g. Aim 3 below), by analyzing directly the fusion activity of synaptic vesicles isolated from knock-out/knock-in mice. This approach compliments electrophysiological analysis because it reports the intrinsic fusion properties of vesicles lacking specific proteins. These experiments will also make it possible to address the function of synaptic vesicle proteins that we have been unable to reconstitute in an active form in Aim 1. In Aim 3, we will test the hypothesis that different isoforms of syt have diverged to impart synapses with distinct kinetic components of release. We predict that syt isoforms with fast kinetics mediate synchronous transmission, while syt isoforms with slower kinetics mediate asynchronous transmission. These studies will provide new insights into information flow in neuronal circuits. Together, the experiments proposed here will provide critical information regarding our understanding of membrane fusion at synapses. This will aid efforts to alter communication between neurons in disease states where synaptic transmission is impaired.