Local calcium signalling in the postsynaptic density

在大脑内部，数百亿个电活动细胞在称为突触的连接处相互通信。突触强度的长期变化被认为是学习和记忆的基础。研究的重点是了解突触可塑性是如何在模式生物的分子水平上实现的，其中包括海参和大鼠。突触可塑性“核心程序”的一些组成部分已经出现。值得注意的是，突触的长时间增强和抑制都是由钙的流入引起的。两种钙敏感酶在这一过程中至关重要：一种是在大量钙离子流入后修饰突触蛋白以加强突触，另一种是在少量钙离子流入后产生相反的作用。然而，这两种酶对试管中的钙表现出同样的敏感性，所以为什么它们对突触中的钙的反应不同并不明显。一个重要的考虑因素是，钙在其入口点附近的细胞中局部集中。我们的研究将追求两个主要目标。首先，我们建议确定这两种酶从钙入口点在称为突触后密度(PSD)的大规模蛋白质集合中的相对位置是否解释了为什么其中一种酶需要更大的钙内流才能激活。其次，我们的目标是确定为什么抑制突触强度的酶在大量钙流入后是不活跃的。我们将采用互补的方法来检测这两种酶在PSD中相对于钙入射点的位置。我们将对PSD样品进行化学交联，并绘制出蛋白质组装中不同蛋白质之间的确切交联点。这将使我们能够建立准确的结构模型，以便我们可以比较关键酶与钙进入点的相对位置。我们还将测量钙传感器在与PSD内的不同蛋白质共同定位时，对实验控制的钙流入的反应。在追求我们的第二个主要目标时，我们将专注于了解锚定蛋白如何调节抑制突触强度的钙敏感酶的活性。我们将确定锚定蛋白片段的高分辨率三维结构，以了解它是如何受到PSD中钙的调节的。我们还将使用一种新的酶分析方法来检测锚定蛋白是否能够调节导致长期突触抑制的酶，使其在低浓度但不是高浓度的钙离子下活跃。这项研究将受益于与德国蛋白质组学专家的合作，从而巩固国际合作伙伴关系。除了解决大脑如何储存记忆的一个基本方面外，我们开发的新技术方法将广泛适用于生物学的其他领域。这项研究与人类大脑疾病高度相关，因为突触是导致神经疾病和精神疾病的突变的场所，而异常的突触可塑性可能是包括阿尔茨海默氏症和自闭症在内的许多大脑疾病的基础。钙敏感酶和锚定蛋白都是拟议研究的重点，都是药物靶标。总体而言，这项研究可以在分子水平上提供见解，为药物设计提供信息。

Within the brain, tens of billions of electrically active cells communicate with each other at junctions called synapses. Long-lasting changes in the strength of synapses are thought to underlie learning and memory. Research has focused on understanding how synaptic plasticity is brought about at the level of molecules in model organisms including sea slugs and rats. Some components of a 'core program' for synaptic plasticity have emerged. Notably, long-lasting potentiation and depression of synapses both follow from influx of calcium. Two calcium-sensitive enzymes are critical for this process: one modifies synaptic proteins to strengthen the synapse following large influxes of calcium whereas the other has the opposite effect following small influxes of calcium. However, both enzymes exhibit equal sensitivity to calcium in the test tube so it is not obvious why they respond differently to calcium within the synapse. An important consideration is that calcium is locally concentrated in cells near to its points of entry. Our research will pursue two major goals. First, we propose to determine whether the relative positions of the two enzymes from calcium entry points, within a massive protein assembly called the postsynaptic density (PSD), explain why one of the enzymes requires larger influxes of calcium for activation. Second, we aim to determine why the enzyme that depresses synaptic strength is inactive following large influxes of calcium.We will apply complementary approaches to examine the positions of the two enzymes relative to calcium entry points in the PSD. We will chemically crosslink PSD samples and map the exact sites of crosslinking between different proteins within the protein assembly. This will enable us to build accurate structural models so we can compare the relative positions of the key enzymes with calcium entry points. We will also measure how a calcium sensor responds to experimentally controlled influxes of calcium when co-localised with different proteins within the PSD. In pursuit of our second major aim, we will focus on understanding how an anchoring protein regulates the activity of the calcium-sensitive enzyme that depresses synaptic strength. We will determine the high-resolution three-dimensional structure of a fragment of the anchoring protein to understand how it is regulated by calcium within the PSD. We will also use a novel enzymatic assay to examine whether the anchoring protein is able to tune the enzyme that brings about long-term synaptic depression such that it is active at low but not high concentrations of calcium.The research will benefit from collaboration with proteomics specialists in Germany thereby cementing an international partnership. In addition to addressing a fundamental aspect of how the brain lays down memories, the novel technological approaches that we develop will be widely applicable to other areas of biology. The research is highly relevant to human brain disease as the synapse is a locus for mutations that cause neurological disease and psychiatric disorders, and aberrant synaptic plasticity may be fundamental to many brain disorders including Alzheimer's and autism. Both the calcium-sensitive enzymes and the anchoring protein that are the focus of the proposed research are drug targets. Overall, the research can provide insights at a molecular level that is informative for drug design.