Synaptic Calcium Channels

突触钙通道

• 批准号: 8728039
• 负责人: Sean Aric Merrill
• 金额: $ 2.87万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728039
• 关键词: Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animals; Behavioral; Caenorhabditis elegans; Calcium; Calcium Channel; Candidate Disease Gene; Cells; Cessation of life; Data; Defect; Disease; Docking; Electron Microscopy; Engineering; Fiber; Fluorescence; Freezing; Gated Ion Channel; Genes; Genetic Screening; Grant; Homeostasis; Image; Individual; Light; Location; Membrane; Modeling; Molecular; Mono-S; Morphology; Mutation; Neurodegenerative Disorders; Neuromuscular Junction; Neuronal Plasticity; Neurons; Pain; Peripheral; Plastics; Probability; Protein Isoforms; Proteins; Regulation; Relative (related person); Retina; Role; Ryanodine Receptor Calcium Release Channel; Site; Stimulus; Synapses; Synaptic Vesicles; Synaptic plasticity; Techniques; Testing; Toxic effect; Up-Regulation; Vesicle; calcium indicator; fluorophore; gain of function; light microscopy; millisecond; mutant; neuronal cell body; neurotransmission; neurotransmitter release; novel; public health relevance; release of sequestered calcium ion into cytoplasm; response; synaptic function; synaptic inhibition; trafficking; voltage

DESCRIPTION (provided by applicant): Calcium is the critical trigger that fuses synaptic vesicles. So, the placement of calcium channels at the synapse can determine the site and probability of vesicle fusions. Importantly, cytosolic calcium is toxic to cells. The entry of calcum into and out of the cell must be carefully regulated to maintain homeostasis. Defects in synaptic calcium regulation underlie some neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer's Disease. Calcium indicators have been used to study the flux of calcium in cell bodies, but synapses are beyond the resolving power of most standardized techniques. Classically, synaptic calcium was thought to enter the through a single type of channel that localized to the center of the synapse. Cells could increase the total amount of calcium by inserting more channels, or increasing the stimulus. However, our preliminary results indicate that several calcium channels coordinate to control distinct regions of the synapse in a multi-type calcium channel model. I propose to study the location of each type of calcium channel in the synapse, and determine its region of assigned synaptic vesicles relative to the dense projection at the center of the synapse. Different channel types may open only in response to specific types of stimulus, suggesting of an additional level of synaptic plasticity. While some channels may localize to the dense projection, it is predicted that other types will localize to peripheral regions of the synaptic active zone. I will test this model in the following three aims. Aim 1) Where are calcium channels in the synapse? I will use nanoscopic fluorescence correlated electron microscopy to identify the precise location of each calcium channel type involved in neurotransmission at the synapse. Aim 2) Which vesicles are assigned to a channel? I will use flash-and-freeze electron microscopy to stimulate synaptic vesicle fusions in calcium channel mutants only milliseconds before freezing. Aim 3) Which proteins localize specific calcium channels to a region? I will use a candidate gene approach and a forward genetic screen to identify specialized proteins involved in synaptic calcium channel placement.

描述（由申请人提供）：钙是融合突触囊泡的关键触发剂。因此，钙通道在突触上的位置可以决定囊泡融合的位置和概率。重要的是，胞质钙对细胞有毒。钙进入和离开细胞必须仔细调节，以维持稳态。突触钙调节的缺陷是一些神经退行性疾病的基础，例如肌萎缩性侧索硬化症和阿尔茨海默病。钙指示剂已被用于研究细胞体中钙的流动，但突触超出了大多数标准化技术的分辨率。传统上，突触钙被认为是通过位于突触中心的单一类型的通道进入的。细胞可以通过插入更多的通道或增加刺激来增加钙的总量。然而，我们的初步结果表明，几个钙通道协调控制突触的不同区域中的多类型的钙通道模型。我建议研究每种类型的钙通道在突触中的位置，并确定其区域的分配突触囊泡相对于致密的突起在突触的中心。不同类型的通道可能只在对特定类型的刺激做出反应时才打开，这表明突触可塑性的额外水平。虽然一些通道可能定位于致密突起，但预测其他类型的通道将定位于突触活动区的周边区域。我将在下面测试这个模型 三个目标。目的1）突触中的钙通道在哪里？我将使用纳米荧光相关电子显微镜来确定在突触处参与神经传递的每种钙通道类型的精确位置。目的2）哪些囊泡被分配到通道？我将使用闪光和冷冻电子显微镜来刺激突触囊泡融合在钙通道突变体只有毫秒前冻结。目的3）哪些蛋白质将特异性钙通道定位于某一区域？我将使用候选基因方法和正向遗传筛选来鉴定参与突触钙通道放置的专门蛋白质。