Synaptic Calcium Channels

突触钙通道

• 批准号: 8874329
• 负责人: Sean Aric Merrill
• 金额: $ 2.91万
• 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/8874329
• 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; Health; Homeostasis; Image; Individual; Light; Location; Membrane; Modeling; Molecular; Mono-S; Morphology; Mutation; Neurodegenerative Disorders; Neuromuscular Junction; Neuronal Plasticity; Neurons; Neurotransmitters; 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; light microscopy; millisecond; mutant; neuronal cell body; neurotransmission; novel; release of sequestered calcium ion into cytoplasm; response; synaptic function; synaptic inhibition; trafficking; vesicular release; 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(申请人提供)：钙是融合突触小泡的关键触发物。因此，钙通道在突触的位置可以决定囊泡融合的位置和概率。重要的是，细胞内钙对细胞是有毒的。钙进入和离开细胞必须仔细调节，以维持内环境平衡。突触钙调节缺陷是一些神经退行性疾病的基础，如肌萎缩侧索硬化症和阿尔茨海默病。钙指示剂已被用来研究细胞体内钙的流量，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定位于突触中心的单一类型的通道进入突触的。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，在一个多类型钙通道模型中，几个钙通道协同控制突触的不同区域。我建议研究每种类型的钙通道在突触中的位置，并确定其指定的突触小泡相对于突触中心致密投射的区域。不同的通道类型可能只对特定类型的刺激做出反应，这暗示着突触可塑性的额外水平。虽然一些通道可能定位于致密投射，但据预测，其他类型的通道将定位于突触活动区的外围区域。我将在以下几个方面测试这个模型 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参与突触神经传递的每种钙通道类型的准确位置。目标2)将哪些小泡分配给一个经络？我将使用闪光和冷冻电子显微镜来刺激钙通道突变体在冷冻前几毫秒的突触囊泡融合。目的3)哪些蛋白质将特定的钙通道定位于某个区域？我将使用候选基因方法和正向遗传筛选来识别与突触钙通道放置有关的特殊蛋白质。