Synaptic Calcium Channels

描述（由申请人提供）：钙是融合突触囊泡的关键触发器。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，胞质钙对细胞有毒。钙的进出细胞必须仔细调节以维持体内平衡。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。钙指示剂已被用于研究钙在细胞体中的通量，但突触超出了大多数标准化技术的分辨能力。传统上，突触钙被认为是通过一种定位于突触中心的单一通道进入大脑的。细胞可以通过插入更多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，在多类型钙通道模型中，几个钙通道协调控制突触的不同区域。我建议研究每种钙通道在突触中的位置，并确定其相对于突触中心密集投影的分配突触囊泡的区域。不同的通道类型可能只在特定类型的刺激下打开，这表明突触具有额外的可塑性。虽然一些通道可能定位于密集投射，但预计其他类型的通道将定位于突触活跃区的外周区域。我将在下面测试这个模型

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.