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Mechanisms of sodium channel clustering at the neuromuscular junction

神经肌肉接头钠通道聚集的机制

基本信息

批准号：
10324587
负责人：
MATTHEW N RASBAND
金额：
$ 34.85万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-20 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10324587
关键词：
ANK1 gene Acetylcholine Actins Action Potentials Affinity Chromatography Agrin Ankyrins Axon Binding Biotinylation Cell Adhesion Molecules Cells Cholinergic Receptors Complex Cytoskeleton Development Disease Escherichia coli Extracellular Matrix Functional disorder Generations Ion Channel Knock-out Knockout Mice Label Lead Link Location Maintenance Mass Spectrum Analysis Mediating Membrane Methods Mind Modeling Molecular Molecular Genetics Motor Neurons Mus Muscle Muscle Contraction Muscle function Myasthenic Syndrome Myotonia Nervous system structure Neuregulins Neuroglia Neuromuscular Diseases Neuromuscular Junction Neurons Neuropathy Nodal Proteins Proteomics Ranvier&apos s Nodes Receptor Activation Role Scaffolding Protein Site Skeletal Muscle Sodium Channel Spectrin Streptavidin Synapses Toxin conditional knockout design extracellular improved insight loss of function periodic paralysis recruit voltage

项目摘要

Na+ channels are highly clustered at the neuromuscular junction (NMJ) deep in the junctional folds below acetylcholine receptors (AchRs). Whereas AchRs respond to the release of acetylcholine from the motor neuron and are responsible for the initial membrane depolarization, the clustered Na+ channels are responsible for the muscle action potential. AchR clustering depends on a combination of agrin, neuregulin, and activity dependent mechanisms that have been described in great detail. However, little is known about the mechanisms responsible for NMJ Na+ channel clustering. Diseases including myotonia, periodic paralysis, and myasthenic syndrome all disrupt the NMJ. Na+ channel clustering occurs at two other locations in the nervous system including nodes of Ranvier and axon initial segments (AIS). Here, both cytoskeletal and extracellular interactions participate in channel clustering and the mechanisms have been described in detail. Remarkably, many of the same proteins involved in Na+ channel clustering at nodes and AIS are also found at the NMJ. By analogy to nodes and AIS, we propose that NMJ Na+ channel clustering depends on similar cytoskeletal and extracellular interactions. Aim 1 will consist of two parts designed to determine the cytoskeletal interactions important for NMJ Na+ channel clustering. First, we will conditionally knockout (specifically in skeletal muscle) the three Na+ channel-binding ankyrins (Ank1-3) singly and in combination. Second, we will conditionally knockout the 4 different spectrins known to be expressed in muscle and that are thought to link ankyrins (and Na+ channels) to the actin cytoskeleton. In both ankyrin and spectrin deficient mice we will evaluate muscle function and Na+ channel clustering. In Aim 2 we will identify the extracellular interactions that participate in NMJ Na+ channel clustering. First, we will generate muscle-specific knockouts of the cell adhesion molecule Nfasc given its location at the NMJ and its important role mediating extracellular interactions at the AIS and nodes. Second, since much less is known about the cell adhesion molecules and extracellular matrix molecules that may underlie NMJ extracellular interactions, we will use proximity biotinylation methods and proteomics to identify these proteins. We will then validate potential candidates for their localization to the NMJ, and using gain and loss of function strategies determine their functions. The aims proposed here will dramatically improve our understanding of the molecular mechanisms controlling Na+ channel clustering at the neuromuscular junction and may lead to important insights into the pathophysiology of neuromuscular diseases and neuropathies where NMJs degenerate or function is compromised.

Na+ 通道高度聚集在下方连接褶皱深处的神经肌肉接头 (NMJ) 乙酰胆碱受体（AchR）。而 AchRs 对运动中乙酰胆碱的释放做出反应神经元并负责初始膜去极化，簇状的 Na+ 通道是负责肌肉动作电位。 AchR 聚类取决于集聚蛋白、神经调节蛋白、以及已详细描述的活动依赖机制。然而，人们对此知之甚少负责 NMJ Na+ 通道聚类的机制。疾病包括肌强直、周期性麻痹、和肌无力综合征都会破坏 NMJ。 Na+通道聚集发生在该区域的另外两个位置神经系统包括 Ranvier 节点和轴突初始段 (AIS)。在这里，细胞骨架和细胞外相互作用参与通道聚类，其机制已被详细描述。值得注意的是，在节点和 AIS 处也发现了许多参与 Na+ 通道聚集的相同蛋白质国家医学杂志。通过类比节点和 AIS，我们提出 NMJ Na+ 通道聚类依赖于相似的细胞骨架和细胞外的相互作用。目标 1 将由两部分组成，旨在确定细胞骨架相互作用对于 NMJ Na+ 通道聚类很重要。首先，我们将有条件淘汰赛（特别是在骨骼肌中）三个 Na+ 通道结合锚蛋白 (Ank1-3) 单独或组合。其次，我们将有条件地敲除已知在肌肉中表达的 4 种不同的血影蛋白，它们是被认为将锚蛋白（和 Na+ 通道）与肌动蛋白细胞骨架连接起来。锚蛋白和血影蛋白均缺乏我们将评估小鼠的肌肉功能和 Na+ 通道聚类。在目标 2 中，我们将识别细胞外参与 NMJ Na+ 通道聚类的相互作用。首先，我们将产生肌肉特异性的敲除细胞粘附分子 Nfasc 位于 NMJ 及其介导细胞外的重要作用 AIS 和节点的交互。其次，由于对细胞粘附分子和细胞外基质分子可能是 NMJ 细胞外相互作用的基础，我们将使用接近度生物素化方法和蛋白质组学来鉴定这些蛋白质。然后我们将验证潜在的候选人其定位于 NMJ，并使用功能获得和丧失策略确定其功能。这这里提出的目标将极大地提高我们对控制 Na+ 的分子机制的理解神经肌肉接头处的通道聚集，可能会导致对病理生理学的重要见解 NMJ 退化或功能受损的神经肌肉疾病和神经病。