Mechanisms of sodium channel clustering at the neuromuscular junction

神经肌肉接头钠通道聚集的机制

• 批准号: 10542388
• 负责人: MATTHEW N RASBAND
• 金额: $ 35.2万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 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/10542388
• 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; Intrinsic drive; Ion Channel; Knock-out; Knockout Mice; Label; 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; Neuregulins; Neuroglia; Neuromuscular Diseases; Neuromuscular Junction; Neurons; Neuropathy; Nodal; Proteins; Proteomics; Ranvier' s Nodes; Receptor Activation; Role; Scaffolding Protein; Site; Skeletal Muscle; Sodium Channel; Spectrin; Streptavidin; Synapses; Toxin; conditional knockout; design; extracellular; gain of function; improved; insight; loss of function; periodic paralysis; recruit; voltage

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+通道高度聚集在神经肌肉接头（NMJ），位于下面的连接褶皱深处 乙酰胆碱受体（AchRs）。而乙酰胆碱受体对运动神经释放的乙酰胆碱有反应， 神经元，并负责初始膜去极化，集群Na+通道是 负责肌肉动作电位。AchR聚集依赖于聚集蛋白，神经调节蛋白， 以及已经详细描述的活性依赖性机制。然而，人们对 NMJ Na+通道聚集的机制。包括肌强直，周期性麻痹， 和肌无力综合征都会破坏NMJ Na+通道聚集发生在细胞膜的另外两个位置。 神经系统，包括朗维尔结和轴突起始段（AIS）。在这里，细胞骨架和 细胞外相互作用参与通道聚集，并且已经详细描述了其机制。 值得注意的是，许多参与节点和AIS处Na+通道聚集的相同蛋白质也在 NMJ。通过类比节点和AIS，我们提出NMJ Na+通道聚类依赖于类似的 细胞骨架和细胞外相互作用。目标1将包括两个部分，旨在确定 细胞骨架相互作用对NMJ Na+通道聚集很重要。首先，我们将有条件地淘汰 （特别是在骨骼肌中）单独和组合的三种Na+通道结合锚蛋白（Ank 1 -3）。 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， 被认为将锚蛋白（和Na+通道）连接到肌动蛋白细胞骨架。在锚蛋白和血影蛋白缺乏 小鼠，我们将评估肌肉功能和Na+通道聚集。在目标2中，我们将确定细胞外 参与NMJ Na+通道聚集的相互作用。首先，我们将产生肌肉特异性敲除 细胞粘附分子Nfasc在NMJ的位置及其介导细胞外 在AIS和节点处的交互。其次，由于对细胞粘附分子的了解少得多， 细胞外基质分子可能是NMJ细胞外相互作用的基础，我们将使用邻近 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 为他们的本地化NMJ，并使用增益和功能丧失的策略决定他们的功能。的 这里提出的目标将大大提高我们对Na+控制分子机制的理解 通道群集在神经肌肉接头，并可能导致重要的见解的病理生理学 神经肌肉疾病和神经病变，其中NMJ退化或功能受损。