Mechanisms of sodium channel clustering at the neuromuscular junction

NA通道高度聚集在下方交界处的神经肌肉交界处(NMJ) 乙酰胆碱受体(AchRs)。而AchRs对从发动机释放的乙酰胆碱做出反应 神经元负责初始的膜去极化，聚集的Na通道是 负责肌肉动作电位。Achr聚集性依赖于集聚蛋白、NeuRegin、 以及已经非常详细地描述的活动依赖机制。然而，人们对此知之甚少。 NMJ钠通道聚集的机制。疾病包括肌强直，周期性瘫痪， 和肌无力综合症都扰乱了NMJ。NA通道群集发生在 神经系统包括兰维尔结节和轴突起始段(AIS)。在这里，细胞骨架和 细胞外相互作用参与通道聚集，其机制已被详细描述。 值得注意的是，许多与节点和AIS的钠通道聚集有关的相同蛋白质也在 《NMJ》。通过对节点和AIS的类比，我们提出了NMJ Na通道聚集依赖于相似 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NMJ钠通道聚集很重要。首先，我们将有条件地淘汰赛 (特别是在骨骼肌中)三个钠通道结合蛋白(ANK1-3)单独或组合在一起。 其次，我们将有条件地敲除已知在肌肉中表达的4种不同的幽灵蛋白，它们是 被认为将骨架蛋白(和钠通道)与肌动蛋白细胞骨架联系在一起。在锚蛋白和幽灵蛋白缺乏的情况下 我们将评估小鼠肌肉功能和钠通道聚集性。在目标2中，我们将识别细胞外 参与NMJ Na通道聚集的相互作用。首先，我们将生成特定于肌肉的击倒 细胞黏附分子NFasc在NMJ的定位及其在细胞外调节中的重要作用 在AIS和节点上的交互。第二，由于对细胞黏附分子和 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鉴定这些蛋白质。然后我们将验证潜在的候选人 由于它们定位于NMJ，并使用功能的得失策略决定了它们的功能。这个 这里提出的AIMS将极大地提高我们对钠的分子控制机制的理解 神经肌肉交界处的经络聚集，可能导致对病理生理学的重要见解 神经肌肉疾病和神经性疾病，其中NMJ退化或功能受损。

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.