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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：
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
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/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&apos 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

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+通道高度聚集在神经肌肉交界处（NMJ）深处的交界处皱褶下面