Mechanisms of sodium channel clustering at the neuromuscular junction

Baylor College Of Medicine, Houston TX

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Abstract

Project Summary Na+ channels are highly clustered at the neuromuscular junction (NMJ) deep in the junctional folds below acetylcholine receptors (AChRs) and in a sub-micron, highly specialized perijunctional zone (PJZ) surrounding the AChRs and synapse; intriguingly, dystrophin is also enriched in this perijunctional zone. The NMJ/PJZ Na+ channels function to resist neuromuscular synapse fatigue. Although we discovered that Na+ channel clustering and maintenance requires ankyrins and spectrins, little else is known about the mechanisms that drive Na+ channel and dystrophin clustering at the NMJ and PJZ. Aging and disease (including myotonia, periodic paralysis, and myasthenic syndrome) all disrupt AChR clustering at the NMJ. However, how aging and disease affect the molecular organization and function of the PJZ is unknown. To overcome these gaps in our knowledge we will perform three specific aims. In aim 1, using several mouse models newly developed in our laboratory, we will use proximity biotinylation (e.g. TurboID) to perform a molecular dissection and proximity proteomics on the PJZ and neuromuscular synapse. We will validate and test candidates using a muscle specific, AAV-guided and CRISPR/Cas9-mediated genome editing approach. In Aim 2, we will determine the functions of two new validated PJZ proteins: Plin4 and Speg. This will be done using knockout and conditional knockout mice. In the final aim of this project, we will again use our newly developed NMJ/PJZ-localized TurboID mice to perform a molecular dissection of the PJZ/NMJ in aged and mdx mice (a model of Duchenne muscular dystrophy). The aims proposed here will reveal the molecular mechanisms underlying assembly of the Na+ channel/dystrophin protein complex in the PJZ and NMJ and may give important insights into the pathophysiology of neuromuscular diseases and aging.