Spatial proteomics identifies FXYD6 as a dual-site protein of neuromuscular junction in the diaphragm.

Morphological studies of the diaphragm have provided a detailed view of its architecture, which consists of parallel skeletal muscle fibers and a central ring of neuronal innervation that includes neuromuscular junction (NMJ) units. NMJs are disease-vulnerable synapses; thus, analysis of the NMJ is essential to understand its function in both healthy and disease-related conditions. The diaphragm was analyzed by spatial proteomics and 115 proteins were enriched at the NMJ. Comparison of the protein signatures of the NMJ and myotendinous junction (MTJ) revealed 31 shared proteins, suggesting partially conserved structures between these junctions. Key mediators of synaptic transmission and extracellular matrix organization were observed among the NMJ-enriched components, which indicates the molecular complexity and regulatory potential of the NMJ. A focused study of the uncharacterized NMJ protein FXYD6 demonstrate enhanced FXYD6 expression in type IIa fibers of the diaphragm, which exhibit a unique balance of oxidative and glycolytic capacity. FXYD6 interacts with Na⁺/K⁺-ATPase subunits in the diaphragm, which supports the function of FXYD6 in the ionic homeostasis required for continuous, fatigue-resistant contraction. Overall, the dataset provides a comprehensive molecular atlas of the NMJ in the diaphragm and opens new opportunities to dissect the synaptic mechanisms underlying respiratory function and neuromuscular diseases.