Ankyrin-dependent Na⁺ channel clustering prevents neuromuscular synapse fatigue

Skeletal muscle contraction depends on activation of clustered acetylcholine receptors (AchRs) and muscle-specific Na⁺ channels (Nav1.4). Some Nav1.4 channels are highly enriched at the neuromuscular junction (NMJ), and their clustering is thought to be essential for effective muscle excitation. However, this has not been experimentally tested, and how NMJ Na⁺ channels are clustered is unknown. Here, using muscle-specific ankyrinR, ankyrinB, and ankyrinG single, double, and triple-conditional knockout mice, we show that Nav1.4 channels fail to cluster only after deletion of all three ankyrins. Remarkably, ankyrin-deficient muscles have normal NMJ morphology, AchR clustering, sarcolemmal levels of Nav1.4, and muscle force, and they show no indication of degeneration. However, mice lacking clustered NMJ Na⁺ channels have significantly reduced levels of motor activity and their NMJs rapidly fatigue after repeated nerve-dependent stimulation. Thus, the triple redundancy of ankyrins facilitates NMJ Na⁺ channel clustering to prevent neuromuscular synapse fatigue.