β-Spectrin Is Colocalized with Both Voltage-gated Sodium Channels and Ankyrin_G at the Adult Rat Neuromuscular Junction

Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, β-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that β-spectrin is precisely colocalized with both VGSCs and ankyrin_G, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, β-spectrin, VGSCs, and ankyrin_G have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that β-spectrin is precisely colocalized with both VGSCs and ankyrin_G at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that β-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrin_G, but not of dystrophin. These observations suggest that interactions with β-spectrin and ankyrin_G help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.