Voltage-Gated Sodium Channels and AnkyrinG Occupy a Different Postsynaptic Domain from Acetylcholine Receptors from an Early Stage of Neuromuscular Junction Maturation in Rats

Abstract

Spatial segregation of membrane proteins is a feature of many excitable cells. In skeletal muscle, clusters of acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na V 1s) occupy distinct domains at the neuromuscular junction (NMJ). We used quantitative immunolabeling of developing rat soleus muscles to study the mechanism of ion channel segregation and Na V 1 clustering at NMJs. When Na V 1s can first be detected, at birth, they already occupy a postsynaptic domain that is distinct from that occupied by AChRs. At this time, Na V 1s are expressed only in a diffuse area that extends 50 -100 m from the immature NMJ. However, in the region of the high-density AChR cluster at NMJ itself, Na V 1s are actually present in lower density than in the immediately surrounding membrane. These distinctive features of the Na V 1 distribution at birth are closely correlated with the distribution of ankyrinG immunolabeling. This suggests that an interaction with ankyrinG plays a role in the initial segregation of Na V 1s from AChRs. Both Na V 1 and ankyrinG become clustered at the NMJ itself 1-2 weeks after birth, coincident with the formation of postsynaptic folds. Syntrophin immunolabeling codistributes with AChRs and never resembles that for Na V 1 or ankyrinG. Therefore, syntrophin is unlikely to play an important part in the initial accumulation of Na V 1 at the NMJ. These findings suggest that the segregation of Na V 1 from AChRs begins early in NMJ formation and occurs as a result of the physical exclusion of Na V 1 and ankyrinG from the region of nerve-muscle contact rather than by a process of active clustering.

A system for studying mechanisms of neuromuscular junction development and maintenance

“…We found that ankyrin G was clustered in close proximity to the NMJ and was found at the muscle membrane but not actually at the NMJ (Fig. 6C), suggesting that at this time of development of the system, the protein localization has not reached a fully mature status as previously reported in vivo (Bailey et al, 2003), further supporting this method for the study of different phases of NMJ maturation.…”

“…The following antibodies were used: mouse anti-βIII tubulin (R&D systems, MAB1195, clone #TuJ1, 1/400), rabbit anti-ryanodine receptor (Braubach et al, 2014) (Millipore, AB9078, 1/200), goat anti-VaChT (Atasoy et al, 2014) (Millipore, ABN100, 1/100), goat anti-ChAT (Sümbül et al, 2014) (Millipore, AB144, 1/100), chicken anti-neurofilament H (NFH) (Wainger et al, 2015) (Millipore, AB5539, 1/400), mouse anti-dihydropyridine receptor (DHPR) (Bradley et al, 2014) (Abcam, Ab2864, 1/400), mouse anti-synaptotagmin (Wong et al, 2014) (Abcam, ab13259 clone ASV30, 1/100), mouse anti-α-actinin (Falcone et al, 2014) (Sigma-Aldrich, A5044 clone BM-75.2, 1/500), mouse anti-sodium channel pan (Bailey et al, 2003) (Sigma-Aldrich, clone K58/35, S8809, 1/100), mouse anti-ankyrin (Bailey et al, 2003) (Thermo Scientific, 33-8800, clone 4G3F8, 1/100), mouse antibassoon (Jing et al, 2013) (Abcam, ab82958, 1/100), rabbit anti-glial fibrillary acidic protein (GFAP) (Achtstätter et al, 1986) (Dako, Z0334, 1/100), mouse anti-oligodendrocytic marker O4 (Paintlia et al, 2004) (Sigma-Aldrich, O7139, clone O4, 1/400), rabbit anti-MuSK (serum T194, gift from Markus Ruegg, Biozentrum, University of Basel, Switzerland, 1/500), mouse anti-rapsyn [Abcam, ab11423 (1234), 1/200], mouse antiSyne1 (clone 8c3, gift from Glenn Morris, Keele University, UK, 1/200).…”

Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes

“…Ankyrin-G may also be required for targeting of Na v 1.1 to specialized sites in neurons and of Na v 1.4 to the neuromuscular junction in skeletal muscle. It is of interest in this regard that ankyrin-G is located at the neuromuscular junction in the postsynaptic domain enriched in Na v channels (16,19,20).…”

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner

“…This interaction maintains the polarized distribution of Na + /K + ATPase in epithelial cells (Nelson and Veshnock, 1987). At developing rat NMJs, voltage-gated sodium channels (Na v 1) and ankyrinG occupy a distinct postsynaptic domain from the AchR clusters (Bailey et al, 2003). At the C. elegans NMJ, the sodium channel could be replaced by Na + /K + ATPase, because there is no candidate gene corresponding to a voltage-gated sodium channel in the C. elegans genome.…”