Overexpression of nPKC ? is inhibitory for agrin-induced nicotinic acetylcholine receptor clustering in C2C12 myotubes

In mammalian skeletal muscle, neuronal-type nitric oxide synthase (nNOS) is found to be enriched at neuromuscular endplates. Here we demonstrate the colocalization of the nicotinic acetylcholine receptor (nAChR, stained with α-bungarotoxin) and nNOS (stained with a specific antibody) in murine C2C12 myotubes. However, coimmunoprecipitation experiments demonstrated no evidence for a direct protein-protein association between the nAChR and nNOS in C2C12myotubes. An antibody to the α1-subunit of the nAChR did not coprecipitate nNOS, and an nNOS-specific antibody did not precipitate the α1-subunit of the nAChR. Treatment of mice with bacterial LPS downregulated the expression of nNOS in skeletal muscle, and treatment of C2C12 cells with bacterial LPS and interferon-γ markedly decreased nNOS mRNA and protein expression. In contrast, mRNA and protein of the nAChR (α-, γ-, and ε-subunits) remained unchanged at the mRNA and protein levels. These data demonstrate that nNOS and the nAChR are colocalized in murine skeletal muscle and C2C12 cells but differ in their expressional regulation.

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The submembrane machinery for nicotinic acetylcholine receptor clustering.

The Journal of Cell Biology ◽

10.1083/jcb.114.1.1 ◽

1991 ◽

Vol 114 (1) ◽

pp. 1-7 ◽

Cited By ~ 108

Author(s):

S C Froehner

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Receptor Clustering ◽

Nicotinic Acetylcholine

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Caveolin-3 Promotes Nicotinic Acetylcholine Receptor Clustering and Regulates Neuromuscular Junction Activity

Molecular Biology of the Cell ◽

10.1091/mbc.e09-05-0381 ◽

2010 ◽

Vol 21 (2) ◽

pp. 302-310 ◽

Cited By ~ 28

Author(s):

Michael Hezel ◽

William C. de Groat ◽

Ferruccio Galbiati

Keyword(s):

Neuromuscular Junction ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Molecular Mechanisms ◽

Molecular Level ◽

Structural Protein ◽

Receptor Clustering ◽

Nicotinic Acetylcholine ◽

Functional Studies ◽

Muscle Specific Kinase

The molecular mechanisms that regulate the organization and activity of the neuromuscular junction remain to be fully identified. Caveolae are invaginations of the plasma membrane. Caveolin-3 is the structural protein component of caveolae in muscle cells. We show that caveolin-3 is expressed at the neuromuscular junction, that it associates with the nicotinic acetylcholine receptor (nAChR), and that a lack of caveolin-3 inhibits clustering of the nAChR in myotubes. At the molecular level, we demonstrate that caveolin-3 is a novel muscle-specific kinase (MuSK) binding protein and that altered nAChR clustering in caveolin-3–lacking myotubes results from inhibition of agrin-induced phosphorylation/activation of MuSK and activation of Rac-1. Functional studies in caveolin-3 null mice show abnormal neuromuscular junction activity that is consistent with altered nAChR localization at the sarcolemma. Together, these data identify caveolin-3 as a critical component of the signaling machinery that drives nicotinic acetylcholine receptor clustering and controls neuromuscular junction function.

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