Nitric oxide synthase (NOS-1) coclustered with agrin-induced AChR-specializations on cultured skeletal myotubes

Previously we reported that neuronal nitric oxide synthase type-1 (NOS-1) i s expressed in skeletal myotubes in vitro. In the present paper we sought t o determine whether agrin-induced membrane specializations known to include the nicotinic acetylcholine receptor (AChR) on cultured myotubes may also contain NOS-1 and related molecules. After treatment with various agrin con structs containing the full C-terminally AChR-clustering domain (fragments N2, N4), but not with fragment C2 (truncated), NOS-1 expressed in the cytos ol of mouse C2C12 skeletal myotubes coclustered with AChR, 43K rapsyn, MuSK , and the dystrophin/utrophin glycoprotein-complex (DUGC), Agrin-induced sp ecializations also included coaggregates of N-methyl-D-aspartic acid (NMDA) -receptor, alpha-sodium (NaCh), or Shaker-type K+ channel (KCh)/PSD-95 comp lexes, and NOS-1. We conclude that agrin is crucial for recruitment of prea ssembled multimolecular membrane clusters, including AChR, NMDAR, and ion c hannels linked to NOS-1. Coassembly of NOS-1 to postsynaptic molecules may reflect site-specific NO-signaling pathways in neuromuscular junction forma tion and functions.