How do CHRND mutations disrupt AChR cluster formation? (360G-Wellcome-206866_Z_17_Z)

The project aims to utilise mutations identified in congenital myasthenic syndromes to study the interactions of the muscle acetylcholine receptor (AChR) and its anchoring protein, Rapsyn.Widely quoted publications suggest that Rapsyn interacts with the M3-M4 intracellular loop of the AChR alpha, beta and epsilon subunits, however we have identified several kinships in which mutations in the M3-M4 intracellular loop of the AChR delta subunit underlie a phenotype that mimics myasthenic syndromes caused by mutations in RAPSN. The project will use in vitro mutagenesis and cell culture experiments to investigate how mutations in CHRND impair agrin induced-AChR clustering in C2C12 myotubes. Variants identified in CMS patients, ie. p.(Glu381Lys) or p.(Arg376His) will be incorporated into expression constructs and transfected in chrnd-/- C2C12 cell that have been created using CRIPR/Cas9 techniques. Similar experiments will be performed following in vitro mutatagenesis that is designed to disrupt potential PKA, PKC and tyrosine kinase phosphorylation sites within the M3-M4 intracellular loop of the delta subunit. The effects of the mutations on agrin-induced AChR cluster formation will be assessed by using fluorecent-labelled alpha bungarotoxin and microscopy. This short project will provide novel data on how mutations within the AChR itself can impair the cluster formation.