Agrin induces whereas acetylcholine (ACh) disperses ACh receptor (AChR) clusters during neuromuscular synaptogenesis.Such counteractive interaction leads to eventual dispersal of non-synaptic AChR rich-sites and formation of receptor clusters at the postjunctional membrane.However,the underlying mechanisms are not well understood.Here we show that calpain,a calcium-dependent protease,is activated by the cholinergic agonist carbachol (CCh) and is required for CCh-induced dispersion of AChR clusters.Interestingly,the AChR-associated protein rapsyn interacted with calpain,in an agrin dependent manner,and this interaction inhibited the protease activity of calpain.Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters.Moreover,the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via over-expressing calpastatin,an endogenous calpain inhibitor,or injecting calpeptin,a cell-permeable calpain inhibitor.These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters,and rapsyn stabilizes AChR clusters by suppressing calpain activity.