Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes

The effects of calpain inhibitors on the total number of acetylcholine rece ptors (AChRs) on cultured rat myotubes and on the stability of AChR cluster s in these myotubes were investigated. The degradation rate of total AChRs labeled with I-125-alpha-bungarotoxin was assessed from radioactivity remai ning in the myotubes as a function of time, Treatment with calpain inhibito rs resulted in a two- to three-fold increase in the half-life of total AChR s. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation, The rate loss of AChR s from the clusters was assessed by measuring the loss of fluorescence inte nsity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatm ent with calpain inhibitors resulted in two-fold increases in cluster half- life. Thus, there was generally no change in total surface receptors with t he calpain inhibitors, whereas cluster half-life was substantially increase d. Furthermore, with a low dose of calpeptin there was no change in turnove r of total cellular AChRs, whereas cluster half-life was doubted, Taken tog ether, these results suggest that the increased half-life of clusters produ ced by the calpain inhibitors may be due to retardation of the lateral move ment from AChRs in the clusters. (C) 2000 John Wiley & Sons, Inc.*