Coated and smooth vesicles participate in acetylcholine receptor transport

The removal of the acetylcholine receptors (AChRs) from the surface of muscle cells serves as an important mechanism in the regulation of the AChR turnover rate. Our previous studies have shown that cultured myotubes contain coated pits and vesicles bearing alpha-bungarotoxin (alpha BTX)-binding sites (Bursztajn 1984; Bursztajn and Fischbach 1984). In this study we have used alpha BTX conjugated to horseradish peroxidase (HRP) and quantitative electron microscopy to determine the intracellular pathway(s) of acetylcholine receptors during the internalization process. To accomplish this, cultured rat myotubes were incubated with alpha BTX-HRP at 4 degrees C after which cells were washed and incubated at 37 degrees C for 0 min to 2 h. After warming the cells, coated pits, coated vesicles and smooth membraned vesicles containing the peroxidase reaction product were present. A threefold increase in coated vesicles containing the reaction product was observed 1 min after warming the cells. The number of smooth-membraned vesicles remained constant at this time point. However, 5 to 15 min after warming the cells, a fivefold increase in the number of smooth membraned vesicles was observed. After 1 h at 37 degrees C the reaction product was present in the lysosomal like bodies, but was not observed in the Golgi complex or the small coated vesicles associated with the Golgi complex. Our observations indicate that there is a size segregation between those coated vesicles containing alpha BTX-HRP reaction product and those in which reaction product is absent. Our studies also suggest that within minutes of AChR internalization coated vesicles lose their coat and become smooth-membraned vesicles.