The long term objective of these studies is to understand molecular events at the neuromuscular junction (nmj). Specifically these studies will investigate the regulation of acetylcholine receptor (ACHR) turnover (degradation and synthesis) and redistribution (clustering), and the role played by the nerve in that regulation. In addition, studies will be directed towards the functional consequences of the unique molecular organization and geometry of the nmj. Tissue and organ culture procedures will provide controlled environments for the study of ACHR regulation and the results compared with those obtained in vivo. Data will be evaluated using morphological and molecular techniques. These win include autoradiography (on the light microscope, TEM and SEM levels) Northern analysis, in situ hybridization and protein purification. Information on the time course of miniature endplate currents (obtained by voltage clamp), and values for AChR and AChE site densities (obtained from quantitative EM autoradiogaphy), will provide the experimental base for studies on neuromuscular function. A newly devised Monte Carlo procedure will then model the kinetics of diffusion and binding, of individual ACh molecules in the clefts of the intact neuromuscular junction. These studies should provide new insights on the functioning of the neuromuscular junction in normal animals, and in animals whose junctional geometry or molecular organization is altered as in diseases such as Myasthenia or Muscular Dystrophy.
