This project will investigate the causes, molecular mechanisms, and effects of clustering of acetylcholine receptors on developing muscle membranes. Clustering of acetylcholine receptors is a central phenomenon in both the formation of neuromuscular junctions in an embryo and in the regeneration of muscular control after an injury. Therefore, the results of this investigation will have a bearing on understanding of genetic neuromuscular defects, neuromuscular disease, and recovery after an injury to the motor system. In addition, the optical techniques (mainly fluorescence) to be employed are rather novel and powerful and should find application in a wide range of problems in cell biology and membrane function. In particular, the effect of neurotrophic factors and physical contact on the rotational mobility of acetylcholine receptors will be examined on developing rat myotubes in culture. Endogenous clusters of receptors form on these myotubes in the absence of neural influence; the locations, time, and mode of their formation will be monitored. The molecular structure of endogenous receptor clusters will be studied by observaing the effect of specifically targeted treatments and drugs on rotational mobility and by examining the codistribution of these receptors with other particular cytoplasmic, membrane, or exocellular proteins. The self-aggregating properties of acetylcholine receptors will be examined in solution and in reconstituted membranes and then compared with the size of receptor microclusters that occur in all regions of the myotube membrane. Rotational mobility of receptors on embryonic myotubes in culture will be compared to that on developing and adult synapses of rat. Lastly, clustering of receptors may affect their function, in particular their chemical kinetic rates of binding to agonists. A new fluorescence technique for measuring chemical kinetics at equilibrium in biological systems will be applied to examine this possibility and to find correlations with the kinetics of ion channel opening and closing of acetylcholine receptors.
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