The transport of acetylcholine receptors (AChR) will be studied in vitro using cultured skeletal muscle. The appearance of AChR cluster; will be observed with Alpha-bungarotoxin (Alpha-BTX) conjugated with the tetramethyl rhodamine (TMR). The distribution and topography of AChR clusters will be quantitated in mononucleated cells and myotubes. The density of AChR at define stages of cluster development will be determined by autoradiography. The surface topography of myotubes showing significant alteration at any given stage will be studied on the ultrastructural level. The surface and intracellular distribution of Alpha-BTX binding sites will be studied with Alpha-BTX conjugated to ferritin. Alpha-BTX binding sites will be quantitated according to the basic morphometric principles published by Weibel (64). Experiments are designed which will allow us to determine whether there are specific domains in the plasma membranes at which receptors cluster and whether new receptors are added at AChR clusters, and how these new receptors are inserted into the plasma membrane. The internal pool of AChR and its route of transport will be studied using immunocytochemistry and immunoelectron microscopy. The pathway of AChR internalization will be studied with respect to membrane specificity at which internalization occurs. The role of coated pits and coated vesicles in the transport of AChR with respect to membrane structure and cluster formation and internalization will be examined at clustered (as visualized by Alpha-BTX-ferritin) and at non-AChR clustered region of the plasma membranes. Our proposed studies may be of considerable value in understanding how the AChRs once synthesized are transported to the plasma membrane, aggregate, and are removed from the cell surface. Ultimately, these studies may not only elucidate the insertion-internalizatio pathway of AChR, but also shed some light on other surface molecules that are precisely expressed on the surface of the plasma membrane.
