The high densities of receptors found in synapses are crucial for effective transmission between a nerve and its target cell. At the neuromuscular junctions, acetylcholine is the transmitter substance and its receptor, the acetylcholine receptor (AChR) is found at very high concentrations in the endplate on the postsynaptic membrane. A large array of molecules is necessary to form the clusters of AChR during development and to keep up the highly structured arrangement of the endplate. One of these molecules is rapsyn. Rapsyn is a peripheral membrane protein. It has binding sites for several of the proteins in the endplate and is closely associated with the AChR. Without this protein’s presence no endplate structures form. Rapsyn can also induce clustering of AChR in several non-muscle cell lines when cotransfected with the subunits of the receptor.
In this project a plasmid containing rapsyn tagged with the enhanced green fluorescent protein (EGFP) was electroporated into the extensor digitorum longu (edl) muscle of mice. The resultant rapsyn-EGFP chimera localises to the endplate in exchange for and in addition to, wild type rapsyn. With the argon laser of a confocal microscope a portion of the rapsyn-EGFP in the endplates was photobleached. The subsequent fluorescent recovery was studied using a fluorescent microscope with a SIT camera. The hypothesis was that the recovery of rapsyn-EGFP would mimic the recovery seen when bleaching labelled AChR in the endplate consistent with the idea that rapsyn forms a complex with AChR already before clustering
Contrary to our expectations, we found that rapsyn-EGFP had a recovery rate, after photobleaching, which was about 14-68-fold higher than that established for the AChR. There seemed to be two pools of rapsyn-EGFP in the endplates with a fast recovering pool constituting about 60-80% of the endplate population and a non-recovered portion consisting of the remaining 20-40% of the endplate population. The recovering pool had a higher turnover and/or mobility than the receptor leading us to suppose that such molecules are either not associated with receptors, or associated with receptors for only shorter periods of time. Furthermore, it is possible to suppose that the non-recovered pool act as complexes with receptors throughout their lifetime, or these might also associate and disassociate with receptors.