Our long-term objectives are to further define the role of the nicotinic acetylcholine receptor (AChR) in synaptic transmission, synaptogenesis, and various motor neurone diseases. Our approach is to molecularly dissect the neuromuscular junction (NMJ) and to establish an artificial system in which individual components of the NMJ can be precisely defined and manipulated. We have recently achieved our goal of establishing such a system (stable expression of cloned Torpedo californica AChRs in mouse fibroblast cells). Not only is this the first stable expression of a receptor/channel, but it is the first stable expression of a protein composed of four different subunits. These subunits are synthesized, assembled into the correct pentameric complex, and the complex is inserted into the plasma membrane and functional. With such a system, three powerful approaches can be employed that will help us to define the different properties of the AChR and to define the role of the AChR in different processs and diseases. 1) By expressing the AChR in a foreign environment, we have isolated it away from other muscle specific gene products, thus allowing the specific properties of the AChR to be determined. 2) By introducing the AChR into foreign cells via cloned cDNAs, the AChR can be manipulated at the DNA level using site-directed mutagenesis techniques. 3) By expressing the AChR in non-muscle and non-neuronal cell types, components of the NMJ can be added to the system in a controlled fashion such that their effect on the AChR can be determined. In this grant application, we are proposing to undertake three major projects. Project #1 will be a thorough physiological characterization of Torpedo AChRs in mouse fibroblast cells. Our system has provided us with the unique opportunity for performing physiological studies of this AChR that have not been possible previously. Once characterized, we will then add to the system (and thereby study the effects of) circulating neurotoxic factors in sera from patients with motor neurone diseases, neurotrophic viruses, antibodies, and toxins. Project #2 will attempt to elucidate the molecular mechanism of AChR clustering on cell surfaces. Agents will be added to our cell lines in order to induce cluster formation. Using site-directed mutagenesis techniques, specific deletions of cytoplasmic domains of each of the receptor subunits will be made to further define the role of each subunit in the clusteing process. If necessary, other proteins thought to play a role in clustering will be added to the system. Project #3 will study the processes of AChR subunit biosynthesis and assembly. Some of the questions we will address include: are there specific subunit-subunit interactions, where are the subunits assembled and degraded, do the subunits interact with heavy chain binding protein, what role do the oligosaccharides play in AChR assembly and AChR function?
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