The long-term objective of this proposal is to understand the role of protein phosphorylation of the nicotinic acetylcholine receptor (nAChR) in modulating signal transduction at the neuromuscular junction. The nAChR mediates signal transduction at this model synapse by virtue of its dual functions as a neurotransmitter receptor and an ion channel. Protein phosphorylation plays a key role in regulating the function of neurotransmitter receptors and ion channels. The nAChR isolated from Torpedo electric organs and mammalian muscle has been shown to be phosphorylated by at least three different protein kinases. Phosphorylation of the nAChR has been demonstrated to increase the rate of receptor desensitization. Information regarding the functional significance of nAChR phosphorylation improves our understanding of mechanisms underlying the modulation of synaptic transmission. In general, questions pertaining to the modulation of synaptic transmission are central to an appreciation of nervous system function under both normal and pathological conditions.
The specific aims focus on characterizing the regulation of nAChR phosphorylation as a necessary preliminary step in realizing the long-term objective. The first specific aim is to characterize the regulation of nAChR phosphorylation by protein kinases. This includes an identification of the intercellular and intracellular signals that stimulate protein kinases to phosphorylate the nAChR. An investigation will be made into the interactions between nerve and muscle in regulating nAChR phosphorylation. The activity of protein kinases will be specifically inhibited as an alternative approach to characterizing nAChR phosphorylation. These studies will also entail a biochemical characterization of the specific sites of nAChR phosphorylation to determine the phosphorylation events that are functionally significant. The second specific aim is to characterize the regulation of nAChR dephosphorylation by protein phosphatases including their regulation by intercellular and intracellular signals and a biochemical characterization of the specific sites of nAChR dephosphorylation . The third specific aim is to correlate functional subpopulations of the nAChR with levels and specific sites of nAChR phosphorylation. A comparison will be made between newly synthesized and preexisting nAChRs and nAChRs with different functional distributions in the cell membrane. The experimental strategy that has been devised to study the regulation of nAChR phosphorylation in situ is to use rat myotubes in culture metabolically labeled with radioactive phosphate. Myotubes will be exposed to treatments which potentially affect the regulation of nAChR phosphorylation. Cocultures will be established between myotubes and a nerve-derived cell line. Muscle cell lines will be transfected with genes encoding inhibitory proteins for protein kinases. The nAChR will then be isolated and subjected to biochemical analysis. Functional subpopulations of the nAChR will be obtained by modifications of the isolation procedure that has been developed.
