The goal is to determine the mechanisms regulating axon terminal excitability and correlative transmitter release. Attention will focus primarily upon the role of ATP, which is released pre- and postsynaptically during motor activity. Using high-resolution techniques, the relative contributions form pre- and postsynaptic sources in rat will be assayed. Moreover, ATP liberation will be studied in innervated and denervated muscle to ascertain the role of nerve stimulation on ATP release. Elevated levels of extracellular ATP cause quantal content to decrease. It is unknown, however, whether exogenous ATP is required for normal synaptic transmission. Therefore, terminal excitability, transmitter release, and single-channel conductances will be assayed using """"""""loose"""""""" and """"""""gigaseal"""""""" patch-clamp techniques in the presence of varying concentrations of ATP and its nonhydrolyzable analogs and following depletion of extracellular ATP by apyrase. Moreover, excitability will be tested in the absence of transmitter release to determine whether ATP derived from pre- or postsynaptic sources affects conduction. In crayfish nerve, low (o.5 mM) concentrations of ATP reduce presynaptic Ca2+ uptake during nerve stimulation, while high concentrations (5 mM) cause a massive increase in Ca2+ influx. The specificity of these effects will be assayed by measuring 45Ca accumulation radiochemically and Ca2+ currents directly using """"""""loose""""""""-patch recording techniques in the presence of different ATP concentration. Protein kinase activity which requires extracellular ATP has been identified on the external surface of a hybrid cell line of neuronal origin. Using gel electrophoresis and autoradiography, the possibility of similar ectokinase activity will be explored at denerrvated, innervated, and neonatal rat neuromuscular junctions. Exogenous ATP is also incorporated into the presynaptic metabolic pool. A major aim is to determine the route. Three likely possibilities will be examined: endocytosis during synaptic vesicle recycling, transport through anion channel pathways, and active transport. These studies should elucidate the source and the functional role of exogenous ATP at the neuromuscular junction.
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