The goal of the proposed research is to understand how receptors for neurotransmitter function, and how cells control receptor function. Acetylcholine receptors or mammalian clonal muscle cells in vitro will be studied by biophysical methods, in particular patch clamp, to generate a kinetic scheme which describes receptor activation. The data will be analysed to obtain rate constants for steps in that scheme. The importance of membrane/receptor interactions will be examined by growing the cells in conditions which alter membrane lipid composition. The function of receptors will be analysed and any alterations interpreted in terms of the kinetic scheme for receptor activation. Further experiments will examine the effects of chemical treatments on receptor function, to gain insight into the structure-activity relationship for the acetylcholine receptor. The observation that the function of receptors on these cells changes with time in culture will be exploited to examine the mechanisms by which cells control the function of the receptors which they express. Additional experiments will analyse the function of acetylcholine receptors on developing and adult mammalian skeletal muscle fibers. The data will be used to determine the number of functionally distinct classes of acetycholine receptors expressed by muscle fibers in different conditions. The data will be analysed to determine the appropriate kinetic scheme to describe receptor activation and estimates for rate constants in that scheme will be obtained. The information provided will give insight into the choices the cell has in changing the function of its receptors and the mechanism it uses to control receptor function. The results of these studies are required to form a quantitative picture of the function of receptors for acetylcholine and to determine how the cell controls the function of receptors. Such a picture is necessary to understand the mechanism of synaptic transmission at the neuromuscular junction and to uderstand the mode of action of pharmacological agents. It is also required before changes in synaptic function during development or after pathological processes can be evaluated. In more general terms, the insight gained are applicable to other chemically transmitting synapses and to the overall question of the control of the ionic permeability of biological membranes.
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