The overall goals of the proposed research are to understand how transmitter-gated membrane channels function and the mechanisms by which cells modulate the functional properties of these receptors. To reach these goals, it is necessary to determine the steps and rates in receptor activation, to determine the rates which are altered in cases in which function is altered, and to determine the likely structural basis for the changes. Further, physiological studies must be performed on cells which may be experimentally manipulated and on which biochemical studies may be done. Recent developments in eukaryotic gene expression allow generation of clonal lines which produce receptors of known subunit composition. These lines are ideal for biophysical, pharmacological and biochemical studies. The proposed work focusses on nicotinic acetylcholine receptors (AChR) which are the best studied representatives of an extended gene family which includes several other transmitter-gated channels. Biophysical methods will be used to study AChR function, biochemical methods to examine structure, and molecular biological methods to generate clonal cell lines. Our previous work has generated clonal cell lines which express mammalian adult and fetal muscle AChR. These cells provide an accessible system in which these receptors may be directly compared. Critical functional properties of these receptors will be determined, including the affinity of the receptor for acetylcholine and the intrinsic channel opening rate. The pharmacology of neuromuscular blocking agents on adult and fetal muscle AChR will then be examined. The work will be extended to examine modulation of adult muscle AChR. Neurons express structurally related nicotinic receptors, but studies of the physiology and pharmacology of these receptors have been hampered by the lack of stable sources of cells which express receptors of defined subunit composition. Therefore, the project proposes to generate stable clonal lines which express neuronal AChR and to use these cells to determine the physiological and pharmacological properties of the receptors. Further work will examine mechanisms for modulation of these neuronal receptors. Finally, the basis for the modulation of neuronal nicotinic AChR expressed by clonal PC12 cells will be determined. This work will provide precise information on the function of nicotinic acetylcholine receptors and on the mechanisms by which a cell can alter the functional properties of these receptors. This knowledge is required to understand both synaptic transmission and the actions of pharmacological agents, in normal and disease states. The understanding will provide insight into the mechanisms of blocking agents and the etiology of disease states involving nicotinic AChR, which are thought to include nicotine dependence and possibly Alzheimer's disease.
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