The overall goal of this project is to determine the extent to which membrane dependent activities of neuronal cells are altered by changes in membrane lipid composition or chronic exposure to agonists for the neuronal nicotinic acetylcholine receptors. Changes in membrane lipid composition will be induced by physiologically-relevant manipulation of the availability of membrane precursors in the culture medium of clonal neuronal cells. Initial experiments have demonstrated that changes in the availability of unsaturated fatty acids causes changes in depolarization-dependent exocytosis, action potential generation and receptor binding characteristics. The specific aims related to membrane lipid modification will be: 1. study the effects of exogenous fatty acids on muscarinic receptors on both NG108-15 and PC12 cells, and alpha-adrenergic receptors on NG108-15 cells, 2. continue developing a fast and efficient method for isolation of purified plasma membrane fragments from the cells, 3. once plasma membranes have been purified, examine their lipid composition and the effects of unsaturated fatty acids on that composition, 4. begin studying the effects of exposure to exogenous fatty acids on transport systems for glucose and catecholamines, 5. begin looking at the effects of membrane lipid modification on the single channel properties of nicotinic acetyl-choline receptors on PC12 cells, voltage-sensitive Na+ channels in NG108-15 cells, and voltage sensitive Ca++ channels in both cell lines. Initial studies have shown that chronic exposure of PC12 cells to a nicotinic receptor agonist causes a time-and concentration-dependent functional down regulation of the subsequent responsiveness of the cells. The specific aims related to this project will be: 1. use various receptor antagonists and channel blocking drugs to determine if functional down regulation results from simple agonist occupancy or also requires ion flux through the open channel, 2. use the patch-clamp technique to determine if the individual channel properties of the receptors are the same or different after chronic receptor occupancy by an agonist, 3. study the effects of chronic membrane depolarization on the regulation of the acetylcholine receptor, 4. devise other schemes for determining the nature of the change in cells chronically exposed to agonists and the mechanisms for induction of these changes.
NIH ExPorter
NIH ExPorter