This research will develop a method for tracking the cholesterol content of cell plasma membranes in real time, thus providing needed mechanistic information that cannot be obtained using traditional protocols. Specifically, the process by which, the integral membrane protein, ABCA1, mediates cholesterol efflux from macrophages (cells that become loaded with cholesterol in atherosclerosis) will be characterized. Current literature indicates that a method for evaluating the cholesterol content of the plasma membrane as a function of ABCA1-mediated cholesterol efflux is needed. The primary aim of this proposal is the further development and implementation of microelectrodes capable of oxidizing cholesterol contained in the cell plasma membrane. Platinum microelectrodes are modified with a lipid bilayer membrane containing cholesterol oxidase, an enzyme that oxidizes cholesterol. The electrode-supported lipid bilayer membrane provides two vital functions; it immobilizes the enzyme on the electrode surface in a near native environment, and extracts cholesterol from the cell plasma membrane. The platinum microelectrodes are recessed a few micrometers in the tip of a glass capillary (cavity microelectrodes) to control the distance between the cell plasma membrane and the electrode surface. Initial single cell experiments show that the enzyme-modified microelectrodes can be used to track the cholesterol content of the cell plasma membrane. Experiments are proposed to track the cholesterol content of the macrophage plasma membrane in real time during ABCA1- mediated cholesterol efflux. Experiments aimed at detecting exocytosis of HDL are also proposed. The data will be used to evaluate the validity of two proposed mechanisms that have been put forth to model the function of ABCA1 in the removal of cellular cholesterol.