The plasma membrane is the largest subcellular cholesterol pool and complex trafficking pathways exist within cells to maintain this distribution. 1 Common methodologies for cellular cholesterol analysis such as fluorescent labeling do not yield precise determination of the physiological cholesterol content of the plasma membrane (ca. 800 mM) relative to that of internal compartments. Furthermore, the isolation of subcellular compartments is prone to contamination, and cholesterol may get transferred between compartments during separation. 2 This lack of spatial resolution in traditional cholesterol analysis of subcellular compartments has led to questions regarding how plasma membrane cholesterol is affected under conditions of altered intracellular cholesterol transport. This Communication reports microelectrodes that allow direct real time measurements of plasma membrane cholesterol content in live mammalian cells at physiological temperature. The measurements are rapid and localized and thus do not significantly perturb the total amount of cholesterol in the plasma membrane.A state-or-the-art assay to estimate plasma membrane cholesterol is the method developed by Rothblat and co-workers where cellular radio-labeled cholesterol is removed by a cyclodextrin solution. 3 Although there is a question as to what cellular cholesterol pools are removed, Rothblat has reported that the initial rate of cellular cholesterol efflux (within ca. 15 s) can be assigned to that from the plasma membrane. Variations of this method have been implemented without regard to this stipulation. Estimates of plasma membrane cholesterol have also been obtained by exposure of cells to cholesterol oxidase solution. A concern with this method is that the enzyme may gain access to internal cholesterol through endocytosis, membrane breakage, and movement of cholesterol during the assay. 2 Our direct electrochemical measurements of plasma membrane cholesterol are localized to the cell surface and require only about 5-10 s thus addressing these caveats.We recently reported electrochemical detection of cholesterol in the lipid bilayer membrane of a giant vesicle 4 and at the surface of a single oocyte 5 at room temperature using a Pt microelectrode (10 µm diameter) modified with a lipid bilayer membrane containing cholesterol oxidase. This surface architecture is not stable at 37°C. Here we report direct covalent attachment of cholesterol oxidase to Pt microelectrodes (4 µm diameter) to achieve a surface structure that is stable at physiological temperatures (Supporting Information). 6 Placing the electrode surface in contact with the plasma membrane ( Figure 1a) results in enzymatic oxidation of cholesterol at the cell surface. The plateau current increase observed in the first several seconds upon positioning the electrode in contact with the cell surface (for example, Figure 1b) is believed to be limited by the rate of enzymatic catalysis and is proposed to be a measure of plasma membrane cholesterol content. Control experiments using bare P...
Platinum microelectrodes are modified with a lipid bilayer membrane incorporating cholesterol oxidase. Details for electrode surface modification are presented along with characterization studies of electrode response to cholesterol solution and to cholesterol contained in the lipid bilayer membrane of vesicles. Ferrocyanide voltammetric experiments are used to track deposition of a submonolayer of a thiol-functionalized lipid on the platinum electrode surface, vesicle fusion for bilayer formation on the thiolipid-modified surface, and incorporation of cholesterol oxidase in the electrode-supported thiolipid/lipid bilayer membrane. The data are consistent with formation of a lipid bilayer structure on the electrode surface that contains defects. Experiments for detection of cholesterol solubilized in cyclodextrin solution show steady-state current responses that correlate with cholesterol concentration. Direct contact between the electrode and a vesicle lipid bilayer membrane shows a response that correlates with vesicle membrane cholesterol content.
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