Alterations of δ-opioid receptor mobility and function by acute and chronic depletion of plasma membrane cholesterol content of live cells
Detailed analysis of the effect of acute and chronic depletion of cell cholesterol level on δ-opioid receptor mobility and function in plasma membrane of model cell lines. For this aim, HEK293 and NG108-15 cells transiently expressing δ-OR-eCFP and δ-OR-eYFP will be analyzed by advanced techniques of fluorescence microscopy FRAP, RICS, FCS, TIRF and 2PPM. Subsequently, alterations of δ-OR mobility will be correlated with alterations of agonist ligand binding, and function of δ-OR measured by agonist-stimulated [35S]GTPγS binding. These experiments will be performed in HEK293 cells stably expressing FLAG-δ-OR and NG108-15 cells. "Antibody feeding" method will be used to distinguish internalized and cell surface pools of δ-OR. Functional studies of δ-OR will be performed in isolated plasma membranes and detergent-untreated, low-density membrane domains. Project will extend the insight into the toxic side-effects of statin-based therapies and bring new knowledge about correlation between lateral mobility of δ-OR in plasma membrane of live cells and its functional significance.