Alterations of δ-opioid receptor mobility and function by acute and chronic depletion of plasma membrane cholesterol content of live cells

Grant Agency
Grant Agency of the Czech Republic
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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.

Mgr. Sýkora Jan Ph.D.

+420 26605 3745