Alterations of Î´-opioid receptor mobility and function by acute and chronic depletion of plasma membrane cholesterol content of live cells
17-05903S, Grant Agency of the Czech Republic (associated contractor - project leader is affiliated with another institution)
Detailed analysis of the effect of acute and chronic depletion of cell cholesterol level on Î´-opioid receptor mobility and function in plasma membrane of model cell lines. For this aim, HEK293 and NG108-15 cells transiently expressing Î´-OR-eCFP and Î´-OR-eYFP will be analyzed byÂ advanced techniques of fluorescence microscopy FRAP, RICS, FCS, TIRF and 2PPM. Subsequently, alterations of Î´-OR mobility will be correlated with alterations of agonist ligand binding, and function of Î´-OR measured by agonist-stimulated [35S]GTPÎłS binding. These experiments will be performed in HEK293 cells stably expressing FLAG-Î´-OR and NG108-15 cells. "Antibody feeding" method will be used to distinguish internalized and cell surface pools of Î´-OR. Functional studies of Î´-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 Î´-OR in plasma membrane of live cells and its functional significance.
Mgr. SĂťKORA Jan, Ph. D.
Department: Department of Biophysical Chemistry
Phone: (+420) 26605 3745
Fax: (+420) 286582307