The P-glycoprotein (Pgp) is a membrane protein expressed in the plasma membrane. It functions as an efflux pump which transports cytotoxic drugs out of the cell utilizing energy from ATP hydrolysis. Pgp is responsible for most cases of multidrug resistance (MDR) in tumors and thus limits the success of chemotherapy. Recently, Pgp has also been suggested to be involved in cell volume regulation as a cell-swelling activated chloride channel. How a single protein can have such different function is unknown. My recent studies have shown that at least two distinct topological structures of Pgp coexist in microsome membranes of a cell- free system. Therefore, it is possible that these two structures carry the two different Pgp functions. The general goals of this application are to define how (or if) the two different structures of Pgp relate to the two different functions and how this is regulated in mammalian cells. To this end, (a) Pgp molecules with a single topological structure will be created by genetic engineering, (b) the Cl--channel and drug-transport activities of these molecules will be investigated, and (c) site-directed polyclonal antibodies will be raised to analyze the membrane topology of Pgp expressed in cells. The information and probes obtained from this study will significantly enhance our understanding of the molecular basis of multidrug resistance and thus increase the effectiveness of cancer chemotherapy. Additionally, this study will increase our understanding of how specific protein topological structures are related to transport functions in general.
