Development Of Mini-assays For High Throughput Screening Of Aquaporins Inhibitors

The aquaporins (AQPs) are a family of water-transporting proteins with 13 homologous members in mammals. The AQP family members have 25%-60% homology in protein sequence, and structurally have a general homotetrameric assembly in cell membranes. Members of the mammalian AQP family are expressed diversely in fluid-transporting epithelium, endothelium of various organs, and in other tissues such as skin, white blood cells and fat cells. The research on gene knockout mouse and human gene mutant has showed that AQP exerted important role in physiology and pathology fluid transport of various tissue. AQP are involved in not only various forms of pathological water retention, but also in exocrine disorders, brain edema, glaucoma, and cancer. Discovering AQP inhibitor may provide new way for therapy many diseases involving in fluid metabolism abnormal.Cl- is the main anion in the cell. The instant change of the osmotic pressure of cell evokes the instant change of the Cl- concentration. The capacity of the cell membrane water permeability determine the speed of Cl- concentration change. YFP-H148Q-V163S is a GFP mutant which is sensitive to Cl-. Cerrtain Cl- concentration can quenching the fluorescence of it. So YFP-H148Q-V163S can be indicator of the capacity of the cell membrane water permeability.We set up the cell models of CHO-V163S,FRT-V163S and SMMC-7221-V163S successfully through stable transfection. Observed through the fluorescence microscope and analyzed the cell fluorescence using FluoStar Optima, we confirm that GFP expressed highly, which is suitable as a reporter protein. Also we set up CHO-V163S-AQP4,FRT-V163S-AQP1 on the foundation of CHO-V163S and FRT-V163S successfully. Through the determination of FluoStar Optima and immunofluorescence, we found that the cell membrane water permeability is very high and the expression of the protein AQP4 and AQP1 is stable.When measuring the water permeability, people used to make the cell in the circumstance of hypotonicity. Our research has contrast the other method to make the cell in the circumstance of hyperosmotic using FluoStar Optima in CHO-V163S-AQP4,FRT-V163S-AQP1 and SMMC-7221-V163S. We has confirmed that the latter is more sensitive and accurate than the former in the process of high throughput screening AQP inhibitor drug on the first time.