The long-term goal is to understand regulation of membrane transporters involved in volume regulation. The first specific aim is to study the signal transduction pathways of transporters involved in regulation of cell volume and transepithelial fluid transport, i.e. the mechanisms by which signals of change in cell volume lead to changes in transport. The transporters are K-Cl, Na-K-Cl, and Na-HCO3 cotransport, and Na/H exchange. First, changes will be determined in steady state kinetics following volume changes. These results will provide indications about transduction pathways, and will guide the next experiments, pre-steady state kinetics. These experiments can reveal if changes in transport are due to stimulation of a forward reaction, inhibition of a reverse reaction, or both. The next step, the combined use of kinetics and pharmacological agents, will provide tentative identification of the enzymes (e.g. kinases or phosphatases) promoting the reactions revealed by kinetics. To confirm the identification of the enzymes, exogenous enzymes will be incorporated into resealed ghosts made from dog or human red cells with the candidate enzyme inhibited. Reconstitution of function will confirm a role for the enzyme. These approaches led to a detailed model for regulation of K-Cl cotransport in sheep red cells. The same approach will advance knowledge of the regulation of Na-K-Cl and Na/H exchange in red cells. K-Cl cotransport is activated by a protein phosphatase. Na-K-Cl and Na/H exchange in some systems are activated by a kinase. Experiments in red cells will show whether the kinase or an inactivating phosphatase is volume-sensitive. Protocols are proposed for studying regulation of the kinases and phosphatases by incorporating various enzymes and antibodies into ghosts. Na-HC03 cotransporter, important in proximal tubule function, was recently found in red cells. Fusion of kidney vesicles with red cells delivers kidney Na-HCO3 cotransport into red cells, where the regulation of both transporters will be studied. The hypothesis will be tested that, during maturation of red cells, a decline in phosphatase activity controlling K-Cl cotransport determines the cells' mature volume. If correct, it may that the abnormally low volume of sickle cells is due to abnormal regulation of the phosphatase, perhaps due to interaction of the mutant hemoglobin with the membrane. The second specific aim is to isolate the phosphatase and kinase which regulate K-Cl cotransport in sheep red cells. The strategies for isolating both enzymes are based on their association with the membrane. The phosphatase appears permanently bound. Cell shrinkage appears to activate the kinase by promoting its binding to the membrane. The isolated enzymes will tested for modulation of K-Cl cotransport in ghosts. Antibodies made against the enzymes will permit determination of their class and characterization of their subunits. The third specific aim is to determine the structure of the K-Cl cotranspofler. Strategies are proposed to obtain full length cDNA based on the functional similarity between K-Cl, Na-K-Cl, and Na-Cl cotransporters and the known structures of the latter two. Expression cloning may also be tried in frog eggs. Structurefunction studies are proposed on the K-Cl cotransporter stably expressed in human embryonic kidney cells.
NIH ExPorter
NIH ExPorter