Three related transport systems and their regulation will be studied. The first, K-C1 cotransport, is of interest because of its role in regulation of cell volume. Swelling of cells activates K-C1 cotransport. There must be a signal of swelling, a sensor of the signal, transduction of the signal to the cotransporter, and changes in transport. None of these aspects of regulation of K-C1 cotransport is well understood. These will be studied in intact LK sheep red blood cells and inside-out vesicles (IOVs) made from these cells. In intact cells, studies of steady state kinetics of transport and time course of change in transport after volume changes will show the nature of changes in transport and provide clues about transduction. In particular these experiments will test the hypothesis that swelling stimulates transport by relief of effects of two endogenous inhibitors, one controlling Jmax and the other controlling affinity for K. K-C1 cotransport in IOVs is volume-sensitive, and provides a convenient model system for the study of regulation because the sensor and transducer are presumably on the cytoplasmic membrane surface. Experiments on IOVs win be guided in part by results from intact cells, and by the hypothesis that the cytoskeleton and its phosphate metabolism are crucial to regulation. It will be tested in IOVs if Mg is the endogenous inhibitor controlling Jmax, and if a mechanical signal from swelling relieves inhibition by Mg by reducing availability of Mg binding sites. The second system is the regulation of the Na/K pump in LK sheep red cells by an endogenous inhibitor, the Lp antigen. Anti-Lp antibody stimulates the pump by relieving this inhibition. Preliminary results suggested that insulin and insulin-like growth factor 1 stimulate the pump in sheep red cells. The hypothesis will be tested that the Lp antigen is a hormone receptor. The kinetic basis of stimulation by hormones will be determined and compared with that of anti-Lp. Attempts will be made to clone the gene for the Lp antigen using a purified anti-Lp to screen an expression library which has been made from LK erythroid precursor cells. The structure of Lp will be compared to that of hormone receptors. Attempts will be made to express Lp in Xenopus eggs. Anti-Lp will also be used to purify the Lp antigen. The hypothesis will be tested using pure antigen and NaK-ATPase from rat kidney that the antigen inhibits the pump by complexing with it, and that stimulation by antibody or hormones is by dissociation of the complex. The third transport system is Na-K-C1 cotransport, also involved in volume regulation. The primary goal is to determine the structure of proteins of the cotransporter by gene cloning. An antiserum to the cotransporter has been made and one protein has been cloned. Others will be and their expression in Xenopus eggs will be studied to confirm their function and to make tentative assignments of their functions. The K-C1 cotransporter may have sufficient structural similarity to the Na-K-C1 cotransporter that it can also be cloned using cDNAs for and anstiserum to the Na-K-C1 cotransporter and the LK library (made from tissue lacking Na-K-C1 cotransport). Expression of this transporter will also be attempted in Xenopus eggs.
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