The long term goals of this proposal are to understand the mechanism, regulation and physiological importance of the bumetanide-sensitive ion cotransport system. In the differentiated kidney epithelial cell line, MDCK, under carefully controlled conditions the bumetanide- (and furosemide sensitive system catalyzed the tightly coupled cotransport of 1 Na plus, 1 K plus and 2 Cl minus. In a wide variety of tissues, this transport system has been implicated in the regulation of cell volume. In many epithelia, bumetanide-sensitive transport also functions in chloride reabsorbtion. The MDCK cell line was many advantages as a system with which to study ion transport, including the availability of three independent mutants defective in the bumetanide-sensitive transport system. One of these mutants has less than 10 percent of the normal levels of bumetanide-sensitive Na plus and K plus fluxes, while another mutant has lost 50 percent of the K plus flux activity but retained nearly 100 percent of the Na+ flux activity. The third mutant has multiple defects and is likely to have a regulatory mutation. The role of this transport system in transepithelial transport and cellular volume regulation will be investigated using these three mutants. In addition, the relationship between cellular volume regulation and growth regulation will be examined. The role of a variety of hormones and other agents in regulating transport will also be investigated. Concurrently, the number and location of the bumetanide binding sites in polarized cellular monolayers will be determined using radioactive bumetanide. Photoaffinity derivatives of bumetanide will be synthesized and tested for specific labeling of the polypeptide component(s) of the transport system in the parental and mutant cell lines. Plasma membrane vesicles will be utilized to corroborate and extend the observations made in whole cells. Attention will be directed at detecting uncoupled NaCl and KCl transport and at determining the role of intracellular ATP in supporting bumetanide-sensitive transport.
