The Na-K-Cl cotransporter is a plasma membrane protein that plays a vital role in cellular and systemic electrolyte homeostasis. In non-polarized cells it is involved in regulation of cell volume and possibly of local extracellular potassium concentration; in transporting epithelia, it is a key element in balancing the transcellular flow C1. In secretory epithelia, the cotransporter functions in concert with C1 channels (CFTR), the Na pump, and K channels to bring about regulated salt movement; in the mammalian kidney another isoform of the transporter mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. Previous work from this laboratory has demonstrated that the Na-K-C1 cotransporter is a glycosylated membrane protein 150 to 195 kDa in size, depending on tissue and species. The goal of this project is to understand the molecular mechanism of the cotransporter including the structural and functional features underlying ion translocation and its regulation, as well as the role of the transporter in various tissues. The proposed studies will be carried out using mammalian cell lines with protein expressed from cDNA's encoding the secretory form of the Na-K-C1 cotransporter, as well as with membranes isolated from a salt-secreting gland of the shark, and with rabbit tissues. The experiments will be greatly aided by probes and technique that are available from the recent work of this laboratory. The Specific Aims of the project are: 1) The membrane topology of the Na- K-C1 cotransport protein will be determined by identifying the sequence location and membrane sidedness of proteolytic cleavage sites, antibody epitopes, glycosylated residue(s), and specific regions labeled by the insertion of epitope tags. 2) Structure/function relationships in ion translocation and bumetanide binding will be determined by a combined approach utilizing photoaffinity labeling and mutagenesis strategies; the hypothesis that transport is mediated by a single polypeptide will also be tested. 3) The mechanism by which the Na-K-C1 cotransporter is activated by intracellular stimuli will be addressed, with particular attention to a role of intracellular [C1] in modulation of the level of protein phosphorylation. Phosphorylated residues of the cotransporter will be located, and the kinase involved will be identified; these studies will also address the question as to whether intracellular ions affect phosphorylation status by binding to the kinase, to a phosphatase, to a modulatory site on the transporter, or to the transport sites. 4) The role of the Na-K-C1 cotransporter in gastrointestinal secretory epithelia, vascular endothelium, nerve, and muscle, will be examined using cDNA and antibody probes to determine cellular distribution of cotransporter isoforms.