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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK047661-04
Application #
2016777
Study Section
Physiology Study Section (PHY)
Project Start
1994-01-01
Project End
1998-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Pedersen, Meike; Carmosino, Monica; Forbush, Biff (2008) Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume. J Biol Chem 283:2663-74
Scott, Graham R; Richards, Jeff G; Forbush, Biff et al. (2004) Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am J Physiol Cell Physiol 287:C300-9
Schomberg, Stacey L; Bauer, James; Kintner, Douglas B et al. (2003) Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-. J Neurophysiol 89:159-67
Flemmer, Andreas W; Gimenez, Ignacio; Dowd, Brian F X et al. (2002) Activation of the Na-K-Cl cotransporter NKCC1 detected with a phospho-specific antibody. J Biol Chem 277:37551-8
Darman, Rachel B; Forbush, Biff (2002) A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277:37542-50
Payne, J A; Ferrell, C; Chung, C Y (2001) Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line. Am J Physiol Cell Physiol 280:C1607-15
Darman, R B; Flemmer, A; Forbush, B (2001) Modulation of ion transport by direct targeting of protein phosphatase type 1 to the Na-K-Cl cotransporter. J Biol Chem 276:34359-62
Sangan, P; Brill, S R; Sangan, S et al. (2000) Basolateral K-Cl cotransporter regulates colonic potassium absorption in potassium depletion. J Biol Chem 275:30813-6
Haas, M; Forbush 3rd, B (2000) The Na-K-Cl cotransporter of secretory epithelia. Annu Rev Physiol 62:515-34
Jacoby, S C; Gagnon, E; Caron, L et al. (1999) Inhibition of Na(+)-K(+)-2Cl(-) cotransport by mercury. Am J Physiol 277:C684-92

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