Previous studies from this laboratory have shown that the Na-K-Cl cotransporter of vascular endothelial cells plays an essential role in regulation of intracellular volume and may also function in vectorial transport of Na and Cl across the blood-brain barrier. Recent studies have shown the existence of a family of cation-chloride cotransporters which includes the secretory and absorptive epithelial Na-K-Cl cotransporter isoforms (NKCC1 and NKCC2. respectively). In order to clarify the function and regulation of endothelial cotransporters at a molecular level, we cloned Na-K-Cl cotransporter cDNA from bovine aortic endothelial (BAE) cells. A 1.0 kb fragment of human NKCC1 DNA encoding the transmembrane region was used to screen a BAE cDNA library. Two overlapping clones spanning the entire coding region were identified and sequenced. The BAE cotransporter amino acid sequence was 86% identical to shark and human NKCC1, particularly in the transmembrane domains although significant variations were found at the Nterminus and in transmembrane segment 2. A survey of bovine tissue RNA using a BAE cotransporter cDNA probe revealed high expression of the cotransporter in brain, colon, stomach, and kidney. A BAE cDNA fragment was cloned into an expression vector and was stably integrated into human embryonic kidney (HEK) cells to evaluate functional expression. Several lines exhibited markedly elevated cotransport activity compared to nontransfected HEK cells. Clones were also evaluated for Na, K. and Cl affinities and bumetanide sensitivity. These functional assays demonstrate that the cDNA cloned is indeed a member of the Na-K-Cl cotransporter family and is highly homologous with NKCC1.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology