Molecular characterization of the Na-K-Cl cotransporter of bovine aortic endothelial cells

The Na-K-Cl cotransporter is an important regulator of endothelial cell volume and may also contribute to flux of Na and Cl across the endothelium of the blood-brain barrier. To date, two Na-K-Cl cotransport isoforms have been identified, the cotransporter in secretory epithelia, NKCC1, and that in absorptive renal epithelia, NKCC2. Our previous studies showed that a monoclonal antibody to the cotransporter of human colonic T84 epithelial cells, an NKCC1 isoform, recognizes a 170-kDa glycoprotein from endothelial cells. The molecular identity of the Na-K-Cl cotransporter present in endothelial cells, however, has been unknown. In addition, although evidence has been provided that phosphorylation of the endothelial cotransporter plays a role in regulating its activity, little is known about potential sites for protein kinase interaction with the cotransporter. The present study was conducted to determine the molecular structure of the endothelial Na-K-Cl cotransporter. Using a 1.0-kilobase (kb) cDNA fragment from a conserved region of the T84 cell cotransporter, we screened a bovine aortic endothelial cell cDNA library and subsequently identified and sequenced two overlapping clones that together spanned the entire coding region. The endothelial cotransporter is a 1,201-amino acid protein with 12 putative transmembrane segments and large amino and carboxy termini, each containing several consensus sites for phosphorylation by protein kinases. Comparison of the endothelial cotransporter amino acid sequence with known NKCC1 and NKCC2 sequences revealed a 96% identity with NKCC1. Northern blot analysis using a cDNA probe from the endothelial cotransporter revealed high expression of approximately 7.5-kb transcripts in a number of bovine tissues. Finally, a prominent expression of Na-K-Cl cotransporter was found by Western blot analysis in both cultured and freshly isolated endothelial cells of bovine aorta and cerebral microvessels.