MOLECULAR CHARACTERIZATION OF THE NA-K-CL COTRANSPORTER OF BOVINE AORTIC ENDOTHELIAL-CELLS

The Na-K-CI cotransporter is an important regulator of endothelial cel l volume and may also contribute to Aux of Na and Cl across the endoth elium of the blood-brain barrier. To date, two Na-K-CI cotransport iso forms have been identified, the cotransporter in secretary epithelia, NKCC1, and that in absorptive renal epithelia, NKCC2. Our previous stu dies showed that a monoclonal antibody to the cotransporter of human c olonic T84 epithelial cells, an NKCC1 isoform, recognizes a 170-kDa gl ycoprotein from endothelial cells. The molecular identity of the Na-X- Cl cotransporter present in endothelial cells, however, has been unkno wn. In addition, although evidence has been provided that phosphorylat ion of the endothelial cotransporter plays a role in regulating its ac tivity, little is known about potential sites for protein kinase inter action with the cotransporter. The present study was conducted to dete rmine the molecular structure of the endothelial Na-K-CI cotransporter . Using a 1.0-kilobase (kb) cDNA fragment from a conserved region of t he T84 cell cotransporter, we screened a bovine aortic endothelial cel l cDNA library and subsequently identified and sequenced two overlappi ng clones that together spanned the entire coding region. The endothel ial cotransporter is a 1,201-amino acid protein with 12 putative trans membrane segments and large amino and carboxy termini, each containing several consensus sites for phosphorylation by protein kinases. Compa rison 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 r evealed high expression of similar to 7.5-kb transcripts in a number o f bovine tissues. Finally, a prominent expression of Na-K-Cl cotranspo rter was found by Western blot analysis in both cultured and freshly i solated endothelial cells of bovine aorta and cerebral microvessels.