Corneal endothelial NKCC: molecular identification, location, and contribution to fluid transport

Abstract

Although Na+-K+-2Cl− cotransport has been demonstrated in cultured bovine corneal endothelial cells, its presence and role in the native tissue have been disputed. Using RT-PCR we have now identified a partial clone of the cotransporter protein in freshly dissected as well as in cultured corneal endothelial and epithelial cells. The deduced amino acid sequence of this protein segment is 99% identical to that of the bovine isoform (bNKCC1). [3H]bumetanide binding shows that the cotransporter sites are located in the basolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral location of the cotransporter. We calculate the turnover rate of the cotransporter to be 83 s−1. Transendothelial fluid transport, determined from deepithelialized rabbit corneal thickness measurements, is partially inhibited (30%) by bumetanide in a dose-dependent manner. Our results demonstrate that Na+-K+-2Cl− cotransporters are present in the basolateral domain of freshly dissected bovine corneal endothelial cells and contribute to fluid transport across corneal endothelial preparations.