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

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.