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

AlthoughNa⁺-K⁺-2Cl cotransport has beendemonstrated in cultured bovine corneal endothelial cells, its presenceand role in the native tissue have been disputed. Using RT-PCR we havenow 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 sitesare 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 thecotransporter. We calculate the turnover rate of the cotransporter tobe 83 s¹. Transendothelial fluid transport, determinedfrom deepithelialized rabbit corneal thickness measurements, ispartially inhibited (30%) by bumetanide in a dose-dependent manner.Our results demonstrate thatNa⁺-K⁺-2Cl cotransporters arepresent in the basolateral domain of freshly dissected bovine cornealendothelial cells and contribute to fluid transport across cornealendothelial preparations.