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 e ndothelial and epithelial cells. The deduced amino acid sequence of this pr otein segment is 99% identical to that of the bovine isoform (bNKCC1). [H-3 ]bumetanide binding shows that the cotransporter sites are located in the b asolateral membrane region at a density of 1.6 pmol/mg of protein, close to that in lung epithelium. Immunocytochemistry confirms the basolateral loca tion of the cotransporter. We calculate the turnover rate of the cotranspor ter to be 83 s(-1). Transendothelial fluid transport, determined from deepi thelialized 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 fres hly dissected bovine corneal endothelial cells and contribute to fluid tran sport across corneal endothelial preparations.