Inhibition of Na+-K+-2Cl- cotransport by mercury

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH-) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl- channels. In this study, we determined whether the Na+-K+-Cl- cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+ inhibits Na+-K+-Cl- cotransport, with inhibitor constant (K-i) values of 25 mu M for the shark carrier (sNKCC1) and 43 mu M for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697-->Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+ binding. We also show that modification of additional SH- groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.