Inhibition of Na1-K1-2Cl2 cotransport by mercury

Jacoby, Steven C., Edith Gagnon, Luc Caron, John Chang, and Paul Isenring. Inhibition of Na1-K1-2Cl2 cotransport by mercury. Am. J. Physiol. 277 (Cell Physiol. 46): C684–C692, 1999.—Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH2) groups. The inorganic form (Hg21) is toxic to epithelial tissues and interacts with various transport proteins including the Na1 pump and Cl2 channels. In this study, we determined whether the Na1-K1-Cl2 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 Hg21 on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg21 inhibits Na1-K1-Cl2 cotransport, with inhibitor constant (Ki) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg21 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 Hg21 binding. We also show that modification of additional SH2 groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.