Abstract: | Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg2+) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na+ pump andCl channels. In this study,we determined whether theNa+-K+-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg2+ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg2+inhibitsNa+-K+-Clcotransport, with inhibitor constant(Ki) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg2+ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane. |