Inhibition of Na(+)-K(+)-2Cl(-) cotransport by mercury.
Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH(-)) groups. The inorganic form (Hg(2+)) 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 Hg(2+) on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg(2+) inhibits Na(+)-K(+)-Cl(-) cotransport, with inhibitor constant (K(i)) values of 25 microM for the shark carrier (sNKCC1) and 43 microM for the human carrier. In further studies, we took advantage of species differences in Hg(2+) 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 Hg(2+) 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.
Jacoby
S C
SC
Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Gagnon
E
E
Caron
L
L
Chang
J
J
Isenring
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D002460:Cell Line; D002678:Chimera / Q000235:genetics / Q000502:physiology; D004284:Dogfish; D006801:Humans; D008628:Mercury / Q000494:pharmacology*; D009154:Mutation / Q000502:physiology; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D028021:Sodium-Potassium-Chloride Symporters; D008628:Mercury