Mercury binding site on Na+/K(+)-ATPase: a cysteine in the first transmembrane segment.
Mercury is an element of great pharmacological and toxicological importance. It reacts with sulfhydryl groups on proteins to form mercaptides. Mercuric mercury (Hg2+), a form that shows primarily epithelial toxicity, can inhibit Na+/K(+)-ATPase at low concentration, but its molecular target site on the protein is not known. To investigate the interaction of Hg2+ with Na+/K(+)-ATPase, we studied the inhibition of Na+/K+ pump activity by inorganic mercury (HgCl2) in Xenopus laevis oocytes expressing wild-type and mutant forms of Na+/K(+)-ATPase. Na+/K+ pump potassium-activated current was inhibited with first-order kinetics (Kon = 7 x 10(3) M-1.sec-1) and an estimated Kd of < or = 170 nM. To study the hypothesis that the cysteine (C113) of the first transmembrane segment of the alpha subunit participates in a Hg2+ binding site, we investigated the inhibition of Na+/K+ pump activity produced by a 1-min exposure to 5 microM HgCl2. Wild-type and C113S and C113Y mutant Na+/K+ pumps were inhibited by 43 +/- 7%, 12 +/- 2%, and 5 +/- 3%, respectively. Because C113 is a component of the cardiac steroid binding site, we studied the interaction of mercury with strophanthidin by exposing oocytes for 2 min to 5 microM HgCl2 in the presence or absence of 50 microM strophanthidin. Strophanthidin reduced the inhibition by mercury from 68 +/- 5% to 30 +/- 7%. Based on the position of C113 in the first transmembrane segment, these results suggest that Hg2+ binding to C113 from the extracellular side is one of the mechanisms by which mercury inhibits Na+/K(+)-ATPase.
Wang
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X
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Horisberger
J D
JD
D000818:Animals; D001665:Binding Sites; D002025:Bufonidae; D003545:Cysteine; D004594:Electrophysiology / Q000379:methods; D005260:Female; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008627:Mercuric Chloride / Q000494:pharmacology*; D008628:Mercury / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D017354:Point Mutation; D011188:Potassium / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012044:Regression Analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013327:Strophanthidin / Q000494:pharmacology; D014982:Xenopus laevis
D011994:Recombinant Proteins; D008627:Mercuric Chloride; D013327:Strophanthidin; D000254:Sodium-Potassium-Exchanging ATPase; D008628:Mercury; D003545:Cysteine; D011188:Potassium