S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

• Published in: The Journal of biological chemistry 2012-09, Vol.287 (38), p.32195-32205
• Main Authors: Petrushanko, Irina Yu, Yakushev, Sergej, Mitkevich, Vladimir A., Kamanina, Yuliya V., Ziganshin, Rustam H., Meng, Xianyu, Anashkina, Anastasiya A., Makhro, Asya, Lopina, Olga D., Gassmann, Max, Makarov, Alexander A., Bogdanova, Anna
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Animals; Binding Sites; Catalysis; Catalytic Domain; Cysteine - chemistry; Dose-Response Relationship, Drug; Glutathion; Glutathione Disulfide - chemistry; Glutathione Disulfide - metabolism; Heart; Hypoxia; Male; Membrane Biology; Molecular Conformation; Myocardium - enzymology; Na,K-ATPase; Oxidation-Reduction; Oxidative Stress; Protein Structure, Tertiary; Rats; Rats, Wistar; Redox Regulation; Redox Signaling; Sodium-Potassium-Exchanging ATPase - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.391094

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity. Background: Na,K-ATPase activity is extremely sensitive to changes in the redox state. Results: Binding of glutathione to the regulatory cysteine residues of the catalytic subunit completely inhibits the Na,K-ATPase by blocking the ATP-binding site. Conclusion:S-Glutathionylation of the catalytic subunit is revealed as a mechanism controlling the Na,K-ATPase function. Significance: Regulatory S-glutathionylation adjusts Na,K-ATPase activity to the changes in intracellular redox state and ATP levels.