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

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 alpha subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the alpha 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 alpha 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 alpha 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 alpha 2 isoform was more sensitive to GSSG than the alpha 1 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.