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 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.
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 11999 Moscow, Russia.
Irina Yu
IY
Petrushanko
Sergej
S
Yakushev
Vladimir A
VA
Mitkevich
Yuliya V
YV
Kamanina
Rustam H
RH
Ziganshin
Xianyu
X
Meng
Anastasiya A
AA
Anashkina
Asya
A
Makhro
Olga D
OD
Lopina
Max
M
Gassmann
Alexander A
AA
Makarov
Anna
A
Bogdanova
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D019803:Glutathione Disulfide / Q000737:chemistry* / Q000378:metabolism; D000860:Hypoxia; D008297:Male; D008968:Molecular Conformation; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine; D019803:Glutathione Disulfide