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(Circulation Research. 2009;105:185.)
© 2009 American Heart Association, Inc.

Cellular Biology

Reversible Oxidative Modification

A Key Mechanism of Na⁺-K⁺ Pump Regulation

Gemma A. Figtree^*, Chia-Chi Liu^*, Stephanie Bibert, Elisha J. Hamilton, Alvaro Garcia, Caroline N. White, Karin K.M. Chia, Flemming Cornelius, Kaethi Geering, Helge H. Rasmussen

From the North Shore Heart Research Group (G.A.F., C.-C.L., E.J.H., C.N.W., K.K.M.C., H.H.R.), Kolling Institute, University of Sydney, Australia; Department of Cardiology (G.A.F., A.G., K.K.M.C., H.H.R.), Royal North Shore Hospital, Sydney, Australia; Department of Pharmacology and Toxicology (S.B., K.G.), University of Lausanne, Switzerland; and Department of Physiology and Biophysics (F.C.), Aarhus University, Denmark.

Correspondence to Helge H. Rasmussen, Department of Cardiology, Royal North Shore Hospital, University of Sydney, St Leonards NSW 2065, Australia. E-mail helger{at}med.usyd.edu.au

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na⁺-K⁺ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of β₁, but not ₁, subunits in rabbit ventricular myocytes at baseline. β₁ Subunit glutathionylation was increased by peroxynitrite (ONOO^–), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased ₁/β₁ subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β₁ subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na⁺-K⁺ pump current (I_p). Cysteine (Cys46) of the β₁ subunit was the likely candidate for glutathionylation. We expressed Na⁺-K⁺ pump ₁ subunits with wild-type or Cys46-mutated β₁ subunits in Xenopus oocytes. ONOO^– induced glutathionylation of β₁ subunit and a decrease in Na⁺-K⁺ pump turnover number. This was eliminated by mutation of Cys46. ONOO^– also induced glutathionylation of the Na⁺-K⁺ ATPase β₁ subunit from pig kidney. This was associated with a 2-fold decrease in the rate-limiting E₂E₁ conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na⁺-K⁺ pump occurs via glutathionylation of its β₁ subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na⁺ levels.

Key Words: glutathionylation • glutaredoxin • Na⁺-K⁺ pump • angiotensin • NADPH oxidase