title: Reversible oxidative modification: a key mechanism of Na+-K+ pump regulation.
abstract: 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 beta(1), but not alpha(1), subunits in rabbit ventricular myocytes at baseline. beta(1) Subunit glutathionylation was increased by peroxynitrite (ONOO(-)), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased alpha(1)/beta(1) subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with beta(1) subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na(+)-K(+) pump current (I(p)). Cysteine (Cys46) of the beta(1) subunit was the likely candidate for glutathionylation. We expressed Na(+)-K(+) pump alpha(1) subunits with wild-type or Cys46-mutated beta(1) subunits in Xenopus oocytes. ONOO(-) induced glutathionylation of beta(1) 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 beta(1) subunit from pig kidney. This was associated with a approximately 2-fold decrease in the rate-limiting E(2)-->E(1) 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 beta(1) subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na(+) levels.
authors: Department of Cardiology, Royal North Shore Hospital, University of Sydney, St Leonards NSW 2065, Australia.
authors: Gemma A
authors: GA
authors: Figtree
authors: Chia-Chi
authors: CC
authors: Liu
authors: Stephanie
authors: S
authors: Bibert
authors: Elisha J
authors: EJ
authors: Hamilton
authors: Alvaro
authors: A
authors: Garcia
authors: Caroline N
authors: CN
authors: White
authors: Karin K M
authors: KK
authors: Chia
authors: Flemming
authors: F
authors: Cornelius
authors: Kaethi
authors: K
authors: Geering
authors: Helge H
authors: HH
authors: Rasmussen
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D027682:Cation Transport Proteins / Q000378:metabolism; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism; D003545:Cysteine; D054477:Glutaredoxins / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D009154:Mutation; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D019255:NADPH Oxidases / Q000378:metabolism; D009865:Oocytes; D010084:Oxidation-Reduction; D010269:Paraquat / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012756:Sheep; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000378:metabolism; D013552:Swine; D014982:Xenopus laevis
chemical_list: C503551:ATP1B1 protein, human; C504190:ATP1B2 protein, human; C519304:ATP1B3 protein, human; D027682:Cation Transport Proteins; D015816:Cell Adhesion Molecules, Neuronal; D054477:Glutaredoxins; D000804:Angiotensin II; D030421:Peroxynitrous Acid; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D011493:Protein Kinase C; C499076:ATP1A1 protein, human; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione; D003545:Cysteine; D010269:Paraquat