Protein Carbonylation of an Amino Acid Residue of the Na/K-ATPase α1 Subunit Determines Na/K-ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells

Background: we have demonstrated that cardiotonic steroids,such as ouabain,signaling through the na/k-atpase,regulate sodium reabsorption in the renal proximal tubule. by direct carbonylation modification of the pro222 residue in the actuator (a) domain of pig na/k-atpase α1 subunit,reactive oxygen species are required for ouabain-stimulated na/k-atpase/c-src signaling and subsequent regulation of active transepithelial 22na+ transport. in the present study we sought to determine the functional role of pro222 carbonylation in na/k-atpase signaling and sodium handling. methods and results: stable pig α1 knockdown llc-pk1-originated py-17 cells were rescued by expressing wild-type rat α1 and rat α1 with a single mutation of pro224 (corresponding to pig pro222) to ala. this mutation does not affect ouabain-induced inhibition of na/k-atpase activity,but abolishes the effects of ouabain on na/k-atpase/c-src signaling,protein carbonylation,na/k-atpase endocytosis,and active transepithelial 22na+ transport. conclusions: direct carbonylation modification of pro224 in the rat α1 subunit determines ouabain-mediated na/k-atpase signal transduction and subsequent regulation of renal proximal tubule sodium transport. © 2016 the authors. published on behalf of the american heart association,inc.,by wiley blackwell.