Regulation of erythrocyte Na/K/2Cl cotransport by an oxygen-switched kinase cascade

Many erythrocyte processes and pathways, including glycolysis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na/K-ATPase activity, ankyrin– band 3 interactions, and nitric oxide (NO) release, are regulated by changes in O₂ pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O₂ dependence of glycolysis, PPP, and ankyrin– band 3 interactions (affecting RBC rheology) are controlled by O₂-dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O₂ dependence of Na/K/2Cl cotransport (catalyzed by Na/K/2Cl cotransporter 1 [NKCC1]) might similarly originate from competition between deoxyHb and a protein involved in NKCC1 regulation for a common binding site on band 3. Using three transgenic mouse strains having mutated deoxyhemoglobin-binding sites on band 3, we found that docking of deoxyhemoglobin at the N terminus of band 3 displaces the protein with no lysine kinase 1 (WNK1) from its overlapping binding site on band 3. This displacement enabled WNK1 to phosphorylate oxidative stress-responsive kinase 1 (OSR1), which, in turn, phosphorylated and activated NKCC1. Under normal solution conditions, the NKCC1 activation increased RBC volume and thereby induced changes in RBC rheology. Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O₂ regulation pathway may also occur in the regulation of other O₂-regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O₂ modulation of ion transporters in erythrocytes.