Abstract
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.
Highlights
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
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
Because the deoxyhemoglobin-mediated WNK1 displacement from band 3 in this O2 regulation pathway may 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
Summary
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 reversible O2-dependent association of deoxyHb with the major erythrocyte membrane protein, band 3, has been shown to constitute a molecular switch that controls the association of glycolytic enzymes with inhibitory sites on band 3, shifting the flux of glucose from glycolysis at low O2 pressures to the pentose phosphate pathway at high O2 pressures [5, 6] This shift in glucose metabolism is thought to be adaptive, because the resulting increase in NADPH in oxygenated conditions can help protect the erythrocyte from the oxidative stress that accompanies high O2 pressures [5, 6]. We further show that this displaced WNK1 kinase activates OSR1, which in turn phosphorylates and activates NKCC1, leading to an influx of NaCl and KCl into the cell whenever O2 levels decrease In this manner, the O2 content of the erythrocyte can modulate red cell volume during RBC transit in the vasculature
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