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

Abstract

Edited by Mike ShipstonMany 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 O 2 pressure that occur as a red blood cell (RBC) transits between the lungs and tissues. The O 2 dependence of glycolysis, PPP, and ankyrin-band 3 interactions (affecting RBC rheology) are controlled by O 2 -dependent competition between deoxyhemoglobin (deoxyHb), but not oxyhemoglobin (oxyHb), and other proteins for band 3. We undertook the present study to determine whether the O 2 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 O 2 regulation pathway may also occur in the regulation of other O 2 -regulated ion transporters, we hypothesize that the NKCC1-mediated regulatory mechanism may represent a general pattern of O 2 modulation of ion transporters in erythrocytes.

The effect of the antisickling compoundGBT1118 on the permeability of red blood cells from patients with sickle cell anemia

“…; Zheng et al. ), who propose that enhanced binding of deoxygenated Hb to band 3 displaces glycolytic and other enzymes, notably WNK1, leading to inhibition of the transporter. In sickle cells, as O 2 levels are reduced, the activity of KCC goes through a nadir at about half saturation of Hb for O 2 (Gibson et al.…”

The role of WNK in modulation of KCl cotransport activity in red cells from normal individuals and patients with sickle cell anaemia

“…In the case of red cell NKCC, WNK1 appears to be responsible for increased activity following deoxygenation [63]. KCC activity is also oxygen dependent, although in a reciprocal fashion being activated by oxygenation rather than deoxygenation.…”

“…Knockout studies in mice have indicated a role for the Src and Syk tyrosine kinases [13, 41, 42], with the conjugate phosphatases identified as PP1, PP2A, or PP2B [2, 3]. More recent publications provide molecular evidence for a role for WNK1 and possibly the functionally redundant WNK substrates SPAK/OSR1 in inhibition of KCC and stimulation of coordinate cotransport, NKCC, in red cells [15, 50, 63]. This paper provides the first demonstration of a functional role for WNKs in control of KCl cotransport in red cells from patients with sickle cell anaemia (SCA).…”

“…Working principally with the HEK293 cell line, but also with human red cells [50], showed that WNK1 inhibition played a role in stimulation of red cell KCC by swelling. Latterly, Low’s group has used transgenic mice to identify an excitatory role for WNK1 for OSR1 and, in regulation of the coordinate transporter, NKCC, upon deoxygenation [63].…”

Regulation ranges and patterns of adaptation to hyponatremia by cells of various organs and tissues of vertebrate animals

“…Synchronous decrease in sodium and potassium concentration in erythrocytes and muscles of roaches and breams at the initial period of hyponatremia indicates that the possible mechanism of their excretion from cells may be connected to the activation of Na + -K + -2Clcotransporter. This carrier is found on the membranes of various types of cells, including those of epithelium, endothelium, nerves, muscles, fi broblasts, and red blood cells (58)(59)(60)(61)(62). The participation of the Na +-K +-2Clcotransporter in the regulation of cellular volume has been studied in vitro.…”