Regulation of erythrocyte Na+/K+/2Cl— cotransport by an oxygen-switched kinase cascade
Many erythrocyte processes and pathways, including glycoly- sis, the pentose phosphate pathway (PPP), KCl cotransport, ATP release, Na+/K+-ATPase activity, ankyrin– band 3 interac- tions, 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 con- trolled by O2-dependent competition between deoxyhemoglo- bin (deoxyHb), but not oxyhemoglobin (oxyHb), and other pro- teins for band 3. We undertook the present study to determine whether the O2 dependence of Na+/K+/2Cl— cotransport (cat- alyzed by Na+/K+/2Cl— cotransporter 1 [NKCC1]) might simi- larly originate from competition between deoxyHb and a pro- tein 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 dock- ing 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 hypothe- size that the NKCC1-mediated regulatory mechanism may rep- resent a general pattern of O2 modulation of ion transporters in erythrocytes.
During transit from the lungs to the tissues, the human erythrocyte (red blood cell [RBC])4 experiences changes in the partial pressure of oxygen that decrease from ~100 mm Hg on the arterial side to <5 mm Hg in metabolically active tissues (1). Although these transitions in O2 pressure have long been known to facilitate O2 unloading in the tissues, they have more recently been established to participate in the regulation of important RBC properties (2–4). For example, the reversible O2-dependent association of deoxyHb with the major erythro- cyte 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 phos- phate 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). The same oxygen-dependent association of deoxyHb with band 3 has also been found to modulate the flex- ibility of the RBC membrane by disjoining a fraction of the band 3–ankyrin interactions at low O2 pressure (7–9).
The improved membrane deformability associated with this rupture of membrane– cytoskeletal interactions has been hypothesized to facilitate the return of deoxygenated RBCs from the microvas- culature to the lungs (8, 10, 11). Finally, hypoxia has also been shown to promote ATP release from circulating erythrocytes, leading to activation of P2Y receptors on endothelial cells and the consequent vasodilation that facilitates blood flow back to the lungs (12, 13).Changes in O2 pressure have also been reported to modulate ion transport in human erythrocytes (14, 15). The Na+/K+/ 2Cl— cotransporter (NKCC1) catalyzes the electroneutral, pas- sive symport of one Na+ plus one K+ plus two Cl— ions across a plasma membrane, promoting an osmotically driven flow of water in the same direction (16 –18). In the human erythrocyte, this cotransport is largely inactive in oxygenated cells but acti- vated in deoxygenated RBCs (15). This O2 regulation has been suggested to involve a kinase, because NKCC1 activation bydeoxygenation can be blocked by kinase inhibitors (19 –23), although the responsible kinase (if any) has not been identified in erythrocytes. In the kidneys NKCC activity has been shown to be regulated by a WNK (With-No-K (Lys)) kinase that is inhibited by intracellular Cl— (24) and activated by an increase in osmotic pressure.
The latter somehow induces phosphoryla- tion of the oxidative stress-responsive 1 (OSR1) kinase, which then phosphorylates and activates NKCC (25–32). Whether the osmotically regulated WNK–OSR1 signaling cascade is also involved in O2 modulation of NKCC1 in erythrocytes remains to be examined.In this paper, we explore the mechanism of O2-dependent control of NKCC1 by investigating the impact of oxygen pres- sure on NKCC1 activity in erythrocytes from transgenic mice that express different mutations in the deoxyHb-binding site on erythrocyte band 3. We demonstrate that deoxyHb (but not oxyHb) binds to the cytoplasmic domain of band 3 and dis- places the WNK1 kinase from its docking site on band 3. 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.
Results
To evaluate the role of the band 3– deoxyHb interaction in regulating Na+/K+/2Cl— cotransport through the NKCC1 transporter, we introduced mutations into band 3 that were known to affect its association with deoxyHb. Based on previ- ous studies that mapped the deoxyHb-binding site on the iso- lated cytoplasmic domain of band 3 (33, 34), we generated a transgenic mouse in which the sequence encompassing the deoxyHb-binding site (residues 1– 45) on murine band 3 was replaced with the homologous sequence from human band 3 (residues 1–35; i.e. which includes the human deoxyHb-bind- ing site). The resulting mouse provided us with a humanized model for analysis of the regulatory role of deoxyHb binding to band 3 in RBCs (34). In a second transgenic mouse, the same human sequence was inserted into murine band 3, except the amino acids responsible for deoxyHb binding (residues 12–23) were deleted, allowing us to determine how the lack of a deoxyHb-band 3 affects O2 regulation of RBC properties. In the present study, we introduced a third mutation into murine band 3 (deletion of residues 1–11) that endows band 3 with a significantly higher affinity for deoxyHb (33, 35) (Fig. 1). The representative schematic strategy is shown in Fig. S1. Hemato- logic analyses of these transgenic mice revealed that their RBCs display essentially normal indices, except their sodium contents are slightly elevated, and their potassium contents are some- what reduced (Table S1).
Further analyses of their osmotic fra- gilities indicate that they also are slightly elevated (Fig. S2), which we interpret to derive from reduced retention of band 3 in the mutant membranes, as shown in Fig. S3. Although RBC membranes from a human patient in which band 3 residues 1–11 are similarly missing also contain reduced levels of band 3(i.e. analogous to the mouse with the high-affinity deoxyHb- binding site on band 3), the mutation in the human RBCs appears to cause much greater membrane destabilization, per- haps because it promotes retention of much less band 3 than seen in the murine RBC membranes (36).O2 regulation of NKCC1 is inhibited by altered deoxyHb– band 3 interactionsAlthough the activity of NKCC1 has been shown to be O2-regulated in erythrocytes from several vertebrate species, including humans (1, 14, 15), O2 regulation has not been reported in mice. As shown in Fig. 2A, cotransport of NaCl and KCl in WT mouse erythrocytes is elevated ~2.5-fold upon deoxygenation, i.e. comparable with the effect reported in human RBCs (15). A similar increase in NKCC1 activity is seen in murine erythrocytes in which the human deoxyHb-binding sequence has been substituted for the homologous mouse sequence, suggesting that the human and murine sequences interact similarly with deoxyHb. In contrast, when the murine deoxyHb-binding sequence is either deleted or replaced with a high-affinity sequence that prevents reversible deoxyHb bind- ing over the physiological range of O2 pressures, oxygen regu- lation of NKCC1 activity is abrogated.
These data suggest that reversible association of deoxyHb with the cytoplasmic domain of band 3 constitutes the molecular switch that controls NKCC1 activity in erythrocytes and that mutations that either decrease or increase the affinity of deoxyHb for band 3 inhibit this regulation of NaCl plus KCl cotransport.Although this interpretation of O2 regulation is consistent with both the transport data and the previously established mechanism for O2 regulation of glycolysis and ankyrin binding (34), a replotting of the data showing absolute rather than rel- ative Na+/K+/2Cl— transport rates argues for a more complex interpretation. As seen in Fig. 2B, RBCs in which deoxyHb is always bound (high-affinity mutant) or always dissociated (Hb site deletion mutant) both display elevated transport rates,regardless of O2 pressure. In contradistinction, data from the same study in WT mice demonstrate that deoxyHb binding to band 3 enhances Na+/K+/2Cl— transport, whereas dissocia- tion of deoxyHb from band 3 reduces Na+/K+/2Cl— transport, suggesting that erythrocytes in which the deoxyHb site has been deleted should have shown reduced rather than elevated NKCC1 activity.O2-dependent regulation of NKCC1 activity involves NKCC1 phosphorylationTo reconcile the above data, we explored whether the path- ways of NKCC regulation present in other tissues are similar to the O2 regulation seen in RBCs.
As demonstrated by Moriguchiet al. (25) and Vitari et al. (26), regulation of NKCC in the kidneys involves a signaling cascade in which an increase in osmotic pressure activates WNK kinase, which in turn phosphor- ylates OSR1 kinase. Activated OSR1 then phosphorylates NKCC on threonines 212 and 217, leading to NKCC activation and the consequent volume changes that protect the kidneys from osmotic stress and facilitate transepithelial salt resorption (37).To determine whether changes in O2 pressure might simi- larly induce changes in NKCC1 phosphorylation in murine erythrocytes, we examined the O2 dependence of NKCC1 phos- phorylation in murine RBCs using an antibody directed against phosphorene's 212 and 217 of human NKCC1 (equivalent to Thr206/Thr211 on murine NKCC1) that are involved inNKCC1 activation in response to osmotic stress in the kidneys(38). As shown in Fig. 2C, deoxygenation of erythrocytes con- taining a functional band 3 deoxyHb-binding site (i.e. either WT or humanized) promotes a ~2-fold increase in phosphor- ylation of threonines 206 and 211, whereas deoxygenation of RBCs either lacking this site or containing the high-affinity deoxyHb site has no significant effect on NKCC1 phosphoryla- tion. This O2 dependence correlates with the O2-insensitive and constitutively activated NKCC1 activity that we observed in RBCs containing either the high deoxyHb affinity mutant and the deoxyHb site deletion mutant, i.e. the two mutanterythrocytes that display O2 independent and constitutively high phosphorylation of threonines 206 and 211 (Fig. 2C).
Deoxygenation of erythrocytes activates both WNK1 and OSR1To determine whether the catalytic activity of WNK1 and/or OSR1 might be regulated by O2 in RBCs, we lysed oxygenated and deoxygenated murine RBCs in Triton X-100 and immuno- blotted the lysates with antibodies to OSR1 phosphorylated on threonine 185 and serine 325 (26, 39, 40), i.e. two conserved resi- dues on OSR1 known to be phosphorylated by WNK1 (25, 26). Asshown in Fig. 3A, similar levels of total OSR1 were present in both oxygenated and deoxygenated cells; however, the level of OSR1 phosphorylation on Thr185 and Ser325 was much higher in deoxy- genated than oxygenated RBCs. These data suggest that deoxy- genation promotes OSR1 phosphorylation/activation.To determine whether WNK1 might catalyze the above O2-dependent OSR1 phosphorylation as it does osmotically induced OSR1 phosphorylation in kidneys, we immunoprecipi- tated WNK1 from both oxygenated and deoxygenated RBCs and examined its ability to phosphorylate exogenously added OSR1 on Thr185 and Ser325. As shown in Fig. 3 (C and D), respectively, phosphorylation of OSR1 on Thr185 and Ser325 was significantly higher when performed with WNK1 immuno- precipitates from deoxygenated than oxygenated cells. Impor- tantly, more than 90% of WNK1 proteins were localized to the membrane in oxygenated erythrocytes, whereas more than 50% of WNK1 was translocated into the cytoplasm in deoxygenated erythrocytes (Fig. 3, E and F). These data suggest that WNK1 is predominantly membrane-bound and inactive in oxygenated RBCs but cytosolic and more active in deoxygenated RBCs, similar to behavior observed for glycolytic enzymes (41, 42).
To confirm that the deoxyHb-binding site on band 3 is involved in WNK1 binding, we used an anti-WNK1 antibody to pull down the WNK1 kinase from detergent extracts of murine erythrocytes to determine whether band 3 is coprecipitated with it. As shown in Fig. 4, similar amounts of WNK1 were pelleted from WT erythrocytes and erythrocytes from all three mutant mice, suggesting that the kinase is similarly expressed in all four mice. However, the amount of band 3 copelleted with WNK1 was 2-fold higher in RBCs containing a functional deoxyHb-binding site (i.e. WT and humanized) than in mutant erythrocytes either lacking the deoxyHb-binding site or expressing the high-affinity binding site. These data argue that mutations in the deoxyHb site on band 3, regardless of whether they enhance or eliminate deoxyHb binding, cause a concur- rent loss in WNK1 binding, confirming that at least some of the deoxyHb binding residues at the N terminus of band 3 are involved in WNK1 binding. Because displacement of WNK 1 from band 3 induces its activation (Fig. 3), these results explain why both deoxyHb-binding site mutants (i.e. mutants without a Hb site and mutants containing the high-affinity Hb site) exhibit a similar constitutive O2-independent activation (Fig. 2B) and phosphorylation (Fig. 2C) of NKCC1. Taken together, our data demonstrate that RBC deoxygenation induces deoxyHb binding to band 3 (residues 12–23 (43)), which in turn promotes displacement and activation of WNK1.
To determine whether the same regulatory pathway for O2 modulation of NKCC1 activity in the mouse might be operative in human RBCs, we expressed GST fusion constructs of the intact cytoplasmic domain of human band 3 (cdb3; residues 1–379) containing no mutation, the high-affinity deoxyHb- binding site mutation, or the deoxyHb-binding site deletion mutation and examined their relative abilities to copellet WNK1 from crude extracts of HEK293 cells expressing WNK1 (Fig. 5). Although GST pulldown of WT human cdb3 was found to copellet WNK1, none of the cdb3s containing a mutateddeoxyHb-binding site was able to copellet WNK1. These data confirm that deoxyHb and WNK1 share an overlapping bind- ing site on both murine and human band 3 and that mutation of this site to either decrease or increase deoxyHb affinity leads to loss of WNK1 binding in both species.WNK1 is believed to respond to osmotic stress in the kidneys by regulating the locations and activities of multiple ion trans- porters and channels (27). Important to this regulatory role appears to be the translocation of WNK1 to intracellular com- partments, mediated by its C-terminal domain in response to elevated osmotic pressure (27). To determine whether the same domain of WNK1 might be involved in regulating WNK1’s O2-dependent translocation in RBCs, we expressed intactWNK1, the WNK1 N-terminal kinase domain, and the WNK1 C-terminal “translocation domain” in HEK293 cells and exam- ined which construct associated with a GST fusion of human cdb3. As shown in Fig. 6, both the intact and C-terminal domain of WNK1 were readily pulled down by GST-cdb3, whereas the N-terminal domain of WNK1 was not. These data argue that a similar domain and mechanism is involved in regulating osmotic translocation of WNK1 in the kidneys and O2 translo- cation of WNK1 in erythrocytes.
Discussion
Orskov and others (1, 15, 22, 44, 45) have shown that several erythrocyte cation transporters are O2-regulated. Initially, a deoxyHb-binding site competition mechanism was dismissed, because erythrocyte cation transporters are not thought to associate with band 3. The binding site competition mechanism then became even more implausible when either elimination or augmentation of deoxyHb’s binding affinity for band 3 yielded the same enhancement of NKCC1 transport (Fig. 2B). Recon- ciliation of these data finally occurred when we discovered that (i) band 3 interacts directly with WNK1, (ii) deoxyHb-induced displacement of WNK1 from band 3 initiates a signaling cas- cade resulting in phosphorylation/activation of OSR1 and phos- phorylation/activation of NKCC1 (Figs. 2 and 3), and (iii) muta- tions leading to either elimination or augmentation of the deoxyHb-binding site on band 3 cause dissociation of WNK1 from band 3 (i.e. accounting for its activation in both mutant RBCs; Fig. 4). Thus, similar to oxygen’s regulation of ankyrin affinity and glucose metabolism, the molecular mechanism for oxygen’s control of NKCC1 reduces to a simple competition between deoxyHb and another protein (i.e. WNK1) for docking on band 3. To the best of our knowledge, this report constitutes the first description of a molecular mechanism by which O2 regulates any solute transporter.
With the O2 switch for regulation of glycolysis, ankyrin bind- ing, and NKCC1 activity now all involving a competition between deoxyHb and another protein for a shared binding site on band 3, the question naturally arose of whether other still uncharacterized O2-regulated pathways (e.g. ATP release (13), KCl cotransport (1, 15, 46), NO release (47), the Na+/K+- ATPase (48, 49), and Na+/H+ exchanger (50, 51), etc.) might be similarly modulated by competition between deoxyHb and another band 3-binding protein for a common site on band 3. Based on the regulatory mechanism elucidated here, it seems possible that membrane-spanning proteins like Piezo-1, the KCl cotransporter, the Na+/H+ exchanger, and the Na+/K+- ATPase may not directly compete with deoxyHb for association with band 3, but rather might be regulated by other signaling proteins that do directly compete for binding to band 3. The most obvious candidate would be the KCl cotransporter (KCC), because KCl cotransport has been shown to be inhibited by a signaling pathway involving WNK1 activation of OSR1 fol- lowed by OSR1 phosphorylation of the KCC (29, 52, 53). Because the effect of O2 on KCl cotransport is exactly the oppo- site of its effect on Na+/K+/2Cl— cotransport (1, 54 –56) (i.e. oxygenation of RBCs activates KCl cotransport while it inhibits Na+/K+/2Cl— cotransport and vice versa) and because the phosphorylation sites on the KCC and NKCC are homologous (57, 58), it seems reasonable to posit that the same deoxyHb displacement of WNK1 might be responsible for the reciprocal inhibition/activation of KCl and Na+/K+/2Cl— cotransport, respectively. Indeed, in view of the plethora of signaling pro- teins reported to bind band 3 in RBCs (e.g. Fgr, Hck, Lyn, Syk, SHP2, and casein kinase I (59 –61)), it is not inconceivable that many of the erythrocyte’s known O2-regulated processes might be controlled by a similar deoxyHb-mediated displacement mecha- nism involving release of a signaling enzyme from band 3.
It is interesting that oxygen regulation of Na+/K+/2Cl— transport in the erythrocyte turns out to be so similar to osmotic regulation of Na+/K+/2Cl— transport in the kidneys. Except for the transition from osmotic stimulation to oxygen stimulation, the signaling cascade appears to be almost identi- cal. However, evolutionary implementation of this regulatory capability in erythrocytes may not have been mechanistically trivial. Thus, although band 3 is expressed in the kidneys, kid- ney band 3 lacks the first 65 amino acids present in erythrocyte band 3 (62), and these amino acids are precisely those that are required for O2 regulation (Figs. 2 and 4). Because these addi- tional 65 amino acids are not homologous to any other sequence reported in the protein sequence databases (except other erythrocyte band 3’s), it is also unlikely that the new N terminus of band 3 could have derived from an exon normally present elsewhere in the genome. Rather, the fact that addition of these amino acids induces global changes in band 3’s struc- ture (63, 64) and function that endow band 3 with the ability to bind ankyrin, multiple glycolytic enzymes, several kinases, and deoxyhemoglobin, etc. (64 –66), suggests that the added N ter- minus evolved to improve erythrocyte function. Although fur- ther studies will be required to define how O2 regulation of Na+/K+/2Cl— transport enhances the erythrocyte’s evolution- ary fitness, the above considerations suggest that the ability of O2 to modulate RBC cation content must somehow improve the red cell’s function. It will be important in the future to deter- mine how O -regulated changes in Na+ and K+ concentrations benefit the erythrocyte’s performance. However, the fact that catecholamine’s have been found to regulate NKCC activity in avian RBCs (21, 67) but not in mammalian erythrocytes) and that catecholamine regulation of avian NKCC has been shown to be modulated by oxygen tension and osmolarity (21, 68) sug- gests that regulation of NKCC in erythrocytes probably occurred early in erythrocyte evolution and that participation of oxygen in this regulation was also an early event. It would be interesting to determine whether the catecholamine-mediated control of NKCC activity might also involve band 3-WNK1 interactions.
Finally, with >20 proteins thought to bind band 3 in red blood cells (AQP1, GAPDH, LDH, PFK, PK, aldolase, Syk, casein kinase 1, SHP2, adducin, ankyrin, protein 4.1, protein 4.2, carbonic anhydrase 2, RhAG, Rh, CD47, glycophorin A, Lyn, Hck, Fgr, and WNK1), the question naturally arises of whether sufficient copies of band 3 exist to accommodate them all. Rough estimates of the number of copies of band 3’s most prominent protein ligands suggest that the answer is affirma- tive. Thus, there are 1.2 × 106 copies of band 3 per RBC (69, 70). If one assumes that there are also 120,000 ankyrins (71), 30,000 adducin dimers (72), 350,000 glyceraldehyde-3-phosphate de- hydrogenases (73), 100,000 aldolases, 30,000 phosphofructokinases (74), 200,000 proteins 4.1 (75), and 200,000 proteins 4.2 (76), ~300,000 copies of band 3 should still remain to accom- modate all of the above signaling enzymes, which far exceeds their numbers in RBCs. Thus, even if all known band 3 periph- eral protein ligands were to compete for the same deoxyHb- binding site, there should still be sufficient deoxyHb sites avail- able to accommodate them all. As a consequence, the potential for regulation of still other erythrocyte pathways by O2 via com- petition for the deoxyHb-binding site on band 3 is still not exhausted. All mouse studies were approved by the National Human Genome Research Institute Animal Care and Use Committee (protocol number (G-04-2)).
The National Institutes of Health Intramural Research Program is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International. Transgenic mice expressing a “humanized” band 3 with the human deoxyHb-binding site, a band 3 lacking deoxyHb-binding site, or a band 3 with high-affinity deoxyHbbinding site were generated through standard homologous recombination techniques in embryonic stem cells as described previously (34). Detailed protocols are included in the support- ing Materials and methods. Blood was collected by retro-orbital bleeding from 6 –10-week-old WT mice or transgenic mice with homozygous band 3 mutations. Complete blood counts were performed on an automatic hematology analyzer (Sie- mens ADVIA 120 hematology system) following the manufa- cturer’s instructions. Reticulocyte WNK463 counts were determined using Syto RNASelect green fluorescent cell stain (Invitrogen). Osmotic fragility was determined by measuring hemolysis of erythrocytes placed in sodium chloride solutions of varying concentrations as described previously (77).