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Am J Physiol Cell Physiol 291: C880-C886, 2006. First published June 14, 2006; doi:10.1152/ajpcell.00436.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

Alicia Rivera,1 Lucia De Franceschi,2 Luanne L. Peters,3 Philippe Gascard,4 Narla Mohandas,5 and Carlo Brugnara1

1Department of Laboratory Medicine, Children’s Hospital Boston, and Department of Pathology, Harvard Medical School, Boston, Massachusetts; 2Department of Clinical and Experimental Medicine, Section of Internal Medicine, University of Verona, Verona, Italy; 3The Jackson Laboratory, Bar Harbor, Maine; 4Lawrence Berkeley National Laboratory, Berkeley, California; and 5New York Blood Center, New York, New York

Submitted 26 August 2005 ; accepted in final form 28 May 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1–/–; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1–/– mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1–/– erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 ± 3.2 in 4.1–/– vs. 9.8 ± 1.3 mmol/1013 cell x h in control mice), with an abnormal dependence on osmolality (EC50 = 417 ± 42 in 4.1–/– vs. 460 ± 35 mosmol/kgH2O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered (K0.5 = 489.7 ± 0.7 vs. 537.0 ± 0.56 nM in control mice), the Vmax of the H-induced Na/H exchange activity was markedly elevated in 4.1–/– erythrocytes (Vmax 91.47 ± 7.2 compared with 46.52 ± 5.4 mmol/1013 cell x h in control mice). Na/H exchange activation by okadaic acid was absent in 4.1–/– erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically downregulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of 4.1–/– erythrocytes.

spherocytosis; cell Na; Na/H exchange

THE ERYTHROCYTE CYTOSKELETON is a complex arrangement of proteins including spectrin, actin, ankyrin, adducing, and protein 4.1 and 4.2. This multi-component structure is responsible for many physiological functions and mechanical properties of the erythrocyte. Deficiency or functional abnormalities of one or more of these cytoskeletal proteins decreases deformability of the erythrocyte plasma membrane, leading to hemolysis. Several hemolytic anemias have been associated with defects in the erythrocyte membrane function due to altered cytoskeletal proteins. These anemias have been classified as hereditary spherocytosis (HS), hereditary pyropoikilocytosis, and elliptocytosis (30).

Chronic hemolysis is the major characteristic of HS. Spherocytic erythrocytes lose mechanical strength and deformability while increasing plasma membrane vesiculation and trapping, leading to their destruction in the spleen. Several studies have shown that defects in either band 3, ankyrin, or spectrin account for a large number of HS cases (24, 29). Spherocytes are also characterized by a lower cellular K and increased Na content with dehydration (32). It has been suggested that these cation alterations are related to increased Na flux pathways such as Na pump, Na-K-2Cl cotransport (NKCC), and Na/Li exchanger. Recent studies have suggested that the increased permeability of the spherocytes is due to a loss of cellular skeleton integrity (10, 16). An increase in cation permeability due to deficiency of spectrin has been observed in a mouse model of spherocytosis (16). Dehydration and microspherocytosis have been observed in mouse erythrocytes lacking either erythroid band 3 protein (26) or beta-adducin cytoskeletal proteins (13). However, there has been no detailed functional characterization of the permeability pathways that mediate changes in cellular Na and K transport in murine spherocytosis.

We have previously described an upregulated response of volume regulatory systems such as NKCC, Na/H exchanger (NHE), and Na leak pathways due to hypertonic shrinkage in mice lacking protein 4.2 (25). We have shown also that the Ca2+-activated K channel (Gardos channel) is functionally upregulated in 4.1 knockout mouse erythrocytes (4.1–/–) and that this channel plays an important role in protecting spherocytes from colloidosmotic lysis (11). Protein 4.1 stabilizes the spectrin-actin complex by anchoring it to the plasma membrane. In erythrocytes, protein 4.1 interacts with spectrin and actin to regulate the mechanical properties of the erythrocyte membrane via a calmodulin-dependent binding site (20). However, little is known about the role that protein 4.1 may play in modulating volume regulatory increase systems such as the NHE. In the present study, we examine the ion transport pathways and volume regulatory responses of mouse erythrocytes devoid of protein 4.1 and identify a novel functional interaction between protein 4.1 and the erythroid NHE.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice with targeted deletion of 4.1 (4.1–/–) and 4.2 (4.2–/–) have been described previously (13, 25, 30). Control mice (C57Bl/6J and 129/SvEv) were purchased from The Jackson Laboratory. These studies were part of the research protocol A04-09-131R, which was submitted to, and approved by, Children’s Hospital’s Animal Care and Use Committee. Chemicals and inhibitors were purchased from Sigma (St. Louis, MO).

Erythrocyte Cation Content and Transport Studies Erythrocyte Na and K contents were determined in fresh mouse erythrocytes by atomic absorption spectrophotometry as previously described by Armsby et al. (1). Briefly, blood was colleted from isofluorene-anesthetized mice into heparinized tubes. After five washes in 172 mM choline chloride washing solution (1 mM MgCl2, 10 mM Tris-MOPS, pH 7.4, at 4°C), an aliquot of 50% cell suspension in washing solution was used for determinations of hematocrit, cell Na (1:50 dilution in 0.02% Acationox), and cell K (1:500 in double-distilled water).

Na-K Pump, NKCC, and NHE Erythrocytes were loaded with equal amounts of Na and K in the presence of nystatin (40 mg/ml), and fluxes were measured as described by Armsby et al. (1). Briefly, erythrocytes were loaded with equal amounts of Na and K using a nystatin solution (in mM): 77 NaCl, 77 KCl, and 55 sucrose. Na-K pump activity was estimated as the ouabain-sensitive fraction (1 mM ouabain) of Na efflux into a medium containing 155 mM choline chloride and 10 mM KCl. NKCC was estimated as bumetanide-sensitive Na and K efflux into medium containing 174 mM choline chloride with 1 mM ouabain in the presence and absence of 10 µM bumetanide. NHE activity was estimated as hydroxyl methyl amiloride-sensitive Na efflux in hypertonic shrinkage stimulation with a solution containing (in mM) 175 mM choline chloride, 1 MgCl2, 10 glucose, 1 ouabain, 0.01 bumetanide, and 10 Tris-MOPS (pH 7.4 at 37°C). Inhibitors were dissolved in DMSO and added to each media as described in figure legends.

K-Cl Cotransport The K-Cl cotransport (KCC) activity was determined as Cl-dependent K influx (using 86Rb as a tracer for K) in erythrocytes exposed to hypotonic swelling. The flux was calculated by subtracting total K influx into hypotonic NaCl medium (255–265 mosmol/kgH2O; 115 mM NaCl, 5 mM KCl, 1 mM ouabain, 10 µM bumetanide) from K efflux in hypotonic Na sulfamate medium (115 mM Na sulfamate, 5 K sulfamate, 1 mM ouabain, 10 µM bumetanide) and expressed as volume-dependent K influx.

Volume Regulation Experiments Fresh erythrocytes were incubated at 37°C for 5 h with gentle shaking in an isotonic medium with ionic composition similar to that of normal mouse plasma. The medium contained 160 mM NaCl, 2 mM KCl, 10 mM Tris-MOPS, pH 7.4, at 37°C, 2 mM Na-biphosphate, 1 mM MgCl2, and 5 mM glucose. To assess the role of various transport pathways in response to hypertonic shrinkage, fresh erythrocytes were incubated at 37°C for 2 h with gentle shaking in a hypertonic medium containing 220 mM NaCl, 2 mM KCl, 10 mM Tris-MOPS, pH 7.4, at 37°C, 2 mM Na-biphosphate, 1 mM MgCl2, and 5 mM glucose. Specific transport inhibitors were added at the specified final concentrations to assess the role of specific ion transport pathways under isotonic and hypertonic conditions.

H-Induced Na Influx Na/H exchange activity was measured by determining the initial rates of net Na influx when outward H gradients were imposed (27). Briefly, cell Na was removed by loading the erythrocytes with 145 K solution in the presence of nystatin (10 µg/ml) at 4°C. The ionophore-free K-loaded erythrocytes were incubated at 10% hematocrit with a K loading solution (pH 5.8–7.4) containing 1 mM ouabain and 10 µM bumetanide for 20 min at 37°C. To clamp the intracellular pH, DIDS (100 µM) and acetazolamide (200 µM) were added to the cell suspension and incubated for another 30 min at 37°C. Because intracellular acidification induces cell swelling and alkalinization induces shrinkage, the loading medium osmolarities were adjusted between 380 (acid) and 310 (alkaline) by varying sucrose between 0 and 60 mM and KCl between 180 and 160 mM. The cell volume was estimated by comparing the hemoglobin per liter of the loaded cells (301–370 g/l) with the fresh cells (320–345 g/l) for each intracellular pH. Furthermore, all flux media contained 1 mM ouabain and 10 µM bumetanide to avoid contribution of the Na pump or NKCC system. Proton-loaded erythrocytes were washed four times and resuspended at 50% hematocrit with unbuffered washing solution at 4°C, osmotically balanced to prevent volume changes. Erythrocytes (200 µl) were added to 2 ml of influx media either at pH 6.0 or pH 8.0 at 37°C. At specified time points, aliquots of 250 µl of the cell suspension were transferred into ice-cold Eppendorf tubes containing 0.7 ml of buffer (85 mM choline-Cl, 85 mM KCl, 0.25 mM MgCl2, 10 mM Tris-MOPS, pH 7.4, at 4°C, 0–60 mM sucrose) layered over 0.3 ml of phthalate oil and spun down. Supernatants and oil were removed, and the erythrocyte pellets were lysed with 1 ml of 0.02% Acationox detergent. Aliquots of the hemolyzed erythrocytes were used to determine hemoglobin concentration by optical density at 540 nm and Na concentration using atomic absorption spectrophotometry. Linear regression of the Na concentration vs. time was used to calculate net Na influx at pH 6.0 and 8.0. The difference between these two conditions is the H-induced Na influx (expressed as mmol/1013 cell x h). Mean cellular volume was measured to adjust flux values to a constant number of erythrocytes.

Density Phthalate Protocol Density distribution curves were obtained using phthalate esters in micro-hematocrit tubes as described in detail by Brugnara et al. (3). Briefly, phthalate solutions were prepared to give a range of densities between 1.08 and 1.11 g/ml. The hematocrit tubes were filled with 30 µl of red cell suspension and 10 µl of different phthalate solutions. Tubes were centrifuged at 12,200 rpm for 10 min at room temperature. The amount of denser cells was calculated from the amount of dense cells (lower layer) over the total amount of cells and expressed as a percentage.

Statistical Analysis All values are presented as means ± SD.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Control of Cell Na Content in Plasma-Like Isotonic Conditions To assess the possible determinants of the increased cell Na of 4.1–/– erythrocytes, freshly collected 4.1–/–, 4.2–/–, and control erythrocytes were incubated for 5 h in a plasma-like medium in the absence and presence of specific transport inhibitors. In control erythrocytes, Na content is relatively stable in the absence of transport inhibitors; cell Na increases with the use of the Na-K pump inhibitor ouabain (1 mM), while there are no significant changes with either bumetanide (10 µM), an inhibitor of the NKCC, or HOE-642 (10 µM), an amiloride analog inhibitor of the NHE (Fig. 1). Various combinations of these three inhibitors did not produce additional changes to those induced by ouabain alone, suggesting that the Na-K pump is the main regulator of cell Na content in these experimental conditions and that NKCC and NHE play a very minor role in determining the steady-state ionic content of normal mouse erythrocytes (Fig. 1, left). Results essentially similar to those of control erythrocytes were obtained in 4.2–/– erythrocytes (Fig. 1, middle). This is not surprising, since the transport abnormalities that we had described previously in 4.2–/– erythrocytes require hypertonic shrinkage (25).


Figure 1
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  		Fig. 1. Changes in erythrocyte Na content in normal control, 4.2–/–, and 4.1–/– mice after a 5-h incubation in isotonic plasma-like medium. Changes in cell Na content from baseline are presented in the absence or presence of the transport inhibitor ouabain, bumetanide, or HOE-642 or various combinations of the three. Data are expressed as means ± SD of 3 separate experiments. Statistical analysis compared with 0 drugs (ANOVA): *P < 0.05, n = 3.

 
In 4.1–/– erythrocytes, there was a much greater increase in cell Na after treatment with ouabain, an indication that the passive net Na entry in these erythrocytes is greatly enhanced and contributes to the increased steady-state cell Na (Fig. 1, right). There were no appreciable effects of either bumetanide alone or HOE-642 alone, suggesting that, in the presence of an active Na-K pump, blockade of either of these pathways does not result in a measurable change in cell Na. Interestingly, when ouabain and bumetanide were combined, the cell Na gain was greater than with ouabain alone, suggesting that the NKCC in 4.1–/– erythrocytes performs net Na extrusion, probably driven by the increased cell Na. The net gain in cell Na was reduced when erythrocytes were exposed to both HOE-642 and ouabain, suggesting that Na entry via the NHE is an important component of the Na influx into 4.1–/– erythrocytes under basal conditions.

Control of Cell Na Content in Hypertonic Conditions Hypertonicity is a known activator of the NHE, which mediates a regulatory volume increase (RVI) upon shrinkage in a variety of erythrocytes. In a first set of experiments, controls and 4.1–/– erythrocytes were incubated at 37°C in a hypertonic NaCl solution, and erythrocyte densities were measured with the phthalate density technique at baseline and after 2-h incubation in the presence or absence of the NHE inhibitor HOE-642. As shown in Fig. 2, incubation in hypertonic media induced a marked shrinkage of the erythrocytes at baseline compared with isotonic conditions. When control erythrocytes were incubated in hypertonic medium for 2 h, there were minimal changes in cell density, with only a small component of erythrocytes showing reduction in cell density, which was slightly inhibited by HOE-642. In 4.1–/– erythrocytes, a substantial decrease in cell density took place over 2-h incubation in hypertonic medium: this reduction in density was completely blocked by HOE-642, which also seemed to induce additional cell shrinkage (Fig. 2). Similar experiments were carried out in control, 4.2+/–, 4.2–/–, and 4.1–/– erythrocytes, measuring cell Na at baseline and after 2-h incubation in hypertonic medium with and without HOE-642. As shown in Fig. 3 and in our earlier studies (25), 4.2–/– erythrocytes exhibit increased NHE activity under hypertonic conditions that is blocked by HOE-642. A much greater activation of the NHE was found in 4.1–/– erythrocytes, which exhibited a twofold greater increase in HOE-642-sensitive cell Na compared with 4.2–/– erythrocytes (Fig. 3).


Figure 2
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  		Fig. 2. Effects of a 2-h incubation in hypertonic media on phthalate density profile. Phthalate density profiles for control (A) and 4.1–/– (B) erythrocytes are presented at baseline for isotonic conditions and hypertonic conditions and after 2-h incubation in the absence or presence of the Na/H exchanger (NHE) inhibitor HOE-642.

 

Figure 3
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  		Fig. 3. Effects of a 2-h incubation in hypertonic media on erythrocyte Na content. Data for cell Na content of control, 4.2+/–, 4.2–/–, and 4.1–/– erythrocytes are presented at baseline and after a 2-h incubation in the absence or presence of the NHE inhibitor HOE-642. Statistical analysis between control and the presence of HOE-642: *P < 0.05, n = 3.

 
Functional Properties of the Na/H Exchange in 4.1–/– Erythrocytes To assess the effects of protein 4.1 on the kinetic properties of the NHE, we determined in control and 4.1–/– erythrocytes the dependence of Na/H exchange activity on osmolarity and internal pH.

Dependence of NHE Activity on Osmolarity In control erythrocytes, the activity of NHE slowly increased as a function of media osmolarity in a sigmoidal pattern with a nominal saturation at 550 mosmol/kgH2O, with a maximal velocity of 9.8 ± 1.3 mmol/1013 cell x h (Fig. 4). One-half of the exchanger activity was reached at 460 ± 35 mosmol/kgH2O. In contrast, 4.1–/– erythrocytes showed a significantly elevated exchanger activity (18.2 ± 3.2 mmol/1013 cell x h; n = 3, P < 0.01), which reached nominal Vmax around 500 mosmol/kgH2O in a hyperbolic pattern. Half-maximal activation was achieved at 417 ± 42 mosmol/kgH2O in 4.1–/– erythrocytes, a value significantly lower (P < 0.01) than that of control erythrocytes. Thus the volume sensitivity of the Na/H exchange is altered in 4.1–/– erythrocytes, resulting in functional upregulation of the system.


Figure 4
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  		Fig. 4. Osmotic activation curve of NHE in control and 4.1–/– (knockout; KO) erythrocytes. Na efflux was measured in the presence or the absence of 10 µM HMA in Na-loaded erythrocytes from control and 4.1–/– erythrocytes as described in METHODS. Kinetic analysis of the curves gave a Vmax of 9.8 ± 1.3 mmol/l cell x h and EC50 of 460 ± 35 mosmol/kgH2O for control data. For 4.1–/– erythrocytes, corresponding values were found: Vmax of 18.2 ± 3.2 mmol/l cell x h and EC50 of 417 ± 42 mosmol/kgH2O. Data are expressed as means ± SE of triplicate experiments (n = 3).

 
Dependence of Na/H Exchange Activity on Internal pH Proton-induced Na influx via NHE was measured in control and 4.1–/– mouse erythrocytes (Fig. 5). In control erythrocytes, NHE activity increased with intracellular acidification, with a maximal velocity of 46.52 ± 5.4 mmol/1013 cell x h and proton affinity constant of 489.7 ± 0.7 nM (pH 6.31). In 4.1–/– erythrocytes, a significant increase in maximal velocity was observed (91.47 ± 7.2 mmol/1013 cell x h; n = 3, P < 0.003), while the affinity constant for intracellular H was unaffected (537.0 ± 0.56 nM; n = 3, pH 6.27). These data suggest that the increase in maximal velocity of the exchange is not mediated by alteration on the internal proton allosteric site but is due rather to an increase in flux rate.


Figure 5
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  		Fig. 5. H-induced NHE in control and 4.1–/– erythrocytes. Na influx was measured in acid-loaded erythrocytes as described in METHODS. Kinetic analysis of the curve gave a Vmax of 46.52 ± 5.4 mmol/1013 cell x h and a K0.5 of 489.8 ± 0.06 nM (pH = 6.31) for control and a Vmax of 91.5 ± 7.2 mmol/l cell x h and a K0.5 of 537.0 ± 0.56 nM (pH = 6.27) for 4.1–/– erythrocytes. Data represent means ± SE of triplicate experiments (n = 3).

 
Pharmacological Modulation of NHE Activity NHE is regulated by phosphorylation-dephosphorylation events via protein kinase C (PKC) and protein phosphatases (18). PKC inhibition (with either chelerythrine or calphostin C) did not affect the volume-sensitive NHE activity in either control or 4.1–/– erythrocytes (Fig. 6). The presence of protein phosphatase inhibitors such as okadaic acid (OKA) and calyculin A (CA) significantly stimulated the volume-stimulated NHE activity in control mouse erythrocytes. This was in accord with previous observations of OKA-induced activation of the NHE in human erythrocytes (27). In contrast, in 4.1–/– erythrocytes, NHE was paradoxically inhibited by the presence of either OKA or CA (Fig. 6). Interestingly, both phosphatase blockers inhibited the NHE up to basal values in 4.1–/– mice, suggesting a marked functional disregulation of this pathway.


Figure 6
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  		Fig. 6. Effects of phosphatase and kinase inhibitors on NHE activity. HMA-sensitive Na efflux was measured in control (solid bars) and 4.1–/– (open bars) erythrocytes. The inhibitors (100 nM) were added to the flux media at time 0. OKA, okadaic acid; CH, chelerythrine; CC, calphostin C; CA, calyculin A. *P < 0.04 and **P < 0.03. Data are expressed as means ± SE of 3 experiments in duplicate determinations (n = 3).

 
NHE activity as a function of media osmolarity was measured in the presence or absence of 100 nM OKA concentration (Fig. 7). In control erythrocytes, the presence of 100 nM OKA resulted in a small shift in the activation of the system by osmolarity (EC50 changed from 460 ± 35 to 494 ± 34 mosmol/kgH2O; P < 0.041, n = 3). In the 4.1–/– erythrocytes, exposure to 100 nM OKA resulted not only in inhibition of the system but also in a complete loss of the activation by osmolarity, with a paradoxical inhibition of the system by hypertonic shrinkage. Thus the absence of protein 4.1 results in a profound alteration of the volume regulatory loop of the NHE.


Figure 7
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  		Fig. 7. Effect of OKA on the volume-stimulated NHE activity. 5-(N,N-Hexamethylene)-amiloride (HMA)-sensitive Na efflux was measured in control and 4.1–/– erythrocytes. Left: control erythrocytes in the presence ({circ}) or absence (bullet) of 100 nM OKA. Right: 4.1–/– erythrocytes in the presence ({circ}) or absence (bullet) of 100 nM OKA. Values are expressed as means ± SE of triplicate experiments (n = 3).

 
Other Transport Pathways of 4.1–/– Erythrocytes: Na-K Pump, NKCC, and KCC Na-K pump and NKCC. The maximal rate of the Na-K pump did not appear to be different in 4.1–/– erythrocytes compared with controls, whereas it was reduced in 4.2–/– and 4.2+/– erythrocytes (Fig. 8) (25). Because the percentage of reticulocytes is significantly greater in 4.1–/– erythrocytes, the maximal activity of the Na-K pump could be considered to be abnormally low in 4.1–/– mice when compared with cell age-matched controls.


Figure 8
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  		Fig. 8. Ion transport via the ouabain-sensitive Na-K pump and the bumetanide-sensitive Na-K-2Cl cotransport (NKCC) in control, 4.2+/–, 4.2–/–, and 4.1–/– mouse erythrocytes. For Na-K pump and NKCC, erythrocytes were treated with the nystatin technique to obtain similar intracellular concentrations of Na and K as described in METHODS. Data are expressed as means ± SD of 3 separate experiments. Statistical analysis compared with control animal: *P < 0.05, n = 3.

 
No significant changes were observed in the maximal rate of NKCC compared with normal controls measured as Na or K flux (Fig. 8). NKCC activity was significantly elevated in 4.2–/– erythrocytes, as previously shown (25). Because the NKCC is highly dependent on intracellular Na (2), the net Na extrusion carried out by this system will be increased in 4.1–/– erythrocytes, as shown by the net changes in Na content of 4.1–/– erythrocytes incubated in isotonic conditions (Fig. 1).

KCC. Studies of the dependence of KCC on cell swelling were hampered by the extreme fragility of 4.1–/– erythrocytes, with significant lysis when osmolarity was decreased below isotonic conditions and associated inability to measure K efflux in a reliable manner. Measurements of K influx using 86Rb showed better reproducibility and demonstrated an increase in K efflux, which can be accounted for by the significant reticulocytosis. Interestingly, the passive permeability of 4.1–/– erythrocytes to K, estimated from the K influx in isotonic chloride-free medium, was increased two- to threefold compared with control erythrocytes (Fig. 9).


Figure 9
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  		Fig. 9. Ion transport via K-Cl cotransport (KCC) in control and 4.1–/– mouse erythrocytes. K influx was measured in isotonic Cl, hypotonic Cl, and isotonic sulfamate (SFA) media. Volume-stimulated and Cl-dependent influxes were calculated as hypotonic Cl minus isotonic Cl and isotonic Cl minus isotonic SFA, respectively. Data are expressed as means ± SD of 3 separate experiments. Statistical analysis for isotonic medium vs. different conditions for control and 4.1–/–: *P < 0.05, n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Erythroid protein 4.1-deficient mice exhibit a unique set of membrane transport abnormalities, some of them shared by other mouse models of cytoskeletal disorders and others unique to this mouse model. Cellular dehydration due to K loss and marked increases in cellular Na content are features seen in both human and mouse spherocytosis. The increased cell Na is due to an increased Na entry via ouabain- and bumetanide-resistant pathways. Our studies extend these observations and indicate that a major mediator of this abnormal Na entry in 4.1–/– erythrocytes is the NHE.

The NHE isoform present in erythroid cells is NHE1, which is the prototype for all other members of this family (4, 6, 19). In erythrocytes and other cell types, NHE activity is activated by phosphorylation (23) and is modulated by insulin and osmotic stress (5, 9). NHE function seems to be markedly altered in 4.1–/– erythrocytes. Our studies suggest that NHE is constitutively activated in 4.1–/– erythrocytes (see Fig. 3), with an increased sensitivity to activation by hyperosmotic shrinkage (Fig. 4) and normal regulation by intracellular H (Fig. 5). In addition, the upregulated NHE of 4.1–/– erythrocyte is paradoxically inhibited by OKA, a known stimulator of this system in normal mouse erythrocytes (Fig. 7). These alterations suggest that there must be a functional interaction between 4.1 protein and NHE in normal mouse erythrocytes that contributes to modulation and regulation of this transporter. These alterations are different from previously described functional abnormalities of the erythroid NHE: alterations of the internal pH NHE regulatory sites have been described in patients with essential hypertension and insulin-resistant diabetes (7, 8), while the affinity of the internal H site was not altered in 4.1–/– erythrocytes.

Pharmacological blockade of phosphatase activity by OKA has been shown to stimulate NHE activity. While the absence of additional stimulation by OKA in 4.1–/– can be rationalized by the upregulated state of NHE, the paradoxical inhibition of the system by OKA remains unexplained (Fig. 7). It remains to be determined whether this paradoxical effect of OKA in 4.1–/– erythrocytes may indicate the presence of a previously unknown modulator that is functionally silent in normal erythrocytes and becomes active in the absence of 4.1 protein.

The Ca-activated phosphatase calcineurin homologous protein (CHP) has been shown to downregulate NHE activity (17, 21). Perhaps in the absence of erythroid protein 4.1 protein, this interaction is altered, resulting in constitutive activation of the exchanger, as shown for CHP2, an isoform of CHP (22). Previous reports on interactions of protein 4.1 with other noncytoskeletal membrane proteins have shown an interaction between the 30-kDa FERM (Four4.1/Ezrin/Radixin/Moesin) domain of erythroid protein 4.1, which mediates cytoskeletal-membrane interactions, and pICln (protein that induces an outwardly rectifying, nucleotide-sensitive chloride current), a cytosolic protein believed to regulate volume-sensitive anion channels (28, 31). The crystal structure of this region of protein 4.1 has been solved recently, and the specific sites of interaction with band 3, glycophorin C/D, and the PDZ (PSD95/Dlg/ZO-1) domain containing the protein p55 binding site have been mapped. Erythroid protein 4.1 interaction with its binding partners is markedly affected by the binding of calmodulin to two separate binding regions of FERM domain in the presence of calcium (14). Calmodulin also regulates NHE-1 function and response to hypertonicity and acidification (12, 15, 34).

The NHE regulatory factor (NHERF) is an essential element in PKA-mediated inhibition of the predominantly renal NHE3 isoform (35–37). The COOH terminus of NHERF specifically interacts with protein of the family of membrane-cytoskeletal adapters ERM (ezrin-radixin-moesin) (33). This interaction is critical for the inhibition on NHE3 activity mediated by cAMP via PKA-dependent phosphorylation (38). The occurrence of such an interaction in mouse erythrocytes and its involvement in the abnormal regulation of NHE1 in 4.1–/– erythrocytes remains to be investigated.

The data presented in this manuscript do not provide conclusive evidence on the mechanisms leading to the reduced K content and dehydration of mouse 4.1–/– erythrocytes. We have reported increased activities of KCC (see Fig. 9) and Ca-activated Gardos channel (11), but the role of these two pathways in generating K loss and dehydration is still unclear. Human studies have also provided inconclusive evidence for the pathophysiology of K loss, with little evidence supporting a role for the KCC in this process (10). However, we have recently demonstrated that the K loss mediated by the Gardos channel is an important compensatory mechanism for the reduction in surface membrane area of HS erythrocytes that protects erythrocytes from premature destruction due to colloidosmotic lysis (11).

In conclusion, 4.1–/– erythrocytes exhibit distinct functional abnormalities, indicative of an important functional interaction between this cytoskeletal protein and the transmembrane ion transport protein NHE. The constitutive upregulation of NHE in 4.1–/– erythrocytes provides an explanation for their elevated baseline cell Na content.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants DK-069388 and HL-067699 (to A. Rivera), DK-50422 (to C. Brugnara), HL-64885 (to L. L. Peters), HL-31579 and DK-32094 (to N. Mohandas), DK-56355 (to P. Gascard) and by Italian Funds for Basic Research (FIRB) Grant RBNE01XHME-003 (to L. De Franceschi). Funding was also provided for A. Rivera by the Center of Excellence in Minority Health and Health Disparities at Harvard Medical School.


   ACKNOWLEDGMENTS
 
We thank Lin-Chie Pong, Michelle Langlois, Michelle Rotter, Maria Argos, and Sarah Sheldon for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Rivera, Children’s Hospital Boston, Dept. of Laboratory Medicine, 300 Longwood Ave., BA 760, Boston, MA 02115 (e-mail: alicia.rivera{at}childrens.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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