Regulation of Cell Volume by Active Cation Transport in High and Low Potassium Sheep Red Cells

A model cell which controls its cation composition and volume by the action of a K-Na exchange pump and leaks for both ions working in parallel is presented. Equations are formulated which describe the behavior of this model in terms of three membrane parameters. From these equations and the steady state concentrations of Na, K, and Cl, values for these parameters in high potassium (HK) and low potassium (LK) sheep red cells are calculated. Kinetic experiments designed to measure the membrane parameters directly in the two types of sheep red cells are also reported. The values of the parameters obtained in these experiments agreed well with those calculated from the steady state concentrations of ions and the theoretical equations. It is concluded that both HK and LK sheep red cells control their cation composition and volume in a manner consistent with the model cell. Both have a cation pump which exchanges one sodium ion from inside the cell with one potassium ion from outside the cell but the pump is working approximately four times faster in the HK cell. The characteristics of the cation leak in the two cell types are also very different since the HK cells are relatively more leaky to sodium as compared with potassium than is the case in the LK cells. Both cell types show appreciable sodium exchange diffusion but this process is more rapid in the LK than in the HK cells.     

Delayed haemolytic action of palytoxin. General characteristics

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as hog greater than or equal to rat, mouse greater than rabbit greater than guinea-pig greater than man. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1--2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42 degrees C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED50 of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.     

Refractoriness of cation transport in turkey erythrocytes to stimulation by cyclic adenosine 3':5'-monophosphate.

In turkey erythrocytes bidirectional fluxes of sodium and potassium develop a time-dependent refractoriness to stimulation by endogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP). The refractoriness of potassium influx and potassium outflux (both of which require extracellular sodium and potassium for stimulation by cyclic AMP) depends on the extracellular concentrations of sodium and potassium. In contrast, the refractoriness developed by sodium outflux (which does not require extracellular sodium or potassium for stimulation by cyclic AMP) does not depend on the extracellular concentrations of sodium or potassium. The refractoriness of these fluxes to cellular cyclic AMP reflects a decrease in the amount by which they can be maximally stimulated and appears to be proportional to the extent to which the transport system is utilized during the course of the incubation. Ouabain significantly reduces the rate at which cation transport in turkey erythrocytes becomes refractory to endogenous cyclic AMP. This effect of the glycoside is independent of the extracellular concentrations of sodium or potassium and does not correlate with how it alters the initial response of the transport systems to cyclic AMP.     

LK sheep reticulocytosis: effect of anti-L on K influx and in vitro maturation

After massive hemorrhage, adult sheep with genotypically low potassium (LK) red cells temporarily produce high potassium (HK) cells with ouabain-sensitive K+ pump fluxes equivalent to mature HK red cells. In light of recent reports of different red cell volume populations accompanying the HK-LK transition also occurring in newborn LK sheep and the unresolved controversy over the effect of anti-L on K+ transport in these immature red cells, we have reexamined the K+ transport changes and the effect of anti-L in the newly formed HK cells at various times after anemic stress and under in vitro conditions. We found that approximately 7 d after bleeding, maximum reticulocytosis occurred in the peripheral blood. After separation by density centrifugation, the top 10% cell fraction contained 100% reticulocytes, with a mean cell volume 2.5 times larger than that of mature erythrocytes. These immature red cells were of HK type, and their K+ pump and leak fluxes were 30 and 10 times higher, respectively, than those found in mature LK cells. The new cells may possess HK- and LK- type pumps because K+ pump influx was significantly stimulated by anti- L. When separated by density centrifugation on days 9, 17, and 23 after bleeding, some of the cells apparently maintained their large size while gaining higher density. Large cells from day 9, kept in vitro for 22 h, showed anti-L-sensitive K+ pump and leak fluxes that declined within hours, paralleling the behavior of these cells in vivo, whereas cellular K+ levels changed much less. It is concluded that the newly formed red cells may belong to a stress-induced macrocytic cell population that does not acquire all of the characteristics of adult LK cells.     

Delayed haemolytic action of palytoxin general characteristics

1. Palytoxin is a haemolysin. The erythrocytes from various species can be classified into a sensitive and a hardly sensitive group. The former contain potassium as their main inside cation and are arranged according to their sensitivity as $\text{hog}\text{?}\text{rat, mouse}\text{>}\text{rabbit}\text{>}\text{guinea-pig}\text{>}\text{man}$. The latter, comprising those from sheep and cattle, have sodium as their main inside cation. In addition, chicken erythrocytes are relatively insensitive. 2. Haemolysis of rat erythrocytes is preceded by a lag period of 1 – 2 h. With increasing temperature the haemolysis proceeds more quickly but reaches the same final range between 25 and 42°C. The pH optimum in Britton-Robinson buffer supplemented with saline is between 7 and 8. Washing off palytoxin during the prelytic period reduces the haemolytic power. 3. The sensitivity of rat erythrocytes decreases with increase of osmolarity between 235 and 415 mosM. Accordingly, their osmotic resistance is lowered by palytoxin in a concentration-dependent manner. 4. With both rat and sheep erythrocytes, potassium loss by far precedes the haemolysis due to palytoxin. Potassium loss is measurable already after 1 min and increases with time. After 2 hours the quotient between the ED₅₀ of haemolysis and that of potassium loss is around 200. Thus palytoxin is an unusually strong but slow haemolysin of the osmotic type. The extreme prelytic potassium loss and the correlation between susceptibility and potassium content of erythrocytes points towards the relevance of ionic fluxes.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

The Response of Duck Erythrocytes to Nonhemolytic Hypotonic Media : Evidence for a volume-controlling mechanism

Duck erythrocytes were incubated in hypotonic media at tonicities which do not produce hemolysis. The cells'' response can be divided into two phases: an initial rapid phase of osmotic swelling and a second more prolonged phase (volume regulatory phase) in which the cells shrink until they approach their initial isotonic volume. Shrinkage associated with the volume regulatory phase is the consequence of a nearly isosmotic loss of KCl and water from the cell. The potassium loss results from a transient increase in K efflux. There is also a small reduction in Na permeability. Changes in cell size during the volume regulatory phase are not altered by 10^-4 M ouabain although this concentration of ouabain does change the cellular cation content. The over-all response of duck erythrocytes is considered as an example of "isosmotic intracellular regulation," a term used to describe a form of volume regulation common to euryhaline invertebrates which is achieved by adjusting the number of effective intracellular osmotic particles. The volume regulatory phase is discussed as the product of a membrane mechanism which is sensitive to some parameter associated with cell volume and is capable of regulating the loss of potassium from the cell. This mechanism is able to regulate cell size when the Na-K exchange, ouabain-inhibitable pump mechanism is blocked.     

Functional Separation of the Na-K Exchange Pump from the Volume Controlling Mechanism in Enlarged Duck Red Cells

Previous publications have described a "volume controlling mechanism" in duck erythrocytes that returns both enlarged and shrunken cells to their original isotonic volume. Enlarged cells return to their original size by readjusting their K content. To study the specificity of this aspect of the mechanism for K, we prepared enlarged cells with various Na and K contents. Only cells containing a high K content resume their original size in the standard isotonic medium. The process of regulation resembles that described above. In contrast, cells containing a high Na content fail to reestablish this volume, but shrink instead until they reach a limiting minimal volume (four-fifths of normal). Here, another mechanism, the cation pump rather than the volume controlling mechanism, removes Na and is responsible for the changes in cell size. Enlarged cells with an intermediate Na and K content utilize both mechanisms to reduce their cation content. Only if Na is prevented from leaving the cell and sufficient K is present initially, will these cells reestablish their original size. These studies demonstrate that the cation pump and volume controlling mechanism function independently and, when cells enlarge, only K can effectively traverse the pathway associated with the volume controlling mechanism. This route differs from the one used by the cation pump to eject Na.     

The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.     

Development of hormonal regulation of ion transport in embryonic chick red cells

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8- or 9-day embryos are stimulated little or not at all. The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport. The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na-K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na-K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain-sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.     

Effect of lysophosphatidylcholine on salt permeability through the erythrocyte membrane under haemolytic conditions

Human erythrocytes were incubated in haemolytic salt or sucrose media and the amount of potassium and haemoglobin released were monitored. In hypotonic NaCl and KCl solutions potassium release and haemolysis increased with time showing that the cell membrane had been injured and became permeable to intra- and extracellular cations which, due to intracellular haemoglobin, causes water influx and continuous haemolysis. Both potassium release and haemolysis remained, however, at their 2-minute level in the presence of LPC. Thus, LPC could reseal the membrane and prevent continuous salt fluxes. It protected erythrocytes from hypotonic haemolysis and the protection was more efficient in NaCl than in sucrose media. This suggests that the increase in the critical volume of erythrocytes caused by LPC occurs both in electrolyte and sucrose media, and the additional protection observed in electrolyte media is due to the resealing of the injured cell membrane by LPC. The repairing mechanism was mediated via the membrane lipids or integral proteins, since the time-course of haemolysis of erythrocytes swollen in NaCl media at the spectrin-denaturing temperature of 49.5 degrees C was similar to that at room temperature with and without LPC. LPC did not protect erythrocytes from colloid osmotic haemolysis caused by ammonia influx in an isotonic NH4Cl medium, but protected the cells from colloid osmotic haemolysis caused by sodium influx through nystatin-channels in NaCl media without any area or volume increase. Hence, LPC could not prevent ammonia influx through the lipid bilayer, but suppressed sodium influx through nystatin-channels presumably via LPC interference with cholesterol.     

Furosemide-sensitive sodium and potassium fluxes in human neonatal erythrocytes

During the course of incubation, in vitro, in a saline medium, we found an increase of cell volume and Na+ content in human neonatal erythrocytes (NNE), from the umbilical cord. The increased cell volume was dependent on the major anion in the medium in that replacement of Cl- by NO-3 abolished the cell volume increase. In erythrocytes from adults neither the cell volume nor the sodium content were altered under similar incubation conditions. Furosemide-sensitive Na+ and K+ fluxes were at variance from those reported from adult erythrocytes. The differences here presented between both cell types would be another instance of changes observed to occur in erythrocytes during the postnatal period.     

Evidence for chloride dependent potassium and water transport induced by hyposmotic stress in erythrocytes of the marine teleost,Opsanus tau

Summary Red blood cells of the marine teleost,Opsanus tau (oyster toadfish), were characterized as to their normal hemoglobin, ion and water contents. Cells were exposed to ouabain containing, hyposmotic salt solutions (osmolarity reduced to 2/3 of normal) in which the cation or anion composition was varied. It was found that the initial cell volume expansion due to water influx was independent of the anion present. However, a secondary volume reduction was dependent on the presence of chloride or bromide anions. During volume reduction, cellular potassium and chloride ion contents fell by about equal amounts. Potassium loss was commensurate to the total amount of potassium ions detected extracellularly about 1.5h after the initial osmotic shock. No major changes were seen in the cellular sodium ion contents. When chloride ions within the cells and in the suspending medium were replaced by nitrate, iodide or thiocyanate, the cells failed to return to volumes close to those of isosmotically suspended controls, and the cellular potassium content also remained constant. In hypotonic potassium chloride the cells failed to extrude potassium chloride and water, and hence retained their expanded volume. Neither potassium loss nor volume decrease occurred in cells swollen in hypotonic sodium chloride media containing furosemide or 4,4 diisothiocyano-2,2-stilbene-disulfonic acid (DIDS). These two compounds are known inhibitors of monovalent cation cotransport and anion self exchange, respectively, in mammalian red cells. Hence toadfish red cells respond to osmotic swelling primarily by activation of an ouabain-insensitive, chloride dependent potassium transport system which is sensitive to inhibition by furosemide and DIDS.     

Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes

Summary

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptideinduced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.     

Membrane Cholesterol Depletion and K+ Transport in High and Low Potassium Sheep Red Cells

The effect of cholesterol depletion on potassium tracer fluxes was studied in sheep red cells. Removal by the plasma incubation method (5, 12, 30) of approximately 31 and 34% membrane cholesterol from high-potassium (HK) and low-potassium (LK) sheep red cells, respectively, did not induce significant changes in the steady-state cation composition of these cells nor in their passive (leak) and active (pump) K⁺ influxes. In cholesterol-depleted LK sheep red cells, there was no impairment nor augmentation of the L_p an tibody stimulated K⁺ pump flux and L₁-antibody-mediated reduction of K⁺ leak flux indicating that the removed cholesterol does not contribute to the activity of the L_p and L₁ antigens.     

Potassium release from erythrocytes of different age rats in 9 simulated lactic acidosis

The methods are described for determination of potassium content in erythrocytes by means of a K+ selective electrode, for evaluation of changes in potassium permeability through their membranes and for detection of the Na+, K+-ATPase contribution to spontaneous and induced cation release from cells. Preincubation of erythrocytes with 5 mM lactate has no effect on the potassium release rate and content in cells of young and adult rats. Under the effect of 25 mM lactate the rate of K+ release from erythrocytes of adult animals lowers and sensitivity to ouabaine of this process stimulated by valinomycin increases. The latter effect is less pronounced in erythrocytes of young rats and is accompanied by a rise in potassium total content in cells.     

Uphill and downhill water transport in rat glioma cells

The cell water content determines the cell volume, which in turn controls numerous cellular functions. The mean volume of rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two types of isotonic solutions were used containing either high or low concentrations of NaCl, KCl or N-methylglucamineCl. In low salt solutions, osmolarity was maintained constant by the addition of sucrose or mannitol. Anisotonicity was induced by changing the concentration of electrolytes. As expected, the cell volume increased when the concentration of electrolytes was decreased from a high (165 mM) monovalent cation concentration. In contrast, the cell volume decreased when the concentration of electrolytes was decreased from a low (85 mM) monovalent cation concentration. Reciprocally and unexpectedly, the cell volume increased during a hyperosmotic challenge when the initial cation concentration was low, whereas it decreased when the initial cation concentration was high. These opposite volume changes observed during similar anisotonic challenges but starting from different electrolyte concentrations provide the first evidence that H2O is not only passively transported (downhill) through aquaporins but also follows ion fluxes (uphill).     

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.     

Phorbol esters and gene expression: the role of rapid changes in K+ transport in the induction of ornithine decarboxylase by 12-O- tetradecanoylphorbol-13-acetate in BALB/c 3T3 cells and a mutant cell line defective in Na+K+Cl- cotransport

A BALB/c 3T3 preadipose cell line defective in Na+K+Cl- cotransport (3T3-E12a cells) has been used to study the relationship between phorbol ester-induced rapid changes in cation fluxes and changes in expression of a gene known to be modulated by this agent. In contrast to its effect on parental 3T3 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) did not inhibit either furosemide-sensitive 86Rb+ influx or the rate of 86Rb+ efflux from preloaded mutant cells. TPA-induced changes in intracellular K+ content were diminished in 3T3-E12a cells as compared with parental cells. Thus, mutation of the Na+K+Cl- cotransport system renders overall potassium transport in mutant cells largely insensitive to modulation by TPA. The morphological and functional responses of 3T3 and 3T3-E12a cells to TPA were also compared. In contrast to the extensive and long-lasting changes in morphology of 3T3 cells after 0.16 microM TPA addition, only slight and shorter-lived morphological effects of TPA were observed in 3T3-E12a cells. The transport properties of mutant cells were not totally unresponsive to TPA since hexose transport (2-deoxyglucose uptake) could be stimulated in both cell types. To establish a possible link between early changes in cation fluxes and activation of gene expression by TPA, the induction of the enzyme ornithine decarboxylase (ODC) was studied in detail. Addition of fresh medium containing serum or exposure to hypoosmotic conditions resulted in the induction of ODC in both 3T3 and 3T3-E12a cells. However, TPA failed to cause an increase in ODC activity in mutant cells, although a substantial induction of the enzyme was seen in parental cells. These results suggest that rapid changes in ion fluxes mediated by the Na+K+Cl- cotransport system are necessary for at least one of the phorbol ester-induced changes in gene expression in responsive cells.     

The M-L blood group system and active potassium transport in sheep reticulocytes

Immature and mature red cells from anaemic homozygous and heterozygous low potassium (LK) type sheep were tested for the presence of the L antigen and for active potassium uptake. Evidence was obtained for the presence of L antigen on immature as well as mature cells, but immature cells had a higher intracellular potassium concentration and increased rate of active potassium transport which was only slightly enhanced after sensitization with L antiserum. The red cells which entered the circulation in the later phases of recovery from anaemia were not haemolysed by anti-L as readily as normal cells, but showed normal haemolytic reactions with other blood typing reagents.
It is suggested that, if the L antigen is directly concerned with suppressing part of the potassium pump in mature LK erythrocytes, its effect must be mediated by changes which occur during the final maturation processes of the red cell.