Interaction of HK and LK Goat Red Blood Cells with Ouabain

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (K_c) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low K_c cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal K_c, it does not alter the apparent number of sites in LK cells when K_c has been reduced.     

The effect of anti-L on ouabain binding to sheep erythrocytes.

Binding of 3H-ouabain was studied in high potassium (HK) and low potassium (LK) sheep red cells. In particular, we investigated the effect of anti-L, an antibody raised in HK sheep against L-positive LK sheep red cells, on 3H-oubain binding and its relation to K+ -pump flux inhibition in LK cells. HK cells were found to have about twice as many 3H-ouabain binding sites and a higher association rate for 3H-ouabain than homozygous LL-type LK cells. The number of 3H-ouabain molecules bound to heterozygous LM-type LK cells is lower than that on LL cells, but the rate of ouabain binding is between that of HK and LL red cells. A close correlation was observed between the rates of 3H-oubain binding and fraction K+-pump inhibition. Exposure of LM and LL cells to anti-L did not affect the number of 3H-ouabain molecules bound at saturation, but increased the rates of glycoside binding and K+ -pump inhibition proportionately, so that for LK cells in the presence of anti-L, the rates of the two processes approximate those of HK cells. These data exclude the possibility that anti-L generates entirely new pump sites in LK sheep cells, but suggest that the antibody increases the affinity of the existing -a+ -K+ pumps for the glycoside.     

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.     

Passive potassium transport in LK sheep red cells. Effects of anti-L antibody and intracellular potassium

The passive K influx in low K(LK) red blood cells of sheep saturates with increasing external K concentration, indicating that this mode of transport is mediated by membrane-associated sites. The passive K influx, iMLK, is inhibited by external Na. Isoimmune anti-L serum, known to stimulate active K transport in LK sheep red cells, inhibits iMLK about twofold. iMLK is affected by changes in intracellular K concentration, [K]i, in a complex fashion: increasing [K]i from near zero stimulates iMLK, while further increases in [K]i, above 3 mmol/liter cells, inhibit iMLK. The passive K influx is not mediated by K-K exchange diffusion. The effects of anti-L antibody and [K]i on passive cation transport are specific for K: neither factor affects passive Na transport. The common characteristics of passive and active K influx suggest that iMLK is mediated by inactive Na-K pump sites, and that the inability to translocate Na characterizes the inactive pumps. Anti-L antibody stimulates the K pump in reticulocytes of LK sheep. However, anti-L has no effect on iMLK in these cells, apparently because reticulocytes do not have the inactive pump sites which, in mature LK cells, are a consequence of the process of maturation of circulating LK cells. The results also indicate that anti-L alters the maximum velocity of both active and passive K fluxes by converting pumps sites from a form mediating passive K influx to an actively transporting form.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells.

Binding of highly purified 125I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75-100 and 500-750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3-6 x 10(3) in HK (MM) and 1.5-4 x 10(3) in LK (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2-1.8 x 10(3) in LL and less in LM(LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na+K+ pumps measured previously in these cells by 3H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na+K+ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and /or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics, which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na+K+ pump molecules is more complex than that in goat red cells previously reported by others.     

Antibody-Induced Alterations in the Kinetic Characteristics of the Na:K Pump in Goat Red Blood Cells

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K_c), increasing K_c at first stimulates the active K influx in HK cells, but at higher K_c the pump is inhibited. These results suggest that in HK cells K_c acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na_c) at the Na translocation sites. In LK cells, K_c inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K_c. The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K_c LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

Active Cation Transport and Ouabain Binding in High Potassium and Low Potassium Red Blood Cells of Sheep

Red cells from high K sheep contained 82 mM K/liter cells and had a pump flux of 0.86 mM K/liter cells x hr; similarly, LK cells had 16.5 mM K/liter cells and a pump flux of 0.12 mM K/liter cells x hr. Using [³H]-ouabain, the relation between the number of ouabain molecules bound per cell and the concomitant per cent inhibition of the pump was found to be approximately linear for both HK and LK cells. The number of glycoside molecules necessary for 100 % inhibition of the pump was 42 for HK cells and 7.6 for LK cells, after correction for six nonspecific binding sites for each type of cell. The ratio of ouabain molecules/cell at 100 % inhibition was 5.5, HK to LK, and the ratio of the normal K pump fluxes was 7.2, HK to LK. The similarity of these ratios suggests that an important difference between HK and LK cells, determining the difference in pump fluxes, is the number of pump sites. The turnover times (ions/site x min) are 6000 and 4800 for HK and LK cells, respectively. The results also indicate a high specificity of binding of ouabain to pump sites.     

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.     

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.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells

Summary Binding of highly purified¹²⁵I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75–100 and 500–750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3–6×10³ in HK (MM) and 1.5–4×10³ in LM (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2–1.8×10³ in LL and less in LM (LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na⁺K⁺ pumps measured previously in these cells by³H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na⁺K⁺ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and/or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na⁺K⁺ pump molecules is more complex than that in goat red cells previously reported by others.     

L-antigen and active potassium transport in HK and LK red cells of Barbary sheep (Ammotragus lervia)

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.     

Enzymatic modification of the L and M antigens in LK and HK sheep erythrocytes and their membranes

Summary This paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K⁺-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K⁺-pump stimulating antibody present in the anti-L serum.     

Different red cell populations in newborn, genetically low potassium sheep: relation to hematopoietic, immunologic and physiologic differentiation

Three red cell populations have been distinguished in genotypically low potassium (LK) newborn sheep by an improved electrical sizing method and were best approximated by a logarithmic normal distribution. Labeling studies with ⁵¹Cr and ⁵⁹Fe exclude transformation of the three red cell populations into each other. Population I, consisting of large red cells (mean volume 36 μm³), with a comparatively slow electrophoretic mobility is present at birth and disappears within three to four weeks from circulation. These cells possess a high potassium (HK) steady state concentration, a K⁺ pump influx activity at least 5-fold greater than observed in adult LK red cells, very low amounts of the L antigens generally associated with the LK property, and do not respond to the stimulatory action of the L antibody. The first population is gradually replaced by population II comprising small red cells (mean volume 28 μm³) of intermediate electrophoretic mobility and with a peak production around day 20 after birth. The potassium concentration, [K⁺]_c, in these cells appears to be lower than in the cells of population I but the L antigen content is increased. Formation of population III (mean volume 30 μm³ and comparatively fast electrophoretic mobility) follows closely that of population II and is preceded by a sharp increase in reticulocytosis. The red cells of population III exhibit parameters characteristic for adult LK cells: low [K⁺]_c and K⁺ pump activity, fully developed L antigen content, and an almost maximal response to the K⁺ pump stimulating effect of anti-L. In L and M antigen positive LK red cells of newborn sheep, the development of the M antigen parallels that of the L antigen. The data are consistent with the hypothesis that cellular replacement and not maturation is the major factor in controlling the HK-LK transition in newborn sheep.     

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.     

Cation transport in different volume populations of genetically low K+ lamb red cells

During the first three months after birth lambs produce sequentially three erthryocyte populations of different mean volume as demonstrated by electric sizing methods (Valet, Franz, and Lauf, J. Cell. Physiol. 94 (1978) 215). We separated by centrifugal elutriation the small volume population (type II) red cells of a genotypically low K⁺ (LK) lamb from the population containing the larger volume type I and III cells, an admixture of fetal (I) and adult (III) erythrocytes. The cells were separated at various time intervals after birth and analyzed with respect to their volumes, cation contents, and cation flux properties by means of ⁸⁶Rb uptake. The effect of anti-L on K⁺ pump and leak fluxes was ascertained in unseparated and separated red cells. It was found that the small red cells of population II, transiently present for several weeks, were fully developed LK cells with K⁺ pumps responding characteristically to the stimulatory action of anti-L. In constrast, the larger cells of population I and III were of high K⁺ (HK) nature at early time points, the K⁺ pump activities approximately ten times higher than adult LK cells. These cells constitute an admixture of type I fetal HK cells, and type III reticulocytes which are precursors for the final type III adult LK cells, since anti-L had a small stimulatory effect. At later times, however, only adult type III LK cells predominated. The data directly support our earlier finding that the HK-LK transition in genotypically LK lambs is primarily governed by cellular replacement.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

On the mechanism of stimulation of the Na/K pump of LK sheep erythrocytes by anti-L antibody

Studies were undertaken to explore the mechanism of stimulation of the Na/K pump in LK sheep erythrocytes by anti-L antibody. First, the numbers of functioning pump sites were determined by correlating [3H]ouabain binding with levels of inhibition of the pump. Untreated (control) cells had approximately 41 pumps per cell, and anti-L treatment caused an increase in the number of functioning pumps to approximately 85 per cell. Reducing the intracellular K concentration, [K]c, to near zero caused an increase in the number of pumps in control cells, but not in anti-L cells, such that the numbers of pumps per cell were about the same in the two cell types. These results led to the prediction that Kc is a noncompetitive inhibitor of the pump in control cells, and that anti-L stimulates the pump and increases number of functioning pumps by reducing noncompetitive inhibition by Kc. Kinetic studies were undertaken to test this prediction: activation of the pump by increasing [Na]c was measured at three fixed levels of [K]c. In control cells, the apparent maximum velocity of the pump (J'max) was reduced approximately threefold by raising [K]c from 0.2 to 9 mmol/liter cells, demonstrating noncompetitive inhibition by Kc. In anti-L cells, J'max did not vary with [K]c, which shows that, as predicted, anti-L abolishes the noncompetitive inhibition by Kc. The modification of the kinetic properties of the pumps by the antibody is highly specific in that affinities for Nac and Ko as substrates are unaffected. However, the effect of the antibody on noncompetitive inhibition by Kc does not explain the stimulation of the pump fully since there is significant stimulation at near-zero [K]c.     

The number of Na:K pump sites on red blood cells from HK and LK lambs

Lambs of known genotype with respect to the locus determining cation composition of red cells were obtained by selective matings. Numbers of K⁺ pump sites per cell were determined on HK and LK lambs 10–20 days postnatal by simultaneously determining [³H]ouabain binding and inhibition of active K⁺ transport. Red cells from HK lambs were indistinguishable from adult HK cells with regard to the K⁺ pump flux and number of pump sites. Cells from genetically LK lambs had pump fluxes and numbers of pump sites intermediate between those from adult HK and LK sheep. The results suggest that the change in cation composition and in the K⁺ pump during the first 60 days in genetically LK lambs can be correlated with a reduced number of K⁺ pump sites.     

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.