Intracellular potassium promotes antibody binding to an antigen associated with the Na/K pump of sheep erythrocytes

Anti-Lp antibody is known to bind to sheep red cells and to stimulate the Na/K pump. The antibody acts by reducing the affinity of the pumps for intracellular K as a noncompetitive inhibitor. We now show that intracellular K enhances the extent of anti-Lp binding to the pump-associated antigens. Cells made with approximately 60 mmol/1 K bound approximately 60% more anti-Lp than cells with O K; binding was assayed by measuring the extent of stimulation of the pump-mediated K influx.     

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.     

K-Cl cotransport in LK sheep erythrocytes: Kinetics of stimulation by cell swelling

Summary The effects of osmotic cell swelling were studied on the kinetics of Cl-dependent K⁺ influx, K–Cl cotransport, in erythrocytes from sheep of the low K⁺ (LK) phenotype. Swelling 25% stimulated transport by increasing maximum velocity (J _max) 1.5-fold and by increasing apparent affinity for external K (K _o ) nearly twofold. Dithiothreitol (DTT) was shown to be a partial, reversible inhibitor of K–Cl cotransport. It inhibited in cells of normal volume by reducingJ _max more than twofold: apparent affinity for K _o was increased by DTT, suggesting that DTT stabilizes the transporter-K _o complex. Cell swelling reduced the extent of inhibition by DTT:J _max was inhibited by only about one-third in swollen cells, and apparent affinity was only slightly affected. This result suggested that DTT does not act directly on the transporter, but on a hypothetical regulator, an endogenous inhibitor. Swelling relieves inhibition by the regulator, and reduces the effect of DTT. Reducing intracellular Mg²⁺, Mg _o , stimulated cotransport. Swelling of low-Mg²⁺ cells stimulated transport further, but only by raising apparent affinity for K _o nearly threefold:J _max was unaffected. Thus effects of swelling onJ _max and apparent affinity are separable processes. The inhibitory effects of Mg _o and DTT were shown to be additive, indicating separate modes of action. There appear to be two endogenous inhibitors: the hypothetical regulator, which holds affinity for K _o , low; and Mg _o , which affectsJ _max perhaps by holding some transporters in an inactive form. Swelling stimulates transport by relieving both types of inhibition.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.     

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.     

Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes

The activity of the ouabain-insensitive Na⁺/Na⁺ exchange system was assessed by measurements of Li⁺ net-uptake in LK and HK sheep erythrocytes in the absence and presence of the L-antibody and various inhibitors. N-ethylmaleimide, p-chloromercuribenzoic sulfonate and phloretin inhibited the exchange by about 50%. Anti-L, while stimulating the K⁺ pump flux in LK cells, did not alter Na⁺/Li⁺ countertransport. The activity of the exchange system with fully saturated internal and external loading sites was estimated to be identical in LK and HK sheep red cells. Hence the Na⁺/Na⁺ exchange system seems to be molecularly unrelated to the ouabain-sensitive Na⁺K⁺ pump in these cells and not under genetic control of the HK/LK and M/L genes.     

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.     

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.     

Charge movement by the Na/K pump in Xenopus oocytes

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage- clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage- independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three- state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.     

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.     

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.     

The polyene antibiotic amphotericin B inhibits the Na+/K+ pump of human erythrocytes

The polyene antibiotic Amphotericin B is known to induce K+ loss from human erythrocytes. In the present study it is shown that this efflux is not solely due to the formation of pores through the membrane but also to the inhibition of the Na+/K+ pump. At 5 microM this inhibition is total. The interaction of Amphotericin B with membrane enzymes is therefore to be taken into consideration when trying to explain its mechanism of action.     

Synchronization of Na/K pump molecules by an oscillating electric field

Synchronization of the Na/K pump molecules in a cell membrane was studied in frog skeletal muscle fibers using double Vaseline-gap voltage-clamp techniques. We found that the pumping rate of naturally random-paced pump molecules can be artificially synchronized by a pulsed, symmetric, oscillating membrane potential with a frequency comparable to the physiological turnover rate. The synchronized pump currents show separated outward and inward components, where the magnitude of the outward component is about three times the randomly-paced pump currents, and the magnitude-ratio of the outward to inward pump currents is close to 3:2, which reflects the stoichiometric ratio of the pump molecules. Once synchronized, the pumping rate is restricted to the field frequency, and the pump currents are mainly dependent on the field frequency, but not the field strength. In contrast to previous work, which by restraining the pumps at a presteady state succeeded in triggering the steps of the pump cycle only individually and between interruptions, here we synchronize the pumps running continuously and in a normal running mode.     

Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process

K-Cl cotransport in LK sheep erythrocytes is activated by osmotic swelling and inhibited by shrinkage. The mechanism by which changes in cell volume are transduced into changes in transport was investigated by measuring time courses of changes in transport after osmotic challenges in cells with normal and reduced Mg concentrations. When cells of normal volume and normal Mg are swollen, there is a delay of 10 min or more before the final steady-state flux is achieved, as there is for swelling activation of K-Cl cotransport in erythrocytes of other species. The delay was shown to be independent of the extent of swelling. There was also a delay after shrinkage inactivation of cotransport. Reducing cellular Mg concentration activates cotransport. Swelling of low-Mg cells activates cotransport further, but with no measurable delay. In contrast, there is a delay in shrinkage inactivation of cotransport in low-Mg cells. The results are interpreted in terms of a three-state model: [formula see text] in which A state, B state, and C state transporters have relatively slow, intermediate, and fast transport rates, respectively. Most transporters in shrunken cells with normal Mg are in the A state. Swelling converts transporters to the B state in the rate-limiting process, followed by rapid conversion to the C state. Reducing cell Mg also promotes the A-- >B conversion. Swelling of low-Mg cells activates transport rapidly because of the initial predominance of B state transporters. The results support the following conclusions about the rate constants of the three-state model: k21 is the rate constant for a Mg-promoted process that is inhibited by swelling; k12 is not volume sensitive. Both k23 and k32 are increased by swelling and reduced by shrinkage; they are rate constants for a single process, whereas k12 and k21 are rate constants for separate processes. Finally, the A-->B conversion entails an increase in Jmax of the transporters, and the B-->C conversion entails an increase in the affinity of the transporters for K.     

On the Na+,K+ pump in fluctuating membrane potentials

The present work investigates the usefulness of noise in the activity of the Na+,K+ pump. Random gating activity of the neighboring ion channels causes local fluctuations of the electric potential. They are modeled by a Markovian symmetric dichotomic noise, added to the membrane potential. The noise-averaged pump current is calculated for a general rectangular voltage signal and the model parameters of the effective two-state enzyme cycle are tuned to fit experimental results. Then, using these parameters, the amount of transported charge is calculated, and studied as a function of noise intensity. Signal and noise characteristics are identified at which fluctuations enhance pump activity. The biological impact of this phenomenon seems to be absent in physiological conditions for it occurs at noise amplitudes over 50 mV, which are unlikely to appear due to ion channels. However, under some conditions, externally applied dichotomic noise of intensity about 150 mV may sensibly increase the quantity of transported charge.